Basic Research—Technology
Atomic Absorption Spectrometry and Scanning Electron
Microscopy Evaluation of Concentration of Calcium Ions
and Smear Layer Removal With Root Canal Chelators
Júlio César Emboava Spanó, DDS, MSc, PhD,* Ricardo Gariba Silva, DDS, MSc, PhD,*
Débora Fernandes Costa Guedes, BCH, MSc, PhD,† Manoel Damião Sousa-Neto, DDS, MSc, PhD,*
Carlos Estrela, DDS, MSc, PhD,‡ and Jesus Djalma Pécora, DDS, MSc, PhD*
Abstract
Aim. The aim of this study was to evaluate the concen-
tration of calcium ions and smear layer removal by using
root canal chelators according to flame atomic absorp-
tion spectrophotometry and scanning electron micros-
copy. Forty-two human maxillary central incisors were
irrigated with 15% ethylenediaminetetraacetic acid
(EDTA), 10% citric acid, 10% sodium citrate, apple
vinegar, 5% acetic acid, 5% malic acid, and sodium
hypochlorite. The concentration of calcium ions was
measured by using flame atomic absorption spectrom-
etry, and smear layer removal was determined by scan-
ning electron microscopy. Mean  standard deviation,
one-way analysis of variance, Tukey-Kramer, Kruskal-
Wallis, Dunn, and kappa tests were used for statistical
analysis. The use of 15% EDTA resulted in the greatest
concentration of calcium ions followed by 10% citric
acid; 15% EDTA and 10% citric acid were the most
efficient solutions for removal of smear layer. (J Endod
2009;35:727–730)
Key Words
Apple vinegar, chelators, irrigants, scanning electron
microscopy, smear layer
From the *Department of Endodontics and †Department of
Chemistry, University of São Paulo, Ribeirão Preto, SP; and
‡
Department of Endodontics, Federal University of Goiás, Goiâ-
nia, GO, Brazil.
Address requests for reprints to Professor Carlos Estrela,
Centro de Ensino e Pesquisa Odontológica do Brazil (CEPO-
BRAS), Avenida C-198, Quadra 487, Lote 9, Jardim América,
CEP: 74.270-040, Goiânia, GO, Brazil. E-mail address:
estrela3@terra.com.br.
0099-2399/$0 - see front matter
Copyright ª 2009 American Association of Endodontists.
doi:10.1016/j.joen.2009.02.008
JOE — Volume 35, Number 5, May 2009
T he success of root canal treatment depends on the control of microorganisms in
infected root canals. Root canal treatment is performed in several steps; one of
the most important phases is cleaning and shaping, when instruments and irrigating
solutions are used. During this phase, organic and inorganic matter remaining in the
root canal must be removed so that root canal filling might adapt to the canal perfectly
(1–6). McComb and Smith (1) reported that the smear layer adheres weakly to the root
canal walls and, therefore, reduces sealer adhesion and affects sealing negatively. It
should be removed before obturation to ensure that close contact of the sealer with
the dentin surface is obtained.
Chelating agents are believed to aid root canal irrigation and removal of the
inorganic smear layer (1–4) because the action of sodium hypochlorite (NaOCl) on
its organic structure is positive (6–8). Østby (9) suggested that the use of ethylenedia-
minetetraacetic acid (EDTA) at a neutral pH promoted chelation of calcium in dentin.
This substance is the one most frequently recommended for the removal of the smear
layer in endodontics (3, 4, 9, 10).
Other substances to remove smear layer have been suggested, such as citric acid,
EDTAC, REDTA, RC-Prep, Glyde file, and apple vinegar (3, 4, 10–16). However, the
effectiveness of chelating agents depends on some factors such as time of application,
pH, concentration, and amount of solution available in the root canal (10–14,
17–23).
Torabinejad et al (10) investigated the effect of a mixture of a new solution
containing tetracycline isomer, a citric acid, for the removal of the smear layer and
a detergent (MTAD) used as a final rinse of the surfaces of instrumented root canals.
The presence or absence of smear layer and the amount of erosion on the surface of the
root canal walls at the coronal, middle, and apical portions of each canal were evaluated
by scanning electron microscopy. The results showed that MTAD was an effective solu-
tion for the removal of smear layer when used as a final rinse. This root canal irrigating
solution does not significantly change the structure of the dentinal tubules when used in
conjunction with 5.25% NaOCl. Estrela et al (13) compared root canal surface cleaning
with apple vinegar, 2.5% sodium hypochlorite, 2% chlorhexidine gel, and EDTA. The
combination of EDTA with irrigating solutions significantly increased smear layer
removal in all cases studied. The best result was obtained with the use of apple vinegar
in combination with EDTA. The vinegar has been indicated as an antiseptic agent
(24–26) and has been used for the treatment of infected wounds (24). The effect of
vinegar on the endodontic microbiota was recently investigated (12). It is important
to know the potential of cleaning associated with its action on tissue healing. Distilled
white vinegar and wine vinegar are composed mainly of acetic acid, whereas apple
vinegar is composed mainly of malic acid. Vinegar has been indicated for centuries
as a result of its medicinal properties. Acetic acid and vinegar have been used to treat
infections (24).
Most studies analyzed only the ability of chelating solutions to remove smear layer
(27–30); therefore, it is important to determine the concentration of calcium ions that is
removed from teeth when different chelators are used. This study evaluated the concen-
tration of calcium ions and smear layer removal by using root canal chelators according
to flame atomic absorption spectrophotometry and scanning electron microscopy.
Concentration of Calcium Ions and Smear Layer Removal With Chelators 727
Basic Research—Technology
Materials and Methods
Experimental Teeth
Forty-two recently extracted single-rooted maxillary central inci-
sors were kept in 0.1% thymol solution and under refrigeration and
later randomly divided into 4 groups. Standard access to the pulp
chamber was performed with diamond burs in a high-speed handpiece;
cervical shoulder removal and compensatory wear were performed with
stainless steel LA Axxess burs (SybronEndo, Glendora, CA). The working
length was checked with a #10 K-file (Dentsply Maillefer, Ballaigues,
Switzerland) introduced into the root canal of each tooth up to the point
when it was visualized at the apex and then pulled back 1 mm. The
anatomic diameter of the canal at working length was also determined
by introducing first series K-type instruments of successively larger
diameters. The anatomic diameter of the root canal was recorded
when the instrument showed resistance to be removed from working
length. All the teeth with a canal diameter at working length greater
than 40 hundredths of a millimeter were discarded and replaced with
others. A standard wear of 20 hundredths of a millimeter in the apical
third was, therefore, ensured. A crown-down technique (Free Tip
Preparation technique) (30) was used with nickel-titanium instruments
(Quantec; SybronEndo Corporation, West Collins, Orange, CA) until
a #60 file (.04 taper) reached working length. All procedures were per-
formed with a NiTi control handpiece (Anthogyr, Sallanches, France).
During preparation, the root canals were irrigated with 8 mL of 1.0%
sodium hypochlorite at each file change. Root canals were rinsed
with 20 mL of deionized water, obtained by using the Milli-Q water
purification system (Millipore, Temecula, CA), for removal of possible
dentin chips. Subsequently, the apical foramen was sealed with
composite resin to keep test irrigating solutions inside the root canal.
Test Irrigating Solutions
After cleaning and shaping, specimens were randomly divided into
7 groups of 6 teeth each according to final irrigating solution: 15%
EDTA (group 1), 10% citric acid (group 2), 10% sodium citrate (group
3), apple vinegar (group 4), 5% acetic acid (group 5), and 5% malic
acid (group 6). In group 7 (control group), teeth were instrumented
and irrigated with 1% sodium hypochlorite, but no final irrigation
solution was used. Each solution remained 5 minutes in the root canal.
All the solutions were prepared in the Research Laboratory of the
Department of Restorative Dentistry (University of São Paulo, Ribeirão
Preto, SP, Brazil). The 1% sodium hypochlorite solution was obtained
by dilution of a 12% solution. Apple vinegar was obtained from Castelo
(Castelo Alimentos S. A., Jundiaı́, SP, Brazil); 15% EDTA, 10% citric
acid, 10% sodium citrate, 5% acetic acid, and 5% malic acid solutions
were prepared by using Sigma Aldrich (St Louis, MO) reagents.
Concentration of Calcium Ions (Atomic Absorption
Spectrometry)
An experimental device was prepared to collect samples of the test
irrigating solutions. The coronal portions of the teeth were glued to
a device attached to a 30-mL plastic tube by using adhesive putty squares
(Pritt Multi-Tak; Henkel Ltda, Itapevi, São Paulo, Brazil). A Luer Slip
syringe (Becton Dickinson, Franklin Lakes, NJ) with a 30-gauge needle
previously fixed to the tooth was used for irrigation. Five milliliters of test
irrigants was used for 5 minutes to irrigate the root canals (1 mL/min).
For irrigation, the 30-gauge irrigating needle (Hawe Neos, Bioggio,
Switzerland) was introduced 1 mm short of working length. To collect
the irrigating solution sample, the syringe was disconnected from the
tooth, and the device was removed from the plastic platform, sealed,
labeled, and prepared for atomic absorption spectrometry by using
an air-acetylene flame (6 mA, 422.7 nm, 0.5 mm; Shimadzu AA-680,
728 Emboava Spanó et al.
Tokyo, Japan) to determine the concentration of calcium removed
from each tooth. Considering that spectrometry is a conventional
method, it was decided just to use 5 repetitions.
Scanning Electron Microscopy
After the procedures described above, all teeth were dried with
paper points. Grooves were prepared by using a metallic disk with
water-refrigerated (KG Sorensen Ind. Com., São Paulo, SP, Brazil) care-
fully and by using a surgical chisel to create a split along the long axis
buccolingually to expose the entire extent of the root canal and were
examined under scanning electron microscopy (JEOL JSM5410, Tokyo,
Japan).
The root canal was measured, and the central part of each middle
third was evaluated. Three calibrated, blinded examiners analyzed all
the images, which were obtained at 1000 magnification.
The scanned samples were evaluated for the amount of smear layer
by using the following scores: 1, no smear layer (0%); 2, a few areas
(nearly 33.3%) covered by smear layer, and many dentinal tubules
visible; 3, most areas (nearly 66.6%) covered by smear layer, and
only a few dentinal tubules visible; 4, all areas (100%) covered by smear
layer, and no dentinal tubules visible. The evaluation was made quali-
tatively, considering these data.
All the results of concentration of calcium ions and smear layer
removal were analyzed statistically by using the software GraphPad
Prism 4.00 (San Diego, CA) and Minitab (State College, PA).
Spectrometry results were described as mean  standard devia-
tion (SD) values. One-way analysis of variance and the Tukey-Kramer
test for multiple comparisons were used to determine statistical
differences between groups. The Kruskal-Wallis and Dunn tests were
used to analyze the nonparametric results of smear layer removal.
Interobserver agreement was analyzed by using kappa statistics.
This study was approved by the Ethics in Research Committee of
the institution where it was conducted.
Results
Spectrometry data were described as mean  SD, respectively, as
following: 15% EDTA, 5.678  1.493; 10% citric acid, 4.298  0.494;
10% sodium citrate, 0.103  0.045; apple vinegar, 2.481  0.312; 5%
acetic acid, 2.259  0.379; 5% malic acid, 2.520  0.508 (Fig. 1).
Statistical differences between groups were determined by one-way
analysis of variance (F = 36.74, R2 = 0.8845; P < .0001). The
Tukey-Kramer multiple comparisons test showed that 15% EDTA had
the greatest concentration of calcium ions in solution, followed by
10% citric acid (second position), apple vinegar, 5% acetic acid, and
5% malic acid (third position), and 10% sodium citrate (fourth
position).
Figure 1. Histogram of frequencies of calcium concentration values in
solutions.
JOE — Volume 35, Number 5, May 2009

Figure 2. (A) Root canal preparation with 1% NaOCl as irrigating solution and treatment with 15% EDTA for 5 minutes as a final rinse resulted in the removal
of the smear layer in the middle portions of the root canal (original magnification, 1000). (B) Root canal preparation with 1% NaOCl as irrigating solution
and treatment with 10% citric acid for 5 minutes as a final rinse resulted in the removal of the smear layer in the middle portions of the root canal (original
magnification, 1000).
In the analysis of smear layer removal, the Kruskal-Wallis test
showed statistically significant differences between samples (P <
.0001). The results of the Dunn test for multiple comparisons presented
that 15% EDTA and 10% citric acid were more efficient to remove smear
layer than the other irrigating solutions (Fig. 2).
Interobserver agreement was good (kappa, 0.792).
Discussion
The solutions containing 15% EDTA and 10% citric acid had
greater concentrations of calcium ions than apple vinegar, 5% acetic
acid, 5% malic acid, and 10% sodium citrate; 15% EDTA and 10% citric
acid were the most efficient irrigants to remove smear layer.
The association of scanning electron microscopy and atomic
absorption spectrophotometry also was used previously by Marques
et al (27), who verified the smear layer removal and the amount of
calcium ions present in 17% EDTAC, 17% CDTA, and 17% EGTA. The
results obtained from the quantification of chelated calcium ions
showed that the root canals irrigated with 17% EDTAC and 17%
CDTA were statistically similar to each other, and both were different
from the specimens irrigated with 17% EGTA, which chelated the least
amount of calcium ions. The same relation was found for smear layer
removal, with 17% EGTA presenting the highest smear layer scores
(high scores indicate less smear layer removal). Considering the differ-
ences between the solutions and concentrations used, correlation with
the results of present study was not possible.
The experimental models used in this study were validated in
previous studies (1, 10, 16, 19, 20, 27–29). The teeth were selected
according to the amount of irrigating solution necessary to fill the
root canal and to enable the collection of irrigating solution samples
to determine the concentration of calcium ions. The sample size was
coherent with complexity to obtain teeth for studies. Experimental solu-
tions were chosen according to the need to compare conventional
chelators (EDTA, citric acid) with nonconventional solutions that might
also remove smear layer such as apple vinegar (12, 13). Different types
of apple vinegar might have acetic acid or malic acid in their constitu-
tion; therefore, this study analyzed commercial apple vinegar, acetic
acid, and malic acid separately. Distilled white vinegar and wine vinegar
are composed of acetic acid primarily, whereas the main component of
apple vinegar is malic acid. The percentage of acetic acid in white
distilled vinegar is higher than in the other solutions. Malic acid found
in apple vinegar is what confers on it its therapeutic properties (12, 13,
24–26, 31).
Zehnder et al (19) evaluated the effect of reducing surface tension
of endodontic chelator solutions (15.5% EDTA, 10% citric acid, or 18%
JOE — Volume 35, Number 5, May 2009
Basic Research—Technology
1-hydroxyethylidene-1, 1-bisphosphonate [HEBP] prepared with and
without 1% [wt/wt] polysorbate [Tween] 80 and 9% propylene glycol)
on their ability to remove calcium from instrumented root canals. Their
results showed that there was no increase in calcium chelating ability
when the surface tension of chelator solutions was lowered with wetting
agents. Citric acid removed significantly more calcium than EDTA or
HEBP solutions (P < .05). EDTA solutions showed a trend to chelate
more calcium than HEBP solutions; this difference was, however, not
significant at the 5% level.
Their results (19) are in disagreement with the results obtained in
this study, in which 15% EDTA showed a better performance to remove
calcium ions than citric acid. These differences might be attributed to
method differences and irrigation time. We used 5 mL of test irrigants
for 5 minutes (1 mL/min), a considerable time and volume of irrigation.
These aspects were discussed in another study conducted by Zehnder et
al (20), which evaluated interactions of EDTA and citric acid with
sodium hypochlorite. Those authors reported that chelation during
root canal irrigation was not necessarily an equilibrium reaction, deter-
mined by a standard stability constant, because rate effects and ligand
exchange reactions might considerably affect complex formation. Irri-
gating times of 1 minute were relatively short, but longer irrigation times
with effective chelators, such as EDTA, might affect dentin structure if
root canals are prepared after removal of smear layer (21).
Torabinejad et al (10) found that MTAD, a solution containing
citric acid as one of its components, effectively removes the smear layer
when used as a final rinse. It does not significantly change the structure
of dentinal tubules when used in conjunction with 5.25% NaOCl as
a root canal irrigating solution. The authors reported that some of
the main disadvantages of the use of EDTA were its destructive effect
on coronal and middle thirds of root dentin and its limited antibacterial
action. In contrast to the destructive effect of 5-minute EDTA exposure,
no significant dentinal erosion was found in a pilot study in which the
surface of the root canals was in contact with MTAD for times ranging
from 1–20 minutes.
The results of our study showed that when used for 5 minutes as
an irrigating solution, EDTA removed more calcium ions than citric acid
or other irrigants. Calt and Serper (21) evaluated the effects of EDTA
on smear layer removal and on the structure of dentin after 1 and
10 minutes. At 1 minute, EDTA irrigation was effective in removing
the smear layer. However, a 10-minute application of EDTA caused
excessive peritubular and intertubular dentinal erosion.
The effects of citric acid and EDTA solutions on the microhardness
and roughness of human root canal dentin were also recently studied
(32). Significant differences in microhardness were found between
Concentration of Calcium Ions and Smear Layer Removal With Chelators 729
Basic Research—Technology
test groups, and the citric acid group had the lowest microhardness.
When the results of surface roughness values were analyzed, the differ-
ences between the EDTA and citric acid groups and citric acid and
control groups were statistically significant. Comparisons showed that
the increase in dentin roughness was significantly greater in the citric
acid group than in the EDTA or control groups. The structure of
mechanically instrumented intraradicular dentin after irrigation with
NaOCl as the initial rinse and BioPure MTAD as the final rinse was
examined in the coronal, middle, and apical parts of root canal walls
with transmission electron microscopy by Tay et al (33), who found
that 17% EDTA and BioPure MTAD were equal in their capacity to
completely remove endodontic smear layers when used as the final
rinse with mechanical agitation. BioPure MTAD is comparatively more
aggressive in demineralizing intact intraradicular dentin and exposed
collagen matrices that were 1.5–2 times as thick as those produced
when EDTA was used. In another study, Tay et al (34) reported that
the identification of microporous, demineralized collagen matrices
from instrumented canal walls with the use of a more biologically
oriented dehydration protocol suggests that these entities probably
existed without prior recognition, when 17% EDTA or MTAD was
used as final irrigants. In addition, the authors emphasized that the reca-
pitulation of an analogous scenario of compacting filling materials
against predentin is not going to be reiterated; the presence of demin-
eralized collagen matrices on smear-free instrumented radicular dentin
stimulates additional food for thought.
De-Deus et al (35) quantitatively and longitudinally analyzed the
smear layer dissolution kinetics of 18% etidronate (HEBP), 9%
HEBP, and 17% EDTA on human dentin by using a single-tooth compar-
ative model. It can be concluded that the demineralization kinetics
promoted by both 9% HEBP and 18% HEBP were significantly slower
than those of 17% EDTA. In a recent review about the use of chelating
agents in root canal treatment, Hülsmann et al (4) found that a final
intensive rinse with a 17% chelating solution reduces the extent of
the remaining smear layer and results in better adaptation of filling to
the canal wall. The order in which the NaOCl and the chelator should
be used has not yet been defined. Solutions that contain EDTA should
be used for 1–5 minutes.
Therapeutic options to treat root canals must be continuously
evaluated and discussed to provide treatment guidelines. Ideally, any in-
tracanal medication should be studied for different types of applications
and according to its antimicrobial, chemical, biologic, and clinical
properties (36, 37), considering the level of evidence (38).
Conclusions
Under these study conditions and within the limitations of this
investigation, 15% EDTA solutions removed the highest concentration
of calcium ions followed by 10% citric acid, when compared with
10% sodium citrate, apple vinegar, 5% acetic acid, and 5% malic acid.
Smear layer removal was the most efficient when 15% EDTA and 10%
citric acid were used.
References
1. McComb D, Smith DC. A preliminary scanning electron microscopic study of root
canal after endodontic procedures. J Endod 1975;1:238–42.
2. Dautel-Morazin A, Vulcain JM, Bonnaure-Mallet M. An ultrastructural study of the
smear layer: comparative aspects using secondary electron image and backscattered
electron image. J Endod 1994;20:531–4.
3. Torabinejad M, Handysides R, Khademi AA, Bakland LK. Clinical implications of the
smear layer in endodontics: a review. Oral Surg Oral Med Oral Pathol Oral Radiol
Endod 2002;94:658–66.
4. Hülsmann M, Heckendorff M, Lennon Á. Chelating agents in root canal treatment: mode
of action and indications for their use—a review. Int Endod J 2003;36:810–30.
5. Zehnder M. Root canal irrigants. J Endod 2006;32:389–98.
730 Emboava Spanó et al.
6. Sen BH, Wesselink PR, Türkün M. The smear layer: a phenomenon in root canal
therapy. Int Endod J 1995;28:141–8.
7. Estrela C, Estrela CRA, Barbin EL, Spanó JC, Marchesan MA, Pécora JD. Mechanism
of action of sodium hypochlorite. Braz Dent J 2002;13:113–7.
8. Estrela C, Estrela CRA, Decurcio DA, Hollanda ACB, Silva JA. Antimicrobial efficacy of
ozonated water, gaseous ozone, sodium hypochlorite and chlorhexidine in infected
human root canals. Int Endod J 2007;40:85–93.
9. Østby NB. Chelation in root canal therapy: ethylenediamine tetraacetic acid for
cleaning and widening of root canals. Odontol Tidskrift 1957;65:3–11.
10. Torabinejad M, Khademi AA, Babagoli J, et al. A new solution for the removal of the
smear layer. J Endod 2003;29:170–5.
11. Cruz-Filho AM, Sousa-Neto MD, Saquy PC, Pécora JD. Evaluation of the effect of ED-
TAC, CDTA, and EGTA on radicular dentin microhardness. J Endod 2001;27:183–4.
12. Estrela C, Holland R, Bernabé PFE, Souza V, Estrela CRA. Antimicrobial potential of
medicaments used in healing process in dog’s teeth with apical periodontitis. Braz
Dent J 2004;15:181–5.
13. Estrela C, Lopes HP, Elias CN, Leles CR, Pécora JD. Limpeza da superfı́cie do canal
radicular pelo vinagre de maçã, hipoclorito de sódio, clorexidina e EDTA. Rev Ass
Paul Cir Dent 2007;61:117–22.
14. Hill PK. Endodontics. J Prost Dent 1959;9:142.
15. Loel A. Use of acid cleanser in endodontic therapy. J Am Dent Assoc 1975;90:145–51.
16. Machado-Silveiro IF, González-López S, González-Rodrı́guez MP. Decalcification of
root canal dentine by citric acid, EDTA and sodium citrate. Int Endod J 2004;37:365–9.
17. Baumgartner JC, Brown CM, Mader CI, Peters DD, Shulman JD. A scanning electron
microscopic evaluation of root canal debridement using saline, sodium hypochlo-
rite, and citric acid. J Endod 1984;10:525–31.
18. Baumgartner JC, Ibay AC. The chemical reactions of irrigants used for root canal
debridement. J Endod 1987;13:47–51.
19. Zehnder M, Schicht O, Sener B, Schmidlin P. Reducing surface tension in
endodontic chelator solutions has no effect on their ability to remove calcium
from instrumented root canals. J Endod 2005;31:590–2.
20. Zehnder M, Schicht O, Sener B, Waltimo T. Chelation in root canal therapy recon-
sidered. J Endod 2005;31:817–20.
21. Calt S, Serper A. Time-dependent effects of EDTA on dentin structures. J Endod
2002;28:17–9.
22. Saquy PC, Maia-Campos GM, Sousa-Neto MD, Guimarães LF, Pécora JD. Evaluation
of chelating action of EDTA in association with Dakin’s solutions. Braz Dent J 1994;
5:65–70.
23. Grawehr M, Sener B, Waltimo T, Zehnder M. Interactions of ethylenediamine tetraace-
tic acid with sodium hypochlorite in aqueous solutions. Int Endod J 2003;36:411–7.
24. Thacker E. The vinegar. São Paulo: Pacific Post Com Ldta; 2000.
25. Valnet J. Aromathérapie. Paris: Librairie Maloine Éditeur; 1973.
26. Phillips I, Lobo AZ, Fernandes R, Gundara NS. Acetic acid in the treatment of super-
ficial wounds infected by Pseudomonas aeruginosa. Lancet 1968;6:11–2.
27. Marques AA, Marchesan MA, Sousa-Filho CB, Silva-Souza YT, Sousa-neto MD, Cruz-
Filho AM. Smear layer removal and chelated calcium ion quantification of three
irrigating solutions. Braz Dent J 2006;17:306–9.
28. Lui JN, Kuah HG, Chen NN. Effect of EDTA with and without surfactants or ultrasonics
on removal of smear layer. J Endod 2007;33:472–5.
29. Silva LA, Sanguino AC, Rocha CT, Leonardo MR, Silva RA. Scanning electron micro-
scopic preliminary study of the efficacy of smear clear and EDTA for smear layer
removal after root canal instrumentation in permanent teeth. J Endod 2008;34:1541–4.
30. Pécora JD, Capelli A, Seixas FH, Marchesan MA, Guerisoli DMZ. Biomecânica rota-
tória: realidade ou futuro. Rev Ass Paul Cirur Dent 2002;56:4–6.
31. Caligiani A, Acquotti D, Palla G, Bocchi V. Identification and quantification of the
main organic components of vinegars by high resolution 1H NMR spectroscopy.
Analyt Chim Acta 2007;585:110–9.
32. Eldeniz AU, Erdemir A, Belli S. Effect of EDTA and citric acid solutions on the micro-
hardness and the roughness of human root canal dentin. J Endod 2005;31:107–10.
33. Tay FR, Pashley DH, Loushine RJ, et al. Ultrastructure of smear layer-covered intra-
radicular dentin after irrigation with BioPure MTAD. J Endod 2006;32:218–21.
34. Tay FR, Gutmann JL, Pashley DH. Microporous, demineralized collagen matrices in
intact radicular dentin created by commonly used calcium-depleting endodontic
irrigants. J Endod 2007;33:1086–90.
35. De-Deus G, Zehnder M, Reis C, et al. Longitudinal co-site optical microscopy study
on the chelating ability of etidronate and EDTA using a comparative single-tooth
model. J Endod 2008;34:71–5.
36. Estrela C, Silva JA, Alencar AHG, Leles CR, Decurcio DA. Efficacy of sodium
hypochlorite and chlorhexidine against Enterococcus faecalis: a systematic review.
J Appl Oral Sci 2008;16:364–8.
37. Barbin LE, Saquy PC, Guedes DFC, Souza-Neto MD, Estrela C, Pécora JD. Determi-
nation of para-chloroaniline and reactive oxygen species in chlorhexidine and
chlorhexidine associated with calcium hydroxide. J Endod 2008;34:1508–15.
38. Hargreaves KM. From consent to CONSORT: clinical research in the 21st century.
J Endod 2005;31:1–3.
JOE — Volume 35, Number 5, May 2009