Response of the periapical tissue of dogs' teeth to the action of citric acid and EDTA

Abstract

The purpose of this study was to analyze the inflammatory response of dog's periapical tissues to 17% trisodium EDTA salt (pH 8.0) and 1% citric acid (pH 2.0). Saline was used as a control. Six adult dogs were used as the biological model of the study. The experimental units comprised 56 roots of mandibular molars (first and second) and premolars (first, second and third). After coronal opening, pulpectomy and root canal instrumentation were performed using the above-mentioned irrigating solutions. After 24 and 48 hours, the animals were euthanized and the teeth and their supporting tissues were removed and histologically processed. The sections were stained with hematoxylin and eosin and analyzed histopathologically with a light microscope at x100 magnification. The histological analysis focused on the occurrence of acute inflammatory response. The presence of swelling, vasodilatation and inflammatory cells were evaluated and the degree of inflammation was determined for each case. Data were analyzed by Fisher's exact test using the SPSS software with a confidence interval of 95% (p<0.05). 17% EDTA and 1% citric acid caused inflammatory responses in dog's periapical tissues with no significant differences to each other or to saline (control) at either the 24-hour (p=0.482) or 48-hour (p=0.377) periods. It may be concluded that the inflammatory response was of mild intensity for the tested substances.

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ABSTRACT
RESPONSE OF THE PERIAPICAL TISSUE OF DOGS’
TEETH TO THE ACTION OF CITRIC ACID AND EDTA
Cristina Berthold SPERANDIO1, Luiz Fernando Machado SILVEIRA2, Lenita Aver de ARAÚJO2,
Josué MARTOS3, Ashwin MALSHE4
1- DDS, MSC, Department of Clinics, Dental School, Federal University of Pelotas, Pelotas, RS, Brazil.
2- DDS, MSC, PHD, Professor, Department of Clinics, Dental School, Federal University of Pelotas, Pelotas, RS, Brazil..
3- DDS, PHD, Professor, Department of Clinics, Dental School, Federal University of Pelotas, Pelotas, RS, Brazil.
4- BS, Electronics Engineering, University of Mumbai, India; PhD Student, State University of New York, New York, NY, USA.
Corresponding address: Prof. Dr. Luiz Fernando Machado Silveira, Faculdade de Odontologia de Pelotas, Universidade Federal de Pelotas -
Rua Gonçalves Chaves 457 - Pelotas, RS, Brazil - 96015-560 - Phone/Fax: 55-53-2224439 - e-mail: lufersil.sul@terra.com.br
Received: June 11, 2007 - Modification: September 11, 2007 - Accepted: October 24, 2007
he purpose of this study was to analyze the inflammatory response of dog’s periapical tissues to 17% trisodium EDTA salt
(pH 8.0) and 1% citric acid (pH 2.0). Saline was used as a control. Six adult dogs were used as the biological model of the study.
The experimental units comprised 56 roots of mandibular molars (first and second) and premolars (first, second and third). After
coronal opening, pulpectomy and root canal instrumentation were performed using the above-mentioned irrigating solutions.
After 24 and 48 hours, the animals were euthanized and the teeth and their supporting tissues were removed and histologically
processed. The sections were stained with hematoxylin and eosin and analyzed histopathologically with a light microscope at
x100 magnification. The histological analysis focused on the occurrence of acute inflammatory response. The presence of
swelling, vasodilatation and inflammatory cells were evaluated and the degree of inflammation was determined for each case.
Data were analyzed by Fisher’s exact test using the SPSS software with a confidence interval of 95% (p<0.05). 17% EDTA and
1% citric acid caused inflammatory responses in dog’s periapical tissues with no significant differences to each other or to
saline (control) at either the 24-hour (p=0.482) or 48-hour (p=0.377) periods. It may be concluded that the inflammatory
response was of mild intensity for the tested substances.
Uniterms: Citric acid. Ethylenediaminetetraacetic acid. Biocompatibility.
INTRODUCTION
The cleaning and disinfection role of the irrigating
solutions in the complex protocol of the endodontic therapy
is essential for treatment success. As McComb and Smith10
(1975) demonstrated the presence of a dentin layer attached
to the root canal walls, the irrigating solutions should act
on both organic and inorganic compounds.
Among the irrigants used for removal of inorganic
compounds are the chelating agents and inorganic acids
represented by ethylenediaminetetraacetic acid (EDTA) and
citric acid, respectively7,10,11,13. Some authors have focused
their studies on the biological effects of citric acid
solutions1,8.
The biocompatibility of citric acid and EDTA in the pulp
and periapical tissues induces an inflammatory response of
variable intensity and depth depending on the methodology
used. Cotton and Siegel5 (1978) and Baumgartner, et al.3
observed no severe or irreversible damage on the human
dental pulp. Sterrett, et al.12 (1993) and Malheiros, et al.8
(2005) stated that the effects observed on the human dentin
may vary according to the concentration and application
time.
McInnes-Ledoux, et al.9 (1985) reported that, in monkeys,
1% citric acid showed severe effects on the pulpal tissues.
Leonardo, et al.6 (1984) observed a good tolerance of the
periapical tissues to EDTA on dog’s teeth, while Zina, et
al.15 (1981) using trisodium EDTA in dog’s teeth did not
observe any damage on the healing process of the pulp
stump and periapical tissues.
Chan, et al.4 (1999) observed that in human pulp cell
cultures, the toxicity of 0.5% and 1% citric acid is proportional
to the concentration and exposure time, whereas other
studies observed in rats that citric acid was less irritating
than EDTA and ethyleneglycol-bis(2-aminoethoxy)-
tetraacetic acid (EGTA)14. Amaral, et al.1 (2007) observed
that both EDTA and citric acid showed cytotoxic effects on
macrophages, though citric acid produced less cytotoxic.
Malheiros, et al.8 (2005) demonstrated in cultured fibroblasts
and in rats (rattus novergicus)in vivo, respectively, that, if
applied at high concentrations, citric acid leads to a delay in
cell growth and has an irritating effect in vivo.
59
J Appl Oral Sci. 2008;16(1):59-63

Based on the endodontic biologic principles, the purpose
of this study was to evaluate, histopathologically, the
periapical tissue response of dog’s teeth submitted to root
canal therapy with two demineralizing chemical substances:
1% citric acid and 17% trisodium EDTA salt.
MATERIAL AND METHODS
Three different solutions were used in this study: 17%
trisodium EDTA salt (, pH 8.0; Chemical Lab. UCPel, Pelotas,
RS, Brazil), 1% citric acid (pH 2.0; Curativa Pharmacy, Pelotas,
RS, Brazil) and saline (Texon Co., RS, Brazil) as the control.
Six adult dogs from the central laboratory of UFPel were
selected as the biological model of the study. The
experimental sample comprised 56 roots of mandibular molars
(first and second; n= 13) and premolars (first, second and
third; n= 22). Seven out of the 35 teeth were lost during the
histological processing. Twenty-eight teeth with two roots
remained, thus totaling 56 roots. Each root was considered
as sample unit.
The animals were sedated with an intravenous injection
of 2% Virbaxyl (Virbac Co., SP, Brazil) and then anesthetized
with Thiopental (Cristália Chemical Products, SP, Brazil).
After oral asepsis with 0.12% chlorhexidine, coronal opening
was performed under rubber dam isolation. Access to the
pulp chamber was gained with #1012, 1015 and 1016 diamond
burs (KG Sorensen, São Paulo, SP, Brazil) and the
convenience shape was given with an Endo Z bur (Dentsply-
Maillefer, Ballaigues, Switzerland). Pulpectomy was
performed with a Hedströen file and the working length was
determined 1 mm short of the radiographic apex with K-files
(Dentsply-Maillefer).
Root canals were instrumented with four K-files
(Dentsply-Maillefer) of greater sizes than that of file used
for root canal length determination. During biomechanical
preparation, the number of irrigations and volume of the
irrigating solution were standardized in 16 mL for premolars
and 32 mL for molars with the aid of disposable syringes
and 30x3, 30x4 and 30x5 hypodermic needles (Becton-
Dickinson, São Paulo, SP, Brazil). In all phases of
biomechanical preparation, either one of the test or the
control substances were used as auxiliary irrigant.
When instrumentation was completed, the root canal
was filled with the test solution for 5 minutes to allow an
intimate contact. After drying, the canal entrance was sealed
with disinfected gutta-percha followed by composite Prisma
APH (Dentsply Ind. e Com. Ltda, Petrópolis, RJ, Brazil) below
the occlusion line.
After 24 and 48 hours of the treatment, the animals were
euthanized and the teeth removed and fixated in 10% formalin
solution. Thereafter, the teeth were decalcified by immersion
in 1:1 formic acid and sodium citrate solution followed by
5% nitrate acid. The specimens were embedded in fused
paraffin Histosec (Merck KGaA, Darmstadt, Germany), cut
with a microtome at the apical foramen in serial 6-µm-thick
sections, which were stained with hematoxylin and eosin
and mounted on glass slides. The sections that best
represented the area of pulp-periodontium communication
were selected and examined with a light microscopy at x100
magnification.
The histologic analysis considered the occurrence of an
acute inflammatory response, so the presence of swelling,
vasodilatation and inflammatory cells was evaluated and
the degree of inflammation was determined for each case.
For systematic examinationof the specimens, a squared
and numbered (with 100 indexed crosses (+) of equal
dimension) eyepiece grid (NE21 mm; Graticules Ltd, Kent,
UK), was introduced into one of the eyepieces of the
microscope. Of the total of 100 crosses that composed the
net, approximately 50 would cover the periodontal space,
being the remainder occupied by the root, dental canal and
alveolar bone, which were excluded from the analysis. Three
fields of the periodontal ligament were evaluated: its most
central area in relation to the apical foramen and two lateral
margins. For each evaluated field, we considered the number
of points coinciding with the occurrence of the three primary
events of inflammatory reaction: swelling, vasodilatation
and presence of inflammatory cells. From all analyzed fields,
the most representative area of inflammatory degree was
selected. The following criteria were used: mild inflammation
- when each parameter was coincident with a maximum of 4
crosses; moderate inflammation - when at least 2 parameters
were coincident with at least 5 and at most 9 crosses; severe
inflammation - when at least 2 parameters were coincident
with 10 or more crosses.
Chi-square test could have been used to assess
differences in the efficacy of the three substances. However,
as the expected frequencies for a few cells were less than 5,
chi-square test was not deemed appropriate. Alternatively,
Fisher’s exact test was used to overcome the problem due
to small cell frequencies because it requires a non-zero
frequency in each cell. Hence, the category “severe” was
eliminated because there was no observation for that level.
The resulting contingency table was used for Fisher’s exact
test is reproduced as Table 2 and 4. The data were analyzed
with a confidence interval of 95% (p<0.05) using the SPSS
software (SPSS 14.0, SPSS Inc., Chicago, IL, USA).
RESULTS
The two tailed p value generated by Fisher’s exact test
for the above contingency table was 0.482 (for the 24-hour
exposure time) and 0.377 (for the 48-hour exposure time). As
both p values were greater than 0.05, the null hypothesis of
independence was accepted, which means that the three
substances did not have different efficacy at the either of
the study intervals.
The results of the histologic analysis of the inflammatory
response observed in dogs’ teeth after contact with EDTA,
citric acid and saline, after 24 and 48 hours, are presented in
the Tables 1 and 2.
In order to provide the necessary adjustment for the
statistical analysis, the scores 1, 2 and 3 were attributed to
the mild (Figure 2), moderate (Figure 3) and severe (Figure
60
RESPONSE OF THE PERIAPICAL TISSUE OF DOGS’ TEETH TO THE ACTION OF CITRIC ACID AND EDTA

4) inflammation degrees, respectively, which differ from the
normal aspect (Figure 1). There was no significant difference
in the inflammation degree among the tested substances
within the 24 hour (p=0.482) or 48 hour (p=0.377) periods.
Substance Number of repetitions Inflammatory response Score Incidence
EDTA 11 Mild 1 9
Moderate 2 2
Severe 3 -
Citric acid 8 Mild 1 7
Moderate 2 1
Severe 3 -
Saline 10 Mild 1 6
Moderate 2 4
Severe 3 -
TABLE 1- Tested substances according to the inflammatory response after 24 hours
Substance Number of repetitions Inflammatory response Score Incidence
EDTA 9 Mild 1 6
Moderate 2 2
Severe 3 1
Citric acid 11 Mild 1 4
Moderate 2 6
Severe 3 1
Saline 7 Mild 1 5
Moderate 2 2
Severe 3 1
TABLE 2- Tested substances according to the inflammation response after 48 hours
FIGURE 1- Normal aspect of the periodontium (H.E., original
magnification: x100)
FIGURE 2- Mild inflammation. vasodilatation (v);
inflammatory cells (i); swelling (e) (H.E., original
magnification: x100)
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SPERANDIO C B, SILVEIRA L F M, ARAÚJO L A de, MARTOS J, MALSHE A

DISCUSSION
In the present study, the sample comprised the roots
instead of molar and premolar teeth because, after
pulpectomy, the surgical wounds behave independently
even if submitted to the same treatment. The 24- and 48-
hour periods were established because, though presenting
a relative antimicrobial activity, the tested substances, are
not indicated for this use and after longer observation
periods there would be the risk of bacterial contamination,
especially in the control group.
The method proposed for inflammation degree
evaluation consisted of using a net of eyepiece reticular
scale adapted to the ocular of the microscope, which allowed
the quantification of the events composing the inflammatory
process, i.e., swelling, vasodilatation and presence of
inflammatory cells in the apical region. Therefore, the
classification of the inflammatory response in mild, moderate
and severe was determined by the counting.
The results obtained with EDTA in relation to the control
group demonstrated a good tolerance by the periapical
tissues. Similar findings have been reported by Zina, et al.15
(1981) and Leonardo, et al.6(1984), the latter evaluating the
tissue response in dogs’ teeth. The statistical analysis
showed that even though there was a severe degree
inflammation within 48 hours, there was no significant
difference between both periods.
Citric acid showed a similar behavior as that of EDTA
and saline at both periods and, though not statistically
significant, it showed a higher degree of inflammation at 48
hours compared to the shorter period. This result differs
from those of previous studies that used citric acid for cavity
cleaning5,9 and evaluated the response to pulp tissues
instead of to periapical tissues.
The findings of the present study and of other authors1,8
disagree from those reported by Chan, et al.4 (1999) who
observed an increase in the inflammation degree with a
longer exposure time in cell cultures. Citric acid is an organic
acid that takes part in the Krebs cycle in the human cells,
being therefore biologically acceptable.
The results of the control group showed that an
inflammatory response is a natural consequence of
pulpectomy. Comparison of the substances in both studied
periods (24 and 48 hours) did not show statistically
significant differences. This fact indicates that, although
these two substances have a low superficial tension, even
in multiple foramen conditions, other factors referring to
their physicochemical properties and the site of application,
control their diffusion. Regarding EDTA, its activity is limited
by the presence of organic material2, in addition to promoting
the coagulation of proteins, which form of a mechanical
barrier that avoids its diffusion towards the periapical
tissues.
Citric acid has the property of not promoting collagen
selling, which occurs with most acids. This justifies its use
for histological preparations in the form of sodium citrate, in
demineralization processes. This aspect could allow a higher
diffusion of this substance, but its acidic characteristics
provide cauterizing properties, due to its low pH.
Both EDTA and citric acid compared to the control group
showed a potential for a mild inflammation, with no
statistically significant difference to each other. The clinical
use of either EDTA or citric acid should be done with caution
to prevent their extrusion to the periapical tissues. Citric
acid showed similar conditions for clinical use as those of
EDTA, as it has similar characteristics regarding the property
as an inorganic solvent, removal of smear layer,
improvements in NaOCl properties when associated to it
and a satisfactory tissue tolerance. Under the tested
conditions, the outcomes justify the clinical use of citric
acid but in a cautious manner.
CONCLUSIONS
Under the experimental conditions of this study, the
obtained results showed that 17% EDTA and 1% citric acid
caused inflammatory responses in dog’s periapical tissues
with no significant differences from saline within the
evaluated time periods (24 and 48 hours). 17% EDTA and
FIGURE 3- Moderate inflammation. vasodilatation (v);
inflammatory cells (i); swelling (e) (H.E., original
magnification: x100)
FIGURE 4- Severe inflammation. vasodilatation (v);
inflammatory cells (i); swelling (e) (H.E., original
magnification: x100)
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RESPONSE OF THE PERIAPICAL TISSUE OF DOGS’ TEETH TO THE ACTION OF CITRIC ACID AND EDTA

1% citric acid produced similar inflammatory reaction to each
other at both the shorter and the longer time period. It may
be concluded that the inflammatory response to the tested
substances was of mild intensity.
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SPERANDIO C B, SILVEIRA L F M, ARAÚJO L A de, MARTOS J, MALSHE A