Antibacterial effectiveness in vitro of different formulations of calcium hydroxide paste

Eficácia antibacteriana in vitro de diferentes formulações de pastas de hidróxido de cálcio

Israel Alexandre De Araujo SENA Isaac Jordão De Souza ARAÚJO Marquiony Marques Dos SANTOS Isabela Pinheiro Cavalcanti LIMA About the authors

ABSTRACT

Objective:

To evaluate the antibacterial activity of four formulations of calcium hydroxide paste against microorganisms commonly found in infected root canals.

Methods:

To evaluate antibacterial activity through the agar diffusion method, pastes of calcium hydroxide were made from its pro-analysis form, diffused into four separate vehicles: distilled water, camphorated p-monochlorophenol, propylene glycol and Otosporin®, testing the antimicrobial activity of these on strains of Staphylococcus aureus, Bacillus subtilis and Enterococcus faecalis. After the incubation period, the presence or otherwise of inhibition zones were observed and their sizes in three stages: 24h, 48h and 72h. With this data, the median between the four dishes was obtained and the consequent value was submitted to Kruskal-Wallis nonparametric statistical analysis, with post-tests of Mann-Whitney and Bonferroni correction, at a significance level of 5%.

Results:

Only pastes with camphorated p-monochlorophenol and Otosporin vehicles caused the formation of significant inhibition zones, with medians of 8.0 mm. Against the strains of Enterococcus faecalis, only pastes with the camphorated p-monochlorophenol vehicle resulted in the formation of significant inhibition zones, with a median of 3.0 mm.

Conclusion

: Otosporin and CMCP vehicles provide greater antimicrobial potential to calcium hydroxide against the studied bacteria. However, only the Ca(OH)2 and CMCP combination was effective against all the strains, and can thus be regarded as the paste formulation with the greatest antimicrobial effectiveness in this study.

Indexing terms:
Calcium hydroxide; Dental pulp cavity; Dentistry

RESUMO

Objetivo:

Avaliar a ação antibacteriana de quatro formulações de pastas de hidróxido de cálcio contra microrganismos comumente encontrados em canais radiculares infectados.

Métodos:

Para avaliar a ação antibacteriana, através do método de difusão em ágar, foram preparadas pastas de hidróxido de cálcio a partir de sua forma pró-análise dispersa em quatro veículos distintos: água destilada, paramonoclorofenol canforado, propilenoglicol e Otosporin®, testando o potencial antibacteriano destas sobre cepas de Staphylococcus aureus, Bacillus subtilis e Enterococcus faecalis. Após o período de incubação, foi verificada a pre¬sença ou não de halos de inibição e seus respectivos tamanhos em três momentos: 24h, 48h e 72h. Com os dados, foi obtida uma mediana das quatro placas e o valor encontra¬do submetido à análise estatística não paramé¬trica Kruskall-Wallis, com pós-testes de Mann-Whitney e penalização de Bonferroni, ao nível de significância de 5%.

Resultados:

Apenas nas pastas com veículos paramonoclorofenol canforado e Otosporin foi observado a formação de halos de inibição significativos, com medianas de 8,0mm. Sobre as cepas de Enterococcus faecalis, somente a pasta com veículo paramonoclorofenol canforado foi observado formação de halo de inibição significativo, com mediana de 3,0mm.

Conclusão:

Os veículos Otosporin e PMCC possibilitam maior potencial antimicrobiano ao hidróxido de cálcio frente às bactérias estudadas. No entanto, somente a associação Ca(OH)2 e PMCC foi efetiva contra todas as cepas, e assim pode ser considerada como a formulação de pasta de maior efetividade antimicrobiana neste estudo.

Termos de indexação:
Cavidade pulpar; Hidróxido de cálcio; Odontologia

INTRODUCTION

The main aims of endodontic treatment are the elimination of microorganisms and the prevention of reinfection inside the root canal. Due to the complex anatomy of the root canal system and the ability of microorganisms to survive in adverse conditions, resistant microorganisms may remain inside the root canals even after full mechanical endodontic instrumentation and irrigation procedures11 Turk B, Sen B, Ozturk T. In vitro antimicrobial activity of calcium hydroxide mixed with different vehicles against Enterococcus faecalis and Candida albicans. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009;108(2):297-301. doi: 10.1016/j.tripleo.2009.03.029
https://doi.org/10.1016/j.tripleo.2009.0...
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In view of the insufficiency of these procedures in combating all microorganisms present in the infected root canal22 Maia Filho E, Maia C, Bastos A, Novais T. Efeito antimicrobiano in vitro de diferentes medicações endodônticas e própolis sobre Enterococcus faecalis. RGO, Rev Gaúch Odontol. 2008;56(1):21-5., as well as the possibility of bacterial infiltration from temporary restorations, between sessions of endodontic treatment33 Silveira C, Cunha R, Fontana C, Martin A, Gomes B, Motta R, et al. Assessment of the antibacterial activity of calcium hydroxide combined with chlorhexidine paste and other intracanal medications against bacterial pathogens. Eur J Dent. 2011;5 (1):1-7., intracanal medication has been used as an auxiliary resource in endodontic treatment22 Maia Filho E, Maia C, Bastos A, Novais T. Efeito antimicrobiano in vitro de diferentes medicações endodônticas e própolis sobre Enterococcus faecalis. RGO, Rev Gaúch Odontol. 2008;56(1):21-5..

In this regard, several substances have been advocated, with calcium hydroxide (Ca(OH)2) being considered the primary clinical choice44 Kim D, Kim E. Antimicrobial effect of calcium hydroxide as an intracanal medicament in root canal treatment: a literature review - Part I. In vitro studies. Restor Dent Endod. 2014; 39(4):241-52. doi: 10.5395/rde.2015.40.2.97
https://doi.org/10.5395/rde.2015.40.2.97...
. This is due to its antibacterial properties, solvent action on organic materials, inducement to form mineralized tissue55 Garcia LF, Almeida GL, Pires-de-Souza FCP, Consani S. Antimicrobial activity of a calcium hydroxide and Ricinus communis oil paste against microorganisms commonly found in endodontic infections. Rev Odonto Ciênc. 2009;24(4):406-9., alkalizing effect, control of the inflammatory and replacement resorption of the root11 Turk B, Sen B, Ozturk T. In vitro antimicrobial activity of calcium hydroxide mixed with different vehicles against Enterococcus faecalis and Candida albicans. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009;108(2):297-301. doi: 10.1016/j.tripleo.2009.03.029
https://doi.org/10.1016/j.tripleo.2009.0...
, neutralization effect in bacterial lipopolysaccharides (LPS)66 Vianna ME, Zilio DM, Ferraz CCR, Zaia AA, Souza-Filho FJ, Gomes BPFA. Concentration of hydrogen ions in several calcium hydroxide pastes over different periods of time. Braz Dent J. 2009;20(5): 382-388 doi: 10.1590/S0103-64402009000500005
https://doi.org/10.1590/S0103-6440200900...
and, lastly, antifungal action77 Blanscet ML, Tordik PA, Goodell GG. An agar diffusion comparison of the antimicrobial effect calcium hydroxide at five different concentrations with three different vehicles. J Endod. 2008;34(10):1246-8. doi: 10.1016/j.joen.2008.07.012
https://doi.org/10.1016/j.joen.2008.07.0...
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It has been well established that endodontic infections are polymicrobial in nature88 Gangwar A. Antimicrobial effectiveness of different preparations of calcium hydroxide. Indian J Dent Res. 2011;22(1):66-70. doi: 10.4103/0970-9290.79986
https://doi.org/10.4103/0970-9290.79986...
. For some of the bacteria involved in these infections, mainly Enterococcus faecalis99 Mohammadi Z, Dummer PMH. Properties and applications of calcium hydroxide in endodontics and dental traumatology. Int Endod J. 2011;44(8):697-730. doi: 10.1111/j.1365-2591.2011.01886.x
https://doi.org/10.1111/j.1365-2591.2011...
, whose resistance in highly alkaline pH is well established1010 McHugh CP, Zhang P, Michalek S, Eleazer PD. pH Required to Kill Enterococcus faecalis in vitro. J Endod. 2004;30(4):218-9. doi: 10.1097/00004770-200404000-00008
https://doi.org/10.1097/00004770-2004040...
, the effectiveness of calcium hydroxide needs to be enhanced. This can be achieved through the use of particular vehicles in the formulation of calcium hydroxide pastes, which should be capable of improving diffusion through the canals and increasing the antibacterial effect1111 Farhad AR, Barekatain B, Allameh M, Narimani T. Evaluation of the antibacterial effect of calcium hydroxide in combination with three different vehicles: an in vitro study. Dent Res J. 2012;9(2):167-72. doi: 10.4103/1735-3327.95231
https://doi.org/10.4103/1735-3327.95231...
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In view of this, Ca(OH)2 powder has been combined with different vehicles, such as distilled water (DW), saline solution, camphorated p-monochlorophenol (CMCP), chlorhexidine, polyethylene glycol, propylene glycol (PG), Otosporin (O), glycerine1212 Estrela C, Decurcio DA, Alencar AHG, Sydney GB, Silva JA. Efficacy of calcium hydroxide dressing in endodontic infection treatment: a systematic review. Rev Odonto Ciênc. 2008;23(1):82-6., and also with chlorhexidine gel1313 Önçag O, Gogulu D, Uzel A. Efficacy of various intracanal medicaments against Enterococcus faecalis in primary teeth: An in vivo study. J Clin Pediatr Dent. 2006;30(3):233-8. doi: 10.17796/jcpd.30.3.r773j6l674lw0268
https://doi.org/10.17796/jcpd.30.3.r773j...
-1414 Gondim JO, Moreira Neto JJS, Gomes DAS, Azevedo ER, Jeremias F, Giro EMA. In vivo study of an intracanal dressing of calcium hydroxide/chlorhexidine in necrotic primary teeth. Braz J Oral Sci. 2014;13(1):70-5. doi: 10.1590/1677-3225v13n1a14
https://doi.org/10.1590/1677-3225v13n1a1...
, in an attempt to improve its antibacterial activity, biocompatibility, speed of ion dissociation and diffusion1111 Farhad AR, Barekatain B, Allameh M, Narimani T. Evaluation of the antibacterial effect of calcium hydroxide in combination with three different vehicles: an in vitro study. Dent Res J. 2012;9(2):167-72. doi: 10.4103/1735-3327.95231
https://doi.org/10.4103/1735-3327.95231...
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Many studies have compared the effectiveness of calcium hydroxide pastes and the use of various vehicles in several respects, namely: the inhibitory behavior towards bacterial infiltration via the crown1515 Murad C, Fariniuk LF, Fidel S, Fidel RAS, Sassone LM. Bacterial leakage in root canals filled with calcium hydroxide paste associated with different vehicles. Braz Dent J. 2008;19(3):232-7. doi: 10.1590/S0103-64402008000300011
https://doi.org/10.1590/S0103-6440200800...
; the relationship of each vehicle with the potential for the diffusion of the calcium hydroxide paste into the dentin tubules1616 Mori GG, Ferreira FC, Batista FRS, Godoy AMS, Nunes DC. Evaluation of the diffusion capacity of calcium hydroxide pastes through the dentinal tubules. Braz Oral Res. 2009;23(2):113-8. doi: 10.1590/S1806-83242009000200004
https://doi.org/10.1590/S1806-8324200900...
; and the degradation of these pastes over the course of time1717 Gupta S. An in vitro study of diffusibility and degradation of three calcium hydroxide pastes. Int J Clin Pediatr Dent. 2011;4(1):15-23. doi: 10.5005/jp-journals-10005-1075
https://doi.org/10.5005/jp-journals-1000...
that results in a greater or lesser degree of substantivity and effectiveness against endodontic pathogens.

Considering the differences between the types of vehicles used in the formulation of calcium hydroxide pastes, in terms of nature, viscosity, miscibility, differences in the characteristics of these pastes are observed when in contact with the root canal structure. These differences may represent alterations in pH, ion dispersion and even alterations in antimicrobial capacity99 Mohammadi Z, Dummer PMH. Properties and applications of calcium hydroxide in endodontics and dental traumatology. Int Endod J. 2011;44(8):697-730. doi: 10.1111/j.1365-2591.2011.01886.x
https://doi.org/10.1111/j.1365-2591.2011...
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Due to the changes in the antimicrobial potential of some formulations of calcium hydroxide paste against particular bacteria found inside the root canals, the aim of this study was to perform a comparative evaluation of the antibacterial action of four different formulations of calcium hydroxide paste, used as intracanal medication in dentistry, against microorganisms that have been detected in infected root canals.

METHODS

The methodology for this study was adapted from Ganesh et al.1818 Ganesh MR, Chaurasia VR, Masamatti VKS, Mujeeb A, Jhamb A, Agarwal JH. In vitro evaluation of antibacterial efficacy of calcium hydroxide in different vehicles. J Int Soc Prev Community Dent. 2014;4(1):56-60. doi: 10.4103/2231-0762.131268
https://doi.org/10.4103/2231-0762.131268...
, who used the agar diffusion test to determine the antibacterial effectiveness of calcium hydroxide pastes on the strains of bacteria commonly found in infected root canals.

An analysis of the antibacterial action of the tested substances was conducted using the method of agar diffusion in a Brain Heart Infusion (BHI) (DIFCO®, Maryland, USA) culture medium. Pastes were prepared by mixing pro-analysis calcium hydroxide (Inodon Laboratório Industrial de Produtos Odontológicos Ltda., Porto Alegre, Rio Grande do Sul, Brazil) combined with a water-soluble vehicle, distilled water; with an oily vehicle, camphorated p-monochlorophenol (Biodinâmica, Ibiporã, Paraná, Brazil); a viscous vehicle, propylene glycol (Farmafómula® - Farmácia de Manipulação, Caicó, Rio Grande do Norte, Brazil) and Otosporin® (hydrocortisone, neomycin and polymixin B, FQM - Farmoquímica, Rio de Janeiro, RJ, Brazil).

The antibacterial action of the proposed pastes was tested on standard strains obtained from the American Type Culture Collection (ATCC): Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6051 and Enterococcus faecalis ATCC 29212.

The standard strains were initially diluted in 10 ml of distilled water with the help of a sterile platinum strap and test tube together with a Bunsen burner until a turbidity that was visually comparable with standard 0.5 on the McFarland scale was obtained. The strains were then cultivated separately inside the dishes, in which there was 35 ml of BHI culture medium, to a dish depth of 4 mm.

Each of the three strain types was inoculated with the aid of a sterile swab on four dishes containing BHI agar. With the aim of avoiding the formation of isolated colonies, the inoculation was performed in horizontal, vertical and diagonal directions. Following this procedure, on each of the dishes, four depressions were made (diameter of 7 mm and depth of 4 mm) together with the culture medium with the aid of a pre-sterilized glass tube.

Each depression was then uniformly filled with one of the four substances tested, in other words, the four different formulations of calcium hydroxide-based paste were analyzed on the same dish. For each substance, four tests were carried out with each of the cultivated microorganisms.

The tested pastes were handled at the point of use using a sterile glass dish and no. 24 stainless steel spatula. It is worth stressing that all substances were handled with standardized quantities of calcium hydroxide powder (100 mg) and the volume of each vehicle (0.15 ml), such that a paste with a consistency of toothpaste was obtained after mixing, using a precision scale and pipette for the standardization of these values.

With the aid of an insertion spatula, the pastes were placed in each of the wells in quantities sufficient to fill them. After a two-hour wait for the diffusion of the calcium hydroxide paste in the medium to occur, the dishes were kept in an oven for 24 hours at 37°C.

Following this incubation, a check was made of the presence or absence of an inhibition halo with the different paste formulations analyzed, and also the size at three different points in time: 24, 48 and 72 hours. The size of the halo was checked in a horizontal direction, the inhibition zone being considered as the distance, in millimeters, between the edges of the diffusion halo formed by the calcium hydroxide paste and the edge of the inhibition halo arising from the inhibition of bacterial growth caused by the pastes. To perform the measurements, a plastic millimeter ruler was employed.

With the data obtained from the measurement of the inhibition halos, the median value of the four dishes was obtained and the consequent value was then submitted to statistical analysis using the Kruskal-Wallis non-parametric test and Mann-Whitney and Bonferroni correction post-tests, at a level of significance of 5%. The tests were conducted using the software application SPSS® 20.0.

RESULTS

The median values for the inhibition halos, in the different formulations of calcium hydroxide paste, in the samples of the following bacteria: Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6051 and Ente¬rococcus faecalis ATCC 29212 at the different times of analysis, are described in Table 1.

Table 1
Comparison of the median (in millimeters) of the inhibition halo of the different formulations of calcium hydroxide paste in samples of the bacteria Staphylococcus aureus ATCC 25923, Ente¬rococcus faecalis ATCC 29212 and Bacillus subtilis ATCC 6051, Caicó (RN), 2015.

At the second and third moments of analysis, at 48 and 72 hours respectively, it was observed that there was no significant difference in the sizes of the inhibition halos versus the first analysis performed at 24 hours.

A statistically significant difference was observed between the substances evaluated with regard to antibacterial action against the bacteria Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6051 (p<0.05). Of the four substances analyzed, only the calcium hydroxide paste with distilled water and the paste with propylene glycol had not formed an inhibition halo at any of the moments of analysis nor on any of the strains used in this study, whereas the pastes with the vehicles CMCP and Otosporin led to the formation of significant inhibition halos.

The individual susceptibility of microorganisms to calcium hydroxide pastes varied, with Staphylococcus aureus ATCC 25923 being the most susceptible and Enterococcus faecalis ATCC 29212 the most resistant. Only with the calcium hydroxide paste that used CMCP as a vehicle was it possible to see the formation of a significant inhibition halo in the samples with Enterococcus faecalis ATCC 29212 at the different times of analysis.

All of the calcium hydroxide-based paste formulations exhibited diffusion halos, on which there was no bacterial growth, remaining so for the three different moments of analysis.

DISCUSSION

The antibacterial action of the intracanal medications must reach the different types of microorganisms that infect the root canal and also inhibit osteoclastic activity and promote tissue repair. Several substances have been recommended to this end with Ca(OH)2 being the intracanal medication most frequently employed1919 Pacíos MG, Silva C, López ME, Cecilia M. In Antibacterial action of calcium hydroxide vehicles and calcium hydroxide pastes. J Investig Clin Dent. 2012;3(4):264-70. doi: 10.1111/j.2041-1626.2012.00147.x
https://doi.org/10.1111/j.2041-1626.2012...
.

The action of calcium hydroxide is directly influenced by the release of calcium (Ca2+) and hydroxyl (OH-) ions, responsible for the alkalinization of the medium and resulting in a pH above 11. Moreover, this is capable of inactivating the enzymes of the cytoplasmic membrane of the organisms, which chemically alters the organic components and transportation of nutrients, causing toxic effects on the cells66 Vianna ME, Zilio DM, Ferraz CCR, Zaia AA, Souza-Filho FJ, Gomes BPFA. Concentration of hydrogen ions in several calcium hydroxide pastes over different periods of time. Braz Dent J. 2009;20(5): 382-388 doi: 10.1590/S0103-64402009000500005
https://doi.org/10.1590/S0103-6440200900...
,2020 Estrela C, Bammann LL, Pimenta FC, Pécora JD. Control of microorganisms in vitro by calcium hydroxide pastes. Int Endod J. 2001;34:341-5. doi: 10.1046/j.1365-2591.2001.00368.x
https://doi.org/10.1046/j.1365-2591.2001...
. Another action mechanism of this medication is its ability to absorb carbon dioxide (CO2), thereby leading to the death of CO2-dependent bacteria33 Silveira C, Cunha R, Fontana C, Martin A, Gomes B, Motta R, et al. Assessment of the antibacterial activity of calcium hydroxide combined with chlorhexidine paste and other intracanal medications against bacterial pathogens. Eur J Dent. 2011;5 (1):1-7..

Various methods areused to evaluate the antibacterial activity of intracanal medications, the in vitro method having the advantage of ease of execution and speed of results, as well as being free of certain factors that influence them, which are inherent to in vivo studies22 Maia Filho E, Maia C, Bastos A, Novais T. Efeito antimicrobiano in vitro de diferentes medicações endodônticas e própolis sobre Enterococcus faecalis. RGO, Rev Gaúch Odontol. 2008;56(1):21-5.. Of the in vitro preparations, the most common method for evaluating antimicrobial activity is the agar diffusion method88 Gangwar A. Antimicrobial effectiveness of different preparations of calcium hydroxide. Indian J Dent Res. 2011;22(1):66-70. doi: 10.4103/0970-9290.79986
https://doi.org/10.4103/0970-9290.79986...
.

On the other hand, the results of agar diffusion tests have to be carefully examined when using materials such as Ca(OH)2, because the culture medium possesses buffering substances and, even though the calcium hydroxide does diffuse, the magnitude of the pH achieved around the medication may not be sufficient to exert antibacterial activity2121 Dotto SR, Travassos RMC, Ferreira R, Santos R, Wagner M. Avaliação da ação antimicrobiana de diferentes medicações usadas em endodontia. Rev Odonto Ciênc. 2006;21(53):266-9.. Nevertheless, it is important to mention that, a similar effect could be produced by the tissue fluids and dentin in ex-vivo or in vivo conditions11 Turk B, Sen B, Ozturk T. In vitro antimicrobial activity of calcium hydroxide mixed with different vehicles against Enterococcus faecalis and Candida albicans. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009;108(2):297-301. doi: 10.1016/j.tripleo.2009.03.029
https://doi.org/10.1016/j.tripleo.2009.0...
.

Given the low solubility of Ca(OH)2, its inability to diffuse adequately and the fact that it requires aT long period of time to alkalinize the culture medium1919 Pacíos MG, Silva C, López ME, Cecilia M. In Antibacterial action of calcium hydroxide vehicles and calcium hydroxide pastes. J Investig Clin Dent. 2012;3(4):264-70. doi: 10.1111/j.2041-1626.2012.00147.x
https://doi.org/10.1111/j.2041-1626.2012...
, it was decided to keep the dishes at room temperature for 2 hours to allow the diffusion of the medications through the agar, and only afterwards to be incubated under the appropriate conditions.

Due to the difficulty of using calcium hydroxide powder in small or curved canals, it has been used in combination with a liquid vehicle in order to facilitate clinical handling during application88 Gangwar A. Antimicrobial effectiveness of different preparations of calcium hydroxide. Indian J Dent Res. 2011;22(1):66-70. doi: 10.4103/0970-9290.79986
https://doi.org/10.4103/0970-9290.79986...
,2222 Estrela C, Pécora JD, Souza-Neto MD, Estrela CRA, Bammann LL. Effect of vehicle on antimicrobial properties of calcium hydroxide pastes. Braz Dent J. 1999;10(2):63-72.. Moreover, this combination has the objective of improving antibacterial activity and biocompatibility as well as influencing the paste’s pH and viscosity and, therefore, facilitating or inhibiting its ion dispersion1111 Farhad AR, Barekatain B, Allameh M, Narimani T. Evaluation of the antibacterial effect of calcium hydroxide in combination with three different vehicles: an in vitro study. Dent Res J. 2012;9(2):167-72. doi: 10.4103/1735-3327.95231
https://doi.org/10.4103/1735-3327.95231...
,2323 Vianna ME, Gomes BPFA, Sena NT, Zaia AA, Ferraz CCR, Souza-Filho FJ. In vitro evaluation of the susceptibility of endodontic pathogens to calcium hydroxide combined with different vehicles. Braz Dent J. 2005;16(3):175-80. doi: 10.1590/S0103-64402005000300001
https://doi.org/10.1590/S0103-6440200500...
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A number of studies have asserted that the combination of calcium hydroxide with distilled water permits a rapid, efficient dissociation and that paste with propylene glycol has greater viscosity and antimicrobial potential1616 Mori GG, Ferreira FC, Batista FRS, Godoy AMS, Nunes DC. Evaluation of the diffusion capacity of calcium hydroxide pastes through the dentinal tubules. Braz Oral Res. 2009;23(2):113-8. doi: 10.1590/S1806-83242009000200004
https://doi.org/10.1590/S1806-8324200900...
. However, in the present study, pastes that used these substances as a vehicle were unable to produce an inhibition halo in any of the tested strains, corroborating the study by Pacíos et al.1919 Pacíos MG, Silva C, López ME, Cecilia M. In Antibacterial action of calcium hydroxide vehicles and calcium hydroxide pastes. J Investig Clin Dent. 2012;3(4):264-70. doi: 10.1111/j.2041-1626.2012.00147.x
https://doi.org/10.1111/j.2041-1626.2012...
.

The ineffectiveness of calcium hydroxide mixed with these vehicles in vitro may, as already mentioned, also be related, as per Gomes et al.2424 Gomes BP, Ferraz CC, Garrido FD, Rosalen PL, Zaia AA, Teixeira FB, et al. Microbial susceptibility to calcium hydroxide pastes and their vehicles. J Endod. 2002;28(11):758-61. doi: 10.1097/00004770-200211000-00003
https://doi.org/10.1097/00004770-2002110...
, to the fact that the culture medium possesses buffering substances and to the vehicle’s low diffusion capacity. Therefore, although calcium hydroxide may have diffused through the medium, the pH levels attained were not sufficient to present inhibitory activity.

Calcium hydroxide paste with Otosporin exhibited antibacterial activity against Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6051. In concert with our study, Estrela et al.2020 Estrela C, Bammann LL, Pimenta FC, Pécora JD. Control of microorganisms in vitro by calcium hydroxide pastes. Int Endod J. 2001;34:341-5. doi: 10.1046/j.1365-2591.2001.00368.x
https://doi.org/10.1046/j.1365-2591.2001...
demonstrated the antibacterial potential of the combination of Ca(OH)2 and Otosporin on these bacteria via the broth dilution method. Moreover, the literature stresses that the combination of calcium hydroxide and corticosteroids (such as the hydrocortisone present in Otosporin) could act as an enhancer in the reduction of pain and inflammation2525 Tabrizizadeh M, Rasti M, Ayatollahi F, Mossadegh MH, Zandi H, Dehghan F, et al. Antimicrobial activity of calcium hydroxide and Betamethasone on Enterococcus faecalis: An in vitro Assessment. Iran Endod J. 2015;10(3):184-7. doi: 10.7508/iej.2015.03.008
https://doi.org/10.7508/iej.2015.03.008...
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Of all the formulations of calcium hydroxide-based pastes used in this study, only the one using camphorated p-monochlorophenol as the vehicle promoted the formation of inhibition halos against all bacterial strains, thus demonstrating it is the most effective, consistent with other studies in the literature88 Gangwar A. Antimicrobial effectiveness of different preparations of calcium hydroxide. Indian J Dent Res. 2011;22(1):66-70. doi: 10.4103/0970-9290.79986
https://doi.org/10.4103/0970-9290.79986...
,1818 Ganesh MR, Chaurasia VR, Masamatti VKS, Mujeeb A, Jhamb A, Agarwal JH. In vitro evaluation of antibacterial efficacy of calcium hydroxide in different vehicles. J Int Soc Prev Community Dent. 2014;4(1):56-60. doi: 10.4103/2231-0762.131268
https://doi.org/10.4103/2231-0762.131268...
.

Nevertheless, the size of the bacterial inhibition zone may be influenced by the molecular size of the chemical substance, toxicity against the tested bacteria and diffusion. An agent that diffuses more easily will exhibit a larger zone and it has been demonstrated that CMCP diffuses more easily through the agar medium1818 Ganesh MR, Chaurasia VR, Masamatti VKS, Mujeeb A, Jhamb A, Agarwal JH. In vitro evaluation of antibacterial efficacy of calcium hydroxide in different vehicles. J Int Soc Prev Community Dent. 2014;4(1):56-60. doi: 10.4103/2231-0762.131268
https://doi.org/10.4103/2231-0762.131268...
.

Enterococcus faecalis has been linked to persistent endodontic infections, having differential characteristics such as the ability to penetrate into the dentin tubules, growing in an alkaline pH, coping with inanition and resisting the action of antibiotics33 Silveira C, Cunha R, Fontana C, Martin A, Gomes B, Motta R, et al. Assessment of the antibacterial activity of calcium hydroxide combined with chlorhexidine paste and other intracanal medications against bacterial pathogens. Eur J Dent. 2011;5 (1):1-7.,2626 Saha S, Nair R, Asrani H. Comparative evaluation of propolis, metronidazole with chlorhexidine, calcium hydroxide and curcuma longa extract as intracanal medicament against e.faecalis: an in vitro study. J Clin Diagn Res. 2015;9(11): doi: 10.7860/jcdr/2015/14093.6734
https://doi.org/10.7860/jcdr/2015/14093....
-2727 Valera MC, Oliveira SAC, Maekawa LE, Cardoso FGR, Chung A, Silva SFP, et al. Action of chlorhexidine, zingber officinale, and calcium hydroxide on candida albicans, enterococcus faecalis, escherichia coli, and endotoxin in the root canals. J Contemp Dent Pract. 2016;17(2):114-8. doi: 10.5005/jp-journal-10024-1812
https://doi.org/10.5005/jp-journal-10024...
. So, of the pastes tested in our study, only calcium hydroxide paste with the CMCP vehicle was capable of promoting the formation of a significant inhibition halo in the samples with Enterococcus faecalis ATCC 29212, as per the study by Gomes et al.2424 Gomes BP, Ferraz CC, Garrido FD, Rosalen PL, Zaia AA, Teixeira FB, et al. Microbial susceptibility to calcium hydroxide pastes and their vehicles. J Endod. 2002;28(11):758-61. doi: 10.1097/00004770-200211000-00003
https://doi.org/10.1097/00004770-2002110...
.

Despite the low solubility of calcium hydroxide in water thus limiting its diffusibility2121 Dotto SR, Travassos RMC, Ferreira R, Santos R, Wagner M. Avaliação da ação antimicrobiana de diferentes medicações usadas em endodontia. Rev Odonto Ciênc. 2006;21(53):266-9., in our study, the presence of diffusion halos in agar was observed for all paste formulations. Accordingly, it may be assumed that these are capable of killing bacteria through direct contact, which means that the remaining microorganisms in contact with this medication will be eradicated if they are not resistant.

Moreover, as this is an in vitro study, the results must be analyzed with caveats before extrapolating to clinical conditions. Accordingly, the use should also be considered of other research methods, such as in vivo studies, for a better understanding of the antimicrobial action of these calcium hydroxide-based intracanal medications.

Similarly, other analyses, using the same methodology but longer time intervals, may be insightful in the sense of relating the time vs. drug effectiveness curve, which will certainly have largescale clinical implications as a result of the various indications regarding the length of time the intracanal drugs should be applied.

CONCLUSION

Thus, it could be seen from the results of our study that the vehicles Otosporin and CMCP enhance the antimicrobial potential, for the bacteria studied, of calcium hydroxide. Nevertheless, only the combination of Ca(OH)2 with CMCP was effective against all of the strains studied, and thus may be considered as the paste formulation with greatest antimicrobial efficacy given the in vitro study method employed and the substances and strains used.

ACKNOWLEDGMENTS

Our sincere thanks go to the National Council for Scientific and Technological Development (CNPq) for the financial backing for this study, under case reference no. 125219/2014-8, and to the State University of Rio Grande do Norte for allowing us the use of its premises at the Microbiology Laboratory at the Caicó campus.

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Publication Dates

  • Publication in this collection
    Dec 2017

History

  • Received
    10 Nov 2016
  • Reviewed
    10 Jan 2017
  • Accepted
    19 May 2017
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