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Effects of applying glutaraldehyde-containing desensitizer formulations on

reducing dentin permeability

Article  in  Journal of Dental Sciences · June 2012

DOI: 10.1016/j.jds.2012.03.005

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Journal of Dental Sciences (2012) 7, 105e110

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ORIGINAL ARTICLE

Effects of applying glutaraldehyde-containing

desensitizer formulations on reducing dentin
permeability
Hiroshi Ishihata a*, Masafumi Kanehira b, Werner J. Finger b,
Hidetoshi Shimauchi a, Masashi Komatsu b

a
Graduate School of Dentistry, Department of Oral Biology, Division of Periodontology and Endodontology,
Tohoku University, Sendai, Miyagi 980e8575, Japan
b
Graduate School of Dentistry, Department of Restorative Dentistry, Division of Operative Dentistry,
Tohoku University, Sendai, Miyagi 980e8575, Japan

Final revision received 22 November 2011; accepted 10 March 2012

Available online 3 May 2012

KEYWORDS Abstract Background/purpose: The efficacy of dentin-desensitizing agents is commonly

chemiluminescence; evaluated in clinical studies by measuring patients’ pain response upon stimulation. Although
desensitizer; indispensable, such trials are time-consuming, and the results depend on an individual’s
dentin subjective pain rating. Therefore, in vitro efficacy screening prior to clinical testing is highly
hypersensitivity; desirable. The objective of this study was to investigate in vitro dentin permeability of two
dentin permeability; glutaraldehyde-containing desensitizer formulations after different modes and times of appli-
glutaraldehyde cation.
Materials and methods: Coronal tooth slices, 1.3 mm thick, were dissected from 60 freshly ex-
tracted third molars. Specimens were treated with EDTA to remove the cutting smear. The
dentin disks were clamped in a split chamber device to determine the baseline permeability
under a liquid pressure of 2.5 kPa for 2 minutes and 13 kPa for 1 minute, to record liquid flow
through the dentin using a photochemical method. Slices were soaked in a 2% albumin solution
and reevaluated under the same pressure cycles prior to active or passive application for 15,
30, or 60 seconds of either Gluma Desensitizer or Gluma PowerGel (GDP) (Heraeus Kulzer, Ha-
nau, Germany) and then reevaluated. Dentin-disk permeability was determined as the area
under the photo signal output voltage line during the pressurizing period (mV s). The statistical
data analysis used a Kruskal-Wallis ANOVA and Mann-Whitney post-hoc test with the signifi-
cance level set to 5%.

* Corresponding author. Department of Oral Biology, Division of Periodontology and Endodontology, Tohoku University, 4-1 Seiryo-machi,
Aoba-ku, Sendai, Miyagi 980-8575, Japan.
E-mail address: isi@m.tohoku.ac.jp (H. Ishihata).

1991-7902/$36 Copyright ª 2012, Association for Dental Sciences of the Republic of China. Published by Elsevier Taiwan LLC. All rights reserved.
doi:10.1016/j.jds.2012.03.005
106 H. Ishihata et al

Results: Permeability at the baseline and after albumin soaking did not significantly differ. For
both desensitizing compounds, 30 and 60 seconds of active and passive applications resulted in
significantly reduced dentin permeability. After the 15 second application, only the actively
treated samples with GDP showed a significant reduction in permeability.
Conclusions: The liquid and the gel desensitizing agents both significantly reduced dentin
permeability. The obvious advantage of a gel formulation is the controlled application, limited
to the hypersensitive tooth area, thus avoiding inadvertent contact with adjacent gingival
tissues.
Copyright ª 2012, Association for Dental Sciences of the Republic of China. Published by
Elsevier Taiwan LLC. All rights reserved.

Introduction permeability and hydraulic conductance,12 was suggested

Dentin hypersensitivity (DH) is by definition a common
complaint, mainly in adult populations in their third and
fourth decades. DH is characterized by a sharp transient
pain in response to thermal, evaporative, tactile, osmotic,
or chemical stimulation of exposed dentin in teeth, without
evidence of other defects or pathology.1e6 DH is a result of
fluid movement within the dentin complex.7 This phenom-
enon was described as the “hydrodynamic theory”.8,9 Based
on this widely accepted theory, the clinical treatment goal
is to provide a permanent seal of the patent tubules or at
least a reduction in their functional diameter to eliminate
or minimize outward fluid flow from the tubules.10,11
Numerous topical agents were suggested for professional
relief of DH. Examples of frequently used topically applied
agents include oxalates, creating tubule obstruction by
precipitating fine-grained calcium oxalate crystals,12e15
dentin adhesives,16e19 protein-precipitating fixative
agents20e22 and restorative materials.23 Gluma Desensitizer
(GDL; Heraeus Kulzer, Hanau, Germany) is a spin-off of the
Gluma Bonding System (Heraeus Kulzer). According to the
manufacturer, GDL is an aqueous solution of 5% glutaral-
dehyde (GA) and 35% 2-hydroxyethyl methacrylate (HEMA).
Bergenholtz et al24 reported that application of Gluma
Primer (which is identical to GDL) effectively inhibited the
discharge of serum albumin from freshly cut dentin cavities
in monkey teeth. They hypothesized that GA, a biological
fixative and one of the major components of Gluma Primer,
was responsible for the coagulation of plasma proteins and
thus tubular blockage. This explanation was corroborated
by results of morphological and clinical studies with GDL
demonstrating peripheral tubular blockage20 and significant
pain relief following topical application to hypersensitive
dentin.21,22,25e27
In order to improve handling procedures for GDL, the
manufacturer developed an analogous gel formulation,
Gluma Desensitizer PowerGel (GDP), for consistent appli-
cation of the compound to sensitive tooth target sites, and
to avoid or minimize the risk of inadvertent contact with
adjacent gingival tissues.
Although clinical trials, i.e., pain studies, are the ulti-
mate proof of the effectiveness of desensitizing agents,28
carefully designed in vitro trials are considered useful
tools to predict clinical effects of DH treatments, although
such trials cannot fully mimic the complexity of vital
teeth. The dentin-disc model, designed to assess dentin
as a valuable tool for in vitro evaluation of dentin-
desensitizing compounds.15,29e31
The aim of this in vitro investigation was to evaluate and
compare the effects of the duration and mode of applica-
tion of GDL and GDP on the permeability of human dentin,
using a modified dentin-disc method.32 The null hypothesis
was that there would be no difference in permeability
reduction among the liquid and gel formulations.
Materials and methods
Materials investigated
In this in vitro study, the effects of the two desensitizing
agents, GDL (lot 010092, expiration date December, 2012)
and GDP (lot VP180809BQ1, expiration date February,
2010), on reducing fluid flow through human dentin discs
were investigated.
GDL is an aqueous solution of 5% GA and 35% HEMA,
whereas GDP is an analogous aqueous gel formulation
including 5% GA and 35% HEMA, which is thickened with
pyrogenic silica and rendered opaque by the addition of
pigments.
Testing device
The testing device described by Ishihata and colleagues32
was used to determine dentin permeability. The apparatus
(Fig. 1) consists of two cylindrical acrylic chambers (5 mm in
diameter and 5 mm high), mounted and fitted with O-rings
on each side of a dentin disc, and clamped in a metal frame.
Each chamber has a liquid inlet and a drainage outlet. The
chamber mounted on the occlusal side of the disc is sealed
with a clear glass coverslip and filled with a chemical illu-
minant reagent (aqueous solution of 0.02% luminol (5-amino-
2,3-dihydro-1,4-phtalazinedione) and 1% sodium hydroxide).
The opposite chamber, fitted to the pulpal side of the dentin
disc, is filled with an activator liquid (1% potassium ferricy-
anide and 0.3% hydrogen peroxide). Upon pressurizing the
activator liquid, the solution passes through the dentin disc’s
tubules to the illuminant-containing chamber and produces
a luminescence reaction. This photo signal is recorded with
a highly sensitive photo diode (S 9295, Hamamatsu
Photonics, Hamamatsu, Japan), mounted 5 mm from the
coverslip of the occlusal chamber. In order to prevent outer
In vitro evaluation of desensitizing compounds
Figure 1 Schematic illustration of the split-chamber device.
The activator solution is pressurized from the apical side of the
dentin disc. Upon penetration to the occlusal side and contact
with the luminol-containing solution a photochemical signal is
generated and recorded with a photodetector in mV.
light interference with the weak luminescence signal, the
entire equipment is enclosed inside a lightproof box. The
output voltage of the photodiode is recorded with an AD
converter at 1 kHz, stored in a central processing unit
controlling the system, and transferred to a personal
computer for data processing and analysis. The entire
procedure is automated in a programmable sequencer.
Specimen preparation and measuring procedures
This trial was approved (Number 21e15) by the Ethical
Committee of the Graduate School of Dentistry, Tohoku
University, Sendai, Japan. Sixty non-identifiable, freshly
extracted human third molars, free of decay and restora-
tions, were used. Immediately after extraction, the teeth
were frozen until further processing, for 2 weeks at the
longest. Coronal 1.3 mm thick tooth slices were cut under
copious water-cooling with a diamond wafer saw micro-
tome (model SP 1600, Leica Microsystems Nussloch, Nus-
sloch, Germany) perpendicular to the vertical tooth axis
between the occlusal enamel portion and pulp horns. The
cut sides of the specimens were cleaned with a 0.5 M EDTA
solution (pH 7.4), applied using a soaked microbrush with
a slight dabbing action during 60 seconds to remove the
cutting smear and open the dentinal tubules prior to thor-
ough rinsing with deionized water and slight air-drying.
Dentin specimens were mounted between the chambers
of a measuring device. Then the illuminant reagent liquid
was injected into the occlusal chamber, and the activator
solution was injected into the chamber fitted to the pulpal
side. The activator solution was automatically pressurized
for 2 minutes at 2.5 kPa, followed by a 2 minute pressure-
free interval. The resulting photochemical signal was
continuously registered. At the end of this first measuring
cycle, the illuminant liquid was discharged, and new liquid
was injected for the second pressurizing cycle under the
same conditions. Then, a wash cycle with deionized water
was automatically initiated before both chambers were
filled again with the respective reagent solutions, pressur-
ized in two consecutive runs with 13 kPa for 1 minute fol-
lowed by a 1 minute pressure-free interval. This entire
procedure for determining the baseline permeability was
107
executed in duplicate for each dentin specimen. The areas
under the output voltage lines during the pressurizing
periods of the two cycles at each pressure were integrated
(mV$s). Means of the four pressure runs at each of the two
pressures applied served as measures for the dentin spec-
imens’ baseline permeability.
Following the baseline permeability evaluation, speci-
mens were removed from the split-chamber column; the
pulpal sides were covered with a few droplets of 2% bovine
albumin solution (albumin, from bovine serum, Cohn Frac-
tion V, pH 5.2; Wako Pure Chemical Industries, Osaka,
Japan). A vacuum-connected chamber was sealed with O-
rings on the opposite specimen side for aspiration of the
albumin solution into the dentinal tubules. The dentin
samples were then rinsed with deionized water for 3
seconds prior to re-mounting in the split-chamber device at
exactly the same position for the same duplicate pressur-
izing cycles as described above for the baseline
characterization.
In the third test run with the same specimens, one of the
desensitizing agents, GDL or GDP, was applied on the gently
air-dried occlusal dentin side. GDL was applied with
a soaked microbrush, while GDP was dispensed to the
target area from a syringe fitted with a blunt needle tipped
with a small brush. With both desensitizing agents, the
effects of 15, 30 and 60 seconds of dwell times on dentin
permeability were evaluated. During the dwell time, the
agents were either left undisturbed (passive application) or
slightly agitated with a microbrush (active application).
Samples treated with GDL were air-dried with compressed air
for 3w5 seconds, whereas dentin samples treated with GDP
were rinsed with deionized water for 5 seconds and gently
air-dried for 3 seconds. Subsequently, the dentin slices were
re-mounted in the split-chamber device, and the perme-
ability was measured using the same pressurizing cycles as
described above for determining the baseline permeability
and the permeability of the albumin-soaked specimens.
Specimen preparation and permeability measurement
took place in an ambient laboratory atmosphere. Five
samples were used for each of the six variable conditions
tested and each desensitizing agent.
The results were statistically analyzed by a non-
parametric Kruskal-Wallis analysis of variance (ANOVA) and
post-hoc Mann-Whitney test with statistical significance set
to P Z 0.05 (PASW Statistics 18.0 for Macintosh, Chicago,
IL, USA).
Results
Figs. 2 and 3 show the mean values and standard deviations
of the permeability of dentin specimens, challenged with
2.5 kPa hydrostatic pressure at the baseline, after albumin
soaking, and after respective treatment with GDL and GDP.
The Kruskal-Wallis ANOVA revealed that there were no
significant differences among the six baseline groups or
among the related albumin-soaked specimen groups, for
specimens allocated to treatments with either GDL or with
GDP. The ANOVA calculated for all baseline and albumin-
treated samples showed no significant differences
(P Z 0.899 for GDL; P Z 0.749 for GDP). After GDL appli-
cation, 30 and 60 seconds of active and passive dwell times
108
Figure 2 Mean values and standard deviations of integrated
chemiluminescence outputs (mV s) of six sets of dentin discs,
pressurized with 2.5 kPa for 2 minutes (n Z 5). The bars of
each group with identical signature represent consecutive
outputs of the same specimens following cleaning with EDTA
(baseline), soaking with albumin, and application of GDL,
respectively. From left to right the three pairs of bars over
each test condition represent dwell times of 15, 30, and 60
seconds after passive (p) and active (a) application. Bars con-
nected with brackets and denoted with single and double
asterisks are significantly different on the 5% and the 1% level
of significance, respectively.
resulted in significantly reduced permeability compared to
the baseline and albumin soaking, whereas a 15 second
application showed no significant reduction in permeability.
For GDP-treated samples, all groups except that with 15
seconds of passive application showed significant reduc-
tions in permeability compared to data at the baseline and
after albumin soaking.
Figs. 4 and 5, respectively, show the permeability results
for the same specimens as in Figs. 2 and 3, allocated to the
GDL and GDP groups, after 13 kPa of pressurization for 1
minute. As with 2 minutes of 2.5 kPa of pressure, no
significant differences were found among the baseline and
albumin groups, or among the pooled baseline and albumin
groups (P Z 0.998 for GDL, P Z 0.721 for GDP). Thirty and
60 seconds of active and passive dwell times produced
Figure 3 Mean values and standard deviations of integrated
chemiluminescence of dentin discs treated with GDP (2.5 kPa/
2 minutes). Conditions as in Fig. 2.
H. Ishihata et al
Figure 4 Mean values and standard deviations of integrated
chemiluminescence of dentin discs treated with GDL (13 kPa/1
minute). Conditions as in Fig. 2.
significantly reduced permeability compared to the base-
line and albumin soaking, whereas 15 seconds of applica-
tion only showed a significant reduction in permeability for
the active application mode of GDP.
Discussion
Although clinical studies are the ultimate proof of desen-
sitizing agents’ efficiency, in vitro testing is a suitable
alternative to acquire relevant screening results, provided
the test setup simulates the conditions necessary for and
are suitable for individual desensitizers’ modes of action.
Desensitization of hypersensitive tooth areas implies
inhibiting or hampering outward fluid flow from patent
dentin tubules. Therefore, in vitro testing of human dentin
samples should be used to evaluate their hydraulic
conductance or permeability before and after application
of a desensitizing agent. Use of freshly extracted third
molars without decay or restorations, is presumably the
best choice to ensure a reasonably homogeneous substrate,
because such teeth are commonly removed from younger
patients and their tubules are not yet obstructed.
Figure 5 Mean values and standard deviations of integrated
chemiluminescence of dentin discs treated with GDP (13 kPa/1
minute). Conditions as in Fig. 2.
In vitro evaluation of desensitizing compounds
In this study, we prepared coronal sections from
extracted teeth, although hypersensitivity is commonly
related to cervical tooth areas. However, several published
reports confirmed that there was no significant difference
between the permeability of occlusal and buccal dentin,33
or between tubule densities of occlusal and cervical
dentin.34,35
To determine the baseline permeability, the smear
produced during diamond-blade cutting was removed from
both sides of the specimens in order to simulate the
condition of hypersensitive teeth, where tubules are patent
at both ends. The physiologic pulp pressure is reportedly 15
cmH2O (z 1.5 kPa).36 In the present study, we pressurized
the dentin discs from the pulpal side with 2.5 kPa, which is
close to the physiologic pressure, and additionally with
13 kPa to investigate the tubule-occluding effects of the
desensitizing agents under extreme non-physiologic condi-
tions. The results showed that 2 minutes of pressure at
2.5 kPa corresponded to the chemiluminescence output
after 1 minute of pressure at 13 kPa. All pressurizing cycles
were repeated in order to remove any possibly remaining
reagent from the preceding run and to verify the first
reading.
GA is supposedly the main component of GDL and GDP
which is responsible for the tubular plugging effect seen
after topical application.22,24,37 Among the many available
protein cross-linking agents, GA, a fairly small molecule
with two aldehyde groups separated by a flexible chain of
three methylene bridges, rapidly reacts with several func-
tional groups of proteins and is more efficient than other
aldehydes in generating stable crosslinks.38
Knutsson et al39 quantitatively determined the release
of plasma proteins in dentinal fluid from cavities prepared
in healthy young human teeth. Albumin and immunoglob-
ulin G (IgG) were found in all dentin samples, whereas
fibrinogen was seen in only four of 16 dentin samples. The
amount of serum albumin always exceeded IgG by a ratio
of 10.5:1. Based on this analysis, it was reasonable to soak
the dentin discs in albumin to prepare them to evaluate
the effects of the desensitizing agents. Bovine albumin,
selected for our experiments as the protein in the perfu-
sion fluid, has a molecular mass of 66 kDa, low enough not
to significantly reduce the permeability of dentin, in
contrast to globulins and lipoproteins, that can cause
marked reductions in permeability.33,39,40 The perme-
ability of the albumin-soaked discs did not significantly
differ from the same dentin discs at the baseline evalua-
tion. This indicates that the 2% aqueous albumin solution
used had no appreciable effect on the perfusion fluid’s
viscosity.33
Both GDL and GDP significantly reduced the permeability
of the dentin discs to similar extents. Zero permeability was
only registered in a few cases. Fluid flow through the
dentinal tubules can appropriately be described by the
Hagen-Poiseuille equation, where fluid flow through a capil-
lary is proportional to the radius raised to the fourth power.
Therefore, reducing the tubular diameter by one-half would
result in a 16-fold lower hydraulic conductance.11 Hence,
the striking reduction in dentin permeability found after
GDL and GDP application may indicate that such fluid flow
reduction in vivo might be adequate to eliminate or at least
greatly reduce pain sensations perceived by patients.
109
The present data show that the reduction in perme-
ability of albumin-soaked dentin was almost identical after
GDL and GDP application, although it could be hypothesized
that diffusion of the active desensitizer components from
a gel might be lower compared to a liquid compound.
Apparently, there is sufficient GA or GA and HEMA37 avail-
able at the dentin interface and to some depth inside the
albumin-soaked tubules for coagulation and plugging of the
tubules with proteins. In agreement with the manufac-
turer’s instruction for use, the dwell time of the desensi-
tizing agents on dentin should be 30 or 60 seconds rather
than 15 seconds, in order to obtain the most pronounced
reduction in permeability. Although the mode of applica-
tion had no practically significant effect on the in vitro
permeability, we suggest using the active mode, i.e.,
moving the applied desensitizing agent gently throughout
the dwell time with an application microbrush for GDL or
the brush-tip of the application cannula for GDP, as slight
agitation always enhances diffusion at interfaces.
The null hypothesis that there would be no difference in
permeability reduction between applications of GDL or the
GDP gel formulation is therefore accepted.
In summary, this in vitro investigation proved that GDL
and GDP have similar efficiencies of reducing the perme-
ability of human dentin. The obvious advantage of the gel
formulation is well-controllable application to treatment
sites, limiting the risk of inadvertent spread of the desen-
sitizing agent to adjacent gingival tissues.
Acknowledgments
The authors would like to thank Heraeus Kulzer (Wehrheim,
Germany) for donating the Gluma Desensitizer and
Gluma Desensitizer PowerGel.
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