Antimicrobial activity of common endodontic materials on Enterococcus faecalis NEWP 0012

The elimination of the microorganisms in its totality, during the endodontic therapy, although desired, is hard to be achieved. Even after the canal filling, the microorganisms are able to be into the dentinal mass and into the periapical region. This essay aims to evaluate the antimicrobial activity of endodontic materials against to the Enterecoccus faecalis NEWP 0012. The inoculation of this microorganism in the glass plates was done using swabs pressing the cotton against the wall of the pipe. The bacterial suspension was sowed evenly on the sterile surface of the Ágar MH. The plates were incubated by aerobic conditions, in a constant temperature in the range of 35o a 37°c, for 24 hours. The endodontic materials tested were: Sealer 26, Endofill, AH plus, MTA Filapex, calcium hydroxide paste associated to the chlorhexidine, Maxxion R glass ionomer, MTA and MTA HP. After material manipulation, they were impregnated into absorbent paper discs of 5mm o f diameter and distributed sporadically on the plates that contained the microorganism. The experimental data were obtained by the measurement of the inhibition halo in the period of 24h, 72h, 7, 15 and 21 days; thus, the microorganism was classified as resistant or sensible to the different products. From the results, it was possible to verify that the calcium hydroxide paste associated to the chlorhexidine presented superior antimicrobial activity on Enterecoccus faecalis NEWP 0012. It was concluded that the calcium hydroxide paste associated to the chlorhexidine has satisfactory antimicrobial activity against the Enterecoccus faecalis NEWP 0012 in the period between 24h to 21 days. Keywords— Calcium hydroxide, Endodontics,


INTRODUCTION
The most common cause of the pulp commit ment is the infection by microorganisms due to the dental caries. Even the pulp necrosis that occurs by aseptically reasons, sooner or later, it is infected. The necrotic pulp t issue is very easily infected because the cells and the molecules of the host immune system does not function in necrotic tissue conditions and the apical periodontiu m cells and mo lecules cannot reach the inner necrotic root zone [1, 16,19].
Many experiences show that the dental pulp can recover itself of an acute inflammation, this is direct ly dependent of the quantity of microorganis ms that will be invading the pulp. In conventional rats and germ-free the pulp alterations after the exposition in a mouth area are variable. In the germ-free there is formation of dentin bridges and absence of inflammat ion, although they present necrosis in the exposal area surface. So met imes the necrosis occurs in the pulp near the exposition to the mouth area and this is considered as caused by the accumulat ion or impaction of foods. In the conventional rats, after two days there is the presence of an infiltrated of neutrophils in the h igh layer and beginning of necrosis.
After seven days the pulp necrosis is more extensive, with periradicular inflammation [13].
The infecting microb iota has the capability of organize itself in a biofilm, changing the own metabolic ways surviving to nutrients scarcity periods or less improper conditions to the development. This association in bio film do that these microorganisms synthesize and excrete, in the run of the metabolic process, substances with antigenic action that trigger off the host inflammat ion, that traduce itself somet imes in chronicle clin ical man ifestations, sometimes in acute man ifestations. The organization of the microorganisms in biofilm also offers its way of protection against aggressions and, in this way, the elimination of organized microorganis ms in biofilm constitutes a much more complex objective [3,17].
A fundamental stage in the endodontic treatment is the root canals system disinfection. Th is stage is accomplished by the employ of cut instruments that lengthen the root canal aiming, by the use of an auxiliary irrigator solution, to the removal of the microorganisms that colonized the root canal. As the instruments frequently are not able to cut all the walls of the root canal, the not-touched areas can stay colonized, main ly if there is the presence of biofilm [1][2][3][4]16].
In this context the auxiliary irrigation solution takes a fundamental ro le to reach reg ions where the endodontic instrument is not able to reach. Therefo re, this stage of the endodontic treatment is defined as chemical-mechanical prepare, because it demands the employ of instruments with mechanical action (cut) associated to the employ of an irrigator solution with effective antimicrobial action [10][11][12][13][14][15][16].
Many chemical substances have been proposed with the purpose of to provide an effective irrigator solution, but it was not developed still a substance that get together all the desirable qualit ies. Between the various desirable characteristics to an ideal irrigator solution, its antimicrobial efficiency against bacterial bio film seems to constitute a fundamental point. In the aid to the co mbat to the microorganisms to the intracanal medications, endodontic cements and biomaterial can be used [8,12,16,19].
It is observed that the antimicrobial capability of the calciu m hydroxide and of the chlorhexid ine dig luconate is complementary, as the other properties (the virtue of one complement the fails of the other). By this reason, the association of these two substances in the manipulation of the intracanal med ication using between sessions is instigator and seems plausible that an addictive or synergic antimicrobial effect can results of this association [5]. In fact, the co mbination of the calciu m hydroxide with the chlorhexidine dig luconate in the man ipulation of intracanal medications used between seeing being sessions, since a short time, widely analy zed and defended, mainly in cases of persistent apical periodontitis associated to pulp necrosis or to failed endodontic fillings, even with the limited availability of informat ion respect to this association [5, 12,19]. The combination of the calciu m hydro xide with the chlorhexid ine in 2,0% is, at least, as effective as the calciu m hydro xide vehiculed in sterile deionized water in relation to the disinfection ("in v ivo") of the root canal dentine of teeth with failed endodontic filling. The authors reported that, in fact, the co mbination of the two active principles presented better results, however, the reduced sample led to the non-significant statistic difference between the two groups [21].
With the advent of this material, this study aims to evaluate the antimicrobial activ ity of endodontic materials by the diffusion tests in agar and by direct contact. Thus, the objective of this essay was to evaluate the antimicrobial activ ity of endodontic material to the Enterecoccus faecalis NEWP 0012.

II.
MATERIAL AND METHODS It was used reference bacterial strains, used as standard for quality control at Microbiology laboratories and susceptibility tests to antimicrobials, co mmercialized by NewProv. Each microorganis m was stabilized in freeze-dried discs in a concentration above 100.000 UFC/ mL. It was used the Enterococcus faecalis NEWP 0012.
The discs that contained the microorganis ms were revitalized with a flambé and cooled tweezers, the discs were aseptically removed of their original bottles and put into 3 mL of BHI nutritive broth (Brain Heart Infusion). The pipes were identified and incubated in 35°± 2° until the visible turb idity (2 -3 hours). After, with aid of a 100-microliter calib rated handle, it was inoculated the microorganis ms in a non-selective mediu m p late (Blood agar) by the technique of exhaustion and again they were incubated in 35°± 2° by 24 hours.
It was used 5 different types of endodontic materials commercially acquired by the formu lation of gel or solution or freeze-dried: G1wh ite MTA Angelus®, G2 -MTA Angelus® HP, G3 -Filapex Angelus® cement, G4 -AH p lus cement, G5ch lorhexidine gel 2% and sodium hydroxide (Farmácia Formu la &Ação SP-Brazil). The manipulat ion of the white MTA Angelus®, of the MTA Angelus® HP, of the Fillapex Angelus® cement and AH plus was accomplished accord to the fabricant instructions. The paste manipulation, gel 2% and sodium hydroxide was accomplished in the 1/1 proportion. after the manipulat ion of these materials, they were impregnated into absorbent paper discs ( Figure 01).

2.Test of anti microbi al sensibility in vi troanti biogram (Diffusion method wi th B awer and Kirby discs).
The preparation of plates and growth mediu ms: the Mueller-Hinton Agar (MH) mediu m was prepared previously fused, sterilized and cooled in 45-50°C. Then, it should be spilled in petri plate of 150mm of diameter until reach a th ickness of about 4mm. In the sequence, it was accomplished the preparation of the inoculums and the pure culture of bacteria (NEWPROV) were cultivated in nutritive broth in 37°C by about 12 hours after the revitalization, enough time that the bacterial suspension presents moderated turbidity. The density of the inoculums fro m then on, was controlled by dilution with saline to obtain a turbidity density equivalent to that obtained by the addition of 0,5mL of BaCL2-2H2O (0,048M ) solution in 99,5mL of H2SO4 0,36N -McFarland Escale.
For the inoculation of the plates, cotton swabs were submerged into the bacterial suspension and the excess was removed pressing the cotton against the pipe wall. The bacterial suspension was sowed evenly on the sterile surface of the MH Agar and the discs of antibiotics or of endodontic materials will be sporadically d istributed on the inoculums. The antibiogram plates were incubated by aerobic conditions, in a constant temperature in the range of 35-37ºC, for 24 hours and/or 7 days.

III. RESULTS
The obtained results about the antimicrobial act ivity of the endodontic materials, was measured although millimeter rulers. The inhib ition halos were measured and compared in the first 24 hours, and again after 72 hours, they were been evaluated in until 21 days, in three different tests. The Enterococcus faecalis demonstrated that it is resistant to the most part of the materials, that did not present any bacterial inhibition halo, and they are: white MTA Angelus®, MTA Angelus® HP, Filapex Angelus® cement, sealer 26, AH Plus® and Maxxion R. in the first test was observed inhibition halo in the materials: chlorhexidine gel 2% and calciu m hydro xide (Table 1 and Graphic 1). They were the two-last observed in until 14 days, fro m this there were decrease of the antimicrobial act ion. Ho wever, the calciu m hydroxide and chlorhexid ine gel 2% were effect ive in all tests in until 21 measured days (Figure 2). intracanal measure with the purpose to avoid that the survivor bacteria to the chemical-mechanical prepare, in a enough number and in a favorable environment, mu ltip ly themselves in the interval between the treatment sessions [3,25]. Therefore, it beco mes evident the need to maintain the canal disinfection obtained by the chemicalmechanical prepare, and this is possible using a proper intracanal medicat ion that presents antimicrobial properties and that functions as physical barrier, of an ideal filling of the root canal systems and of a proper crown sealing. Besides that, the intracanal medication has the intention to reduce the periradicular inflammat ion, to solubilize o rganic material, to neutralize to xic products, to control the persistent exudation, to control the inflammatory extern dental reabsorption and to stimu late the reparation by mineralized t issue. With this objective, some options of intracanal medicat ions are been researched. Studies prove the efficiency of ones, more than others, which were present in the clin ical routine. It was used the method of diffusion in agar, due to be considerate standard and of easy execution. Other study already used this methodology. The tested materials in this study were Sealer26®, AH Plus®, Fillapex Angelus®, white MTA Angelus®, MTA Angelus® HP, Restorative glass ionomer Maxxion R®, Calciu m hydroxide with Chlorhexidine in gel 2% and Cotosol® [29]. But only the Calciu m hydroxide with Chlorhexid ine has real efficiency against the EnterococcusFaecalis, that was the chose microorganism to the accomp lish ment of the experiments, because it has a considerable resistance to auxiliary chemical substances and commonly used med ications in Endodontic, besides being frequently associated to the presence of periradicular lesions and to the failu re in the endodontic treatment. It is an anaerobic facultative microorganis m, relatively easy to be cultivated and of high clinical relevance [4]. It can be co mmon ly isolated of clinical samp les, presents peculiar resistance to the calciu m hydro xide, especially, to the elevated level of the pH that results of the dissociation of the hydroxy l ions that occurs in aqueous medium [1,21]. Because it is in powder form, the calciu m hydro xide can be associated to other substances to be inserted into the root canal. Usually, the calciu m hydro xide used in the endodontic practice is manipulated with saline that is water soluble and as they are an association, that have together chemical characteristics of dissociation, diffusibility and filling ab ility that are determinant to the biological behavior [13]. However, according to other studies, specific microorganisms, mainly Enterococcus faecalis, have been showed resistant to the Ca(OH)2 [6,10] and besides that, the antimicrobial efficiency of the pastes in a long term has been questioned [24]. In this way, researches as this have been developed adding active vehicles with antimicrobial propert ies associated to the calciu m hydro xide increasing this activity, without losing its other characteristics [16]. Estrela et al. (1995) [13], discussed the mechanisms of action of the calciu m hydroxide in the treat ment of the endodontic infections. The authors highlighted that the calciu m hydro xide effect is linked to the increase of the pH resultant of the dissociation, in aqueous mediu m, in hydroxyl ions and calciu m ions. The calciu m hydro xide actuates in the activation of the host tissue enzymes as the alkaline phosphatase that participates in the recovery of the mineralized tissues affected by the endodontic infection. In the bacteria, it actuates changing the integrity of essential sites promoting the inactivation of the cytoplasmic memb rane enzy mes interfering, in metabolic and homeostatic processes, growth and cell div ision. Therefore, the actions of the calciu m hydro xide occur in two fronts, biological, favoring the defense mechanisms and the host affected tissues reparation, as also bacteriological, through the antimicrobial action. This therapeutic action makes that the calciu m hydro xide prevails between the active principles of the intracanal med ication, but, despite the particular efficiency in the action by direct contact in the main canal, its actuation in the pH into the dentinal tubules and in the cement, region is not so significant, demonstrating a deficiency.
The calciu m hydroxide presents some limitations, as for examp le, a low solubility and low diffusibility, and action only for d irect contact, that impedes the arrival of this substance to the hard to reach places in teeth that present anatomic variat ions, as isthmus, apical deltas, recesses, places where the bacteria are protected of the action of the intracanal medicat ions. The solution of chlorhexid ine dig luconate 2%, when applied for 10 minutes, previously to the endodontic filling, can enter the dentinal tissue and to provide the antimicrobial action for more than 12 weeks, even if this capability is reduced in function of the time, beco ming a great ally to the calciu m hydro xide. The use of the chlorhexid ine digluconate 2% as intracanal med ication can be indicated in cases with primary infection. In retreats, the use of the chlorhexid ine can be further important, using alternately as irrigator solution during the chemical-mechanical prepare or as intracanal medication between the sessions [32].
The activity of the chlorhexidine d igluconate 0,2% in the reduction of the remaining antimicrobial population after the instrumentation of the canal as delay dressing was showed [8] due to its broad antimicrobial spectrum, the chlorhexidine has been widely used in the Endodontic. Its use has been proposed in form of digluconate salt, liquid or in gel, in different concentrations, as intracanal medication. A recent study evaluated the antimicrobial activ ity of six irrigator against anaerobic bacteria and reported that the chlorhexid ine was the most effective. When used as intracanal med ication, the chlorhexidine had a better effect than the calciu m hydroxide in the elimination of Enterococcus faecalis into the dentinal tubules. It is the material that demonstrates most efficiency against this microorganism, in the tests accomplished by this essay [22]. Lenet et al. (2000) [18] co mpared, in vitro, the residual antimicrobial activ ity of the chlorhexid ine gel 0,2% and 2%, in a system of controlled liberation, and of the calcium hydro xide associated to a saline solution, as intracanal medication, in bovine incisors, for 7 days. After the experimental period, the specimens were inoculated in Enterococcus faecalis for 21 days. The results showed that the chlorhexidine gel 2% had absence of viable bacteria in all the dentin depth.
The chlorhexid ine gel 2% presented most antimicrobial activity. The association of the calciu m hydro xide with the chlorhexidine gel 2% decreased the antimicrobial activity of the chlorhexid ine, however, potentialized the activity of the calcium hydroxide [30].
Studies demonstrates that the chlorhexidine presents more capability to eliminate the Enterococcus faecalis than the calciu m hydroxide [31]. Nonetheless, other results were found to be unsatisfactory to chlorhexidine, mainly related to the inability to inactivate the LPS liberated by Gram-negatives bacteria. However, the calciu m hydro xide can neutralize bacterial endotoxins, especially, the lipopolysaccharide (LPS) present in the cell wall of the Gram-negative bacteria through the lip id hydrolysis that composes part of the LPS mo lecule [27]. It highlights that the "Lipid A", that is responsible to the biological or antigenic act ion of the LPS, is hydrolyzed when is submitted to extremely high pH levels, as the generate by the calciu m hydro xide in an aqueous mediu m [13]. The chlorhexid ine digluconate does not change the pH of the med ication with calciu m hydro xide, it presumes that its antimicrobial action and the inactivation capability of the bacterial LPS is not altered [26,28]. Other effect of the association of the calciu m hydro xide to the chlorhexid ine is increase the antimicrobial capability of the calciu m hydro xide, boosting its penetrability capacity into the dentinal tubules. Also, there was observed in this association, the fact that calciu m hydro xide can actuate as physical barrier wh ile the chlorhexid ine, in function of its substantivity, maintains the canal free of microorganism [1, 14,[17][18][19][20][21]28].
Even the calciu m hydro xide presenting low solubility in water (1,2 g/L to 25°C) and this limits its diffusibility, it is observed diffusion halos in agar to all the associations based in calciu m hydro xide. However, it was verified the inefficiency of the calciu m hydro xide itself against the Enterococcus faecalis as was observed by other researchers [1,28].

V.
CONCLUSION It concludes that the calciu m hydro xide paste associated to the chlorhexidine possess satisfactory antimicrobial act ivity against Enterecoccus faecalis NEWP 0012 in a period of 24h to 21 days.