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Endodontic Topics 2012, 27, 35–53 © 2013 John Wiley & Sons A/S.
All rights reserved Published by John Wiley & Sons Ltd

ENDODONTIC TOPICS
1601-1538

Irrigation: beyond the smear layer

MARKUS HAAPASALO, WEI QIAN & YA SHEN

Traditionally, much of the attention placed on irrigation in endodontics has focused on smear layer removal. While
the smear layer continues to be a relevant topic, other areas related to irrigation and irrigants have emerged that
also require a more in-depth analysis and understanding. This review starts with the smear layer, partially from a
new angle, and expands into other topics such as uninstrumented root canal areas, and effect of irrigation in lateral
canals and dentin canals. Advanced microbiological models for irrigation research will be presented and discussed.
The effect of sodium hypochlorite and decalcifying solutions on dentin structure and dentin strength has become
an important topic, which is related to the possible harmful effects of irrigation such as dentin erosion and even
vertical root fracture. The impact of irrigant sequence and time of exposure will also be discussed, and
recommendations for irrigation protocols will be made.

Received 5 February 2013; accepted 6 March 2013.

Introduction Smear layer

Irrigation is regarded as a centrally important part A smear layer always forms when a metallic endodontic
of successful root canal treatment. Irrigation and instrument touches a mineralized dentin wall within a
irrigating solutions have a variety of different root canal (Fig. 1). Therefore, the smear layer always
physical/mechanical, chemical, biological, and consists of mineralized dentin, but often also of
microbiological effects, most of which have been predentin, remnants of pulp tissue, bacteria, and
discussed in other articles of this volume of biofilm (1–3). The smear layer is caused by hand and
Endodontic Topics. Because of the importance of rotary stainless steel and NiTi files as well as by
irrigation, for decades it has been a continuous focus ultrasonic tips and various burs that are used in the root
of interest in endodontic research. Table 1 shows the canal. However, the smear layer is not created by
results of a Medline search using keywords describing instruments which are softer than dentin; for example,
several topics related to root canal irrigation. an EndoActivator tip hitting a canal wall at high
Although this search was far from exhaustive, it gives frequency does not create a smear layer. The smear layer
a rough view of the relative popularity of these is a thin, amorphous layer, ca. 0.5–2 mm thick, that
topics. It is clear that while some areas have been covers the dentin surface and thus hides the openings of
extensively studied, others have gained much less the dentin canals (Figs. 2 and 3). Sometimes, part of the
attention. Removal of the smear layer, for example, smear layer/dentin debris has been pushed into the
has been thoroughly explored, whereas topics such as dentin canals by instruments to a depth of even several
dentin erosion, uninstrumented root canal areas, micrometers. The smear layer that forms in teeth with
predentin, and endodontic biofilm are addressed only pulpitis has one important difference from the smear
in a limited number of studies. The purpose of this layer that forms in teeth with apical lesions: bacteria and
review is to present a summary of the effects of antigenic material are present only in the latter. This
irrigation in the various parts of the root canal difference may affect a dentist’s decision regarding the
environment, starting from the smear layer and then necessity of smear layer removal.
extending beyond the smear layer to the lesser The smear layer should be removed for the following
known areas of root canal irrigation. reasons: (i) it may contain microbial cells and antigens

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Haapasalo et al.

Table 1: Number of results in Medline search using

selected endodontic keywords and their combinations
endodontics 26531

root canal hypochlorite 1448

endodontics hypochlorite 1151

dentin EDTA 907

pulp hypochlorite 827

root canal EDTA 802

dentin hypochlorite 685

endodontics EDTA 638

EDTA hypochlorite 584

smear EDTA 556

pulp EDTA 493

smear layer EDTA 463

smear layer hypochlorite 354

Fig. 1. A collage image of the creation of a smear layer.
pulp tissue hypochlorite 158 The instrument touches some parts of the mineralized
biofilm root canal 158
dentin, causing smear layer formation. In the untouched
areas, mineralized dentin remains covered by predentin
biofilm endodontics 154 and pulp tissue.
dissolution root canal 83

dissolution endodontics 64

pulp tissue EDTA 76

uninstrumented root canal 57

uninstrumented endodontics 55

biofilm faecalis endodontics 45

dissolution root canal hypochlorite 42

dissolution pulp tissue 39

biofilm root canal irrigation 33

biofilm hypochlorite endodontics 31

dissolution endodontics hypochlorite 27

biofilm endodontics irrigation 27

uninstrumented hypochlorite 23

uninstrumented endodontics hypochlorite 20

dentin erosion EDTA 20

predentin EDTA 17 Fig. 2. SEM image of a smear layer on the main root
dissolution root canal EDTA 16 canal wall.
uninstrumented endodontics irrigation 15

dentin erosion hypochlorite 14

dissolution endodontics EDTA 12 (Fig. 4); (ii) it weakens the effects of disinfecting
biofilm EDTA endodontics 12 agents in dentin (4); and (iii) it may affect the quality
predentin hypochlorite 8 of the root filling and bonding to the canal wall. The
uninstrumented EDTA 6 last point is dependent on the type of the sealer;
uninstrumented endodontics EDTA 5
however, most studies have shown that removal of the
smear layer improves bonding of many sealers to
dentin (5–9). Nevertheless, when bacteria or their
components can be part of the smear layer, there is a

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 Irrigation: beyond the smear layer

Fig. 3. SEM image of a fractured root specimen

showing the thin smear layer from the side, covering the Fig. 5. Sodium hypochlorite does not remove the smear
dentin canal openings. layer. It does dissolve the organic part of the smear layer,
but that cannot be visualized in the SEM image.

Fig. 4. SEM of a smear layer with embedded rod-shaped Fig. 6. NaOCl followed by a final rinse of EDTA (or
bacteria. citric acid) completely removes the smear layer.

Therefore, the recommended protocol for smear

strong consensus on the need to remove it (10,11), layer removal is NaOCl followed by EDTA
although some authors have emphasized that the (ethylenediaminetetraacetic acid) or citric acid
question still remains open (12). As with many other (Fig. 6). Despite the often presented views that EDTA
questions in dentistry and medicine, lack of long-term or citric acid remove the smear layer, they cannot do it
clinical studies (effect of smear layer on long-term completely without the preceding use of NaOCl
prognosis) will continue to keep the discussion going. (Fig. 7). Water, saline, chlorhexidine (CHX), or iodine
The ability of irrigating solutions to remove the compounds have no dissolving effect on the smear
smear layer has been extensively studied (13–21). layer (22). Several other chemicals have also been
Because the smear layer contains both inorganic studied and some have been shown to have an effect
and organic material, it cannot be removed by any of on the smear layer. These include peracetic acid,
the presently available root canal irrigants alone, etidronic acid, and EGTA (ethylene glycol tetraacetic
including NaOCl (Fig 5). The sequential use of acid) (16,23–26). Despite showing some promise,
sodium hypochlorite (NaOCl) and a chelating agent especially peracetic acid, these solutions have not
or acid that dissolves inorganic tissue is required. become popular as root canal irrigants.

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Haapasalo et al.

A smear layer is formed in areas where metal ranging between 1 and 5 min. According to several
instruments work on dentin (27,28). The most studies, 1–2 min EDTA irrigation as a final rinse after
common method of irrigant delivery is by syringe and NaOCl is enough (29,30).
needle placed deep into the canal. In most cases the The often ignored advantage of instrumentation and
needle follows the path created by the instruments. creation of a smear layer is that the biofilm structures
Therefore, removal of the smear layer can usually be in the area are either removed by the instrument,
done successfully as the irrigants are released into the loosened into the canal space and washed out of the
very same areas where the smear layer has formed. canal by irrigation, or baked into the smear layer. As
Despite the good predictability of smear layer removal, removal of the smear layer (together with the bacteria
there is no consensus in terms of the exact time and biofilm structures in the layer) can be predictably
required for NaOCl and EDTA (or citric acid) accomplished by irrigation, it can be speculated that
irrigation to completely remove it. Varying results first creating and then removing the smear layer is an
have been reported with irrigation times usually advantage in the effort to eliminate root canal biofilm
in these areas (Fig. 8 drawing). Recently, a new type of
NiTi root canal instrument, the SAF (self-adjusting
file) was introduced (31–33). The file does not rotate
like conventional rotary NiTi files; instead it adapts its
form to the shape of the root canal due to its elastic,
hollow tube structure, and grinds the canal wall using
a fast, small amplitude up-and-down motion. The
design and motion path of the SAF dramatically
increases the percentage of canal wall area touched
(and smeared) by the instrumentation, especially in
canals with challenging cross-sections such as those
that are oval, flattened, or C-shaped (34–38). The
cleaning effect of the SAF is further facilitated by an
in-built irrigation mechanism where the irrigant enters
the pulp chamber and the root canal through the
hollow file structure (31–33). According to micro-CT
studies, the amount of canal wall dentin removed is
Fig. 7. EDTA alone cannot remove the smear layer
completely. The inorganic portion is removed but the
small, and the main purpose of the SAF system is to
organic matter is still left partially blocking the dentin facilitate cleaning rather than removal of much dentin
canal openings. (34,39,40).

Fig. 8. A collage image series of the effect of instrumentation and smear layer removal on biofilm bacteria in the area.
(a) Root canal wall before treatment: dentin, predentin, and biofilm. (b) After instrumentation the pulp tissue and
much of the biofilm have been removed; some bacteria have been “baked” into the smear layer. (c) Irrigation by
NaOCl and EDTA has removed the smear layer, including the microbial matter in the layer.

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 Irrigation: beyond the smear layer

Fig. 10. Inorganic debris and organic matter packed

into the thin isthmus between two joining canals in
maxillary second premolar.

Fig. 9. A histological image of the pulp–dentin

interface. From the left: mineralized dentin, predentin
and calcospherites, necrotic pulp tissue.

Uninstrumented areas in the main

root canal
Practically every root canal contains untouched areas
after complete instrumentation. Micro-CT studies in
molar teeth have shown that, except for the SAF, 35 to
> 50% of the root canal wall area remains untouched
by instruments (Fig. 1) (41–43). This has several
important practical consequences: (i) these areas do not Fig. 11. A close-up image of the previous picture.
have a smear layer, and (ii) the areas can only be cleaned Dentin debris and organic fibers can be seen.
chemically or (iii) they can be cleaned by a combination
of chemical compound and physical energy such as
ultrasound. The untouched areas not only lack the time, the calcospherites grow bigger until they join
smear layer, they are likely to be covered by predentin with each other and with the underlying mineralized
(Fig. 9), remnants of pulp tissue, and possibly also dentin. The goal of irrigation in the untouched areas is
biofilm. In addition, debris from instrumentation may to remove all tissue covering the mineralized dentin.
be packed into these areas (Figs. 10 and 11). This means that the biofilm and pulpal remnants on
A special structural feature of the uninstrumented the surface of the predentin are also removed when the
areas is the presence of calcospherites. These are round clean, mineralized dentin is uncovered. Because
dentin structures representing the mineralization front biofilm, pulpal remnants, and predentin are mainly
of dentin (Fig. 9). Dentin mineralization starts inside organic matter, NaOCl is the key irrigant in the
predentin from the initiation and growth of calcific cleaning of the untouched parts of the root canal.
structures around odontoblast processes (44). Over Figures 12–15 show the gradually advancing effect

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Haapasalo et al.

Fig. 12. A SEM image of the superficial layers of Fig. 14. A SEM image of mid layers of predentin. The
predentin, viewed from the direction of the root canal. calcospherites are clearly bigger than in the superficial
Microscopic calcospherites can be seen in the predentin layers in the previous images. The grooves in the
collagen network. calcospherites are in fact dentin canals: the calcospherites
form around odontoblast processes.

Fig. 13. A close-up image of the previous picture. When

sodium hypochlorite dissolves the collagen bundles, the
smallest calcospherites will be washed out of the canal as Fig. 15. SEM of large calcospherites which have joined
they are not attached to the mineralized dentin. the mineralized dentin. Almost all predentin collagen
has been dissolved by sodium hypochlorite, only a few
remaining fibers can be seen between the calcospherites.

of NaOCl on the untouched area. EDTA and

chlorhexidine and different combination products
such as MTAD, QMiX, SmearClear, Tetraclean, or it is likely that biofilm can grow on the surface of
iodine solutions have no tissue-dissolving effect on predentin, direct histological-bacteriological or other
the organic matter in the untouched parts of the microscopic evidence of this is scarce. Figures 17 and
root canal wall (Fig. 16). Alternating the use of 18 show examples of biofilm in direct contact with
NaOCl and EDTA during instrumentation is a predentin. If thick layers of biofilm grow on predentin,
common practice. However, EDTA effectively they may interfere with the effectiveness of NaOCl
abolishes the tissue-dissolving effect of NaOCl and irrigation in these areas. While the collagen network of
should therefore not be used until at the end of the predentin can be dissolved by high-concentration
treatment as the final rinse (45,46). NaOCl in ca. 1 min, biofilm may offer much stronger
The relationship between predentin and biofilm resistance to NaOCl (47,48). The impact of irrigation
development has not been studied in detail. Although on biofilm will be discussed later in this article.

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 Irrigation: beyond the smear layer

Fig. 16. SEM image of an uninstrumented area of the Fig. 18. Brown and Brenn staining shows biofilm
root canal irrigated with EDTA. The predentin is islands in predentin (light yellow). Notice the darker
unaffected by the EDTA. yellow calcospherites and round dentin canals. Courtesy
of Dr. Domenico Ricucci.

irrigation is to help to remove the residual debris

either by a mechanical washing effect or by dissolving
it with the chemical action of the irrigants. In recent
years, several studies mainly based on micro-CT scans
of instrumented teeth and root canals have revealed
that, especially in teeth with two root canals in one
root and anastomoses between the canals, dentin
debris seems to be packed in considerable amount into
the untouched areas (Figs. 10 and 11). Paque et al.
(49) showed that instrumentation without irrigation
resulted in the reduction of canal area/volume in
particularly in the isthmus areas (between canals)
of molar teeth. Dentin debris loosened by the
instruments and then packed in these areas has the
same radiopacity as normal dentin, which explained
the results as seen in the micro-CT scanned images.
Endal et al. (50) modified the experiment so that
irrigation with high volumes of NaOCl was used
during the rotary instrumentation, and a final rinse
with EDTA was also performed. However, after
irrigation the amount of debris was similar to that in
the study by Paque et al., where irrigation was not
used at all. In another study, Paque et al. (51) reported
Fig. 17. Histological specimen with Brown and Brenn
that EDTA and in particular passive ultrasonic
staining of apical root canal shows biofilm growth (dark
blue) on predentin. Courtesy of Dr. Domenico Ricucci. irrigation were able to reduce the amount of debris,
but even after ultrasonics about half of the debris
remained in the canal system. Recently, the same
Dentin debris
group compared the effect of 2.5% NaOCl irrigation
Hand and rotary endodontic instruments cut dentin to 2.5% NaOCl mixed with 9% etidronic acid on the
and remove part of the debris directly between the amount of residual debris (52). The study showed that
blades of the files (Fig. 8). One of the goals of there was significantly less debris after the use of the

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Haapasalo et al.

combination irrigant than after the regular 2.5% material/sealer or not. Ricucci & Siqueira (56)
NaOCl. All of the above studies on debris were reported histological observations of root-filled,
done using conventional rotary NiTi files in canal extracted teeth where lateral canals were filled partly
preparation. In a recent study, the amount of debris with a root canal sealer and partly by tissue remnants
was compared in canal systems prepared either with and biofilm. Their findings showed that irrigation in
rotary files or by an SAF system, which uses a self- those teeth had not been able to remove all organic
adjusting, hollow NiTi file in an up-and-down motion matter from the lateral canals. At present, there is no
instead of rotation (52). The study found considerably quantitative data from any large study about the
less debris after the use of the SAF than after the rotary cleanliness of lateral canals at the time of root filling.
instrumentation. Therefore, the role and importance of infection
In teeth with pulpitis, the dentin debris left in the root possibly persisting in the lateral canals after root canal
canal system is not supposed to contain microbes. treatment remains an unsolved question.
Therefore, that kind sterile debris is less of a concern The effectiveness of different irrigation systems in
with respect to the long-term prognosis of the root lateral canals or simulated lateral canals has been
canal treatment. However, in teeth with apical evaluated in a few studies. Usually the studies are based
periodontitis, the presence of microbes and microbial on various in vitro models using extracted teeth or
antigenic material in the residual dentin debris can be plastic models with artificial lateral canals prepared by
expected. The few studies on this topic so far indicate small reamers or burs. These standardized lateral canals
that there are no easy solutions to solve the issue of have been filled for instance with a dye or with some
residual debris in infected teeth. Fortunately, if the organic material, and the cleaning ability of different
canal space can be adequately sealed from the irrigation systems or strategies has been compared. In
surrounding tissues, the likelihood that the debris left in some studies, solutions with some level of radiopacity
the canal, even if containing microbial material, would have been used. In general, several of these studies have
cause persisting problems or prevent healing is small. shown that continuous ultrasonic irrigation and passive
ultrasonic irrigation (PUI) help NaOCl penetrate into
the lateral canals better than regular positive pressure
Effect of irrigation in lateral canals
irrigation (PPI) or the use of some other “activation”
Lateral canals can be found in all parts of the root canal device such as S-files (57–60). De Gregorio et al. (61)
system: furcation, cervical third, mid-root, and apical compared irrigant penetration using PPI, negative
third. Lateral canals are most frequent close to the apex pressure irrigation (NPI), and irrigation with the SAF,
(“apical delta”) (53,54). Lateral canals are much larger with and without small amplitude pecking motion.
than dentin canals, 10–200 mm in diameter, and They reported that NPI was the only method that
contain vital tissue such as connective tissue and blood was associated with irrigant penetration in all teeth
vessels (55). Another important difference with dentin in this group to working length. However, none of
canals is that lateral canals pass through the cement the experimental groups demonstrated predictable
layer and connect the root canal to the periodontal irrigation of simulated lateral canals.
ligament space (PDL). In infected teeth, lateral lesions Necrotic bovine pulp tissue was used in one study to
are a sign of the presence of a lateral canal. This indicates fill the artificial lateral canals placed at defined and
that lateral canals in such teeth contain bacteria; different angles to the main canal in an in vitro model
histological studies have in fact clearly shown the (62). The effectiveness of irrigation with 2.5% NaOCl
presence of bacteria and biofilm in lateral canals (56). to dissolve the pulp tissue in the lateral canals was then
Because of their direction and size, lateral canals evaluated. The results clearly showed the superiority of
cannot be cleaned by mechanical instrumentation. PUI as compared to NaOCl with no activation or
Thus, if the goal is to clean lateral canals, chemical activation using rotary S-files. However, somewhat
cleaning is the only method. The necessity of cleaning contrary results were obtained in another study where
lateral canals by irrigation is a matter of some ultrasonic activation was compared to EndoActivator
controversy. In general, lateral lesions seem to heal (EA) (63). Extracted teeth were instrumented to size
well by good quality root canal treatment, irrespective #40/0.06 taper, and final irrigation was performed
of whether the lateral canal is filled by a root filling with either of the two systems. Each unit was used for

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 Irrigation: beyond the smear layer

Fig. 19. SEM image of root canal wall reveals three lateral canals and an attached pulp stone. The most apical (golden
arrow) of the lateral canals has been filled with debris, whereas the lateral canal next to it has no debris (blue arrow).

1 min each with 6.15% NaOCl and 17% EDTA. regarding lateral canals. One can speculate that because
According to the authors, “The EA was significantly lateral canals are often filled with root filling material, in
better in removing debris at all levels when compared particular sealer, it is likely that debris is at least not
with other treatment groups (P < 0.05) and resulted in blocking the lateral canals completely. Although not of
obturation of significantly more numbers of lateral scientific value, the typical observation in SEM studies
canals (p < 0.01)” (63). of instrumented and irrigated root canals is that lateral
The effect of the smear layer on the filling of lateral canal openings on the main root canal wall are open
canals by sealer or gutta-percha has been examined in with no evidence of dentin debris (Fig. 19).
only a few studies. In a study by Fachin et al. (64),
eighty teeth were instrumented and irrigated either Dentin penetration by
with NaOCl only or with NaOCl and EDTA and
irrigating solutions
root-filled using cold lateral condensation. No
statistically significant difference between the two The most common pathway of bacteria from the oral
groups was found in the number of lateral canals filled, cavity into the pulp is via dentin canals. After occupying
which may indicate that lateral canals are so large that the main root canal, bacteria will in most cases invade
they are not covered (blocked) by the smear layer. A the dentin canals, this time in the opposite direction,
similar result was also reported by de Gregorio et al. i.e. from the inside out. It has been reported that
(57) who found that the use of PUI but not EDTA 60–90% of teeth with apical periodontitis have bacteria
improved the penetration of NaOCl into lateral canals. penetrated into the dentin canals (65–67). Peters &
In another study, the same authors reported that Wesselink (68) reported that “in more than half of the
NPI helped the irrigant reach working length better infected roots, bacteria are present in the deep dentin
than PUI; however, the PUI group demonstrated close to the cementum.” Matsuo et al. (67) also
significantly more penetration of the irrigant into the reported that bacteria will remain in the dentin canals in
lateral canals than did the NPI group (58). the vast majority of cases even after the main root canals
There are no studies of lateral canals and dentin have been instrumented wide. The role of the bacteria
debris. While several studies have shown that debris is that remain in the dentinal tubules after treatment
packed into the anastomoses between, for example, is a matter of some controversy. Peters et al. (69)
mesial canals in lower molars, no such data exists concluded “A sound obturation technique immediately

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Haapasalo et al.

following the cleaning, shaping, and disinfection phases seem to be equally well suited to measure dentin
allows the remaining bacteria in the tubules to be either disinfection (80,81).
inactivated or prevented from repopulating the Recently, a new dentin infection model (82) was
(former) canal space. In the vast majority of cases, those introduced to address the two main problems of studies
bacteria appear not to jeopardize the successful of dentin disinfection: the uneven presence of bacteria
outcome of root canal treatment.” However, it should in the dentin canals and the challenges in sampling and
be emphasized that the situation can be different culturing. In the new model, bacteria are forced into
depending on whether root surface dentin has been the dentin canals by centrifugation. Using this method,
resorbed in the area of dentin canal invasion or not. cells of Enterococcus faecalis have been successfully
Histological evidence shows that in the absence of root introduced deep into the dentin canals at high density
surface cement, bacteria can more easily penetrate (82–84). Vitality staining and confocal microscopy
through the entire thickness of the (apical) root and have shown that the bacteria are not killed by the
even grow as biofilms on the root surface (70,71). centrifugation. The bacteria in dentin canals are
Despite the uncertainty of the role of residual bacteria incubated in culture broth for different time periods to
in the dentin canals at the time of root filling, there is a allow them to fully recover and to create a biofilm in the
consensus that, optimally, all bacteria should be dentin canals. After the infection has been established,
eliminated from the root canal system, including those the outer ends of the dentin canals are closed with a
in the lateral canals and the dentin canals. A number varnish to simulate the cement layer, preventing easy
of studies have addressed the killing of bacteria by through-flow of the chemicals (82). The disinfecting
endodontic local medicaments and irrigating solutions. agents are placed on the pulpal surface of the dentin
Both in vivo studies and in vitro studies using different specimens for varying time periods, after which the
dentin infection models have been used. In the majority dentin is fractured and the survival/killing of bacteria in
of the studies, infected dentin has been directly sampled dentin is measured using viability stain and confocal
by, for example, a bur, and the dentin dust has been laser scanning microscopy. The results have shown
collected and cultured to obtain the numbers of excellent penetration of bacteria deep into the dentin in
colony forming units (cfu) in each sample (72). The most dentin canals, and the killing experiments have
comparative antibacterial effect in dentin of calcium shown consistent results with low standard deviation
hydroxide, different concentrations of sodium (Fig. 20). The results have shown that, for example,
hypochlorite and chlorhexidine, and sometimes some
other root canal disinfecting agents have been
examined. The results of these studies have been
largely contradictory, and the ranking of the agent’s
antimicrobial effect has shown variation from one study
to the next; in some no difference was found (73–79).
However, it is the opinion of the authors of this review
that the variable results may be rather a consequence of
the limitations of the sampling and culturing method
than a reflection of the true efficacy of the different
disinfectants. In cultural studies, the collected dentin is
usually vortexed to separate the bacterial cells from each
other and then cultured on agar plates for cfu counting.
However, the bacteria are still likely to be entrapped
inside the dentin chips in dentin canals. Great variation
in cfu counts between parallel samples is typically
observed in these studies. Large standard deviation
results in low significance. While cultural studies are
Fig. 20. Three-dimensional construction of confocal
usually effective in detecting significant differences in microscopy scans of bacteria in dentin canals, in a dentin
planktonic killing experiments, where the differences biofilm model after irrigation. Green color indicates
typically are even several logarithmic steps, they do not alive bacteria, red color killed bacteria.

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 Irrigation: beyond the smear layer

high-concentration NaOCl killed bacteria inside dentin concentration (e.g. 1% vs. 6%), and that temperature
much more effectively that 1 and 2% solutions, which played little role, too. At 2 min, 1% NaOCl at room
showed effectiveness similar to 2% chlorhexidine. A temperature penetrated ca. 75 mm into dentin, while
new root canal irrigant, QMiX, showed equal killing 6% solution advanced ca. 130 mm (85). When heated
to high-concentration NaOCl (84). The obvious to 45°C, the results for the same solutions were 80 mm
advantage of the new dentin infection model and use of and 145 mm. After an exposure of 20 min, the
confocal microscopy is that the measurements can be corresponding distances were 180 mm (195 mm) for
done in situ, without damaging the area. The method 1% solution and 220 mm (280 mm) for 6% solution
also provides the opportunity to evaluate killing (heated solutions in parentheses). The results showed
dynamics and effects at different depths of dentin. that extending the exposure time ten-fold, from 2 to
A prerequisite to any disinfectant effect in dentin is 20 min, helped the NaOCl only double the distance of
that the agent penetrates into the dentin canals. penetration. In a corresponding study, Kuga et al. (86)
Therefore, it is surprising how little is known about reported somewhat lower penetration using 2.5%
the penetration of irrigating solutions into dentin. The sodium hypochlorite solution either alone or mixed
reason for this is probably the technical (and chemical) with EDTA, citric acid, or peracetic acid. For NaOCl
difficulty of measuring such small volumes at exact only, the depth of penetration was 107 mm.
distances e.g. by chemical analysis. In addition, when Interestingly, mixing with EDTA greatly reduced the
dentin is cut or fractured, this in itself is likely to have depth of bleaching, while mixing with peracetic acid
a major effect on liquid movement in dentin canals, seemed to increase the penetration, although due to
making the measurements unreliable. In 2010, Zou the small sample size the difference was not statistically
et al. (85) published a study of the penetration of significant. It should be pointed out that bleaching of
NaOCl into dentin using a dye model. Dentin canals the stained dentin does not necessarily correlate well
of dentin blocks were first stained by a dye solution with the ability of the solutions to kill bacteria in the
(crystal violet) placed in the main root canal, after same area. However, when these results (penetration)
which the standardized canal lumen was filled with are compared to the results (killing) from the new
NaOCl solutions at different temperatures and dentin infection model (82,83), it can be suggested
concentrations for time periods ranging from 2 min to that the results from dye bleaching experiments have
20 min. After the exposure, the NaOCl was washed predictive value for the killing experiments with
away with running water and the blocks were blotted sodium hypochlorite.
dry and fractured, allowing direct observation and
measurement of the bleached area under a microscope Effect of irrigating solutions
(Fig. 21). The results showed that, surprisingly, there
on dentin
was little difference between solutions of different
The purpose of irrigation is to advance healing by
dissolving and removing tissue remnants and debris
from the root canal and by killing bacteria. The
downside of irrigation is that there are some potentially
harmful effects from irrigation, depending on the type
of the chemical, concentration, time of exposure, and in
what sequence the solutions are used in the canal.
Sodium hypochlorite is the most important of the
irrigating solutions as it is the only one that can dissolve
organic tissue. It also has strong antimicrobial activity.
The longer one uses NaOCl in a given canal system and
the higher the concentration, the more effective it is in
cleaning organic matter from the canal and killing the
bacteria. Therefore, it is of great interest whether there
are any harmful effects from the prolonged use of
Fig. 21. NaOCl penetration into stained dentin. NaOCl. Grigoratos et al. (87) studied the effect of 3%

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Haapasalo et al.

and 5% NaOCl on dentin bars cut from human teeth through-flow of the liquids. Studies of NaOCl
root dentin during 2 h long exposure. The results penetration using dentin blocks with the cement layer
revealed a significant (P < 0.001) decrease in the left intact show relatively limited penetration of the
modulus of elasticity and flexural strength of dentin solution into dentin. It may therefore be concluded
with both concentrations. In the same study, 1 week of that the in vitro studies with dentin bars may be
exposure to saturated calcium hydroxide also reduced best suited for ranking different chemicals (and
the flexural strength but did not affect the modulus of concentrations) with regard to their potential effects on
elasticity of dentin. When bars were challenged by dentin rather than for giving exact values for dentin/
calcium hydroxide after the 2 h NaOCl treatment, root weakening in a clinical situation.
additional weakening of dentin did not occur other
than what was already caused by NaOCl alone.
Dentin erosion
Corresponding results with 5.25% NaOCl were also
reported by Sim et al. (88). Marending et al. (89) Erosion of root canal wall dentin is another area of
studied the effect of NaOCl concentration with a growing interest in endodontic research. As with the
similar study design and reported that exposure for 1 h general effects of long-term use of NaOCl on the
to 9% and 5% NaOCl caused marked reduction in the mechanical characteristics of dentin, the concern is that
elastic moduli of the bars while no effect could be weakening of dentin by canal wall surface erosion may
measured from a 1% solution. Electron microscopic in theory also contribute to increased risk of vertical
analysis and backscattered electron micrographs root fracture. Structurally, dentin is made up of
revealed that the effect was due to the impact on the collagen fibers covered by hydroxyapatite coating (93–
organic component of dentin, while the inorganic 95). NaOCl and EDTA (or citric acid) together attack
component was unaffected (89). In another study by both the organic and inorganic components of dentin.
the same group, the authors reported a drop in flexural It is therefore not surprising that some reports have
strength but not in elastic modus after 24 min exposure described a dramatic effect of irrigation on the
to 2.5% NaOCl. Three min exposure to 17% EDTA structural integrity of root canal wall dentin
before or after NaOCl did not cause additional (89,96,97). Eroded root canal surface was already
weakening of the dentin (90). Pascon et al. (91) described by Brännström in 1984 (98) and by Tatsuta
published a critical review of articles focusing on the et al. in 1999 (99). The exact mechanism of erosion was
effects of NaOCl irrigation on dentin mechanical not clear, and calcium hydroxide was also assumed to
properties. Out of 16 articles included originally, 9 were play a role in the greatly widened tubule openings
accepted for the final critical appraisal. The authors observed after calcium hydroxide treatment and
concluded that “sodium hypochlorite adversely alters irrigation with NaOCl and EDTA (99). In an SEM
the mechanical properties of root dentine, when used as study, Niu et al. suggested a relationship between
an endodontic irrigant.” In a recent study, the effect on erosion and EDTA followed by NaOCl (96); they
soft tissue and dentin of stabilizing the pH of the reported that final irrigation with 6% NaOCl
NaOCl solution was examined (92). The authors accelerated dentinal erosion following treatment with
concluded that NaOH-stabilized NaOCl solutions 15% EDTA. The authors also showed that the same
have a higher alkaline capacity and are thus more regimen effectively removed debris from the root canal.
proteolytic than non-stabilized solutions. The effect of the opposite sequence (NaOCl–EDTA)
It is difficult to evaluate how well the above studies was not examined in this study (96). Mai et al. (97)
reflect the events in the root canal and root dentin studied the effect of prolonged NaOCl irrigation
during endodontic treatment. The dentin bars used in (5.25%) followed by 2 min final irrigation with 17%
the studies (89) are thinner than root dentin. In EDTA on radicular dentin erosion. When the NaOCl
addition, when the bars are cut, the small dentin canals was used for 10 min, EDTA caused 0.5-micron-thick
are opened from both sides. This will greatly affect the demineralization fronts in dentin. However, after a
penetration of NaOCl; in fact, the NaOCl penetrates 60 min long NaOCl challenge, extensive surface and
the full thickness of the dentin pieces in a short time. In subsurface erosion was detected after the final 2 min of
the in vivo situation, the root surface is usually covered EDTA irrigation (97). The flexural strength of 200-
by the cement layer, which effectively prevents mm-thick dentin specimens decreased ca. 90% of the

46
 Irrigation: beyond the smear layer

original value. The authors stated that “the decline in

dentin 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.” Zhang et al. (100) studied the effect of
initial irrigation with two different sodium hypochlorite
concentrations on the erosion of instrumented
radicular dentin. The times for NaOCl exposure ranged
from 10 to 240 min, while 17% EDTA was always used
for 2 min. The study showed that 5.25% NaOCl caused
more dissolution of subsurface collagen than the 1.3%
solution. Transmission electron microscopic specimens
Fig. 22. Instrumented root canal wall after irrigation
revealed that the subsurface erosion extended 10– for 1 min with 5% NaOCl and 1 min with 17% EDTA.
15 mm beneath a sealer-bonded dentin surface after the Smear layer has been completely removed; the dentin
use of 5.25% NaOCl for 20 min. The final EDTA rinse wall shows no signs of surface erosion.
removed the collagen-depleted apatite phase, resulting
in an eroded canal wall (100). The opposite sequence of
the two irrigants was not examined in this study.
In a recent study, Qian et al. (101) evaluated the
effect of irrigation sequence, time, and type of
demineralizing agent on root canal wall dentin erosion.
A smear layer was first created on canal walls of
extracted human teeth by a hand file. The study
revealed that the irrigant sequence played a major role
in the development of root canal wall surface erosion.
When 5.25% NaOCl was used first for time periods
ranging from 1 to 5 min, demineralizing agents (17%
EDTA, 17% EGTA, or 10% citric acid) that were used as
the final irrigants caused relatively little surface erosion. Fig. 23. A combined image of root canal wall dentin
When 5 min of 5.25% NaOCl was followed by 5 min of with no erosion (left) and with strong surface erosion
the demineralizing solution, the average area of the (right).
dentin canal opening grew from ca. 8.0 mm to 8.2 mm
with EGTA, 8.7 mm with EDTA, and 9.3 mm with citric was greatest after 10% citric acid and mildest after 17%
acid (101). However, with the reverse sequence, the EGTA.
average areas were 13.3 mm (EGTA–NaOCl), 18.1 mm When NaOCl is used as the first irrigant, the
(EDTA–NaOCl) and > 30 mm (citric acid–NaOCl). In hydroxyapatite coating on collagen seems to protect
the last case, the dentin canal opening had joined so that the collagen fibers and the effect of the NaOCl on
a single opening could no longer be measured and the dentin is limited. However, when decalcifying solution
entire surface was seriously eroded. Even 1 min final is used, the hydroxyapatite is quickly dissolved close to
irrigation with 5.25% NaOCl after 1 min of EDTA the main root canal, exposing the underlying collagen
increased the orifice area from 8.0 mm to 14.2 mm. fibers. If NaOCl is used again at this stage, it can
However, when the initial irrigation by EDTA was directly attack the protein (collagen) and in a relatively
reduced to only 30 sec, the following 5 min NaOCl short time cause considerably destruction of the
irrigation increased the canal opening less, from 8.0 mm collagen backbone of surface dentin. In the study by
to 8.9 mm. Figures 22–26 illustrate typical surface Qian et al. (101), times shorter than 1 min of final
views of dentin after different irrigation sequences NaOCl rinse were not tested; therefore it is not
with the various solutions for different times. Erosion possible to know the minimum time of exposure after
using NaOCl after the demineralizing agents which the erosion becomes prominent (101).

47
Haapasalo et al.

Fig. 24. Strong erosion of root canal wall dentin after Fig. 26. Specimen in the previous image fractured and
1 min irrigation with 17% EDTA followed by 1 min viewed from the side. The image reveals deep
irrigation with 5% NaOCl. penetration of the erosion effect.

Recently, Stojicic et al. (103) expanded the experiment

using oral biofilms from six different subjects, grown
anaerobically for different time periods ranging from 1
day to 8 weeks. Chlorhexidine, sodium hypochlorite,
and iodine potassium iodide were used as the
disinfecting agents. This study (94) showed that the
development of resistance of the biofilm was not
dependent on the source of the oral bacteria; all six
biofilms followed the same pattern and changed from
sensitive to resistant between two and three weeks of
biofilm age. Another interesting result in this study was
that while the three tested disinfecting agents (CHX,
NaOCl, and iodine potassium iodide) have different
Fig. 25. Strong erosion of root canal wall dentin after
30 sec irrigation with 10% citric acid followed by 5 min mechanisms by which they attack the microbial cell,
irrigation with 5% NaOCl. they all became less effective during the same period of
biofilm maturation, between two and three weeks
of biofilm growth. The results of these two studies
Irrigation and biofilm
(103,104) suggest that irrigation studies which mostly
Oral bacterial biofilms have been extensively discussed have used biofilms grown from 12 h to two weeks may
in several review articles in a previous volume (Vol. 22) have given too optimistic a picture of the antimicrobial
of the Endodontic Topics journal. However, some points effects of endodontic irrigating solutions. Wang et al.
of special interest regarding irrigation are worth (84) recently compared killing of bacteria in infected
addressing here as well. During the last few years, dentin after short-term and long-term incubation of
interest in endodontic biofilms has been growing the bacteria before the killing experiments. The results
rapidly, and several studies have measured the also showed decreased susceptibility of bacteria in the
effectiveness of different disinfecting agents against three-week-old dentin infection as compared to one
biofilm bacteria. In 2009, Shen et al. (102) compared week.
the sensitivity to different chlorhexidine preparations of While it is possible that different biofilm models
mixed species of oral biofilms grown in anaerobic have different time curves regarding maturation and
conditions. The result showed that the biofilm changed becoming less susceptible, it is important that the
from sensitive to much more resistant between two and models used in biofilm research are characterized so
three weeks of biofilm growth and maturation. that their behavior is well known at different stages of

48
 Irrigation: beyond the smear layer

maturation. This is a prerequisite to make it easier to irrigating solutions can kill bacteria in infected dentin,
compare results from different studies of disinfection depending on the type and concentration of the
and biofilms. irrigant, and time of exposure. However, serious
concerns have been expressed regarding weakening of
dentin strength by irrigation. Irrigation–dentin
Recommendations interaction will be one of the key areas of future
research in order to optimize the desired effects of
Sodium hypochlorite is the most important irrigating
irrigation and to minimize the harmful effects, which
solution during root canal treatment and should
might lead, at worst, to longitudinal tooth fracture.
be used throughout the instrumentation. High
concentrations of 5–6% kill bacteria and dissolve
organic tissue better than low concentrations of 1–2%. References
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