Australian Dental Journal

The official journal of the Australian Dental Association

Australian Dental Journal 2015; 60: 65–72

doi: 10.1111/adj.12275

Comparative evaluation of the effectiveness of desensitizing

agents in dentine tubule occlusion using scanning electron
microscopy
CL Chen,* A Parolia,* A Pau,* IC Celerino de Moraes Porto†
*School of Dentistry, International Medical University, Bukit Jalil, Kuala Lumpur, Malaysia.
†Department of Restorative Dentistry, Federal University of Alagoas, Macei o, Alagoas, Brazil.

ABSTRACT
Background: Dentine hypersensitivity (DH) occurs on exposed dentine and is dependent on the patency of dentinal
tubules. This study compared the effectiveness of red propolis extract (RPE), calcium sodium phosphosilicate (Novamin)
and arginine-calcium carbonate (ACC) in occluding dentine tubules.
Methods: Eighty dentine discs from extracted human molars were randomly divided into four groups (n = 20): Group 1
– RPE; Group 2 – Novamin; Group 3 – ACC; Group 4 – saline. The discs were etched with 37.5% phosphoric acid and
treated with the test agents. Ten treated discs from each group were then exposed to 6% citric acid challenge. The extent
of tubule occlusion was assessed using scanning electron microscopy (SEM). Three blinded assessors scored each SEM
image on the degree of tubule occlusion. Differences in occlusion were tested using ANOVA and Tukey adjustment.
Results: Discs treated with ACC demonstrated more tubule occlusion, followed by RPE and Novamin, and were greater
in statistical significance when compared to discs treated with saline. Following acid challenge, RPE treated discs main-
tained more occlusion, followed by ACC and Novamin.
Conclusions: All three agents demonstrated tubule occlusion. Although ACC showed more occlusion following treat-
ment, RPE demonstrated a higher degree of occlusion following acid challenge.
Keywords: Arginine-calcium carbonate, calcium sodium phosphosilicate, dentine hypersensitivity, red propolis extract, scanning
electron microscopy.
Abbreviations and acronyms: ACC = arginine-calcium carbonate; DH = dentine hypersensitivity; PBS = phosphate buffered saline;
SEM = scanning electron microscopy; RP = red propolis; RPE = red propolis extract.
(Accepted for publication 14 May 2014.)

drinks in their diet and avoid cleaning their teeth.

INTRODUCTION
Dentine hypersensitivity (DH) is characterized by
short sharp pain arising from exposed dentine in
response to stimuli typically thermal, evaporative,
tactile, osmotic or chemical and which cannot be
ascribed to any other form of dental defect or pathol-
ogy.1 DH is a common occurrence and is often one
of the main reasons why patients seek dental treat-
ment. The reported prevalence of DH varies from
4% to 57%.2,3 The discomfort of DH experienced
by the patients is highly subjective and can vary sub-
stantially between individuals. For some patients, the
discomfort is perceived as a low grade pain, of slight
concern, but for others the discomfort may present
as a disturbing, severe pain which can affect their
quality of life.4 To avert stimulation of the hypersen-
sitive areas, patients tend to avoid certain food and
© 2015 Australian Dental Association
Inability to maintain adequate plaque control may
lead to increased risk of caries development and peri-
odontal problems. Therefore, effective and long-last-
ing treatment is of great interest to both patients and
dental professionals. Three major mechanisms of den-
tinal sensitivity have been proposed in the literature:
neural theory, odontoblastic transduction theory and
hydrodynamic theory. The most widely accepted
theory is the hydrodynamic theory proposed by
Br€annstr€om, who suggested that exposed dentine
with patent tubules allows the movement of tubule
fluid which leads to dentine sensitivity.5 Scanning
electron microscopy (SEM) has shown that tubules in
clinically characterized ‘sensitive’ exfoliated teeth are
eight times more numerous, two times wider in
diameter and more penetrable, whereas tubules in
‘non-sensitive’ teeth are fewer, smaller and usually
65
CL Chen et al.
blocked.6 This suggests that agents which have the
ability to occlude patent dentinal tubules and/or are
adhesive to dentine surfaces and hence achieve sur-
face coverage and seal most tubules, would be ideal
to relieve DH. There has been a significant advance-
ment in understanding DH, and various agents and
therapies have been developed to combat the prob-
lem.7 However, none of the treatment regimens have
been completely efficient in treating DH. Therefore,
the continued development of new desensitizing
agents is required.8 Recently, calcium sodium phos-
phosilicate (Novamin) has been found to be very
effective for occluding dentinal tubules. When intro-
duced into the oral environment, Novamin releases
sodium, calcium and phosphate ions, which then
interact with saliva and result in the deposition of a
crystalline hydroxycarbonate apatite layer over the
exposed dentine and within the dentinal tubules.9–12
Continued efforts have recently resulted in a novel
technology that uses 8% arginine-calcium carbonate
(ACC) for treating DH, which has shown instant and
lasting relief of DH.13 It is unique in that two of its
key components, arginine and calcium, are found
naturally in saliva, and that the arginine and calcium
carbonate work together to accelerate the natural
mechanisms of occlusion to deposit a dentine-like
mineral, containing calcium and phosphate, within
the dentinal tubules and in a protective layer on the
dentine surface.14 Propolis is a natural resinous sub-
stance collected from plant buds or exudates by Apis
mellifera bees. The composition of propolis varies
according to its botanical origin. Generally it con-
tains a mixture of resin, essential oils, wax mixed
with bee glue (the salivary secretions of bees), amino
acids, minerals, ethanol (alcohol), vitamins A, B com-
plex, E, zinc, pollen and the highly active biochemi-
cal substance known as bioflavonoids. Bioflavonoids
are well known plant compounds which have anti-
bacterial, antifungal, antiviral, antioxidant and anti-
inflammatory proprieties.15,16 Brazilian propolis is
classified into 12 types according to their physio-
chemical characteristics and related to geographic
locations.16 Red propolis (RP) is a newly classified
13th type. The RP variety is obtained in north-east
Brazil. Bees collect reddish exudates from Dalbergia
ecastophyllum (L) Taub (Leguminosae) to make red
propolis.17,18 RP has been reported to have a high
concentration of phenolic acids and flavonoids which
have been associated with a variety of health bene-
fits.19,20 Propolis has been used in the treatment of
DH and found to be effective.21–25 However, no
study has compared the effect of red propolis extract
(RPE) with other desensitizing agents in relieving
DH. The objective of this experimental study was
to evaluate and compare the effectiveness of RPE,
Novamin and ACC in occluding dentinal tubules.
66
MATERIALS AND METHODS
Sample preparation
Eighty dentine discs were prepared from extracted
human permanent molars. The crown was sectioned
with a diamond saw (IsoMet® 1000 Percision Saw;
Buehler, Lake Bluff, IL, USA), perpendicular to the
long axis of the tooth, to create dentine discs with a
thickness of 1.0  0.1 mm from the mid coronal den-
tine. The occlusal enamel was removed from each
tooth, exposing the middle dentine. The disc surface
was free of enamel and pulp horns. The dentine discs
were polished using 600 grit silicon carbide paper
(CarbiMetTM, Buehler, Lake Bluff, IL, USA) to create
an even and uniform surface. The polished samples
were then placed in a jar of distilled water and soni-
cated for 10 minutes to remove the polishing abrasive.
After sonication, the samples were rinsed with saline.
The dentinal tubules were opened by etching the den-
tine discs in a Petri dish with 37.5% phosphoric acid
(Gel Etchant; Kerr Corp., Orange, CA, USA) for
30 seconds. After etching, the samples were rinsed
with distilled water, then placed in a jar of distilled
water and finally sonicated once again for five min-
utes. The etched and sonicated samples were stored in
phosphate buffered saline (PBS, pH = 7) for subse-
quent SEM analysis without coating.
Experimental design
The dentine discs were randomly divided into four
groups, each containing 20 discs: Group 1 – 10% red
propolis extract (Apic Flora, Maceio, Alagoas, Brazil);
Group 2 – paste containing Novamin (NUPRO® Sens-
odyne®, Dentsply Corp., Smile Way York, PA, USA);
Group 3 – paste containing arginine-calcium carbon-
ate (Colgate® Sensitive Pro-ReliefTM, Colgate Oral
Pharmaceuticals Inc., New York, NY, USA); and
Group 4 – saline (negative control).
Treatment of samples
Double-sided tape was used to fasten the samples
onto microscope slides with the polished side facing
up. Group 1 samples were wetted with PBS buffer
and then the test product (RPE) was applied onto the
dentine surface using a rubber cup in slow speed
handpiece (GENTLE power LUX 20LP, KaVo Dental
Corp., Fruehauf Drive, Charlotte NC, USA). At low
speed, the test product was gently applied onto the
surface for one minute. For samples in Groups 2 and
3, test products were applied according to the manu-
facturers’ instructions (in-office treatment). The sam-
ples were left undisturbed for five minutes at room
temperature. Thereafter, the samples were immersed
© 2015 Australian Dental Association
 Effectiveness of desensitizing agents in dentine tubule occlusion
in a jar of PBS for five minutes while stirring. The
samples were then gently rinsed with distilled water
to ensure removal of any excess product from the sur-
faces. This treatment was repeated three times. Ten
specimens were randomly selected from each group to
be coated with a layer of gold/palladium using a mini
sputter coater (Emitech SC7620, Quorum Technolo-
gies Ltd, Lewes, East Sussex, UK) for subsequent
SEM analysis.
Acid challenge
The remaining 10 treated samples from each group
were immersed in 6% citric acid (pH 2) in a Petri dish
for one minute, followed by rinsing in saline for two
minutes. The samples were coated with a layer of
gold/palladium for subsequent SEM analysis.
Evaluation of occlusion
The extent of dentinal tubule occlusion was assessed
using SEM (TM3000 Tabletop Microscope, Hitachi,
Tokyo, Japan). Dentine samples were coated to aid
conductivity. The samples were examined at an oper-
ating voltage of 5 kV in Shadow 2 image mode. The
SEM micrographs were obtained at a magnification
of 2000X. Dentine samples were examined after each
of the following situations: (1) after etching with
phosphoric acid – to ensure that the dentinal tubules
are in an open unoccluded state; (2) after application
of test agent – to determine the effect of the test
agents on dentinal tubule occlusion; (3) after acid
challenge – to determine the susceptibility of the
treatment to acid challenge. SEM visual assessment
may present a concern of subjectivity. To minimize
the subjectivity, four representative images of each
treated dentine surface of each specimen were cap-
tured. The images were then assessed independently
by three well trained blinded reviewers to score the
level of tubule occlusion (on a categorical scale of 1–
5), in accordance with the tubule occlusion classifica-
tion scoring system:26,27 (1) occluded (100% of
tubules occluded); (2) mostly occluded (50–<100% of
tubules occluded); (3) partially occluded (25–<50%
of tubules occluded); (4) mostly unoccluded (<25%
of tubules occluded); (5) unoccluded (0%, no tubule
occlusion).
The mean score of tubule occlusion by the three
blinded reviewers was taken and used for analysis.
Figure 1 summarizes the experimental design.
Statistical methods
The primary objective was to evaluate and compare
the effectiveness between the desensitizing agents in
occluding dentinal tubules. Data were entered into
© 2015 Australian Dental Association
SPSS 17.0 (SPSS Inc., Chicago, IL, USA). The mean of
tubule occlusion score was analysed using ANOVA.
All pair-wise treatment comparisons were performed
with a Tukey adjustment for multiplicity; treatment
differences and p-values were presented. The signifi-
cant level was set at p < 0.05.
RESULTS
Treatment with test products from all groups
showed statistically significant differences from each
other. All three test agents presented significantly
superior tubule occlusion and acid resistance when
compared to saline (Tables 1 and 2). Samples that
were treated with ACC demonstrated significantly
more tubule occlusion when compared to RPE
(p = 0.001) and Novamin (p = 0.001) (Table 1).
After treatment with acid challenge, RPE showed
significantly more resistance to acid challenge when
compared to ACC (p = 0.001) and Novamin
(p = 0.001) (Table 2). Post-treatment with acid chal-
lenge resulted in more openings of dentinal tubules
of the treated samples from all groups (Table 3).
However, only RPE showed no statistically signifi-
cant difference between the tubule occlusion before
and after acid challenge (p = 0.664). In contrast, sig-
nificant differences were observed within the group
of Novamin (p = 0.001) and ACC (p = 0.001). The
SEM micrographs of etched dentine surface by phos-
phoric acid demonstrated fully patent dentinal
tubules (Fig 2a). Examination of dentine surface
treated with RPE showed dentine surface coverage
and peritubules particle depositions by the product
(Fig 2b, Group 1). After treatment with Novamin,
some tubules remained completely occluded whilst
some remained open (Fig 2b, Group 2). All the
tubules of the samples treated with ACC were
occluded by crystal-like depositions (Fig 2b,
Group 3). After acid challenge, tubules observed in
the samples treated with RPE remained mostly
occluded (Fig 2c, Group 1). In contrast, more open-
ings of tubules were observed in the samples treated
with Novamin and ACC (Fig 2c, Group 2 and 3).
The tubules of samples treated with saline remained
completely open after treatment as well as after acid
challenge (Fig 2b and c, Group 4).
DISCUSSION
Saliva naturally occludes patent dentinal tubules by
transporting calcium and phosphate ions into the
tubules to induce tubule plugging and by forming a
surface protective layer of salivary glycoprotein
with calcium and phosphate.14 However, this pro-
cess of natural tubule occlusion is very slow and
the tubule plugging is easily removed by dietary
67
CL Chen et al.
Fig. 1 Summary of experimental design to prepare dentine specimens for different treatments and SEM observations.
acid and physical insult, thus rendering it neither
effective nor reliable in providing lasting relief of
DH. Therefore, successful treatment regimens which
can provide rapid, long-lasting tubule occlusion and
resistance to the challenges in the oral environment
are needed. Results from the present study show
that RPE was significantly effective in dentinal
tubule occlusion in comparison to saline. This is in
agreement with in vitro studies by Almas et al.21
and Sales-Peres et al.22 Propolis has also recently
been proven to be clinically effective in reducing
DH.24,25 This may be attributed to the interaction
between the high content of flavonoids in RPE and
the dentine, forming crystals that are able to adhere
to the dentine surface and occlude the dentinal
tubules.22 Other possible factors could be the pres-
ence of natural resinous substances in propolis that
show a bonding mechanism similar to dental adhe-
sive materials such as composite resin or varnish.
Resin adhesives are capable of bonding to the tooth
surface by diffusion and mechanical interlocking in
68
microporosities, and therefore may have the poten-
tial to provide surface coverage, occlude dentinal
tubules and prevent fluid movement.28 Moreover,
propolis may provide a durable occlusion because of
its diffusion deep inside dentinal tubules that may
be difficult to remove and therefore prolong pain
relief.29 However, the mechanism of action of RPE
is still unclear. Propolis has been shown to stimulate
the production of transforming growth factor
(TGF)-b1, a growth factor known to be important
for odontoblast-like cell differentiation.30 Studies
have reported that when direct pulp capping was
performed with propolis-derived flavonoids, partial
dentine bridge formation was detected beneath the
pulp-capping material after four weeks.31,32 These
studies have shown that flavonoids stimulate repara-
tive dentinogenesis which is believed to contribute
to the process of dentinal tubule occlusion.
Novamin in this study has been shown to have a
significant effect in dentinal tubule occlusion com-
pared to saline. This is consistent with previous in
© 2015 Australian Dental Association
 Effectiveness of desensitizing agents in dentine tubule occlusion

Table 1. Mean difference of tubule occlusion score Table 3. Comparison of mean of occlusion within
between groups – after treatment each group – before and after acid challenge
Comparison between Mean difference p-value Group After After treatment Mean p-value
groups (i–ii) treatment followed by difference
acid challenge
i ii
RPE 2.88 2.94 –0.058 0.664
RPE Novamin –0.350 0.006* Novamin 3.23 4.23 –0.992 0.000*
ACC 0.433 0.000* ACC 2.45 3.73 –1.275 0.000*
saline –2.117 0.000* Saline 5.00 4.98 0.025 0.083
Novamin ACC 0.783 0.000*
saline –1.767 0.000* The mean difference is significant at the 0.05 level. *Significant dif-
ACC saline –2.550 0.000* ference. **Very significant difference.
Mean difference is the tubule occlusion score of after treatment
Data represent mean difference and p-value from ANOVA test and minus the score of after treatment followed by acid challenge. Neg-
Tukey adjustment. ative difference favours the group in column (i) whilst positive dif-
The mean difference is significant at the 0.05 level. *Significant dif- ference favours the group in column (ii).
ference. **Very significant difference. Occlusion Classification Scoring (based on three blinded examiners
Mean difference is the group in column (i) minus the group in col- average): 1 = occluded, 2 = mostly occluded, 3 = partially occluded,
umn (ii). Negative difference favours the group in column (i) whilst 4 = mostly unoccluded, 5 = unoccluded.
positive difference favours the group in column (ii). No significant difference (p > 0.05) indicates that there was not
Occlusion Classification Scoring (based on three blinded examiners much change in tubule occlusion by the treatment before and after
average): 1 = occluded, 2 = mostly occluded, 3 = partially occluded, acid challenge.
4 = mostly unoccluded, 5 = unoccluded.

(Ca-P) layer onto dentine surfaces or into tubules,

Table 2. Mean difference of tubule occlusion score resulting in physical tubule occlusion.36,37 Significant
between groups – after treatment followed by acid dentinal tubule occlusions were observed in samples
challenge treated with ACC compared to those treated with
Comparison between Mean difference p-value Novamin and saline. This result is in agreement
groups (i–ii) with in vitro studies by Lavender et al.38 and Li
et al.39 Clinical trials by Nathoo et al.40 and Kap-
i ii
ferer et al.41 have also proven ACC to be effective
RPE Novamin –1.283 0.000* in relieving DH. The mechanism of action of ACC
ACC –0.783 0.000*
saline –2.033 0.000*
was explained by Kleinberg,42 who suggested that
Novamin ACC 0.500 0.000* the combination of arginine and calcium carbonate
saline –0.750 0.000* forms a positively charged complex which readily
ACC saline –1.250 0.000*
binds to the negatively charged dentine surface and
Data represent mean difference and p-value from ANOVA test and within the dentinal tubules. In addition, the alkaline
Tukey adjustment. pH of the ACC is sufficient to facilitate the deposi-
The mean difference is significant at the 0.05 level. *Significant dif-
ference. **Very significant difference. tion of calcium and phosphate from saliva and/or
Mean difference is the group in column (i) minus the group in col- dentinal fluid to form plugs that seal the patent
umn (ii). Negative difference favours the group in column (i) whilst tubules.42 The present study showed that ACC dem-
positive difference favours the group in column (ii).
Occlusion Classification Scoring (based on three blinded examiners onstrated more tubule occlusion compared to
average): 1 = occluded, 2 = mostly occluded, 3 = partially occluded, Novamin. This is inconsistent with the results
4 = mostly unoccluded, 5 = unoccluded. reported by West et al. in an in situ study which
showed 5% Novamin-based toothpaste was signifi-
vitro studies by Wang et al.33,34 Favourable results cantly more superior than 8% arginine-based tooth-
of the efficacy of Novamin in relieving DH have paste.26 In the West study, an electrical toothbrush
also been demonstrated by in vivo studies by Pra- was used for the application of desensitizing agents
deep et al.35 and Rajesh et al.10 The active ingredi- which could create more smear plugs in the dentine
ent of Novamin is a bioactive glass that was by the action of the bristles.43 A successful desensi-
originally developed as a bone regenerative material tizing agent should be able to withstand frequent
which appeared to offer suitable materials for sur- challenges by acidic drinks and food, and still dem-
face reactivity. In an aqueous environment, sodium onstrate superior occluding properties. Citric acid is
ions (Na+) from Novamin begin to exchange with one of the major organic hydroxyl acids found in
hydrogen cations (H+ or H3O+), which allows cal- fruits, fruit juices and soft drinks. Many citric juices
cium ions (Ca2+) and phosphate ions (PO43-) to be are over pH 2.7, but those below are still frequently
released from the filler particle structure. Ca2+ and consumed, e.g. blackcurrant, lemon, grapefruit and
PO43- ions from Novamin, along with mineral ions lime juice.44 Therefore, 6% citric acid at pH 2 was
in saliva are able to form a calcium phosphate chosen as a post-treatment in this study to simulate
© 2015 Australian Dental Association 69
CL Chen et al.
(a)
(b)
(c)
Fig. 2 Representative SEM of dentine surface showing the extent of tubule occlusion (a) after etching with phosphoric acid; (b) after treatment; (c) after
treatment followed by acid challenge. Group 1 – RPE, Group 2 – Novamin, Group 3 – ACC, Group 4 – saline.
the resistance of the three test products to acid
challenge in the oral environment. The results show
that all test products offered statistically significant
resistance to the acid challenge when compared to
saline. Among the test products, RPE was the most
resistant to acid challenge, followed by ACC and
then Novamin. This provides evidence of the poten-
tial of RPE to be used as a sustainable tubule
occluding agent to relieve DH. Various methods
have been used in different in vitro studies to test
the mode of actions and properties of desensitizing
agents including hydrostatic fluid filtration sys-
tems;1,3,5 attenuated total reflection Fourier trans-
form infrared spectroscopy;3 energy dispersive X-ray
analysis;3 confocal laser scanning microscopy21 and
electron spectroscopy for chemical analysis.21 This
present study only focused on using SEM for the
visual assessment of the level of tubule occlusion by
the test products. SEM investigation was selected
because it is a non-destructive approach for surface
analysis. It also provides high-resolution, three-
dimensional images and topographical information.
SEM has been used in many previous investigations
of the effect of desensitizing toothpaste on dentine
tubule occlusion.22,23,26,33,34,45 Other aspects that
were not within the scope of this study include
70
identification of the composition of the products
formed on the dentine discs, analysis of the extent
of the product’s penetration deep into the dentinal
tubules and evaluation of the dentine permeability
(fluid flow). As the present study conducted was an
in vitro experiment, only limited aspects of the nat-
ural oral environment could be replicated. These
include saliva, acquired pellicles, movement of oral
musculature and brushing habit, which can affect
the efficacy of the treatment. Therefore, further in
vitro as well as in vivo experiments comparing
these test products using the parameters mentioned
above should be the future scope of the present
study.
CONCLUSIONS
All three test agents demonstrated dentinal tubule
occlusion. Although ACC showed more occlusion
following treatment, RPE demonstrated a higher
degree of occlusion following acid challenge.
ACKNOWLEDGEMENTS
This study was sponsored by the International Medi-
cal University, Kuala Lumpur, Malaysia. The authors
© 2015 Australian Dental Association
 Effectiveness of desensitizing agents in dentine tubule occlusion
would like to thank Dr Muneer Gohar Babar, Nor-
bazlin Md Marham and Dr Lee Han Hing for their
contribution to the study.
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