Basic Research—Technology
Similar Influence of Stabilized Alkaline and Neutral
Sodium Hypochlorite Solutions on the Fracture
Resistance of Root Canal–treated Bovine Teeth
Erick Miranda Souza, DDS, MS, PhD,* Amanda Martins Calixto, DDS,†
Camila Nara e Lima, DDS,† Fernanda Geraldo Pappen, DDS, MS, PhD,‡
and Gustavo De-Deus, DDS, MS, PhD§
Abstract
Introduction: Stabilizing sodium hypochlorite (NaOCl)
at an alkaline pH is proposed to increase solution stability and tissue dissolution ability; however, a reduction
on the flexural strength of dentin discs has been found
to be a side effect. This study sought to determine
whether a stabilized alkaline NaOCl reduces the fracture
resistance of root canal–treated bovine teeth after root
canal preparation compared with a neutral solution
counterpart. Methods: The 4 anterior incisors were
removed from 20 mandibular bovine jaws, and each 1
was randomly assigned to 1 of 4 groups (20 teeth
each). Teeth were prepared with a sequence of 6
K-type files. The following experimental groups received
a different irrigation regimen: G1: distilled water (negative control), G2: 5% NaOCl at a pH of 7.2, and G3: 5%
NaOCl at a pH of 12.8; in the positive control group (G4),
teeth remained untreated. The time of contact and volume of solution were carefully standardized. After bone
and periodontal ligament simulation, teeth were subjected to a fracture resistance test. Results: A significant
difference was observed among the 4 groups tested
(analysis of variance, P < .05). The 5% NaOCl groups
(G2 and G3) presented significantly lower resistance
to fracture than the control (G1 and G4) (Tukey test,
P < .05). Both NaOCl solutions similarly reduced the
fracture resistance at approximately 30% (Tukey test,
P > .05). No differences were observed between positive and negative control groups (Tukey test, P > .05).
Conclusions: Stabilized alkaline and neutral NaOCl
solutions similarly reduced the fracture resistance of
root canal–treated bovine teeth by about 30%. (J Endod
2014;40:1600–1603)
Key Words
Alkaline NaOCl, bovine teeth, fracture resistance of
endodontically treated teeth
T
he use of sodium hypochlorite (NaOCl) solutions largely remains the mainstream
approach for root canal disinfection because of the unique tissue proteolysis capacity and microbial suppression by NaOCl (1, 2). These essential properties of NaOCl
solutions are predominantly influenced by the amount of available chlorine (3, 4).
In NaOCl solutions, chlorine can take different chemical forms depending on the
solution’s pH. At an alkaline pH, the predominant form is hypochlorite (ClO ),
whereas at a neutral pH the hypochlorous acid form predominates (5). The latter is
considered to be more bactericidal than hypochlorite (6); thus, it seems appropriate
to adjust the pH of NaOCl to a neutral level with the purpose of increasing its antimicrobial effectiveness (7). However, at a pH of 7.5, NaOCl was found to be unstable, which
causes a severe reduction in its shelf life (7, 8), preventing the neutralized solution from
being marketed on a regular basis. In addition, the drop in hypochlorite ion renders
neutralized NaOCl more cytotoxic (8) and less effective in dissolving organic tissue
(9) because the cleaning effectiveness of NaOCl solutions is related to the presence
of ClO (8, 10, 11). Therefore, all NaOCl solutions available for clinical use are
alkaline.
Albeit alkaline, NaOCl solutions rapidly show a drop in pH when active chlorine is
consumed during interaction with tissues and microorganisms (3, 5, 9), which, in turn,
result in a severe decline in the solution’s ability to dissolve organic tissue (9). Recently,
the effect of adding an alkali with the aim of maintaining the stability of the solution and
preserving its capacity to dissolve organic tissue has been investigated (9). Some available household bleach and dental-marketed NaOCl solutions are currently adding alkali
to provide stabilization and to increase the shelf life of NaOCl (12), while also claiming a
superior proteolytic effect.
Unquestionably, tissue proteolysis encompasses a pivotal feature of NaOCl because
either vital or necrotic tissue remnants may became a potential source for root canal
reinfection in cases of incomplete canal disinfection or leakage. Nonetheless, the proteolytic action of NaOCl also negatively impacts on dentin, causing the depletion of its
components of organic nature (13). This highly undesired NaOCl side effect irreversibly
changes the dentin framework, causing a dry weight reduction by 14% (14). Therefore,
the occurrence of physical and mechanical changes in dentin disks, such as microcrack
formation and the reduction in flexural strength, microhardness, and modulus of elasticity after NaOCl use is not a surprise (14–23).
Following a cause-effect rationale, the increase in the proteolytic effect triggered by
a stabilized alkaline NaOCl solution (8–10) may intensify the side effect on the organic
scaffold of dentin, ultimately leading to the undesired end result of root weakening
From the *Department of Dentistry II, Federal University of Maranh~ao, S~ao Luis, Brazil; †Private Practice, S~ao Luis, Brazil; ‡Department of Endodontics, Federal
University of Pelotas, Pelotas, Brazil; and §Department of Endodontics, Grande Rio University, Rio de Janeiro, Brazil.
Address requests for reprints to Prof Erick Miranda Souza, Av dos Holandeses, Cond Sports Garden, ap 1004A–Olho D’agua, S~ao Luis, MA, Brazil 65065-180. E-mail
address: erickmsouza@uol.com.br
0099-2399/$ - see front matter
Copyright ª 2014 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2014.02.028
1600
Souza et al.
JOE — Volume 40, Number 10, October 2014
Basic Research—Technology
(9, 13, 23). In fact, stabilized alkaline NaOCl induced a severe decrease
in the elastic modulus and flexure strength of dentin discs compared
with a nonstabilized counterpart (9). Because weakened roots may
significantly impact tooth survival, the effect of stabilized alkaline NaOCl
over fracture resistance should be investigated.
We aimed to evaluate whether the use of a stabilized alkaline NaOCl
solution influences the strength of root canal–treated bovine teeth
compared with a neutral NaOCl solution counterpart. The null hypothesis is that NaOCl solutions at 2 different pH levels do not influence the
fracture resistance of bovine teeth.
Materials and Methods
Sample Size Calculation
An analysis of variance (ANOVA) fixed-effects model (F family,
G*Power 3.1.1 for Windows, Heinrich-Heine-Universit€at D€usseldorf,
Germany) was used to set the ideal sample size. Based on a pilot study,
the effect size was determined at 0.58; an alpha-type error = 0.05 and
power b = 0.95 were input. The results indicated a minimum total sample size of 56 teeth and a critical F of 2.79 for the ANOVA evaluation.
Sample Selection and Preparation
Twenty mandibular bovine jaws from cattles similar in age were
selected to provide the 4 anterior incisors. Animals were slaughtered
for feeding purposes, and the jaws were donated to this study. With
the aim of providing anatomic matching among the groups, each
selected incisor was randomly assigned to 1 of 4 groups, resulting in
20 teeth per group and a total sample size of 80 teeth. For each jaw,
the 4 incisors were measured at a level 8 mm from the apex to ensure
roots display a 8–10 mm diameter. Root diameters lower or lager than
the established parameters results in the exclusion of the cow. After
extraction, teeth were stored in saline until use.
All teeth were cross-sectioned at levels of 8 mm coronally and
12 mm apically to the cement/enamel junction by means of a lowspeed saw (VC-50 Precision Diamond Saw; Leco, Miami, FL) under
copious water cooling resulting in samples with lengths of 20 mm.
The pulpal tissue was removed with Hedstr€oen files (Dentsply Maillefer,
Ballaigues, Switzerland).
Preparation and Characterization of Irrigating Solutions
Freshly prepared technical-grade NaOCl solutions were obtained
from a pharmacy (Special Farma, S~ao Luis, Brazil). A 2-mol/L NaOH
solution was mixed with a standard 10% NaOCl solution to obtain an
NaOH-stabilized alkaline 5% NaOCl solution (9). The neutral solution
was acquired by mixing a standard 10% NaOCl solution with 1% sodium
bicarbonate (NaHCO3) (24). The available chlorine of the solutions was
certified using a standard iodine/thiosulfate titration method immediately before and after the experiments (20 days later). Before and after
the experiment, the pH of the solutions was verified using a calibrated
pH electrode (Model 6.0210.100; Metrohm, Herisau, Switzerland). The
pH of NaOH-stabilized NaOCl was also determined after 40 minutes of
contact with a bovine root canal to confirm that the stabilization was
effective (9). All root canal preparations were performed in a controlled
room temperature.
Root Canal Instrumentation and Irrigation
The apical portion of each tooth was sealed off with wax, preventing any irrigation liquid from being extruded from the large apical opening created after apical sectioning. Groups G1–G3 received different
irrigation regimes as follows: G1: distilled water (negative control),
G2: 5% sodium hypochlorite with a pH of 7.2, and G3: 5% NaOCl
JOE — Volume 40, Number 10, October 2014
with a pH of 12.8. In the positive control group (G4), teeth remained
untreated.
To ensure irrigated groups (G1–G3) received the same volume
of irrigation, root canals were instrumented using a sequence of 6
hand K-files (Dentsply Maillefer), which were selected after determining the first instrument to bind at 1 mm from the apical opening.
After each hand file, 5 mL irrigation solution was delivered into the
root canal using a 27-G endodontic needle (NaviTip; Ultradent Products, South Jordan, UT) reaching 3 mm from the apex. A constant rate
of 1 mL/min was achieved using a VATEA peristaltic pump (ReDent
Nova, Ra’anana, Israel). After irrigation, the root canals remained
filled with the solution, and the subsequent instrument was used to
prepare the root canal using a step-back technique. Each instrument
was used for 2 minutes. Considering the number of instruments used
(6), the total period that root canal dentin remained in contact with
the solutions was 26 minutes. The extruded solution was aspirated
adjacently to the coronal opening to make sure that any solution
was drawn off the external root surface. All teeth received a final irrigation with 10 mL distilled water for 5 minutes, removing any solution
remnants from the root canal. Furthermore, root canals were dried
with paper points and stored at 37 C with 100% humidity until the
strength tests were performed.
Simulation of the Periodontal Support Apparatus
Teeth were firstly immersed in melted wax (Horus; Herpo Produtos Dentarios, Petropolis, RJ, Brazil) up to 2.0 mm below the cementoenamel junction to create a 0.2- to 0.3-mm-thick wax layer covering the
root. Furthermore, a polystyrene resin (Cristal, Piracicaba, Brazil) was
used to embed the roots in polyvinyl chloride cylinders (a 21-mm diameter and 25-mm high). After the resin was set, the teeth were withdrawn
from the polyvinyl chloride cylinders, and the wax removed from root
surface and resin cylinder ‘‘sockets’’ using a warm water flush for 2 seconds. A polyether impression material (Impregum Soft; 3 M/ESPE, Seefeld, Germany) was delivered to the cylinder hole using a syringe. The
samples were immediately reinserted into the respective cylinder
socket, and any excess of impression material was removed, finally
resulting in a simulated periodontal ligament of 0.2–0.3 mm (25).
Fracture Strength Test
All specimens were subjected to a compressive load at a crosshead
speed of 0.5 mm/min by means of a servo-hydraulic universal testing
machine (EMIC DL2000; EMIC Equipamentos e Sistemas de Ensaio
Ltda, S~ao Jose dos Pinhais, Brazil) until fracture. The specimen was
fixed to an apparatus that allowed a 45 angle formation with the
EMIC loading tip, simulating a traumatic shock on the middle third of
the crowns from a buccal-lingual direction. The ultimate load required
to fracture the specimens was recorded in newtons.
Statistical Analysis
Raw data adhesion to Gaussian distribution and homogeneity of
the variance were studied a priori (Shapiro-Wilk and Levene tests).
Because both assumptions were confirmed (P > .05), 1-way ANOVA followed by the Tukey Honest Significant Difference post hoc test were
selected to verify the effect of the solution in the fracture strength of
bovine teeth. The a-type error was set to 0.05.
Results
Table 1 displays the mean and standard deviations of the tested
groups. One-way ANOVA indicated a significant difference between
the groups (P < .05). The negative control group (distilled water)
behaved similarly to positive controls because no significant change
Root Canal–treated Bovine Teeth
1601
Basic Research—Technology
TABLE 1. Mean and Standard Deviations (SD) of the Ultimate Fracture
Strength (Newtons) of the Groups Tested
Groups
Mean
Positive control
Negative control (distilled water)
pH = 7.2, 5% NaOCl
Stabilized pH = 12.8, 5% NaOCl
a
103.13
105.88a
79.85b
74.09b
SD
24.42
28.13
18.34
18.25
Different letters indicate significant differences at P < .05 (Tukey HSD).
in resistance to fracture was observed (Tukey HSD, P > .05). However,
both NaOCl groups (G2 and G3) significantly reduced the fracture resistance of bovine teeth by approximately 30% compared with controls
(Tukey HSD, P < .05). The 5% NaOCl group with a stabilized pH of
12.8 (G3) presented a similar influence in the fracture resistance as
the 5% NaOCl group with a pH of 7.2 (G2) (Tukey HSD, P > .05).
Discussion
Together with the loss of tooth substance (26), chemical-induced
changes over dentin are largely considered the main reasons for a
reduction in the stiffness of root canal–treated teeth (23). Microhardness, elastic modulus, and flexural strength are some mechanical properties of dentin that are highly modified by treatment with NaOCl (23).
Changes in microhardness, for instance, disclose modifications in both
organic and inorganic parts of dentin, whereas a reduction in elastics
modulus and flexural strength may potentially turn dentin brittle
(23). The NaOCl strong oxidant effect adversely reacts with the dentin,
causing a remarkable depletion of its organic framework (13). This
organic constituent, composing about 22 wt% (13), is mainly constituted by type I collagen and proteoglycans (27). The collagen matrix
forms the major component and organizes itself into a fibrillar frame
around peritubular dentin, whereas proteoglycans connect 1 or more
glycosaminoglycan chains and are responsible for regulating water content and intratubular permeability (28, 29). Overall, the deleterious
effects of NaOCl solutions on the collagen and proteoglycan matrix
might result in dentin contraction, induce an increase in stress
concentration and crack propagation, and ultimately contribute to
the significant reduction in the fracture strength (13). Moreover, the
nonspecific proteolytic action of NaOCl is also capable of affecting carbonate ions (30), an inorganic reinforcing phase that also influences
dentin mechanical properties (31). These known effects of NaOCl
over dentin are certainly reasons for the reduction in root toughness,
which was observed in the NaOCl-treated groups. Therefore, the null
hypothesis presented here is rejected.
However, bearing in mind the reported superior proteolytic effect
of alkalized NaOCl solutions (8–10), the disclosure that the stabilized
alkaline 5% NaOCl solution significantly reduced the fracture
resistance of bovine teeth to the same level as the theoretically less
proteolytic neutral NaOCl (P > .05) is surprising. Even though
neutral NaOCl solutions are currently not marketed as endodontic
irrigants, a pH level of 7.2 has been selected to confirm the
hypothesis that a reduction in the pH level would be beneficial by
decreasing the proteolytic side effect on the organic part of dentin,
which could minimize root weakening. However, both pH solutions
similarly reduced the fracture resistance of bovine roots to about
30%, which is in contrast with a previous report that found an
intensification of the negative effect by stabilized NaOCl solution over
flexure strength and elastic modulus of dentin (9). The present
outcome might be interpreted as the interplay of various factors
including the remarkable differences in the nature of the present study
setup compared with flexural strength and elastic modulus investigation
1602
Souza et al.
designs. In the latter, dentin discs are usually submerged into the irrigant, leading to the discs being affected by the solution from all sides.
Also, the volume of irrigant to dentin ratio is inversed in this assay
because in clinical reality the amount of solution is small compared
with the extent of root dentin (9). In the close to clinical scenario
used here, the solution only affected the inner root canal surface. Gravitational forces also push the solution in a perpendicular direction in
relation to the dentinal tubules, which certainly influences its spreadability into dentin. Penetration into dentinal tubules by syringe-driven
NaOCl is, indeed, very limited (32). Thus, mimicking the clinical scenario, the superior proteolytic effect over dentin by stabilized NaOCl
appeared not to affect root dentin stiffness in the same magnitude as
previously reported using dentin bar designs (9).
Another factor accounting for this outcome is the role of the total
contact time of NaOCl with dentin. Both the proteolytic and antimicrobial effects of NaOCl solution are known to be strongly time dependent
(5). The same can be expected for the deleterious effects on dentin.
Microcracks, for instance, were markedly visible over dentin surface
after 6 hours of immersion in a 5% NaOCl solution (13), and flexural
strength and elastic modulus of dentin were reduced after a minimum of
24 minutes–2 hours of contact with NaOCl (23). The 26-minute average
contact time used in this study, which was selected to mimic a multifile
instrumentation approach, might have been insufficient to make the
stronger oxidant and proteolytic effect over root toughness by the
NaOH-stabilized alkaline NaOCl prominent.
A decrease in the pH level as ClO is consumed (33) could also be
hypothesized as an explanation for the similar results between NaOCl
solutions. Jungbluth et al (9) were unable to find any pH reduction
after 30 minutes of contact with dentin discs, which might be explained
by the fact that NaOH maintains high pH levels despite the consumption
of available chlorine. Here, after 40 minutes of contact of
NaOH-stabilized solution with the roots, no drop in pH was observed.
Because various factors could affect the tissue dissolution ability of
NaOCl solutions, such as the concentration of available chlorine, temperature, and time of contact (5), efforts were made to keep these constant during the experiment. Considering the unreliable concentrations
of chlorine in household bleach and dental NaOCl irrigants (9),
technical-grade NaOCl solutions were ordered from a pharmacy in
order to a priori guarantee the amount of available chlorine content;
this was confirmed by solution titrations before and after experiments.
The temperature of the room was also controlled to avoid any interference with the proteolytic action of solutions.
Surprisingly, the instrumentation-only group (positive control)
did not display any lowering impact on the root strength as largely
observed in other previous studies on human tooth (26). A speculative
explanation could be hypothesized because of the natural morphologic differences between bovine and human teeth. Bovine incisors
present thicker dentinal walls (3–4 mm) compared with human roots
(1–3 mm). After canal instrumentation of human root, the dentin loss
possibly has a superior impact on overall tooth resistance compared
with bovine teeth in which the thicker dentinal walls would be less
impacted by the dentin removed after instrumentation. However, the
lack of studies on the effect of root canal instrumentation in bovine
teeth prevents a further confirmation of this speculative hypothesis.
The use of bovine teeth could be regarded as a limitation. However, some similarities between bovine and human teeth can be viewed as
positive for this study, such as the similar modulus of elasticity and tensile strength (34) and the number and distribution of dentinal tubules
(35–37), which potentially influence the ability of dentin to resist
loading. Also, the possibility to easily standardize bovine teeth among
groups according to age is advantageous. In addition, using a splitmouth design is depicted as an experimental improvement. These
JOE — Volume 40, Number 10, October 2014
Basic Research—Technology
approaches reduced the biasing role of anatomy, which is usually difficult to exclude when using human substrates. Not less important is the
simulation of the periodontal apparatus that has been found to
approach the in vitro setup to clinical reality by distributing the fracture
pattern to radicular areas (25).
The stabilization of alkaline NaOCl is seen as beneficial for helping
to keep the proteolytic-driven chemical debridement active during root
canal treatment. Doubt existed regarding whether the clinical use of this
solution could make roots more brittle. The present results provide laboratorial evidence to minimize this concern. However, as a laboratorialbased study, extrapolations to the clinic cannot be forthright. Pondering
the limitations of the present study, it can be concluded that
NaOH-stabilized alkaline NaOCl solution presented the same level of
influence as a neutral counterpart in the fracture resistance of root
canal–treated bovine teeth.
Acknowledgments
The authors deny any conflicts of interest related to this study.
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