Clinical Research

Quantification of Endotoxins and Cultivable Bacteria in Root Canal Infection before and after Chemomechanical Preparation with 2.5% Sodium Hypochlorite
Frederico C. Martinho, MSc, and Brenda P.F.A. Gomes, PhD
Abstract
This clinical study was conducted to quantify endotoxins and cultivable bacteria in teeth with pulp necrosis and apical periodontitis before and after chemomechanical preparation with 2.5% sodium hypochlorite (NaOCl) and to investigate the possible correlation of endotoxin and cultivable bacteria with the presence of clinical symptomatology. Twenty-four root canals were selected. Samples were collected before (s1) and after chemomechanical preparation (s2). Culture techniques were used to determine the colony-forming unit. A limulus amebocyte lysate (LAL) assay was used to quantify endotoxins (lipopolysaccharide, LPS). LPS and bacteria were detected in 100% of the initial samples (s1), with a median concentration of 139 endotoxin units/mL and 2.64 105 colony-forming units/mL, respectively. Higher levels of LPS were found in teeth with clinical symptomatology (p .05). At s2, mean endotoxin reduction of 59.99% and mean bacterial load reduction of 99.78% were found. Our findings indicated that chemomechanical preparation with 2.5% NaOCl was moderately effective against bacteria but less effective against endotoxins in root canal infection. Furthermore, a statistically significant association was found between higher levels and clinical symptomatology. (J Endod 2008;34:268 272)

Key Words
Chemomechanical preparation, cultivable bacteria, endotoxins, root canal infection, 2.5% sodium hypochlorite

acteria and their by-products play a primary etiologic role in the development and perpetuation of apical periodontitis (1). The bacteria involved in primary endodontic infection are predominantly gram-negative anaerobic species (2) that present lipopolysaccharide (LPS) on the outer layers of their cells walls (2), which functions as an endotoxin in the host organism (3). LPS (a virulence factor), generally referred to as endotoxin, released during disintegration of bacteria after multiplication and death (4), has been detected in teeth with pulp necrosis (5 8). Strong evidence correlates its presence in root canals with inflammatory reactions and bone resorption of the periradicular tissues (9 12). A high content of endotoxins in root canals has been associated with endodontic signs and symptoms such as spontaneous pain, pain on palpation, and tenderness to percussion (6, 7, 13). Furthermore, its egression through the apical foramen into periapex can even perpetuate an apical periodontitis (9, 11, 14). Therefore, the main goal of an endodontic treatment and main property of the irrigant solution is based not only on bacterial load reduction and antimicrobial properties (15), respectively, but also on reducing endotoxins and on inactivating the toxic effects of LPS, which are important for the healing process of periapical tissues (9, 11, 14). Sodium hypochlorite (NaOCl), widely used in endodontic infections at different concentrations (16), demonstrates a high capacity to reduce and eliminate bacteria from infected root canals (17, 18). However, in vitro (19 21) and in vivo (11, 22), studies with induced periapical lesions have shown a low or absent efficacy for NaOCl in neutralizing endotoxins. Such studies were performed with enterobacterial LPS, which displays toxicity that is not comparable to that of endodontic bacteria (23). Therefore, the present clinical study was conducted to quantify endotoxins and cultivable bacteria in root canal infections with pulp necrosis and apical periodontitis before and after chemomechanical preparation with 2.5% sodium hypochlorite and to investigate the possible correlation of endotoxin and cultivable bacteria with the presence of clinical symptomatology.

Material and Methods

From the Department of Restorative Dentistry, Endodontic Division, Piracicaba Dental School, State University of Campinas, Piracicaba, So Paulo, Brazil. Address requests for reprints to Dr Brenda P.F.A. Gomes, Faculdade de Odontologia de Piracicaba, UNICAMP, rea de Endodontia, Avenida Limeira, 901, Piracicaba, So Paulo 13414-903, Brazil. E-mail address: bpgomes@fop.unicamp.br. 0099-2399/$0 - see front matter Copyright 2008 by the American Association of Endodontists. doi:10.1016/j.joen.2007.11.015

Patient Selection Twenty-four patients at the Dental School of Piracicaba, So Paulo, Brazil, for root canal treatment were selected for this study. The age of patients ranged from 1657 years. The selected teeth were single-root, presenting necrotic pulp tissues with radiographic evidence of apical periodontitis. None of the patients reported spontaneous pain. Eleven cases were asymptomatic, and 13 cases presented either tenderness to percussion and/or pain on palpation. A detailed medical and dental history was obtained from each patient. Patients who had received antibiotic treatment during the last 3 months or who had a general disease were excluded from the study. The Human Volunteers Research and Ethics Committee of the Dental School of Piracicaba approved a protocol describing the specimen collection for this investigation, and all patients signed their informed consent to participate in the study. Sampling Procedure The methods followed for the microbiologic procedures performed in this study have been previously described (7, 8, 24). The teeth were isolated from the oral cavity

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with a rubber dam, and the disinfection of their external surfaces and the surrounding structure field was carried out by using 30% hydrogen peroxide followed by 2.5% NaOCl. The solutions were inactivated with 5% sodium thiosulfate to avoid interference with bacteriologic sampling. The sterility of the external surfaces of the crown was checked by taking a swab sample from the crown surface and streaking it on blood agar plates, which was incubated aerobically and anaerobically. NaOCl was prepared by Proderma (Farmcia de Manipulao Ltda, Piracicaba, So Paulo, Brazil). The manufacturer diluted NaOCl in sterile water without preservatives. The solution was prepared 24 hours before the beginning of the experiment, always in small portions. All subsequent procedures were performed aseptically. A 2-stage access preparation was performed. The access cavity was made without the use of water spray but under manual irrigation with sterile apyrogenic saline and by using sterile apyrogenic high-speed diamond bur. This first stage was performed to promote a major removal of the contaminants (microorganisms and endotoxins). In the second stage before entering the pulp chamber, the access cavity was disinfected following the protocol described above. Its sterility was checked by taking swab samples of the cavity surface and streaking on to blood agar plates, with subsequent incubation at 37C under both aerobic and anaerobic conditions. A new sterile apyrogenic bur was used, accomplished by irrigation with sterile apyrogenic saline, to access the canal. A sterile pyrogen-free paper point (size 35; DentsplyMaillefer, Ballaigues, Switzerland) was then introduced into the full length of the canal, which was determined radiographically, and retained in position during 60 seconds for sampling. The procedure was repeated with 5 paper points. Afterwards, one paper point was placed in a pyrogen-free glass for the chromogenic limulus amebocyte lysate (LAL), and the other paper points were pooled in a sterile tube containing 1 mL of VMGA III transport medium for microbial cultivation. The endotoxin samples were frozen at 20C, and microbial cultivation was performed after all sample collections. All samples were collected by using the same technique.

Culture Procedure The transport media containing the root canal samples were shaken thoroughly in a mixer inside an anaerobic chamber for 60 seconds (Vortex, Marconi, Piracicaba, So Paulo, Brazil). Serial 10-fold dilutions were made up to 10 4 in tubes containing fastidious anaerobe broth (LAB M, Bury, UK). Fifty microliters of the serial dilutions was plated, by using sterile plastic spreaders, into 5% defibrinated sheep blood fastidious anaerobe agar (FAA; Lab M) to culture nonselectively obligate anaerobes and facultative anaerobes. The plates were incubated at 37C in an anaerobic atmosphere for up to 14 days. After this period, the colony-forming units (CFUs) were visually quantified for each plate. Determination of Endotoxin Concentration The quantitative chromogenic LAL assayQLC-100 LAL test kit (BioWhitaker, Inc, Walkersville, MD) was used to measure endotoxin concentrations in the root canals before and after chemomechanical procedures. Standard Curve As a parameter for calculation of the amount of endotoxins in root canal samples, a standard curve was plotted with endotoxins supplied in the kit (Escherichia coli 0111:B4) with a known concentration (25 endotoxin units [EU]/mL) and following the manufacturers instructions. After the LAL test procedure (described below), the absorbencies of endotoxin standard solutions with a series of endotoxin concentrations (ie, 0.1, 0.25, 0.5, and 1.0 EU/mL) were measured individually with an enzyme-linked immunosorbent assay plate-reader (Ultramark; Bio-Rad Laboratories, Inc, Hercules, CA) at 405 nm. The standard curve fulfilled the criteria of linearity (r 0.980), as reported by the guideline on validation of LAL tests(25). Spike Concentration To avoid the inhibition or enhancement of LAL, the addition of a known concentration of E. coli endotoxins (spiking procedure) is recommended. For all tests, the spike recovery was 0.4 EU/mL. This activity was chosen because of its lower position on a logarithmic standard curve (its value was set as zero). Test Procedure Initially, serial dilutions of the samples were made to 10 4. 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, Cambridge, MA) was used in a heating block at 37C and maintained at this temperature throughout the assay. Initially, 50 L of the blank was added followed by the standard endotoxin solutions, and the samples were consecutively added to the wells. This was followed by the addition of 50 L LAL to each well, and the microplate was then briefly shaken. Ten minutes later, 100 L of substrate solution (prewarmed to 37C) was added to each well, always maintaining the same sequence. The plate was mixed and incubated at 37C for 6 minutes. Afterwards, 100 L of a stop reagent (acetic acid, 25% v/v) was added to each well, and the absorbance (405 nm) was read by using a spectrophotometer (Ultramark; Bio-Rad Laboratories). Calculation of Endotoxin Concentrations The mean absorbance value of the blank was subtracted from the mean absorbance value of the standards and the value of samples to calculate the mean absorbance of the samples. Because this absorbance value was directly proportional to the concentration of endotoxins present, the endotoxin concentration was determined from the standard curve.
Quantification of Endotoxins and Cultivable Bacteria in Root Canal Infection

Clinical Procedures After the first microbiologic sampling, the pulp chamber was thoroughly cleaned with 2.5% NaOCl. A K-file (Dentsply-Maillefer), size 10 or 15, was placed in the total length of the root canal, as calculated by the preoperative radiograph. The coronal two thirds of each root canal were prepared initially by using rotatory files (GT Rotatory Files size .10/20 and .08/20; Dentsply-Maillefer) at 350 rpm, reaching 4 mm before the total length. Gates-Glidden drills, sizes 5, 4, 3, and 2 (DynaFFDM, Bourges, France) were used until 2 mm before the total length, prepared with GT files. The working length (1 mm from the radiographic apex) was checked with a radiograph after inserting an anatomic file in the canal to the estimated working length, confirmed by the apex locator (Forum Technologies, Rishon Le-Zion, Israel). The apical stop was established by using K-files (Dentsply-Maillefer). The apical stop ended after the use of 3 files larger than the initial one. Step-back flaring of the canal was performed with larger files at intervals manipulated in a filling action. The file used to prepare the apical stop was used to recapitulate. Stepping back ended after the use of 3 files larger than the file that prepared the apical stop (18). The use of each instrument was followed by an irrigation of the canal with 5 mL of 2.5% NaOCl solution. The working time for the chemomechanical procedure was established at 20 minutes for all cases. Before the second sampling (S2), NaOCl was inactivated with 5 mL of sterile 0.5% sodium thiosulfate during a 1-minute period.
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TABLE 1. Distribution of Clinical Features, Endotoxin, and CFU in 24 Root Canals with Pulp Necrosis and Apical Periodontitis Samples
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Median Ranging values Mean reduction

Clinical features Symptomatic TTP POP

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No No No No No No No No No No No Yes Yes Yes No No Yes No Yes Yes Yes Yes No No No No No No No No No No No No No Yes Yes Yes Yes Yes Yes Yes No No No No No No No No No No No No No No No No No

Endotoxin (EU/mL) S1 S2 S1
388 381 635 25 272 110 417 86 270 145 696 224 558 252 519 236 682 46 459 301 608 347 139 3 53 48 145 31 149 8 228 67 157 109 196 78 85 5 121 26 17 11 37 16 103 58 228 117 228 EU/mL 72.50 EU/mL 2.64 17 to 696 EU/mL 3.0 to 381 EU/mL 1.2 59.99%

Number of CFU/mL S2
5

1.48 10 8.0 102 3.96 105 2.0 102 4.30 105 6.0 102 3.94 105 4.0 102 3.26 105 0 2.60 105 8.0 102 5 3.12 10 2.0 102 2.64 105 2.0 102 3.26 105 0 8.60 105 3.0 103 5 1.36 10 6.0 102 1.20 105 0 1.36 105 0 3.26 105 6.0 102 5 3.60 10 4.0 102 2.46 105 6.0 102 3.74 105 6.0 102 1.58 105 2.0 102 1.46 105 0 1.96 105 8.0 102 2.30 105 2.0 102 2.42 105 6.0 102 2.96 105 1.20 103 1.23 105 6.0 102 5.25 102 CFU/mL 105 CFU/mL 105 to 8.6 105 CFU/mL 0 to 3.0 103 CFU/mL 99.78%

CFU, colony forming units; TTP, tenderness to percussion; POP, pain on palpation; s1, initial samples; s2, after chemomechanical preparation.

Statistical Analysis The data collected (CFUs and endotoxin concentrations) were statistically analyzed with SPSS for Windows, version 12.0 (SPSS Inc, Chicago, IL). The Mann-Whitney test was performed to verify any significant difference in the amount of bacteria/endotoxin between symptomatic and asymptomatic teeth. The Spearman test was used to correlate LPS/CFU with a particular symptom (tenderness to percussion or pain on palpation). The Friedman test was performed to compare the amount of bacteria and endotoxin before (s1) and after chemomechanical preparation (s2). When significant differences were found between different sampling times, the Wilcoxon test was used subsequently. Significance levels were always set at 5% (p .05).

Results
No microbial growth was observed in any of the sterility check samples. At the initial sampling, bacteria were recovered from all root canals. The CFU count ranged from 1.2 105 to 8.6 105 (median value, 2.64 105) (Table 1). In the samples from asymptomatic teeth, the median CFU value was 2.27 105, whereas in symptomatic teeth it was 3.50 105 (Table 2). No statistical difference was found in CFU values between symptomatic and asymptomatic teeth (p .05). MoreTABLE 2. Endotoxin and CFU Values in Symptomatic and Asymptomatic Teeth Teeth
Endotoxin Median value Range in values CFU Median value Range in values
CFU, colony forming unit.

over, no correlation was found between CFU and the presence of any particular symptoms (tenderness to percussion or pain on palpation) (p .001). After chemomechanical preparation, the bacterial load dropped to a mean value of 99.78%, with a median value of 5.25 102 CFU/mL (Table 1). A total of 5 cases, 2 with clinical symptomatology and 3 cases that were asymptomatic, presented no bacterial growth after chemomechanical preparation. Analyses of the quantitative data revealed that the number of CFUs in s2 was significantly reduced in comparison with s1 (p .05). Quantitative data are depicted in Table 1. The LAL assay (QLC-1000) indicated that endotoxins were present in 100% of the root canal samples. The concentration of endotoxins in all root canals ranged from 17696 EU/mL, with median value of 228 EU/mL (Table 1). In asymptomatic teeth, the median endotoxin content was 127.53 EU/mL. Higher levels of endotoxin were found in teeth with clinical symptomatology (median value, 519 EU/mL) (Table 2). Statistically significant differences (p .05) were found in LPS content between symptomatic and asymptomatic teeth. No correlation was found between LPS and pain on palpation (p .001). In contrast, a positive correlation between LPS and tenderness to percussion was found (p .001). After chemomechanical preparation, LPS concentration dropped to a value of 59.99%, with a median value of 72.50 EU/mL (Table 1). Analyses of the quantitative data revealed that the LPS content in s2 was significantly reduced in comparison with s1 (p .05). Quantitative data are shown in Table 1.

Symptomatic
519 EU/mL 272696 EU/mL 3.50 105 CFU/mL 1.36 105 to 8.60 105 CFU/mL

Asymptomatic
127.53 EU/mL 17228 EU/mL 2.27 105 CFU/mL 1.20 105 to 3.74 105 CFU/mL

Discussion
The high sensitivity of the LAL test (5) permits the detection and quantification of endotoxins in different oral sites (26 28) including root canal infections (7, 8). However, special attention should be given to its use in endodontic infections, because other biologic materials such as inflammation exudates (29), as well as the presence of grampositive bacteria (30), might interfere in the assay, even though only in

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high concentrations. To avoid these interferences, serial dilutions of up to 10 4 and spiking procedures (recommended by the manufacturers) were performed in the present study. Even though clinical studies (31, 32) have demonstrated high levels of bacterial load reduction after chemomechanical preparation with 2.5% NaOCl, no information was provided on the efficacy of chemomechanical preparation with 2.5% NaOCl for reducing LPS content in human teeth with pulp necrosis and apical periodontitis. Bacteria and LPS were detected in all initial root canal samples, confirming the strong correlation between bacteria (1) and their by-products (3, 9, 12, 22) and the presence of apical periodontitis. The mechanism by which LPS contributes to the initiation of inflammatory response and periapical bone resorption appears to be related to the expression of TLR2 (33) and TLR4 (33, 34) in dental pulp tissue and normal human odontoblast as well as to its capacity of stimulating macrophages in releasing cytokines as necrosis factor (14) and interleukins 1, 6, and 8 (35). The initial number of viable bacteria in the infected root canals was 105, a number that is comparable to previous studies (8, 32). No association was found between the amount of viable bacteria and the presence of clinical symptomatology (pain on palpation and tenderness to percussion). However, our findings, in accordance with previous studies (6, 7, 13), demonstrated a strong correlation between clinical symptoms (pain on palpation and tenderness to percussion) and high levels of endotoxin in the root canal, particularly tenderness to percussion. Such a correlation suggests that an increase in endotoxin levels in infected root canals might be associated with an increased degree of apical periodontitis and, consequently, with the development of clinical symptoms (pain on palpation and tenderness to percussion). The concentration of endotoxin in all root canal samplings ranged from 17696 EU/mL (median value, 228 EU/mL). In asymptomatic teeth, endotoxin contents ranged from 17228 EU/mL (median value, 127.53 EU/mL), similar to the findings of Vianna et al (8), who detected endotoxin levels ranging from 10200 EU/mL (mean value, 151.61 EU/mL). In contrast, higher levels of endotoxin were found in teeth with clinical symptomatology, ranging from 270696 EU/mL (median value, 519 EU/mL). Nevertheless, whether the initial amount of endotoxins found in the present and in previous studies (7, 8) is able to initiate an apical peridodontitis by itself is still not known. Because LPS is released during death and autolysis of gram-negative bacteria (4), an inverse proportion between bacteria load and LPS content in root canal infection after chemomechanical preparation might be expected, unless root canal instrumentation exhibits an ability to remove and/or the auxiliary chemical substance (NaOCl) presents a high capacity to neutralize endotoxin. However, no consistent finding could be observed in this respect in the present study, because both bacterial load and LPS content were reduced after chemomechanical preparation. Chemomechanical preparation with 2.5% NaOCl was able to reduce the bacterial load from 105 to 102, corroborating previous studies (31, 32). However, in contrast to the mean percent value of bacterial load reduction (99.78%), LPS contents were reduced in only 59.99%. Moreover, LPS was recovered from all root canal samplings after chemomechanical preparation. Vianna et al (8) also detected LPS in all root canal samples after chemomechanical preparation with 2.0% chlorhexidine gel. Nevertheless, the amount of endotoxin in the root canal that is sufficient to cause damaging effects has not been established. The high efficacy of chemomechanical preparation in reducing bacterial load and its low ability to remove or inactivate LPS from root canals might not be related only to the irrigating solution (2.5% NaOCl). It might well be due to the fact that LPS can adhere irreversibly to mineralized tissue (14), making it difficult to be removed from dentin walls. Perhaps the use of a chelating substance (eg, ethylenediaminetetraacetic acid) before the placement of a root canal medication (eg, calcium hydroxide) and root canal filling might help to remove LPS from the root canal walls, but this speculation needs a better investigation. Even though the present study demonstrated that chemomechanical preparation with 2.5% NaOCl was able to reduce LPS content by only 59.99%, a successful endodontic treatment can still be achieved in clinical practice. However, high contents of LPS might play an important role in the development and the persistence of endodontic symptoms after root canal treatment.

Acknowledgments
We thank Mr Adailton dos Santos Lima for technical support. This work was supported by grants of the Brazilian agencies FAPESP (04/12436-9, 05/53729-1) and CNPq (305437/2006-2, 470820/ 2006-3).

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