Basic Research—Technology

Effects of Concentrations and Exposure Times of Sodium

Hypochlorite on Dentin Deproteination: Attenuated Total
Reflection Fourier Transform Infrared Spectroscopy Study
Xiaoli Hu, DDS, MS,*k Yanwen Peng, PhD, MS,†k Chee-peng Sum, PhD, BDS, MSc,‡
and Junqi Ling, DDS, MS, PhD§

Abstract
Introduction: The purpose of this study was to quanti-
tatively analyze the effects of different concentrations
and exposure times of sodium hypochlorite (NaOCl) on
dentin deproteination and to determine the suitable
concentrations and exposure times to minimize the
effects of deproteination on dentin. Methods: Intact
human dentin slabs were treated with 0.5%, 1%, or
2.25% NaOCl for 1, 5, or 10 minutes. A 0.9% NaCl solu-
tion served as the control. To investigate the effect of
NaOCl on the surface chemical changes to human
dentin, the attenuated total reflection Fourier transform
infrared (ATR-FTIR) spectroscopy technique was used to
analyze the amide:phosphate ratio and carbonate:phos-
phate ratio. Results: The amide:phosphate ratio
decreased significantly after NaOCl treatment as
compared with the control group (p < 0.05). In the
0.5% NaOCl treatment group, the amide:phosphate
ratio was significantly higher than that in the 1% and
the 2.25% NaOCl treatment groups (p < 0.05). The
different exposure times (1, 5, or 10 minutes) of NaOCl
within the same concentration did not influence the ami-
de:phosphate ratio (p > 0.05). NaOCl treatment did not
affect the carbonate:phosphate ratio (p > 0.05).
Conclusions: NaOCl, 0.5%, is recommended as the
predominant concentration for routine use during root
canal therapy to minimize any NaOCl-induced dentin de-
proteination. It is suggested that a prolonged exposure
to low concentrations of NaOCl is less damaging to
dentin in attempting to achieve antisepsis during instru-
mentation of root canal therapy. (J Endod 2010;36:
2008–2011)
Key Words
Attenuated total reflection Fourier transform infrared,
dentin deproteination, sodium hypochlorite
T he success of root canal therapy depends on the quality of several factors, including
the instrumentation, irrigation, disinfection, and three-dimensional obturation of
the root canal system. Mechanical and chemical effects of the irrigant solutions are
crucial during irrigation. Sodium hypochlorite (NaOCl) solutions are the most widely
recommended irrigation solutions on the basis of their excellent antimicrobial potency
(1, 2) and distinctive capacity to dissolve remnant necrotic tissues (3–5).
General agreement regarding the optimal concentration and duration of NaOCl treat-
ment does not exist. A higher concentration (5.25%) and long exposure time (40
minutes) are needed for eliminating Enterococcus faecalis on contaminated bovine
dentin cylinders that had been incubated in brain-heart infusion broth for 3 weeks (2).
The use of NaOCl had been reported to affect dentin mechanical properties by the degra-
dation of organic components of dentin (6). Various concentrations and time periods have
reportedly affected dentin mechanical properties. A 24-minute exposure to 2.5% NaOCl
causes a significant drop in the flexural strength (7). The 2-hour exposure of 5.25% NaOCl
reduces the flexural strength and elastic modulus of dentin (8). A 15-minute exposure to
1% NaOCl reduces the dentin microhardness (9). A 60-second exposure to 5% NaOCl
caused the reduction in dentin microhardness (10). Ten-minute deproteination with
5% NaOCl reduced the push-out bond strength between dentin surfaces and fiber posts
cemented (11). NaOCl may also adversely influence the sealing ability and the adhesion
of dental materials including resin-based cements and root canal sealers (6). From these
studies, it is clear that the physical properties of dentine are very much affected by the
removal of the organic phase of dentine by the use of a high concentration of NaOCl.
It would be prudent to select a suitable NaOCl concentration and exposure time to
minimize its influence on the deproteination of dentin (12), which consequently influ-
ences the mechanical properties while achieving asepsis and the desired debridement
effects. The time-dependent effects of a high-concentration NaOCl (12%) on dentin de-
proteination from 0 to 48 hours at time intervals of 0.5 hours had been reported (12).
Linear relationships were found to exist between exposure time (10 to 240 minutes) of
NaOCl (1.3% and 5.25%) and ‘‘apatite/collagen ratio’’ of dentin powders (13).
However, to our knowledge, there has been no direct quantitative study about the effects
of the low concentration (0.5%-2.25%) and the short exposure time (1-10 minutes) of
NaOCl on dentin deproteination. As a simple, effective direct, nondestructive, and sensi-
tive technique, the attenuated total reflection Fourier transform infrared (ATR-FTIR)
spectroscopy technique can be used to investigate the effects of NaOCl on the surface
chemistry of human dentin slabs.

From the *Department of Preventive Dentistry, Guanghua College of Stomatology, Sun Yat-sen University, Guangzhou, China; †Department of Immunology, Sun
Yat-sen University, Guangzhou, China; ‡Department of Restorative Dentistry, National University of Singapore, Singapore; and §Department of Operative Dentistry
and Endodontics, Guanghua College of Stomatology, Sun Yat-sen University, Guangzhou, China.
k
Xiaoli Hu and Yanwen Peng contributed equally to this article.
Corresponding author: Dr Junqi Ling, Department of Operative Dentistry and Endodontics, Guanghua College of Stomatology, Sun Yat-sen University, Guangzhou,
China. E-mail address: lingjq@mail.sysu.edu.cn.
0099-2399/$ - see front matter
Copyright ª 2010 American Association of Endodontists.
doi:10.1016/j.joen.2010.08.035

2008 Hu et al. JOE — Volume 36, Number 12, December 2010

 Basic Research—Technology
The aims of the study were to quantitatively analyze the effects of
different concentrations and exposure times of NaOCl on dentin depro-
teination by ATR spectroscopy and to determine if a suitable concentra-
tion and duration of NaOCl treatment can be recommended to minimize
the effect of deproteination on dentin.

Materials and Methods

The Preparation of Dentin Specimens
Thirty fresh intact human premolars extracted for orthodontic
reasons were used in this study with institutional review board approval
and informed consent. The teeth were selected based on dimensions,
similarity in morphology, and absence of any cracks or carious defects,
especially within the root portion. Debris and soft-tissue remnants on
the root were removed with a sharp scalpel. All teeth were stored in
phosphate buffered saline at 4 C until used.
The tooth crowns were removed at the cementoenamel junction Figure 1. A typical ATR spectrum of human dentin without NaOCl treatment.
using a high-speed bur under water coolant. Each root was sectioned The absorption peaks between 800 and 2,000/cm-1 of chemical components
longitudinally into two parallel dentin slabs about 4  4  2 mm by present on human dentin are shown.
a Microslice 2 (Metals Research Limited, Cambridge, England). Sixty
dentin slabs were rinsed and ultrasonicated for 5 minutes with deion- Results
ized water (14). The typical ATR-FTIR spectrum of intact human dentin is shown in
The 60 dentin specimens were randomly assigned to 12 groups Figure 1. The peaks in these spectra (800-2,000/cm-1) have been as-
treated with 0.9% NaCl, 0.5% NaOCl, 1% NaOCl, or 2.25% NaOCl for signed according to the literature (16, 17). Because apatite is
1, 5, or 10 minutes; 0.9% NaCl group was used as the control. Each insoluble in NaOCl, the phosphate stretching vibration peak provides
specimen was placed in one well of a 24-well tissue culture plate con- a constant intensity for the purpose of normalization (12). Therefore,
taining 1 mL of treatment solution and then washed 3 times with deion- the spectra collected were normalized to the phosphate stretching
ized water and air-dried. vibration at 1,026/cm-1. The normalized spectra are presented in
Figure 2. These spectra suggested a clear weakening of the peaks at
1,242, 1,550, and 1,643/cm-1 after NaOCl treatment. However, the
ATR Spectroscopy
1,643/cm-1 peak existed as a weak band after NaOCl treatment. The
ATR-FTIR spectra were collected with a Thermo Nicolet 5700
presence of phosphate peak became more apparent with time (Fig. 2).
spectrometer (Nicolet, Madison, WI) and a smart OMNI sampler acces-
The amide:phosphate ratio decreased after NaOCl treatment
sory (Nicolet, Madison, WI) with germanium (Ge) as an internal reflec-
(Fig. 3). Two-way analysis of variance revealed a statistically significant
tion element. For the ATR-FTIR spectra collection, three different spots
main effect for ‘‘concentration’’ (p < 0.05), a statistically insignificant
on the surface of each specimen was randomly chosen and marked by
main effect for ‘‘exposure time’’ (p > 0.05), and a statistically insignif-
a high-speed handpiece using a fine diamond bur. The specimens were
icant interaction effect of ‘‘concentration’’ and ‘‘exposure time’’
then put onto the face of the Ge crystal of the smart OMNI sampler acces-
(p > 0.05). After the NaOCl treatment with different concentrations
sory. They were carefully adjusted so that the pointed tip of the standard
(0.5%, 1%, and 2.25%), the amide:phosphate ratio decreased signifi-
pressure tower would be just pressed onto the center of the mark.
cantly as compared with the control group (0.9% NaCl) (p < 0.05). In
Spectra were collected in the range from 750 to 4,000/cm-1 at
the 0.5% NaOCl treatment group, the amide:phosphate ratio was statis-
4/cm-1 resolution by using 100 scans. Three randomly selected and
tically significantly higher than that in the 1% and the 2.25% NaOCl treat-
marked locations on each of the dentin slabs were measured.
ment groups (p < 0.05). The amide:phosphate ratio was statistically
The ATR-FTIR spectra of air were obtained and automatically sub-
insignificantly different between the 1% and the 2.25% NaOCl treatment
tracted by the OMNIC 7 software (Nicolet). After baseline correction and
groups (p > 0.05). The different exposure time (1, 5, or 10 minutes) of
normalization, the effect of NaOCl on collagen depletion was evaluated
NaOCl within the same concentrations did not influence the amide:-
using the collagen and apatite ratio (the ratio of absorbance of amide I
phosphate ratio (p > 0.05).
peak to phosphate v3 peak) (12–15). Smaller ratio (amide:phosphate)
Two-way analysis of variance revealed that the main effects of
values corresponded to a greater extent of dentin deproteination.
‘‘concentration’’ and ‘‘exposure time’’ together with the interaction
Another parameter of the ATR-FTIR spectra, the carbonate:apatite ratio
effect between them did not statistically significantly influence the car-
(the ratio of absorbance of carbonate v2 peak to phosphate v3 peak) was
bonate:phosphate ratios (p > 0.05). This meant that NaOCl with
used to evaluate the effects of NaOCl on inorganic structure of dentin
different concentrations and exposure time did not change the
(14).
carbonate: phosphate ratios on dentin surfaces, indicating NaOCl may
not change the inorganic components on dentin.
Statistical Analyses
The dependent variables of this study were the ‘‘amide:phosphate Discussion
ratio’’ and the ‘‘carbonate:phosphate ratio’’ with independent variables In this study, we quantitatively analyzed the effects of NaOCl on the
being ‘‘concentration’’ and ‘‘exposure time.’’ Two-way analysis of vari- amide:phosphate and carbonate:phosphate ratios by ATR-FTIR and
ance and post hoc multiple comparisons with Bonferroni correction found that NaOCl removed the organic components but did not influence
were used to evaluate the significance levels of the main factors and their the inorganic phase of human dentin. This result is in agreement with the
interactions by a statistical package SPSS 16.0 (SPSS Inc, Chicago, IL). findings of others studies (12, 18, 19). Using a photoacoutic FTIR

JOE — Volume 36, Number 12, December 2010 Effects of Concentrations and Times of Sodium Hypochlorite on Deproteination 2009
Basic Research—Technology
0.9% NaCl 1 min
1
0.5% NaOCl 1 min
1% NaOCl 1 min
0.8
2.25% NaOCl 1 min
Absorbance

0.6

0.4

0.2

0
800 1000 1200 1400 1600 1800 2000

Wavenumbers (cm-1)

1 0.9% NaCl 5 min

0.5% NaOCl 5 min
0.8
1% NaOCl 5 min
Absorbance

2.25% NaOCl 5 min

0.6

0.4

0.2

0
800 1000 1200 1400 1600 1800 2000

-1
Wavenumbers (cm )
Figure 3. (A) The amide:phosphate ratio and (B) carbonate:phosphate ratio
on dentin surfaces treated by NaOCl at different concentrations and exposures
1 0.9% NaCl 10 min times. *p < 0.05; **p < 0.01.
0.5% NaOCl 10 min
0.8
1% NaOCl 10 min may complicate the evaluation of the treatment outcome because of the
Absorbance

0.6 2.25% NaOCl 10 min
0.4
0.2
0 produced insignificantly different amide:phosphate ratio. This implied
800 1000 1200 1400 1600 1800
-1
Wavenumbers (cm )
Figure 2. Representative ATR spectra of dentin slabs after NaOCl treatments
with different concentrations and exposure times. (A) ATR spectra of dentin
after NaOCl treatments for 1 minute, (B) 5 minutes, and (C) 10 minutes.
The black, blue, green and red lines represent 0.9% NaCl, 0.5% NaOCl, 1%
NaOCl, and 2.25% NaOCl treatment groups, respectively. The spectra have
been normalized to phosphate v3 (peak at 1,026/cm-1). Five specimens in
each group are analyzed. (This figure is available in color online at www.
aae.org/joe/.)
spectroscopy, the spectra of the NaOCl-treated dentin specimens showed
a slow and heterogeneous removal of the organic phase but kept the
carbonate apatite unchanged (12). However, it had been reported that
NaOCl can remove not only the organic material but also the carbonate
and magnesium ions from dentin crystal structure as well (20). These
differences may be caused by the different techniques used or species
differences in dentin composition. Infrared absorption spectroscopy
was used to detect the influence of NaOCl treatment on dentin mineral
(20). In that study, bovine root dentin, instead of human dentin, was
cut and pulverized to powder, desiccated, and dispersed in potassium
bromide for infrared measurements. The differences of bovine and
human dentin composition need to be further investigated (18). Further-
more, the subsurface dentin powder was included in that study, and this
regional variation of dentin. The chemical components present in dentin
such as water, phosphate, carbonate, and organic material absorb the
infrared radiation strongly. In our study, ATR-FTIR was used as an ideal
method for measurement of surface chemistry changes.
One important finding of this study was that the different exposure
time (1, 5, or 10 minutes) of NaOCl with the same concentration
2000
that the exposure times (between 1 and 10 minutes) of NaOCl produced
the negligible differences of dentin deproteination. Di-Renzo et al (12)
found that the normalized amide:phosphate ratio of etched dentin was
decreased dramatically after 12% NaOCl treatment within 0 to 100
seconds. After 100 seconds, prolonged treatment (up 15 minutes)
did not change the ratio further (12). With 5% NaOCl treatment, the
extent of deproteination was enhanced after 10 seconds, reaching
plateaus between 30 and 60 seconds and attaining a maximum after
120 seconds (19). These results show that the extent of deproteination
of NaOCl is not linearly related to the exposure time in tens of seconds to
several minutes. This is because of the fact that the collagen exposed on
the dentin surfaces can be quickly attacked and removed by the NaOCl
solutions. Then, the deproteination process of NaOCl reverts to the
hydroxyapatite-encapsulated collagen and shows little change over
a period of several minutes (12). Other studies showed that the apati-
te:collagen ratio and exposure time (10-240 minutes) of NaOCl (1.3%
and 5.25%) were linearly related (13). On the other hand, our study
confirms that the NaOCl treatment leads to the concentration-
dependent collagen depletion. The extent of dentin deproteination in
the 1% and 2.25% NaOCl treatment groups is significantly larger than
that in 0.5%. A previous report showed that the use of 5.25% NaOCl re-
sulted in a lower amide:phosphate ratio than that of 1.3% NaOCl (13).
Numerous publications have addressed the antibacterial activity of
NaOCl. The efficacy of antimicrobial action is related to both

2010 Hu et al. JOE — Volume 36, Number 12, December 2010


concentration and exposure time. Previous studies have found that even
at lower concentration NaOCl showed antimicrobial activity against
resistant bacteria such as E. faecalis, Candida albicans, and Pseudo-
monas aeruginosa (21–25). However, in a bovine dentine model, it
took 5.25% NaOCl over 40 minutes to completely eliminate E.
faecalis from blocks of dentine inoculated with E. faecalis and
incubated in brain heart infusion broth for 3 weeks, whereas 1.3%
and 2.5% failed to eliminate all E faecalis in that duration (2). The
differences between this study and other studies may caused by the
reduction of direct contact between NaOCl irrigant and E. faecalis as
a result of tubule infection, the lack of instrumentation, and the use of
deactivator (5% sodium thiosulfate solution) after NaOCl irrigation
(2). NaOCl at a higher concentration (5.25%) was more effective in
disinfection of the dentinal tubules (26). A combined 16S ribosomal
RNA-based reverse-transcriptase polymerase chain reaction and
reverse-capture checkerboard hybridization approach revealed that
the bacterial diversity was overall markedly reduced after chemome-
chanical preparation by using hand files and copious irrigation with
2.5% NaOCl (27). However, detectable levels of ribosomal RNA, repre-
senting viable cells, were observed in 60% of the cases after chemome-
chanical preparation (27). Currently, it remains to be determined the
minimum contact time and concentration of NaOCl required to eliminate
bacteria from the canal system. Concerns mainly remain about the
persistent infection from bacteria left in the depth of dentinal tubules.
NaOCl at various concentrations (1%, 2%, 4%, and 6%) and exposure
times (2, 5, and 10 minutes) can penetrate into the dentinal tubules
between 77 and 300 mm (28). It has been reported that the E. faecalis
rapidly invaded the dentin tubules and the front; the infection reached
1,000 mm in dentin blocks after 3 weeks of incubation (29). Therefore,
it is hard to reach all bacteria in dentin tubules by NaOCl irrigation espe-
cially with lower concentration. The depth of penetration increased with
increasing NaOCl concentration, but the differences were small. The
depth of penetration with 1% NaOCl was about 50% to 80% of the values
with the 6% solution. However, the increased exposure time resulted in
deeper penetration of NaOCl (28). Prolonging the exposure time of
NaOCl has more pronounced effect in dentin penetration compared
with the increased concentration.
Based on the results of the present study, 0.5% NaOCl is suggested
as the predominant concentration for routine use during root canal
therapy to minimize any NaOCl-induced dentin deproteination as
compared with higher concentrations. NaOCl at a higher concentration
(5.25%) and a longer exposure time may be suggested when there is
persistent infection in root canals. Because the exposure time (1-10
minutes) of 0.5% NaOCl produces an insignificant different extent of
dentin deproteination, it is recommended to prolong the exposure
time but not to increase the concentration in order to obtain better anti-
microbial activity during the instrumentation phase of routine root
canal therapy.
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Effects of Concentrations and Times of Sodium Hypochlorite on Deproteination 2011