Residual Antibacterial Activity of Chlorhexidine Digluconate and Camphorated P-monochlorophenol in Calcium Hydroxide-Based Root Canal Dressings

1Department of Dentistry, Federal University of Vales do Jequitinhonha e Mucuri, Diamantina, MG, Brazil 2Departament of Endodontics, School of Dentistry of Araraquara, São Paulo State University, SP, Brazil 3Department of Pediatric Clinic, Preventive and Social Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil 4School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil


INTRODUCTION
Biologically, the formation and persistence of apical abscesses, granulomas and cysts are intimately associated with pulp necrosis secondary to root canal system infection (1).Due to this cause-effect relationship, one of the goals of root canal treatment is to eliminate or significantly reduce the residual necrotic tissue, microorganisms and their byproducts, thus creating favorable conditions for the occurrence of periapical tissue healing (2)(3)(4).
Biomechanical preparation is the first and most important step to eliminate an endodontic infection.However, the cutting action of files as well as the flushing and chemical actions of the irrigating solutions are primarily effective against the microbiota situated in the main root canal (1,5).After meticulous cleaning and shaping of the main root canal, the use of calcium hydroxide[Ca(OH) 2 ]-based pastes as intracanal dressings has been widely recommended for a complementary antiseptic action, among other favorable properties (6)(7)(8)(9)(10)(11).However, the antimicrobial activity of these Braz Dent J (2007) 18(1): 8-15 pastes is still not fully understood and several questions remain unanswered regarding the time of mantainance of these pastes in the canals, the type of vehicle and the need of adding other anti-septic components to Ca(OH) 2 .
Clinically, though satisfactory antiseptic results have been obtained with Ca(OH) 2 -based pastes for up to 7 days (12), studies of hydroxide ion diffusion through dental structure suggest that the minimum time should be 2-3 weeks (9).This is required to reach the threshold alkalinity in order to inactivate or eliminate bacteria and fungi remaining in the root canal system and possibly periapical biofilm microorganisms as well (1,13).
Regarding the vehicles used for preparation of Ca(OH) 2 -based pastes, aqueous (i.e., distilled water, saline and anesthetic solution) or viscous (i.e., polyethylene glycol and glycerin) hydrosoluble vehicles are the most commonly used.These substances are considered inert from an antiseptic point of view (6).
The presence of strictly anaerobic and facultative gram-positive and gram-negative cocci, bacilli and filaments in the root canal system has been demonstrated (1,4,5,12).These microorganisms show a wide range of susceptibility to calcium hydroxide and therefore the addition of other antimicrobial agents to Ca(OH) 2based pastes has been suggested, such as camphorated p-monochlorophenol (CMCP),which has been shown to be effective against resistant root canal microbiota (3,5,10,11,14).In addition, chlorhexidine digluconate has been used as an alternative vehicle for Ca(OH) 2based pastes because of its broad antimicrobial spectrum, diffusibility and substantivity (8,11,14,15).
Due to the effectiveness of Ca(OH) 2 -based intracanal medications for treatment of teeth with pulp necrosis and periapical lesions, it is important to determine the duration of their antimicrobial activity and whether it is influenced by paste composition.Thus, this study evaluated the influence of two hydrosoluble vehicles (anesthetic solution and polyethylene glycol) and two antiseptics (CMCP and 2% chlorhexidine digluconate), on the residual antibacterial activity of Ca(OH) 2 -based pastes placed in root canals of dogs' teeth with experimentally induced chronic periapical lesions.

Induction of Periapical Lesions and Root Canal Therapy
This study was conducted on 72 root canals of upper and lower premolars of 4 1-year-old dogs.The animals were injected intravenously with 3% sodium nembutal at a dose of 15 mg/kg body weight, and occlusal cavities were opened, the pulp tissues were carefully removed with Hedstrom files, and root canals were left exposed to the oral environment for 7 days for purpose of contamination.After this period, the access openings were sealed with a 3-mm-thick layer of zinc oxide-eugenol cement (Pulpo Sam; S.S.White, Rio de Janeiro, RJ, Brazil), until the induction of periapical lesions.Standard radiographs were taken at 15-day intervals to monitor the development of radiolucent periapical areas suggestive of chronic periapical lesions, which usually occurred between 60 and 90 days.
The operative procedures were undertaken under rubber dam isolation, followed by antisepsis of the operative field with 5% iodated alcohol and neutralized with alcohol/ether.After removal of the coronal seal, the septic contents of the initial 2/3 of the root canals were explored and neutralized using 5.25% sodium hypochlorite as irrigating solution.Subsequently, rotary instrumentation was performed with the ProFile rotary System (Dentsply/Tulsa Dental Products: Tulsa, OK, USA) using size 30 to 60 files with 0.04 and 0.06 tapers according to a crown-down preparation technique.Copious irrigation with 3.6 mL of 5.25% sodium hypochlorite was done at each change of file using a syringe and a 27G needle.Pre-enlargement proceeded with orifice shapers for exploration/neutralization of the apical third of the root canal, maintaining the apical foramen patent with a size 30 K-file, followed by rotary apical preparation.The apical stop was enlarged to a diameter of 0.60 mm and was located at 1.5 mm short of the radiographic apex.The smear layer was removed with buffered 14.3% EDTA solution, which was stirred with a K-file for 5 min.Then, the root canals were flushed with 3.6 mL of the irrigating solution and dried with paper points.The canals were filled with Ca(OH) 2based pastes prepared with hydrosoluble vehicles either aqueous (anesthetic solution or 2% chlorhexidine digluconate solution) or viscous (polyethylene glycol).
Calcium hydroxide p.a. (Labsynth Products Ltda, Diadema, SP, Brazil) used in groups 3 and 4 was weighed on an analytical balance and added to the respective vehicles at a ratio of 0.9 g powder/mL solution to obtain a toothpaste consistency.Both pastes were prepared at the time of use.Calen ® and Calen ® / CMCP pastes were placed into the canals using a syringe with a threaded piston (ML; S.S.White Artigos Dentários Ltda) and a long needle (Gengibrás -27G; Ibras CBO Ind. Bras., São Paulo, SP, Brazil) with a silicon stop to limit the penetration to the working length.The other two pastes were applied with a #4 spiral lentulo coupled to a low-speed handpiece.The amount of Ca(OH) 2 -based paste placed in the root canals was that enough to completely fill the canal space, which was confirmed radiographically.

Evaluation of Residual Antibacterial Activity
After 21 days, samples of the Ca(OH) 2 -based pastes were retrieved from the root canals with size 60 K-files and their residual antibacterial activity was assessed against a Micrococcus luteus ATCC 9341 strain.After cultivation in agar nutrient (An-Nutriente Broth; Difco, Detroit, MI, USA), the test strain was inoculated in Müller Hinton broth (MHb-Müller Hinton broth; Difco) and incubated at 37°C for 24 h.Under antiseptic conditions, 1.0 mL of the Micrococcus luteus ATCC 9341 culture, at a concentration of 10 6 cfu/mL and adjusted to 0.5 of the McFarland scale, was added to 20x100 mm Petri plates containing 12 mL of Müller Hinton agar (Mha-Müller Hinton Medium-Difco).Each plate contained 4 files with different intracanal medicament and 1 file with a freshly prepared past (control sample), disposed radially.After pre-diffusion for 2 h at 24ºC, the plates were incubated aerobically at 37ºC for 24 h.To optimize the reading, aliquots of 5 mL of 0.05% 2,3,5-triphenyltetrazolium chloride gel -TTC (Sigma, St. Louis, MO, USA) were added which had been prepared with 1.0% agar (Difco).After solidification, the plates were incubated at 37ºC for 30 min.The halo of inhibition formed around each file was measured at its largest diameter perpendicular to the file using a millimeter rule under stereomicroscopy.Statistical analysis of the data was performed by Mann-Whitney U test at 5% significance level.those retrieved from the root canals exhibited residual antibacterial activity (Tables 1 and 2).In order of increasing residual antibacterial activity the paste can be presented as follows: Ca(OH)

DISCUSSION
After remaining in the root canals for 21 days, all Ca(OH) 2 -based pastes still showed measurable residual antibacterial activity of different magnitudes.The interpretation of these results must take into account the antibacterial action of calcium hydroxide, the vehicles and antiseptics, as well as the variables related to the methodology used.
The antibacterial action of calcium hydroxide results from its high alkalinity (pH [12][13].The hydroxide ions act on molecules or structures that are essential for microbial metabolism or reproduction (9).Furthermore, root canal filling itself is a mechanical barrier to prevent the influx of nutrients and microbial recolonization (1,2).The bactericidal action of CMCP is due to the interaction of chlorine and phenol, its principal ingredients, which inactivate sulphydryl groups (SH) of oxidase and dehydrogenase enzymes and denature the structural proteins, principally the bacterial cellular membrane (3).With respect to chlorhexidine, Jenkins et al. (16) considered that the cationic molecule attaches to the negatively charged bacterial cell wall altering its permeability.Thus, a bacteriostatic effect occurs at low concentrations.At high concentrations, a bactericidal effect is observed by the precipitation or coagulation of citoplasmatic contents.From an antiseptic standpoint, anesthetics and polyethylene glycol are considered inert vehicles (6).
Although no panacea, calcium hydroxide has been proved lethal to microorganisms that compose the endodontic microbiota by direct contact in vitro and in periods that varied from minutes to few days (3,5,10,11).However, in root canals, either in vitro or, especially, in vivo, physicochemical and biological barriers represented by the buffering capacity of dentin and tissue fluid (exudates and blood) reduce the antiseptic efficacy of calcium hydroxide (11,17).As a result, Ca(OH) 2based dressings should remain for a minimum of 2 to 3 weeks in order to obtain satisfactory alkalinity of root dentin.However, some microorganisms present in the dentinal tubules, such as Enterococcus faecalis, remain resistant to calcium hydroxide (3,11).This justifies the need to add antiseptics such as CMCP or chlorhexidine to Ca(OH) 2 -based pastes (11,18).
Using the agar diffusion test, it has been shown that the efficacy of an antiseptic is mainly related to its solubility, constant diffusion of the agar medium, toxicity and test organisms used in each experiment (7,11,18,19).Although the methodology used in this study is well known, several factors might have interfered with the results.Concerning the culture medium, the viscosity must be taken into account, volume and depth of agar medium, the pH of the substrates in the plates, the load and concentration of ions in the medium, the conditions of storage of the agar plates, the contact of the experimental material with the agar, the time, the temperature of incubation of the plates and the optimization of the reading.The variables related to the antiseptic refers to weight, molecular size and form, sensitivity of the medication, as well as its solubility and diffusion in the water-based agar medium.Regarding the test microorganism, its origin (wild strains or collection species), the density of the inoculum and the type of organism should be ascertained.The time and the point or area for reading the halos of inhibition must also be defined.The lack of standardization is possibly the main cause of variations in results when this methodology is used (3,7,10,19,20).The present study used as an indicator of residual antimicrobial activity, the standard strain Micrococcus luteus ATCC 9341, which is a strictly aerobic gram-positive coccus commonly found in root canals and easily cultured in vitro.
In this study, the viscosity, volume, depth of the agar medium, conditions of storage of the agar plates, contact of the experimental material with the agar, density of the inoculum, as well as the time and temperature of incubation of the plates and the location to read the halos of inhibition were standardized.Moreover, after placement of the intracanal medications, there was a 2-h pre-diffusion period at room temperature, which, according to Leonardo et al. (19), corresponde to the long-term diffusion of the dressing through the root dentine.In addition, after 24-h incubation, TTC gel was applied to optimize the reading.The red compound formed by redox reaction of viable or growing microorganisms facilitates the reading of diffusion and inhibition halos (19,20).Thus, it was shown that in 1 sample of Calen ® group and in 3 samples of Calen ® /CMCP and Ca(OH) 2 /anesthetic pastes the diffusion occurred without the formation of inhibition halos.
As with the example of distilled water and glycerin, the anesthetic solution behaved as an inert vehicle, without antibacterial activity, and therefore the antibacterial effect observed by this paste must be due to calcium hydroxide effect.The viability of evaluating the antimicrobial activity of Ca(OH) 2 in an agar medium has been questioned because the bactericidal activity of Ca(OH) 2 is associated with its alkalinity, which would be affected by the buffering capacity of the culture medium associated with its low solubility.Compara-tively, in the root canal system, dentin also exerts a neutralizing action on hydroxyl ions (17), which are progressively replaced by the continuous diffusion of Ca(OH) 2 .Therefore, a period of pre-diffusion must occur before evaluation to verify the antiseptic action of calcium hydroxide in agar, otherwise it will be evaluated as inefficient (3,7).
The anesthetic solution has been used as a vehicle for calcium hydroxide in many clinical situations involving periapical pathology (6).After remaining in the root canals for 21 days, the Ca(OH) 2 /anesthetic solution paste showed a significant reduction in its antibacterial activity, compared to Calen ® , Ca(OH) 2 /chlorhexidine and its respective controls.This behavior may be partially attributed to the pH of the vehicle, which is acid (pH4.5) and perhaps to its greater solubility in the tissue fluids.From an antiseptic point of view, this type of paste must be renewed after approximately 21 days, if a longer treatment is needed.
There have been positive reports of Ca(OH) 2 / CMCP paste (3,7,10,19).However, a negative synergism seemed to have occurred in this study because the residual action of the Ca(OH) 2 /CMCP paste was significantly less than that of the formulation without CMCP, and the control samples showed similar results.The progressive loss of CMCP from the root canal is known, but surprisingly, CMCP seemed to reduce the bactericidal activity of calcium hydroxide.
The Calen ® paste maintained a stable antiseptic action or, in other words, its residual antibacterial activity was similar to that of the control samples.Leonardo et al. (19) observed similar results against Micrococcus luteus and Enterococcus faecalis.In contrast, Gomes et al. (7) noted the smallest zones of inhibition of bacterial growth for Calen ® paste.Possibly, their results are due to the readings being based on the lowest values of inhibition halos and, above all, for not undertaking an optimization stage before reading.A relevant factor related to the methodology used in the present study was the optimization of the culture medium with TTC gel.This procedure, according to Begue and Kline (20) helps distinguishing the halo of inhibition of bacterial growth that frequently is interpreted as a halo of diffusion of material.
The fact that the largest halos of inhibition were observed for the Ca(OH) 2 /chlorhexidine paste may be attributed to the positive synergism between these two antiseptics, as well as to the greater solubility and diffusion of chlorhexidine in the agar medium.Siren et al. (18) reported that chlorhexidine increases the antiseptic action of calcium hydroxide.Chlorhexidine digluconate in a liquid or gel form has been used as an irrigating solution or intracanal medication, in vitro, in situ and in vivo, in concentrations that range from 0.2 to 5% (8,(14)(15)(16).Its marked antimicrobial substantivity as an intracanal medication must also be emphasized (15,18).Comparatively, in the antisepsis of dentinal tubules infected with Enterococcus faecalis, CMCP has shown greater efficacy than Ca(OH) 2 (11), while chlorhexidine in the form of a sustained-release device or solution showed a performance similar to that of CMCP ( 14) and superior to that of Ca(OH) 2 (8,18) .
All control materials showed larger halos of inhibition in relation to the samples retrieved from root canals, especially Ca(OH) 2 /anesthetic and Calen ® /CMCP.Although the precise amount of each long-term dressing used in the diffusion test was not determined, the results suggest that the components of long-term dressings originating from the root canals showed a progressive reduction in their antiseptic capacity, possibly by being consumed in physicochemical and biological processes, for example: 1) calcium hydroxide carbonation; 2) organic material dissolution; 3) reaction with bacterial organic components (proteins and phospholipids); 4) neutralization by dentine buffering system.Additionally, an important fraction of calcium hydroxide is dissolved by tissue fluids, via the apical foramen.
It should be emphasized that the Ca(OH) 2 / chlorhexidine paste showed a notable residual antiseptic activity, whose halos of inhibition were larger than those of the control materials.This behavior may be attributed to the greater diffusibility of chlorhexidine in agar, as well as its concentration.Due to its broad antiseptic spectrum, antibacterial substantivity, low surface tension, dentin diffusibility and relative low cytotoxicity, 2% chlorhexidine has been shown to be a promising vehicle for calcium hydroxide intracanal medication for endodontic treatment and re-treatment of teeth with pulp necrosis associated with periapical lesions (15,16,18).
Although the agar diffusion test has some limitations, according to the methodology proposed and based on the results of this study, it may be conclude that all Ca(OH) 2 -based pastes showed measurable residual antibacterial activity against the standard strain Micrococcus luteus ATCC 9341 after 21 days in the root canals.In contrast to Ca(OH) 2 /chlorhexidine and Calen ® pastes, Calen ® /CMCP and Ca(OH) 2 /anesthetic pastes showed a significant reduction in their residual antibacterial activity.Furthermore, unlike camphorated monochlorophenol, 2% chlorhexidine digluconate increased significantly the antibacterial activity of Ca(OH) 2 .

Table 1 .
Halos of inhibition (in mm) of the Ca(OH) 2 -based pastes that were not applied to root canals (control).

Table 2 .
Halos of inhibition (in mm) of residual antibacterial activity Ca(OH) 2 -based pastes retrieved from the root canals.

Table 3 .
Comparison of the means of inhibition halos relative to Tables1 and 2.