Influence of 2% chlorhexidine gel on calcium hydroxide ionic dissociation and its ability of reducing endotoxin

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1 Influence of 2% chlorhexidine gel on calcium hydroxide ionic dissociation and its ability of reducing endotoxin Fernanda Graziela Correa Signoretti, MSc, a Brenda Paula Figueiredo de Almeida Gomes, PhD, b Francisco Montagner, PhD, c Fernanda Barrichello Tosello, BDS, a and Rogério Castilho Jacinto, PhD, d Piracicaba and Porto Alegre, Brazil ENDODONTIC DIVISION, DEPARTMENT OF RESTORATIVE DENTISTRY, PIRACICABA DENTAL SCHOOL, UNIVERSITY OF CAMPINAS Objective. The aim of this study was to evaluate the influence of 2% chlorhexidine gel (CHX) on ph, calcium release, and Ca(OH) 2 capability of reducing endotoxin. Study design. Calcium release was verified by atomic-absorbance spectrophotometry, and ph was measured with a phmeter. For endotoxin quantification, extracted human teeth previously contaminated with standard endotoxin were filled with: group I, Ca(OH) 2 saline solution; group II, Ca(OH) 2 CHX; and group III, CHX for 14 days. The remaining endotoxin was quantified by using chromogenic quantitative test. Statistical analysis was performed with analysis of variance and Tukey post hoc test (P.05). Results. Group II released more calcium than group I (P.05) after 15 days. Groups I and II showed alkaline ph in all periods, and group I showed higher ph values than group II (P.05) after 30 days. Groups II and III showed significantly more decreased endotoxin than group I. Conclusions. CHX did not interfere with the chemical properties of Ca(OH) 2, in fact even improving its properties of reducing the endotoxin content in root canals in vitro. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;111: ) Supported by the Brazilian agencies FAPESP (06/ , 07/ , 08/ ), FAPERGS ( ) and CNPq ( / , /2008-6, /2009-0). a Postgraduate Student. b Associate Professor. c Adjunct Professor, Endodontic Area, Department of Conservative Dentistry, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. d Adjunct Professor, Endodontic Area, Pelotas Dental School, Federal University of Pelotas; Research Fellow, Endodontic Division, Department of Restorative Dentistry, Piracicaba Dental School, Piracicaba, Brazil. Received for publication May 25, 2010; returned for revision Oct. 5, 2010; accepted for publication Nov. 14, /$ - see front matter 2011 Mosby, Inc. All rights reserved. doi: /j.tripleo Anaerobic bacteria, especially gram-negative, and their by-products (e.g., cell wall compounds) contribute to inflammatory periapical processes and might be related to painful endodontic symptomatology. 1-4 Endotoxins (lipopolysaccharides [LPSs] from gramnegative cell walls) are released during bacterial death or division, inducing inflammatory periapical response and enhancing the sensation of pain in endodontic infections. 5 Previously, extracted endotoxin inoculated in root canals of different experimental animals induced inflammatory reaction and bone resorption on the periapical area of all groups, 6-8 which shows the important role of endotoxins on the pathogenesis of periapical lesions. One of the main objectives of endodontic procedures is to remove bacteria from root canals. Nevertheless, endotoxins released after rupture of bacteria cell walls might remain in the periapical area, inducing or maintaining periapical pathologies, 9,10 which might reflect on the success of the endodontic treatment. Therefore, it is paramount that chemomechanical preparation and root canal dressing application during root canal treatment of infected teeth not only eliminate bacteria but also reduce their endotoxic contents. 6,7 The root canal medications of choice are generally calcium hydroxide pastes, because they show biologic activity against infections of the root canal system. 11,12 Physicochemical properties of calcium hydroxide pastes (ph, calcium release, and solubility) have been extensively studied, because of their importance in the mechanism of action of these medications. 13,14 Ionic dissociation is responsible for calcium hydroxide biologic properties, such as antimicrobial action 6,11 and capability of inducing periapical healing by deposition of mineral tissues. 12 Despite being the most commonly used medication, calcium hydroxide is not effective against some important bacteria, 15 such as Candida albicans and Enterococcus faecalis. Besides, its efficacy in reducing the number of bacterial cells between 653

2 654 Signoretti et al. May 2011 appointments is questionable. 16 Therefore, other substances have been associated with calcium hydroxide to improve its antimicrobial action. Chlorhexidine is an antimicrobial agent with a wide spectrum of action that has been used during root canal preparation. 17,18 It has also been used as an intracanal medication, because it is more effective against Enterococcus faecalis than calcium hydroxide. 19,20 Besides, after a prolonged contact with root canal dentin, chlorhexidine shows substantivity. 21 The association of calcium hydroxide with chlorhexidine enhances calcium hydroxide antimicrobial activity, which results in a more effective activity against resistant microorganisms without interfering with the biologic properties of calcium hydroxide. 20 Several studies have investigated the antimicrobial activity of these medications. However, little is known about the influence of chlorhexidine on calcium hydroxide regarding the chemical properties of calcium hydroxide and its capability of inactivating endotoxin present in infected root canals. Therefore, the objective of the present research was to investigate whether addition of 2% chlorhexidine gel to calcium hydroxide paste interferes with the release of calcium and hydroxide ions as well as with the endotoxin reduction produced by calcium hydroxide. MATERIALS AND METHODS Calcium release and ph The pastes were prepared as follows: group I (n 40): Ca(OH) 2 0.9% physiologic solution (1:1, v/v); group II (n 40): Ca(OH) 2 2% chlorhexidine gel (1:1, v/v); group III (n 40): 2% chlorhexidine gel. A Lentulo drill #40 (Dentsply/Maillefer, Tulsa, OK, USA) was used to insert each paste into polyethylene cylindric tubes (10 mm 1.3 mm; Embramac, São Paulo, Brazil), which had their bottom orifices sealed. Next, the cylindric tube samples were inoculated in Falcon tubes containing 10 ml deionized water. Ten empty tubes (without medication) were used as negative control samples. The ph and calcium ion release were measured after 24 hours and 7, 15, and 30 days. The ph measure was performed by using a ph meter (Procyon digital phmeter model AS 720, electrode A 11489; Procy Instrumental Científica, São Paulo, Brazil). The samples were distributed into 120 falcon tubes. For each of the 4 different periods of the study, 10 different tubes of each group were analyzed, with 100 L being collected from each tube and diluted in 2 ml 0.15% potassium chlorite (Merck, Darmstadt, Germany) to prevent the possible interference of alkaline phosphates and metals. The glass flasks were washed with 5% nitric acid (Merck, Darmstadt, Germany), and 10% EDTA (1 mol/l) was used as blank to calibrate the apparatus to zero. Calcium release was measured with an atomic absorption spectrophotometer (model 403; Perkin-Elmer Corporation, Brandford, CT, USA). Data were calculated by comparing them to the standard curve built from the calcium standard solution. Endotoxin quantification This study was submitted to and approved by the local research committee of the Piracicaba Dental School, São Paulo, Brazil (no. 040/2008). Forty recently extracted maxillary central or lateral incisors were used in the study, all selected on the basis of dimensions and similarity in morphology. Debris, calculus, and soft tissue on the root surfaces were cleaned by using a Gracey curette, and all of the teeth were stored in saline solution (0.9% NaCl) until use. The crown was sectioned to standardize the root length to 14 mm. Working length was determined by subtracting 1 mm from this measurement. Canals were enlarged by using a #60 K-file (Maillefer), with step-back cleaning and shaping performed with recapitulation, followed by irrigation with 2% chlorhexidine gel and saline solution. The cervical third was enlarged by using #3 and #4 Gates-Glidden drills. During instrumentation, the root canals were filled with 2% chlorhexidine gel and irrigated with 5 ml saline solution for each file used. After preparation, they were irrigated with 3 ml 17% EDTA for 3 minutes and then irrigated with 5 ml saline solution. The canals were dried with sterile paper points (Maillefer), and the outer surfaces of the samples were covered with 2 layers of cyanoacrylate, except the cervical opening. Sample sterilization All of the samples were sterilized by autoclaving (20 minutes at 121 C) and randomly assigned to 5-cell culture plates (96-well Cell Culture Plates; Corning and Costar, Corning, NY, USA), 10 teeth for each experimental group and 5 teeth for each control group. The samples and all the materials used in the experiment were irradiated with 60 Co gamma-rays for degradation of preexisting LPS (Embrarad; Empresa Brasileira de Radiações, Cotia, SP, Brazil). Contamination with endotoxin Escherichia coli O55:B5 endotoxin (Lonza, Walkersville, MD, USA) was used for the experiments. Under sterile laminar flow, the standard solution containing endotoxin (20,000 endotoxin units [EU]/mL) was inoculated into the root canals of 35 samples by using a micropipette. Endotoxin solution was not inoculated into the 5 teeth of the negative control group. All of the samples were sealed with pyrogen-free cotton balls, and

3 Volume 111, Number 5 Signoretti et al. 655 the plates containing the samples were closed, sealed, and incubated at 37 C in a humidified atmosphere. Experimental groups After 24 hours, the cervical sealing was removed and the canals were filled with intracanal medicaments and divided into 5 groups, as follows: Group I: paste of calcium hydroxide physiologic saline solution (n 10) Group II: calcium hydroxide 2% chlorhexidine gel (n 10) Group III: 2% chlorhexidine gel (n 10) Group IV (positive control): no medicament (n 5); Group V (negative control): no endotoxin and no medicament (n 5). After a 14-day period, the root canals were irrigated with 5 ml pyrogen-free water to remove all intracanal medicaments, with 2.5 ml of the washing being collected with a nonpyrogenic 3.0-mL plastic syringe (Injex Industrias Cirúrgicas, São Paulo, SP, Brazil) to quantify the remaining endotoxin. Quantitative chromogenic Limulus amoebocyte lysate assay During the procedures, a series of precautions were taken to avoid contamination of the samples. All materials coming into contact with samples were endotoxin free. The endotoxin standard curve for the quantitative chromogenic Limulus amoebocyte lysate (LAL) assay (QCL-1000; Lonza) was generated by following the manufacturer s procedure, and the least-square value (R 2 ) obtained was 1.0. The parameter for calculation of the concentration of endotoxin in the sample was plotted by using the endotoxins supplied in the kit (E. coli O111:B4) with a known concentration [29 EU/mL]. After the LAL incubation, the absorbance of standard endotoxin solutions at a series of endotoxin concentrations (i.e., 0.01, 0.25, 0.5, and 1.0 EU/mL) were measured individually by using an enzyme-linked immunosorbent assay reader (Ultramark Microplate Reader; Bio-Rad Laboratories, Hercules, CA, USA). The absorbance at 405 nm was linear within the concentration range used. Test procedure. Serial dilutions of the samples were made to LAL reagent water (blank) was used as a negative control. All reactions were accomplished in duplicate to validate the test. A 96-well microplate (Corning Costar) was used in a heating block at 37 C, and maintained in it throughout the assay. Initially, 50 L of the blank, 50 L ofthe standard endotoxin solutions, and 50 L of each Table I. Averages and standard deviations for calcium release (mg/l) of groups I, II, and III in the following periods: 24 hours, 7 days, 15 days, and 30 days Group I Group II Group III 24h ( 20.50) ac ( 12.44) ab 0 ba 7d ( 51.89) aab ( 41.22) aa 0 ba 15d ( 36.78) bb ( 53.70) aa 0 ca 30d ( 34.45) aa ( 21.09) aa 0 ba Different lowercase letters indicate significant differences between groups. Different uppercase letters indicate significant differences between periods (analysis of variance: P.05; complemented by Tukey test: P.05). sample were consecutively added to the wells. This was followed by the addition of 50 L LAL to each well by using a multichannel pipette and reagent reservoir, and then the microplate was briefly shaken. Ten minutes later, 100 L of substrate solution (prewarmed to 37 C) was added to each well, maintaining always the same sequence. The plate was mixed and incubated at 37 C for 6 minutes, after which 100 L of a stop reagent (acetic acid, 25% v/v) was added. The absorbance (405 nm) was read by using a spectrophotometer (Ultramark; Bio-Rad). Calculation of endotoxin concentration. The mean absorbance of the blank was subtracted from the mean absorbance of the standards and samples to calculate the mean absorbance of the samples. Because this absorbance value was directly proportional to the concentration of endotoxin existing in the sample, the endotoxin concentration was determined from the standard curve. Statistical analysis The statistical analysis was carried out with BioEstat 5.0 (Belém, Pará, Brazil). The results were statistically analyzed by using analysis of variance and Tukey post hoc test. In all cases, P values of.05 were considered to be statistically significant. RESULTS Calcium release and ph The means and standard deviations of calcium release (mg/dl) and ph are shown in Tables I and II, respectively. The negative control showed no release of calcium) ions in none of the experimental periods and ph values remaining around 7. Endotoxin quantification The initial amount of endotoxin inoculated was 20,000 EU/mL. After the experimental periods, group III had the lowest endotoxin concentration (mean 1,674 EU/mL), followed by group II (mean 2,248 EU/mL),

4 656 Signoretti et al. May 2011 Table II. Averages and standard deviations for ph values of the groups in the different time periods Group I Group II Group III 24h ( 0.32) ab ( 0.19) ab 5.52 ( 0.12) bb 7d ( 0.25) aa ( 0.26) aa 5.50 ( 0.31) bb 15d ( 0.38) ab ( 0.24) aab 6.88 ( 0.79) ba 30d ( 0.11) aa ( 0.08) ba 5.58 ( 0.13) cb Different lowercase letters indicate significant differences between groups. Different uppercase letters indicate significant differences between periods (analysis of variance: P.05; complemented by Tukey test: P.05). and there was no statistical significant difference between these groups (Table III). On the other hand, group I showed the highest endotoxin concentration (mean 3,573 EU/mL), being statistically different from groups II and III (Table III). The negative control did not show any endotoxin detection. The positive control showed presence of endotoxin in all samples (mean 11,000 EU/mL). DISCUSSION The present in vitro study has demonstrated the release of hydroxide and calcium ions from 3 different pastes used as intracanal medication after 30 days of application. The study model, using polyethylene cylindric tubes, was chosen to standardize the amount of medication used in each sample. Atomic absorption spectrophotometry for analyzing calcium ions has been successfully used in earlier studies. 22,23 The ph values of the solutions with samples containing calcium hydroxide did not show considerable differences, whereas the ph of 2% chlorhexidine gel varied from 5.50 to 6.88, as expected and in accordance with values found in the literature. 24 Despite the low ph values shown in group III (2% chlorhexidine gel), the ph of the mixture of chlorhexidine with calcium hydroxide in group II did not differ from the ph values of calcium hydroxide used with an aqueous vehicle (group I), which is corroborated by earlier studies. 14,25 The calcium hydroxide pastes released calcium ions in all periods of the study. However, higher values of calcium ions were observed at the end of 30 days in the group where calcium hydroxide was mixed with an aqueous vehicle. The addition of chlorhexidine digluconate to calcium hydroxide forms calcium digluconate and a precipitate of chlorhexidine molecules (which is not soluble). In the present study, the mixture of calcium hydroxide with 2% chlorhexidine gel showed more constant values of calcium release, with fewer alterations during the periods of study, compared with the paste with aqueous vehicle. Those values were higher than the ones reported by Simon et al., 26 who Table III. Average endotoxin concentration for each group after 14 days (EU/mL) Initial concentration Group I Group II Group III Positive control Negative control 20,000 3,573 b 2,248 a 1,674 a 12, a,b Different letters indicate significant differences between groups (analysis of variance: P.05; complemented by Tukey test: P.05). used a different study model, i.e., human extracted teeth that had been sealed coronally and apically. Therefore, they had the interference of medication diffusion through dentin tubules. In contrast, the 1.3 mm diameter of the polyethylene cylindric tubes used in the present study allowed a direct contact of the paste with aqueous solution, and the experiment was not under the influence of the dentin buffer effect. 27 The standard endotoxin used in this study was the one extracted from E. coli. The fact that the antiinflammatory response induced by E. coli endotoxin might be different from the response promoted by LPS of bacteria normally isolated from the oral cavity 7,28,30 was not relevant, because the objective was to compare the antiendotoxic action of different medications. The endotoxin concentration used in the present study (20,000 EU/mL) was based on the values detected in vivo by Jacinto et al. 4 in necrotic root canals from symptomatic teeth, although the harmful level of endotoxin has not yet been determined. 10 The period between endotoxin inoculation and introduction of medication in the root canals was 24 hours. According to Oliveira et al., 31 the period of 24 hours between endotoxin inoculation and introduction of medication was the minimum amount of time required for endotoxin diffusion through dentinal tubules toward the radicular surface. The reduction in the concentration of endotoxin inoculated in the positive control group was already expected, because of LPS diffusion through dentinal tubules over time. 31 However, the storage conditions were the same for all samples of all of the groups, and the endotoxin reduction found in the positive control group was not significant. In general, earlier research shows an effective action of calcium hydroxide on LPS. 3,32 In addition, histologic and radiographic analyses in dogs have shown that calcium hydroxide is capable of neutralizing endotoxin. 6,7 On the other hand, Oliveira et al. 30 showed that 2% chlorhexidine was not effective against endotoxin over a period of 7 days. In the present study, none of the pastes totally neutralized the endotoxin present in the root canals, which is in accordance with Vianna et al. 10

5 Volume 111, Number 5 Signoretti et al. 657 The medications tested in this research partially reduced the endotoxic content after 14 days of application. Moreover, there were differences between the groups, and the addition of chlorhexidine increased the action of calcium hydroxide on LPS. The combined use of chlorhexidine and calcium hydroxide in the root canal may generate excessive reactive oxygen species, which might explain why the use of such a mixture showed more efficacy in eliminating endotoxin, even though nonsoluble chlorhexidine precipitate is formed. 33 On the other hand, Vianna et al. 10 did not observe differences between calcium hydroxide pastes with or without 2% chlorhexidine gel. These differences in the results of both studies could be due to the different study models (in vitro and in vivo) or to the periods evaluated, because in the study of Vianna et al. 10 the medications were used for 7 days. The action of chlorhexidine in partially reducing the endotoxin inoculated is in disagreement with Tanomaru et al., 28 who found no influence of 2% chlorhexidine on the periapical healing of endodontic lesions induced in dogs teeth by LPS. The explanation could be that after biomechanical preparation the canals were left empty for 60 days, which could have allowed a reinfection, compromising the periapical healing. In the present study, the endotoxin reduction exerted by the pastes containing chlorhexidine could be explained by the fact that this substance is an amphipathic biguanidine, which neutralizes cell wall components (e.g., LPS). 34 The endotoxin reduction shown in the present study, in addition to other properties of chlorhexidine, such as wide antimicrobial spectrum, 20 substantivity, 35 and low cytotoxicity, 36 suggests that it might be an effective substance to be associated with calcium hydroxide in the treatment of root canal infections. In conclusion, none of the pastes evaluated were capable of completely reducing the endotoxin inoculated in the root canals. Furthermore, the addition of chlorhexidine with calcium hydroxide did not interfere with the chemical properties of calcium hydroxide, which is responsible for antimicrobial activity and healing induction of periapical tissues. Chlorhexidine enhanced, in vitro, the capacity of calcium hydroxide to reduce the endotoxic content of root canals. 29 The authors thank Thaís Mageste Duque and Geovania Caldas Almeida for technical support. REFERENCES 1. Farber PA, Seltzer S. Endodontic microbiology. 1. Etiology. J Endod 1988;14: Horiba N, Maekawa Y, Abe Y, Ito M, Matsumoto T, Nakamura H. Correlations between endotoxin and clinical symptoms or radicular radiolucent areas in infected root canals. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1991;71: Safavi KE, Nichols FC. Effect of calcium hydroxide on bacterial lipopolysaccharide. 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