Decalcifying effect of 15% EDTA, 15% citric acid, 5% phosphoric acid and 2.5% sodium hypochlorite on root canal dentine.

Abstract

To evaluate and compare ex vivo the decalcifying effect of 15% EDTA, 15% citric acid, 5% phosphoric acid and 2.5% sodium hypochlorite on root canal dentine.
Two 2-mm-thick slices were cut from the coronal third of the root of 10 human incisors. Each slice was sectioned into two equal parts. Specimens were assigned to one of four groups (n = 10) for immersion in 20 mL of either 15% EDTA, or 15% citric acid, 5% phosphoric acid or 2.5% NaOCl, for three time periods (5, 10 and 15 min). The concentration of Ca(2+) extracted from the dentine was measured by atomic absorption spectrophometry. The amount of calcium extracted was analysed using the Kruskal-Wallis test for global comparisons and the Mann-Whitney U-test for pairwise comparisons.
In the three time periods, 15% EDTA and 15% citric acid extracted the largest amount of calcium, with no significant differences between them. The 2.5% NaOCl solution extracted insignificant amounts of calcium, whereas 15% EDTA extracted 86.72% of the calcium in the first 5 min, and 15% citric acid and 5% phosphoric acid had a similar pattern of calcium removal (77.03% and 67.08% in first 5 min, respectively).
Solutions of 15% EDTA, 15% citric acid and 5% phosphoric acid decalcify root dentine, with most calcium extracted during the first 5 min of action. The efficacy of 15% citric acid and 15% EDTA solutions was significantly greater than that of 5% phosphoric acid solution at each time period (5, 10 and 15 min).

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Decalcifying effect of 15% EDTA, 15% citric acid,
5% phosphoric acid and 2.5% sodium hypochlorite
on root canal dentine
M. Pe ´rez-Heredia1, C. M. Ferrer-Luque1, M. P. Gonza ´lez-Rodrı ´guez1, F. J. Martı ´n-Peinado2&
S. Gonza ´lez-Lo ´pez1
1Department of Dental Pathology and Therapeutics, School of Dentistry, University of Granada; and2Department of Edaphology
and Agricultural Chemistry, School of Sciences, University of Granada, Spain
Abstract
Pe ´rez-Heredia M, Ferrer-Luque CM, Gonza ´lez-Rodrı ´guez
MP, Martı ´n-Peinado FJ, Gonza ´lez-Lo ´pez S. Decalcifying
effect of 15% EDTA, 15% citric acid, 5% phosphoric acid and
2.5% sodium hypochlorite on root canal dentine. International
Endodontic Journal, 41, 418–423, 2008.
Aim To evaluate and compare ex vivo the decalcifying
effect of 15% EDTA, 15% citric acid, 5% phosphoric
acid and 2.5% sodium hypochlorite on root canal
dentine.
Methodology Two 2-mm-thick slices were cut from
the coronal third of the root of 10 human incisors. Each
slice was sectioned into two equal parts. Specimens
were assigned to one of four groups (n ¼ 10) for
immersion in 20 mL of either 15% EDTA, or 15% citric
acid, 5% phosphoric acid or 2.5% NaOCl, for three time
periods (5, 10 and 15 min). The concentration of Ca2+
extracted from the dentine was measured by atomic
absorption spectrophometry. The amount of calcium
extracted was analysed using the Kruskal–Wallis test
for global comparisons and the Mann–Whitney U-test
for pairwise comparisons.
Results In the three time periods, 15% EDTA and
15% citric acid extracted the largest amount of
calcium, with no significant differences between them.
The 2.5% NaOCl solution extracted insignificant
amounts of calcium, whereas 15% EDTA extracted
86.72% of the calcium in the first 5 min, and 15%
citric acid and 5% phosphoric acid had a similar
pattern of calcium removal (77.03% and 67.08% in
first 5 min, respectively).
Conclusions Solutions of 15% EDTA, 15% citric
acid and 5% phosphoric acid decalcify root dentine,
with most calcium extracted during the first 5 min of
action. The efficacy of 15% citric acid and 15% EDTA
solutions was significantly greater than that of 5%
phosphoric acid solution at each time period (5, 10 and
15 min).
Keywords: citric acid, decalcification, EDTA, phos-
phoric acid, sodium hypochlorite, spectrophometry.
Received 7 April 2007; accepted 5 November 2007
Introduction
Many authors have concluded that the smear layer
created during root canal preparation should be
removed from the dentine surface of the canal wall.
The following reasons have been cited: smear layer
harbours bacteria and can be detrimental to effective
disinfection of dentinal tubules by preventing sodium
hypochlorite, calcium hydroxide and other intracanal
medicamentsfrompenetrating
(Clark-Holke et al. 2003, Shahravan et al. 2007); and
its total removal improves the adaptation of filling
materials to the root canal (Karagoz-Kucukay & Bayirli
1994, Sen et al. 1995), increases the bond strength of
dentinaltubules
Correspondence:
Department of Dental Pathology and Therapeutics, School of
Dentistry, University of Granada, Campus de Cartuja, Colegio
Maximo s/n. 18071, Granada, Spain (Tel.: +34 958249655;
fax: +34 958240908; e-mail: cferrer@ugr.es).
ProfessorCarmen Marı ´aFerrer-Luque,
doi:10.1111/j.1365-2591.2007.01371.x
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resin-based endodontic sealers to root dentine (Econo-
mides et al. 1999, Saleh et al. 2002, Gogos et al. 2003);
and reduces apical and coronal microleakage with most
sealers currently used for canal filling (Cobankara et al.
2002, Economides et al. 2004, Khayat & Jahanbin
2005).
Removal of the smear layer requires the use of
irrigants that can dissolve both organic and inorganic
components. Different irrigants have been recom-
mended to remove the inorganic component of root
dentine, e.g. EDTA solutions at a concentration of
15–17% and pH of 7–8 (Garberoglio & Becce 1994,
Calt & Serper 2000, Di Lenarda et al. 2000, O’Connell
et al. 2000), citric acid at a concentration of 5–50%
(Ferrer Luque et al. 1993, Garberoglio & Becce 1994,
Di Lenarda et al. 2000, Haznedaroglu 2003) and
phosphoric acid at different concentrations and applied
in different ways (Garberoglio & Becce 1994, Ayad
2001, Perez-Heredia et al. 2006). Sodium hypochlorite
(NaOCl) solutions are used as the main irrigation agent
for removing the organic component because of their
bactericidal power and capacity to dissolve organic
matter and necrotic tissue (Inaba et al. 1996, Zehnder
et al. 2002). NaOCl is a halogenated compound used as
a nonspecific proteolytic agent capable of removing
magnesium and carbonate ions (Sakae et al. 1988).
Baumgartner & Mader (1987) and Baumgartner &
Cuenin (1992) suggested that its use may expose
inorganic material, which would prevent greater
dentine dissolution, or may leave a smear layer of
mineralized tissue, which would increase the Ca/P ratio
on the dentine surface.
Some reports (Hennequin & Douillard 1995, Dog ˘an
& C ¸alt 2001, Scelza et al. 2003, Ari & Erdemir 2005)
have demonstrated that the mineral content of root
dentine is modified by the use of EDTA and citric acid
solutions to remove the inorganic component from
instrumented canals alongside the use of sodium
hypochlorite to remove the organic component.
Phosphoric acid, used daily in conservative dentistry,
is a strong acid capable of removing the smear layer
from root dentine. Ayad (2001) obtained partial smear
layer removal with a 10% concentration of this acid
andtotal removal with
Garberoglio & Becce (1994) compared 17% EDTA,
3% EDTA and a combination of 24% phosphoric acid
plus 10% citric acid for root canal cleaning and
obtained similar results amongst the three solutions.
A recent study (Perez-Heredia et al. 2006) alternated
aqueous solutions of 2.5% sodium hypochlorite with
demineralizing solutions of 15% citric acid, 15% EDTA
a 32% concentration.
or 5% phosphoric acid, reporting the efficacy of these
agents to remove the smear layer during root canal
preparation. However, there are no data on the
decalcifying capacity of phosphoric acid in root dentine
or on its efficacy in comparison with EDTA and citric
acid solutions.
The hypothesis tested in this study was that there are
no differences in decalcifying capacity of solutions of
15% EDTA, 15% citric acid, 5% phosphoric acid and
that 2.5% sodium hypochlorite does not extract
calcium from root canal dentine; in three immersion
time periods.
The objective of this study was to assess, using
atomicabsorptionspectrometry,
capacity of solutions of 15% EDTA, 15% citric acid,
5% phosphoric acid and 2.5% sodium hypochlorite in
three immersion time periods.
the decalcifying
Materials and methods
Tooth selection
Ten maxillary central incisors, extracted for periodontal
reasonsfrom patientswithin an
40–60 years, were stored in distilled water with thymol
crystals until use. Patients were informed that the teeth
would be used in this study applying the relevant
ethical criteria: all patients consented.
agerangeof
Root canal preparation
Crowns were removed at the cemento-enamel junction
level using an Accutom-50 diamond cutter (Accutom
Hard Tissue Microtome, Struers, Ballerup, Denmark)
under copious water cooling. Root cementum was
removed from the root surface using a fine-grained
diamond bur (Perio-Set, Intensive. Grancia, Switzer-
land) at low speed and under low water cooling.
Root canals were instrumented under constant
water cooling with Peeso burs no. 4 to 6, (Dentsply
Maillefer, Ballaigues, Switzerland) using a contra-angle
handpiece. After each instrument change, root canals
were irrigated with 5 mL of distilled water.
Two 2-mm-thick transverse sections were obtained
from the coronal third of each root with an Accutom
50 automatic pre-programmed machine (Accutom
Hard Tissue Microtome). Each slice was then divided
in two equal halves, obtaining a total of four sections of
each root (S1, S2, S3 and S4).
Sections were weighed using a HM 202 precision
balance (A&D Engineering Inc., San Jose, CA, USA),
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equalizing their weight with disks of 600-grit silicon-
carbide paper (WS 18-B Struers, Ballerup, Denmark),
which were always applied to the same central surface
to avoid altering the geometry of the disks. Sections
were then labelled and stored in flasks with distilled
water at room temperature until use.
Sections of the same root (S1, S2, S3 and S4) had
approximately the same weight, geometry and degree
of calcification, allowing comparison of the decalcifying
capacity of the four irrigation solutions by testing them
on comparable specimens.
The 40 specimens obtained were divided into four
experimental groups (n ¼ 10) for treatment with
different irrigation solutions – group 1 : 15% EDTA,
pH 7; group 2 : 15% citric acid, pH 1.6; group 3 : 15%
phosphoric acid, pH 1.02; group 4 : 2.5% sodium
hypochlorite, pH 11.9. The pH of each solution was
determined by using a pH meter equipped with Micro
PH 2000 electrode (Crisol, Alella, Spain). The accuracy
of the pH meter was £0.01.
The 2.5% sodium hypochlorite solution was pre-
pared by diluting 10% hypochlorite solution (Panreac,
Barcelona, Spain) four times in distilled water; the 15%
citric acid solution by dissolving 30 g of monohydrated
acid (Panreac) in distilled water to a volume of 200 mL;
the 15% EDTA solution by dissolving 30 g of disodium
EDTA (Panreac) in distilled water to a volume
<200 mL, favouring dissolution with the addition of
sodium hydroxide (NaOH) and adding 2 mol L)1
hydrochloric acid (HCl) to obtain a pH of 7; and the
5% phosphoric acid solution by dissolving 10 g of
phosphoric acid (Panreac) in distilled water to a volume
of 200 mL. All solutions were homogenized by con-
stant stirring at18–21ºC
multi-stirrer.
Initially, 20 mL of each solution was prepared as a
blank to determine calcium levels without exposure to
specimens. Each specimen was immersed in 20 mL of
irrigantsolution for
(t1¼ 5 min, t2¼ 10 min and t3¼ 15 min). Every
5 min, 5 mL of irrigant solution was extracted with a
graduated pipette, which was then placed in hermet-
ically sealed and labelled glass vessels.
usinga magnetic
threeimmersiontimes
Spectrometer examination
Three extracts were obtained from each sample and
measured in a SpectrAA 220 FS atomic absorption
spectrometer (Varian Iberica SL, Madrid, Spain) using
an air/acetylene mixture as fuel for the flame. The
spectrometer was calibrated using solutions of 2, 5 and
10 ppm of Ca2+as reference pattern. The concentra-
tion of the original Ca2+solution was 1000 ppm
(Merck Inc., Whitehouse, NJ, USA). Values for extracts
were expressed in mg L)1(ppm).
The mg of Ca2+/g (29)
mg Ca2+/g extracted in each time period were calcu-
lated as follows:
and thepercentage
mg Ca2þ=g ¼ ðppm Ca2þÞ ? ð10?3L=mLÞ
? V=P ðV;volume;P;weight of the specimen in mgÞ
% mg Ca2+/g ¼ mg Ca2+· 100/total mg Ca2+. This
value expresses the % Ca increase in each time interval
with respect to the total Ca2+extracted.
Statistical analysis
First, a full-factorial regression model of repeated
measures was used to assess the significance of the
interaction between two factors (type of irrigation
solution and immersion time in irrigation solution)
for the extracted calcium data (mg Ca2+and % mg
Ca2+) The Kolmogorov–Smirnov test was used to
assess the distribution of the extracted calcium data.
Because results for each group did not follow a
normal distribution, variables were analysed using a
nonparametric test. The amount of calcium extracted
(mg Ca2+and %mg Ca2+) by different irrigating
solutions and in different immersion times was
analysed using the Mann–Whitney U-test (pairwise
comparisons) and the Kruskal–Wallis test (global
comparisons). The level of statistical significance
was set at P < 0.05.
Results
Full-factorial regression analysis of the influence of the
type of irrigation solution (15% EDTA, 15% citric acid,
5% phosphoric acid or 2.5% NaOCl) and of the time of
immersion in solution (5, 10 or 15 min) revealed a
statistically significant interaction between these two
factors in the amount of calcium extracted (P ¼ 0.003)
andin thepercentage
(P < 0.001). Table 1 shows the amount of calcium
extracted (mg Ca2+) for each type of irrigant solution
and immersion time. After a 5-min immersion in
irrigant solution, 15% EDTA had extracted the greatest
amount of Ca2+, followed by 15% citric acid and 5%
phosphoric acid, with a negligible amount of Ca2+
extracted in 2.5% sodium hypochlorite. The differences
amongst solutions were significant (P < 0.05) except in
the comparison between 15% EDTA and 15% citric
of calciumextracted
Root canal decalcifying Pe ´rez-Heredia et al.
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acid (P ¼ 0.820). After a 10-min immersion, there was
no significant difference (P ¼ 0.821) between the
amounts of Ca2+extracted by 15% EDTA and 15%
citric acid, but these were significantly greater than the
amounts extracted by 2.5% NaOCl and 5% phosphoric
acid. After a 15-min immersion, there was again no
significantdifference(P ¼ 0.623)
amounts of Ca2+extracted by 15% EDTA and 15%
citric acid but these were significantly greater than the
amounts extracted by 2.5% NaOCl and 5% phosphoric
acid.The global comparison amongst times shows
statistically significant differences (P < 0.001) in the
calcium extracted amongst the three immersion periods
and amongst the four irrigation solutions.
shows the % calcium extracted during the three time
periods. The most rapid decalcification rate was with
15% EDTA, which extracted 86.72 ± 7.49% of the
calciumduringthe first
between 5 and 10 min, and 5.75 ± 4.19% between
10 and 15 min. A similar behaviour was shown by 5%
citric acid and 5% phosphoric acid, which extracted
77.03 ± 11.98% and 67.08 ± 9.89% during the first
5 min of immersion. No significant differences were
found (P ¼ 0.241) in the % calcium extracted by 2.5%
NaOCl between the 5-min and 10-min immersions.
betweenthe
Table 2
5 min,10.02 ± 6.35%
Discussion
The efficacy of agents used to remove smear layer and
demineralize and soften root dentine during root canal
treatment has been examined by various means,
including microhardness measurements, micro-radio-
graphic assessments, spectrometry studies (Verdelis
et al. 1999, Dog ˘an & C ¸alt 2001, Scelza et al. 2003,
Machado-Silveiro et al. 2004, Ari & Erdemir 2005,
Gonzalez-Lopez et al. 2006) and, especially, electron
microscopy studies (Ferrer Luque et al. 1993, Calt &
Serper 2000, Di Lenarda et al. 2000, O’Connell et al.
2000, Ayad 2001, Haznedaroglu 2003, Perez-Heredia
et al. 2006). The decalcifying efficacy of these acid and
chelating agents depends on the root length, application
time, diffusion in the dentine and, especially, the
solution pH (Sen et al. 1995, Dog ˘an & C ¸alt 2001,
Serper & Calt 2002). The use of a neutral pH of around
7.3 is recommended for EDTA solutions (Serper & Calt
2002). Citric acid has shown to be effective at pH values
of 0.8–1.9 (Hennequin & Douillard 1995, Haznedaro-
glu 2003).In the present study, the amount of
extracted Ca2+increased with time in all solutions and
no significant differences were found between 15%
EDTA and 15% citric acid. These findings are consistent
Table 1 Amount of calcium extracted
(mg Ca2+) as a function of irrigating
solution and immersion time*
Irrigating solution
(? x ± SD
Immersion time
5 min 10 min 15 min
15% EDTA
15% citric acid
5% phosphoric acid
2.5% NaOCl
0.085 ± 0.029b,1
0.075 ± 0.019b,1
0.035 ± 0.015c,1
0.009 ± 0.004a,1
0.094 ± 0.028b,2
0.093 ± 0.024b,2
0.046 ± 0.020c,2
0.015 ± 0.004a,2
0.098 ± 0.028b,3
0.099 ± 0.027b,3
0.052 ± 0.023c,3
0.019 ± 0.004a,3
*In the full-factorial regression model, P values were <0.001 (for irrigating solution),
<0.001 (for immersion time) and 0.003 (for irrigating solution · immersion time
interaction).
Read vertically, the same letters indicate absence and different letters presence of
significant differences.
Read horizontally, the same numbers indicate absence and different numbers presence
of significant differences.
Table 2 Percentage of calcium extracted
(mg Ca2+) as a function of irrigating
solution and immersion time*
Irrigating solution
(? x ± SD)
Immersion time
5 min 5–10 min 10–15 min
15% EDTA
15% citric acid
5% phosphoric acid
2.5% NaOCl
86.72 ± 7.49b,1
77.03 ± 11.98b,c,1
67.08 ± 9.89c,1
43.43 ± 14.15a,1
10.02 ± 6.35b,2
17.23 ± 9.46b,c,2
22.30 ± 7.25c,2
34.04 ± 7.99a,1
3.08 ± 3.01b,3
5.75 ± 4.19b,3
10.64 ± 4.29c,3
22.56 ± 7.97a,2
*In the full-factorial regression model, P values were 0.446 (for irrigating solution),
<0.001 (for immersion time) and <0.001 (for irrigating solution · immersion time
interaction).
Read vertically, the same letters indicate absence and different letters presence of
significant differences.
Read horizontally, the same numbers indicate absence and different numbers presence
of significant differences.
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with previous results using 10% and 20% citric acid and
17% EDTA solutions (Scelza et al. 2003, Gonzalez-Lopez
et al. 2006). However, in all three immersion times
studied, the decalcifying capacity of 15% EDTA and
15% citric acid solutions was higher than that of the 5%
phosphoric acid solution (Table 1). The reason for these
differences may be that the concentration of phosphoric
acid was lower than that of the EDTA and citric acid
solutions. Thus, a higher extraction of Ca2+ions could
be expected if higher concentrations of phosphoric acid
were used. Decalcification may also be higher at a
specific pH, as in the case of citric acid solution at pH 1.1
(Hennequin & Douillard 1995). However, higher con-
centrations of phosphoric acid could cause reprecipita-
tion of hydroxyapatite from the calcium phosphate
solutions formed by the initial dissolution of root
dentine. The formation of new calcium phosphate
complexes would reduce the extraction of calcium ions
from exposed root dentine (Marshall et al. 1993). An
effective irrigation solution must also be able to remove
the inorganic component from dentine, and a recent
study (Perez-Heredia et al. 2006) demonstrated that the
combined use of 5% phosphoric acid and 2.5% sodium
hypochlorite solutions is adequate to remove the smear
layer from the root canal.
extracted was higher during the first 5 min of immer-
sion in all solutions, with the highest percentage
(86.72%) extracted with 15% EDTA solution. These
results are in agreement with the report by Cergneux
et al. (1987) of total removal of the smear layer after
using a 15% EDTA solution for 4 min. Other studies
demonstrated that the highest amount of Ca2+ions is
extracted during the first 3 min of immersion in a 17%
EDTA solution (Scelza et al. 2003, Gonzalez-Lopez et al.
2006) and during the first 5 min in a 10% citric acid
solution (Machado-Silveiro et al. 2004). After 5 min,
the decalcification progressively reduced, and signifi-
cant differences were found between the 10-min and
15-min immersion periods (Table 2). These results
agree with those obtained by Gonzalez-Lopez et al.
(2006) using the same methodology and could be
explained in relation to the acid and chelating solutions
studied, by an increase in the organic material exposed
on root dentine surfaces after action of the demineral-
izing agents. The organic matrix of dentine may act as a
limiting factor in the dissolution of the inorganic
component, thus reducing the decalcifying action of
chelating agents over time (Inaba et al. 1996, Verdelis
et al. 1999, Dog ˘an & C ¸alt 2001).
a 2.5% sodium hypochlorite solution had a small
decalcifying effect. The extraction of Ca2+ions was
In this study, the % Ca2+
In the present study,
significantly lower than achieved with the acid and
chelating solutions assessed. It has been reported that
treatment with sodium hypochlorite causes mineral
accumulation in human root dentine (Inaba et al.
1996). Sodium hypochlorite dissolves organic material
and exposes inorganic material, thereby avoiding a
greater dissolution of root dentine and it leaves a smear
layer of mineralized tissue (Baumgartner & Mader
1987, Baumgartner & Cuenin 1992). It has been
demonstrated that the use of 2.5% sodium hypochlorite
as irrigation solution, either alone or combined with a
17% EDTA solution, significantly increases the Ca/P
ratio of root dentine (Dog ˘an & C ¸alt 2001).
study, 2.5% sodium hypochlorite solution obtained a
higher percentage of calcium ion extraction during the
first 5 min followed by a slow decrease in the extraction
rate, with no significant differences between the 5-min
(43.43%) and 10-min (34.04%) immersion times.
Changes in hydroxyapatite re-crystallization after
sodium hypochlorite treatment (Perdigao et al. 2000)
may be responsible for the decrease in calcium and
phosphorus found in root dentine (Ari & Erdemir 2005).
Furthermore, in removing the organic component from
dentine, sodium hypochlorite also eliminates mineral-
ization inhibitors and increases the porosity of residual
dentine (Sakae et al. 1988, Inaba et al. 1996). Passage
of Ca2+ions into the irrigation solution would explain
the decalcification results obtained with 2.5% sodium
hypochlorite solution in the present study.
In this
Conclusions
Within the limitations of the present study, it can be
concluded that the use of solutions of 15% EDTA, 15%
citric acid or 5% phosphoric acid produces root dentine
decalcification, mainly during the first 5 min of action.
The efficacy of 15% EDTA and 15% citric acid solutions
was significantly higher than that of 5% phosphoric
acid solution in all three immersion periods studied. It
was also observed that 2.5% sodium hypochlorite
solution is capable of extracting small amounts of
calcium from root dentine.
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