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journal of dentistry 38 (2010) 201206

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Differential aggressiveness of ethylenediamine tetraacetic

acid in causing canal wall erosion in the presence
of sodium hypochlorite

Sui Mai a, Young Kyung Kim b, Dwayne D. Arola c, Li-sha Gu a,

Jong Ryul Kim d, David H. Pashley e, Franklin R. Tay f,*
a
Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, China
b
Department of Conservative Dentistry, School of Dentistry, Kyungpook National University, Daegu, Republic of Korea
c
Department of Mechanical Engineering, University of Maryland Baltimore County, Baltimore, MD, USA
d
Department of Conservative Dentistry, School of Dentistry, KyungHee University, Seoul, Republic of Korea
e
Department of Oral Biology, School of Dentistry, Medical College of Georgia, Augusta, GA, USA
f
Department of Endodontics, School of Dentistry, Medical College of Georgia, Augusta, GA 30912-1129, USA

article info abstract

Article history: Objective: This study tested the hypothesis that the use of ethylenediamine tetraacetic acid
Received 7 August 2009 (EDTA) as a final irrigant causes canal wall erosion only after prolonged use of 5.25% sodium
Received in revised form hypochlorite (NaOCl) as the initial irrigant.
13 October 2009 Methods: Two irrigation protocols were employed: (1) 5.25% NaOCl as the initial irrigant for
Accepted 13 October 2009 10 min and 17% EDTA as the final irrigant for 2 min; and (2) 5.25% NaOCl for 60 min and 17%
EDTA for 2 min. Flexural strengths of dentine beams prepared from human mid-coronal
dentine were evaluated using a miniature three-point bending device after they were
Keywords: irrigated with either protocol. Transmission electron microscopy was used to examine
Dentine the conditions of instrumented canal walls irrigated with either protocol after they were
EDTA bonded with methacrylate resin-based sealers.
Erosion Results: Immersion of dentine beams in NaOCl for 60 min instead of 10 min prior to the use
Flexural strength of EDTA resulted in a significant ( p < 0.001) decline in flexural strength. For both NaOCl
Irrigation initial rinsing times, a 2-min final irrigation with EDTA created similar 0.5 mm thick
NaOCl demineralisation fronts. However, extensive surface and subsurface erosion of radicular
dentine was seen only when the dentine was immersed in NaOCl for 60 min.
Conclusion: The apparent aggressiveness of EDTA in causing canal wall erosion is attributed
to the prolonged use of NaOCl. The associated decline in dentine flexural strength has
potential clinical relevance when thin pulp chamber dentine is immersed in NaOCl for
lengthy periods during canal instrumentation. This may render root-treated teeth more
prone to vertical fracture.
# 2009 Elsevier Ltd. All rights reserved.

1. Introduction predictable means for clinicians who encompass removal of

canal wall smear layers as part of their root canal cleaning and
The sequential application of sodium hypochlorite (NaOCl) shaping objectives.1 Nevertheless, canal wall erosion, char-
and ethylenediamine tetraacetic acid (EDTA) provides a acterised by dissolution of intertubular and peritubular

* Corresponding author. Tel.: +1 706 7212031; fax: +1 706 7216252.

E-mail address: ftay@mcg.edu (F.R. Tay).
0300-5712/$ see front matter # 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jdent.2009.10.004
 Author's personal copy
202 journal of dentistry 38 (2010) 201206
dentine and coalescence of widened dentinal tubules, has
been associated with the use of this dual irrigant regime.210 As
these surface features were acquired exclusively using
scanning electron microscopy, little is known about the
subsurface characteristics of canal wall erosion. Decline in
mechanical properties of a bulk specimen is unlikely to be
caused only by surface alteration of the material. Thus, the
recent report that prolonged NaOClEDTA irrigation results in
deterioration of the mechanical properties of radicular
dentine11 requires clarification of the subsurface extent of
canal wall erosion.
As EDTA demineralises radicular dentine,12,13 it is logical to
assume that canal wall erosion is caused by prolonged use of
EDTA or its sustained release from dentinal tubules. Indeed,
attempts have been made to inactivate the chelating effects of
EDTA with NaOCl.14 For short exposure times that are
clinically relevant for dentine bonding (<1 min), the deminer-
alisation action of 17% EDTA is mild when compared to the
more aggressive acid conditioners employed in etch-and-rinse
adhesives.15 Even for longer contact times (12 min) that are
clinically relevant for endodontic irrigation, the calcium
chelation capacity of EDTA is self-limiting16,17 due to the
excellent buffering capacity of dentine.18 Thus, it is unlikely
that canal wall erosion is caused solely by EDTA irrigation. As
the mechanical properties of mineralised dentine are
adversely affected by its prolonged contact with 35%
NaOCl,1921 it is more likely that NaOCl and EDTA interact
synergistically to augment penetration of EDTA into the
intertubular and peritubular dentine to expedite their disin-
tegration. If this hypothesis is correct, increasing the initial
NaOCl exposure time while keeping the final EDTA exposure
time constant should increase the likelihood of inducing canal
wall erosion. Thus, the objective of the present study was to
test the hypothesis that the use of EDTA as a final irrigant
causes canal wall erosion only after prolonged use of 5.25%
NaOCl as the initial irrigant.
2. Materials and methods
2.1. Flexural strength
Flexural strength evaluation was performed using mid-
coronal dentine obtained from 30 non-carious human third
molars to ensure collection of enough testable substrates and
consistency in dentine tubular orientation. A dentine disk was
prepared perpendicular to the longitudinal axis of each tooth
with a slow-speed Isomet saw (Buehler Ltd., Lake Bluff, IL,
USA) under copious water cooling. A 7 mm  3 mm  0.2 mm
beam was prepared from the centre of each disk to ensure that
dentinal tubules were oriented parallel to the plane of
maximum stress during three-point flexure.22 The dimensions
of each beam were measured to the nearest 0.01 mm.
The dentine beams were randomly divided into three
groups (N = 10). Beams in the control group were tested under
water without prior immersion in active root canal irrigants.
The first experimental group was designed to simulate root
canal treatment in single-rooted anterior teeth with large
canals wherein the NaOCl remains in the pulp chamber for
10 min during canal instrumentation. Accordingly, the beams
were first immersed in 5.25% NaOCl (The Clorox Company,
Oakland, CA, USA) for 10 min, rinsed with deionised water and
immersed in 17% EDTA (SigmaAldrich, St. Louis, MO, USA) for
2 min. The beams were thoroughly rinsed with deionised
water again to prevent continuous demineralisation of the
dentine surfaces. The second experimental group was
designed to simulate a more complex treatment scenario in
root canal treatment of posterior teeth wherein the NaOCl
remains in the pulp chamber for 60 min during canal
negotiation and shaping. Accordingly, the beams were
immersed in 5.25% NaOCl for 60 min, rinsed with deionised
water, immersed in 17% EDTA for 2 min and then with
deionised water.
Flexural testing was performed using a miniature three-
point flexure device with a 5-mm support span.23 Each 7-mm
long beam was placed on top of the support span and loaded to
fracture under water using a universal testing machine
(Vitrodyne V100, Liveco Inc., Burlington, VT, USA) at a
crosshead speed of 1 mm/min. Flexural strength (in MPa)
was calculated using the formula 3PL/2bd2, where P = load at
fracture [N], L = length of support span [mm], b = beam width
[mm] and d = beam thickness [mm]. As the data from the three
groups were not normally distributed, they were statistically
analysed using KruskalWallis ANOVA and Dunns multiple
comparison test at a = 0.05.
2.2. Transmission electron microscopy
Two methacrylate resin-based sealers, RealSeal and RealSeal
SE (SybronEndo, Orange, CA, USA) were used to facilitate
ultrastructural examination of the radicular dentine after
irrigation with the aforementioned experimental protocols.
These mild sealers rely more on the ability of EDTA rather than
their inherent self-etching capacities to bond to radicular
dentine.24,25 They were used in lieu of non-bondable sealers to
create continuous sealerdentine interfaces that were amend-
able to ultramicrotomy without separation of the thin
sections. Five single-rooted human teeth were used. A
polyvinylsiloxane (PVS) mould was prepared for each tooth
by inserting the root into mixed, putty-type PVS. Each root was
split into two longitudinal halves, which were re-assembled
back into the mould. Working length was established 0.5 mm
short of the apex. Instrumentation was performed using a
crown-down technique. The canals were prepared initially up
to size 40, 0.06 taper using nickeltitanium rotary instruments
(Profile rotary instruments, Dentsply Maillefer, Ballaigues,
Switzerland). The apical seat was further prepared to size 60
using stainless steel hand files. During cleaning and shaping,
five roots were irrigated with the first experimental irrigation
protocol. The other five roots were irrigated using the second
experimental protocol. The irrigants were delivered to within
2 mm of the working length using 30-gauge ProRinse irrigation
needles (Dentsply Tulsa Dental, Tulsa, OK, USA). As there was
continuous seepage of the irrigants from the fractured roots,
the 60 min NaOCl irrigation protocol was modified by immer-
sing the entire assembly in NaOCl for 50 min after an initial 10-
min period of syringe-mediated NaOCl irrigation.
The root-halves were removed from the mould after
irrigation. Half of the fractured canal was filled with RealSeal
and the other half with RealSeal SE, according to the
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journal of dentistry 38 (2010) 201206 203

manufacturers instructions. A 2-mm thick transverse section

containing the sealerdentine interface was prepared from
each root-half. Sectioning was performed at 5 mm from the
apical foramen, corresponding to the middle third of the canal.
The specimens were fixed, dehydrated in an ascending
ethanol series and embedded in epoxy resin.26 Undeminer-
alised, 90120 nm thick sections were examined without
staining using a JEM-1230 transmission electron microscope
(JEOL, Tokyo, Japan) operated at 110 keV.

3. Results

Flexural strengths (mean  standard error of the mean, in

Fig. 1 Flexural strengths of mineralised dentine beams
(3 mm T 7 mm T 0.2 mm thick) that were tested to failure
under water. Values are mean W standard error of the
mean (N = 10). Groups designated by the same letters
above the error bars are not statistically significant
( p > 0.05).
MPa) for the control, first (10-min NaOCl) and second (60-min
NaOCl) experimental groups were 218.0  7.5, 220.9  20.8 and
22.1  2.7, respectively. Exposure of the dentine beams to
NaOCl for 60 min significantly reduced dentine flexural
strength ( p < 0.001) when compared with those beams that
had not been immersed in active irrigants. By contrast,
exposure of the dentine beams to NaOCl for 10 min did not
result in a significant decline in flexural strength (Fig. 1).

Fig. 2 TEMs of instrumented root dentine that had been treated with 5.25% NaOCl for 10 min and 17% EDTA for 2 min.
Specimens were bonded with RealSeal (RS) that utilised a separate self-etching primer (P), or RealSeal SE (SE), a self-
adhesive sealer. M: mineralised dentine. (A) The RSdentine interface revealed mild etching (open arrowhead) of the
dentine surface without erosion of the subsurface dentin. (B) High magnification of Fig. 2A showing a completely
demineralised zone (between open arrowheads). (C) The SEdentine interface showed a similar mild surface etching effect
(open arrowhead). (D) High magnification of Fig. 2C revealed a partially demineralised zone (between open arrowheads)
with remnant apatite crystallites.
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204 journal of dentistry 38 (2010) 201206

Instrumented canal walls in the 10-min NaOCl experi- 4. Discussion

mental group were completely devoid of smear layer,
irrespective of whether they were bonded with RealSeal that
utilised a self-etching primer (Fig. 2A) or the self-adhesive
RealSeal SE (Fig. 2C). For both methacrylate resin-based
sealers, a 0.5 mm thick, uniform demineralisation front could
be identified along the dentine surface (Fig. 2B and D). There
was no erosion of the subsurface peritubular and intertubular
dentine.
Instrumented canal walls in the 60-min NaOCl experi-
mental group exhibited severe surface and subsurface erosion
that extended 2530 mm beneath the resindentine interfaces
(Fig. 3A and C). This tunneling erosive pattern resulted in a
weakened dentine infrastructure that was characterised by
dissolution of the peritubular dentine, enlarged and inter-
connected tubular spaces and extensive loss of the inter-
tubular dentine. Irrespective of the type of adhesive sealer
(Fig. 3B and D), 0.5 mm thick, demineralisation fronts were
present on all eroded dentine surfaces and subsurfaces.
The results obtained from the present study support the
hypothesis that the use of EDTA as a final irrigant causes canal
wall erosion only when NaOCl is used adjunctively as the
initial irrigant. The reduction in mean flexural strength in the
60-min NaOCl experimental group to 1/10 of that in the control
group has to be interpreted with caution, as both sides of the
200 mm thick dentine flexure specimens were simultaneously
exposed to irrigants. Clinically, we expect a milder scenario
with irrigants diffusing only from the pulpal side of the canal
space. Generation of a 2530 mm thick erosion zone from
either side could have reduced the intact dentine to only 140
150 mm thick. This ultrastructure-based approximation is
likely to underestimate the extent of damage, as NaOCl may
penetrate far deeper into the intact dentine, causing bio-
chemical damage to the organic components of the miner-
alised collagen2730 that are not recognisable at the
ultrastructural level.

Fig. 3 TEMs of instrumented root dentine that had been treated with 5.25% NaOCl for 60 min and 17% EDTA for 2 min before
the application of RealSeal (RS) or RealSeal SE (SE). P: self-etching primer; M: mineralised dentine. (A) The RSdentine
interface exhibited severe surface and subsurface erosion (between open arrows) with extensive loss of peritubular and
intertubular dentine and enlarged, interconnected tubular spaces. A thin demineralisation front was present on all eroded
dentine surfaces (open arrowheads). (B) High magnification of Fig. 3A. The thickness of the demineralisation fronts (open
arrowheads) in the eroded regions was similar to that depicted Fig. 2B. (C) The SEdentine interface exhibited a similar
tunneling erosive pattern (between open arrows) and thin demineralisation fronts (open arrowheads). (D) High
magnification of Fig. 3C showing a thin layer of demineralised dentine (open arrowheads) on all the eroded dentine
surfaces.
 Author's personal copy
journal of dentistry 38 (2010) 201206
The subsurface erosive pattern identified from the radi-
cular dentine specimens in the 60-min NaOCl group has not
been reported in the literature. It had never been observed
when EDTA or NaOCl31 was used separately, even when either
solution was in prolonged contact with mineralised dentine.
Although apatite-encapsulated mineralised dentine is less
vulnerable initially to the destructive effects of NaOCl,32 its
organic phase will eventually be deproteinised by NaOCl in a
time-dependent manner,2730 resulting in increases in brittle-
ness921,28 and permeabililty.21,33,34 Even with these destruc-
tive NaOCl-induced events, preservation of the apatite phase
within the mineralised dentine28,29 implies that erosive
disintegration of intertubular and peritubular dentine will
not occur.31
Conversely, when NaOCl and EDTA are used in sequence,
the two irrigants can interact synergistically to induce an
erosive effect. Unlike dentine demineralisation that creates
uniform demineralisation fronts propagating from the dentine
surface downwards, deproteinisation proceeds non-uni-
formly along deproteinisation channels within the dentinal
tubules.28 Removal of the organic phase in mineralised
dentine by NaOCl enhances the permeability21,33,34 of peri-
tubular and intertubular dentine to EDTA, which in turn,
demineralises the apatite phase and expedites NaOCl infiltra-
tion and destruction. Once all the exposed collagen fibrils are
dissolved, the deproteinisation process reverts to that
observed for the apatite-encapsulated collagen, repeating
the viscious cycle. This series of events also explains why the
thickness of the demineralisation front in the 60-min NaOCl
group did not increase but remained consistently at 0.5 mm
along the dentine surface and eroded subsurface sites (Fig. 3B
and D). Although peritubular dentine is noncollagenous,34 it is
rich in glutamic acid-containing proteins which are also
susceptible to the deproteinisation. Being hypermineralised,
the more rapid demineralisation rate of peritubular dentine35
probably expedites the diffusion of NaOCl into the intertubular
dentine.
5. Conclusion
Within the limits of this study, it may be concluded that the
apparent aggressiveness of EDTA in causing erosion of
radicular dentine is attributed to the prolonged initial
contact of the dentine by NaOCl. The 60-min period of
NaOCl application should not be viewed as an overzealous
use of the disinfecting irrigant. On the contrary, this time
period is clinically realistic in the treatment of teeth with
multiple canals wherein the pulp chamber dentine is
constantly bathed in NaOCl during negotiation and shaping
of the canals. Indeed, clinicians are employing different
means to improve the debridement efficacy within the root
canal system. They include frequent replenishing of
irrigants, warming of NaOCl to improve its tissue dissolu-
tion efficacy and the use of different agitation devices to
enhance irrigant flow and replacement along the canal
walls.36 In the presence of thin pulp chamber dentine or
canal walls, the synergistic action between NaOCl and EDTA
may render these rooted-treated teeth more prone to
vertical fracture.
205
Acknowledgements
The costs of TEM preparation and examination were gener-
ously supported by the Dental Research Center, School of
Dentistry, Medical College of Georgia (P.I. Tay). We thank
Robert Smith for TEM support, Thomas Bryan for resin
embedding and Michelle Barnes for secretarial support.
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