Article_6_1_3
ADHESIVE DENTISTRY
INFLUENCE OF FLUORIDE MOUTHWASH CONTAINING NANOHYDROXYAPATITE ON
Original Article
THE DENTIN INTERFACE OF DIRECT RESTORATIONS: A RANDOMIZED CONTROLLED
CROSSOVER IN SITU STUDY
Dayane Carvalho Ramos Salles de Oliveira1a* , Livia Rodrigues de Menezes2a , Lúcia Trazzi Prieto1a ,
Erick Kamiya Coppini1b , Luís Alexandre Maffei Sartini Paulillo1c , Gisele Damiana da Silveira Pereira2c
1Department of Restorative Dentistry, Piracicaba Dental School, State University of Campinas, Piracicaba, SP, Brazil
2
Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil
a
PhD, Assistant Professor
b
PhD
c
PhD, Associate Professor
ABSTRACT DOI: https://doi.org/10.25241/stomaeduj.2019.6(1).art.3
Introduction: To evaluate the influence of a fluoride mouthwash containing OPEN ACCESS This is an Open
Access article under the CC BY-NC
nanohydroxyapatite on the dentin bonding interface microhardness of direct resin 4.0 license.
composite restorations in situ.
Methodology: Forty human premolars were sectioned and restored with a three-step Peer-Reviewed Article
adhesive system or a self-etch adhesive system and a conventional resin-based composite. Citation: de Oliveira DCRS, de Menezes LR,
After the initial microhardness measurements on the samples’ dentin interface, the Prieto LT, Coppini EK, Sartini Paulillo LAM,
da Silveira Pereira GD. Influence of fluoride
restored samples were fixed in acrylic palate appliances for an in situ experiment. Ten mouthwash containing nanohydroxyapatite
on the dentin interface of direct restorations:
volunteers participated in a randomized double-blinded crossover study using the palate a randomized controlled crossover in situ
appliances and a placebo fluoride mouthwash for one week, one-week wash-out, and study. Stoma Edu J. 2019;6(1):24-28
one-week fluoride mouthwash containing nanohydroxyapatite. The final microhardness Received: February 09, 2019
measurements were evaluated, and data were analyzed by ANOVA and Tukey’s test Revised: February 28, 2019
Accepted: March 18, 2019
submitted for multiple comparisons (α = 0.05; 0.8 power). Published: March 19, 2019
Results: There were statistically significant differences between the mouthwash
*Corresponding author:
treatments with both adhesive systems tested (p < 0.001). The nanohydroxyapatite used Dayane C.R.S. de Oliveira
in the mouthwash was an influencing factor on the dentin interface microhardness of Department of Restorative Dentistry,
Piracicaba Dental School
resin composite restorations. State University of Campinas, 901, Limeira
Conclusion: The fluoride mouthwash containing the nanohydroxyapatite was able to Ave.SP-13414-903 Piracicaba, Brazil
Tel / Fax: +55 (019) 2106-5341
increase the dentin microhardness of the direct resin composite restorations regardless e-mail: dayoli87@gmail.com
of the adhesive system used. Copyright: © 2019 the Editorial Council
Keywords: Dental materials; composite resins; dental adhesive; mouthwashes. for the Stomatology Edu Journal.
1. Introduction enables the infiltration of various harmful agents, as
Dental enamel etching increases the surface energy, well as oral fluids, to infiltrate these gaps. Therefore,
which facilitates the penetration of the adhesive ions, such as calcium and fluoride, present in saliva or
system and the adhesion to this substrate [1]. Adhesion solutions used as mouthwashes [4] can also penetrate
to the enamel substrate is more efficient than to the and fill the micro porosity present in the hybrid layer,
dentin substrate since it is composed of an inorganic thus decreasing its permeability [8-10]. It becomes
matrix, while dentin presents a more significant evident that evaluating whether or not the use of
amount of organic matrix composed mainly of mineralizing agents allows adducing within the hybrid
collagen and water [2]. Moisture control, maintenance layer of ions that can fill the nanopores present in the
of the permeable collagen to the adhesive system dentin interface is essential. Thus, this study assessed
penetration, and demineralization of acid etching are the influence of a fluoride mouthwash containing
essential factors to improve the transition zone, called nanohydroxyapatite on the dentin bonding interface
the hybrid layer [3], which is the primary mechanism microhardness of direct resin composite restorations.
of adhesion to the dentin substrate [4]. Several The null hypothesis was that the use of the fluoride
factors can influence the hybrid layer development. mouthwash containing nanohydroxyapatite will not
When moisture control is performed incorrectly, the influence the dentin interface microhardness of direct
penetration of the monomers into the demineralized resin composite resin restorations.
dentin is incomplete, resulting in failure areas. This
increases the risk of nanoleakage through the pores
present in the adhesive interface of this defective 2. Methodology
hybrid layer. It can cause postoperative sensitivity and 2.1. Ethical aspects
reduce the longevity of the direct restorations [5,6]. This in situ, double-blinded, single center, randomized
The presence of failure areas on the adhesive interface controlled crossover study was authorized by the
24 Stoma Edu J. 2019;6(1): 24-28. http://www.stomaeduj.com
INFLUENCE OF FLUORIDE MOUTHWASH CONTAINING NANOHYDROXYAPATITE ON THE DENTIN INTERFACE OF
DIRECT RESTORATIONS: A RANDOMIZED CONTROLLED CROSSOVER IN SITU STUDY
Original Article
Figure 1. Flow diagram with a systematic illustration of the crossover in situ model and experimental design.
local Institutional Ethics Committee of the Piracicaba UNICAMP, Piracicaba, SP, Brazil. For the three-step
Dental School, Piracicaba, SP, Brazil (protocol adhesive system, the excess water was removed by
#149/2012). A written informed consent was obtained capillary action with an absorbent paper. The dentin
from all volunteers in the study. Volunteers had the etching was performed with 35% phosphoric acid for
right to withdraw from the study at any time and for 15 s, followed by rinsing with abundant water for 30 s.
any reason without prejudice. The moisture was controlled with a moist cotton ball.
Both adhesive systems were used according to the
2.2. Preparation of the specimens manufacturers’ instructions with a microbrush and
Forty human premolars recently extracted because of light cured for 20 s with a LED light source (Radii, SDI,
orthodontic reasons were donated to this study. The Victoria, Australia). The restoration was carried out
teeth were stored for 24 h in a 0.1% thymol solution at with a single 2 mm increment of the resin composite
37°C, then stored in distilled water until the beginning in a 4 x 4 mm acetate pattern to standardize the direct
of the study. The coronal portion was separated from restorations, and then light-cured for 20 s. All samples
the root through a straight section on the long axis were stored in 100% relative humidity at 37°C for 24 h.
of the tooth using a metallographic precision cutter
(Isomet 1000, Buehler Ltd., Buff Lake, IL, USA) with a 2.3. In situ study
diamond saw under constant irrigation. To obtain a This in situ, double-blinded, single center,
flat dentin surface, the occlusal surfaces were ground randomized controlled crossover study was carried
with sandpaper silicon carbide (SiC) #320 and #400 in out for three weeks, including a one-week fluoride
a metallographic polisher sander cooled with water. nanohydroxyapatite-free phase, a one-week washout
Then, 4 x 4 mm dentin blocks were obtained with 2 mm period and a one-week fluoride nanohydroxyapatite-
of thickness. After an ultrasound bath to remove the active phase. A total of 10 adult volunteers (mean age
sander debris, the standardization of the smear layer 23.3 ± 2.3 yrs; 5 females and 5 males) were recruited
was performed on the dentin flat by manually rubbing in the Piracicaba Dental School, UNICAMP, Piracicaba,
with SiC wet sandpaper #1000 and #1200 for 15 s per SP, Brazil and in the Dental School of the Federal
sandpaper grit. The dentin blocks were kept in 100% University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
relative humidity at 37°C. This study used the total All volunteers had confirmed satisfactory oral health,
etching technique with a three-step adhesive system did not use any medication, did not have any systemic
(Scotchbond Multi-Purpose Plus, 3M ESPE, St. Paul, disease or pregnancy, and did not use any mouthwash
MN, USA) and a self-etch adhesive system (Clearfill SE or toothpaste except as provided for the research.
Bond, Kuraray, Tokyo, Japan) with a nanofilled resin In this study, all volunteers completed the study,
composite (Filtek Z350 XT, 3M ESPE, St. Paul, MN, USA). no participants were excluded or withdrawn from
All clinical and laboratory steps were performed at the the study. Impressions of all the volunteers’ mouths
Restorative Department of Piracicaba Dental School, were made to obtain a model to make a palate acrylic
Stomatology Edu Journal 25
INFLUENCE OF FLUORIDE MOUTHWASH CONTAINING NANOHYDROXYAPATITE ON THE DENTIN INTERFACE OF
DIRECT RESTORATIONS: A RANDOMIZED CONTROLLED CROSSOVER IN SITU STUDY
Original Article Table 1. Means (standard deviations) values for microhadness test (KHN).
Microhardness
Experimental Group
Initial Final
Clearfill SE (+ testing mouthwash) 42.59 (0.5) Ba 44.33 (0.6) Aa
Clearfill SE (+ placebo mouthwash) 41.29 (0.5) Aa 40.4 (1.0) Ab
ScotchBond MP (+ testing mouthwash) 42.15 (0.4) Bb 45.42 (0.6) Aa
ScotchBond MP (+ placebo mouthwash) 39.66 (0.6) Aab 40.35 (0.5) Ab
*Means followed by different capital letters in the same line and small letters in the same column were significantly different (p < 0.05).
appliance for the upper arch. In each plate two cavity their teeth using the same toothpaste containing no
sites were fabricated where the specimens were fixed. fluoride during all study. The volunteers should wear
All sites were positioned posterior to the incisive the appliance for more than 20 h per day, removing
papillae to avoid contact between the tongue and it only while eating or brushing. The volunteers were
the specimens. Every site had a 4.5 x 4.5 mm uniform instructed to rinse off their appliance and store it in
gap with 4 mm in depth covered by a plastic mesh artificial saliva while not in use.
to allow free contact of the saliva with the specimens,
but protection from mechanical disturbance. New 2.4. Microhardness readings (KHN)
specimens were inserted into the appliance before The Knoop Microhardness of the dentin interface
phase 2. The specimens were randomly assorted and (illustrated in Fig. 1) was examined using a
allocated into two groups: experimental or placebo. A microhardness tester (HMV-2, Shimadzu, Kyoto,
systematic illustration of the crossover in situ model Japan) with a 50 g load for 15 s in three equally spaced
and the experimental design can be observed in points on each sample. A blinded trained operator
Fig. 1. In the first week half of the volunteers were performed all the microhardness measurements
randomly allocated to use the fluoride mouthwash before and after the crossover in situ experiment. The
containing nanohydroxyapatite, while the other half microhardness of each sample was taken from the
to use the placebo fluoride mouthwash containing arithmetic mean of the three readings.
no nanohydroxyapatite. After the second week
“wash-out” to neutralize the fluoride agent and the 2.5. Statistical analyses
microhardness readings, the groups were reversed The sample size was calculated according to a
and conducted as described in the first week. The pilot test to provide a power of 0.8. The power was
volunteers who used the placebo fluoride mouthwash calculated according to power analysis. The data were
in the first week used the tested mouthwash in the subjected to ANOVA and Tukey’s test with a limit of
second week and vice versa. The study products and 5% probability of making decisions.
tested materials were provided as coded packages
labeled with participants number and study period.
None of the volunteers, in situ study instructors or 3. Results
in situ study monitors that were participating in the ANOVA showed a significant difference between
study knew the type of group (placebo or test) being the tested groups (placebo/tested mouthwash x
tested until all procedures were finalized. The in situ time) (p < 0.001). Mean values taken from Tukey’s
study coordinators were responsible for blinding test are shown in Table 1. As could be observed,
all participants and labeling the study products and the nanohydroxyapatite used in the mouthwash
tested materials. The mouthwashes used in the study was an influencing factor on the dentin interface
were prepared by FGM Produtos Odontológicos microhardness of resin composite restorations.
(89219-501 Joinville, SC, Brasil). The placebo fluoride The fluoride mouthwash containing the
mouthwashes were produced with the same nanohydroxyapatite managed to increase the dentin
color, viscosity and taste compared to the tested interface microhardness of direct resin composite
mouthwash. The placebo mouthwash and the tested restorations regardless of the adhesive system used,
one contained the same components, including the while the placebo fluoride mouthwash solution
fluoride and the potassium nitrate, but only the tested containing no nanohydroxyapatite had no influence
mouthwash contained the nanohydroxyapatite, on dentin interface microhardness.
as in the Desensibilize Nano P (FGM Produtos
Odontológicos, 89219-501 Joinville, SC, Brasil). The
volunteers were instructed to swish 20 mL of the 4. Discussion
mouthwash around their mouth for thirty seconds The present study clinically investigated the
then spit it out with no rinsing. The volunteers were influence of a fluoride mouthwash containing
also instructed to use it twice a day, every day, after nanohydroxyapatite on the dentin bonding interface
brushing and flossing their teeth in the morning microhardness of direct resin composite restorations
and at night. All volunteers were instructed to brush in situ. There are several in vitro studies showing
26 Stoma Edu J. 2019;6(1): 24-28. http://www.stomaeduj.com
INFLUENCE OF FLUORIDE MOUTHWASH CONTAINING NANOHYDROXYAPATITE ON THE DENTIN INTERFACE OF
DIRECT RESTORATIONS: A RANDOMIZED CONTROLLED CROSSOVER IN SITU STUDY
possible effects of mouthwashes in composite should be performed to further evaluate the clinical
Original Article
restorations overtime [11-14]. However, there is no performance of these different treatments using
consensus about this topic, and the reason for this mouthwashes and their real effects in the long term.
is that different mouthwashes contain different By all means, the addition of nanohydroxyapatite
components. The crossover in situ model allowed to seems to increase the efficiency of fluoride with
access the real effects in the oral cavity. Moreover, the regard to mineralization. The remineralizing formula
use of the same fluoride mouthwash composition containing nanostructured calcium phosphate,
varying only the addition or not of nanohydroxyapatite, organized in the crystalline form of hydroxyapatite
allowed to access the real influence of the different allows the releasing of calcium and phosphate ions
mineralizing agents on the dentin bond interface to the demineralized surface of the tooth, which
of composite restorations. The null hypothesis that can be reorganized in the form of hydroxyapatite,
the use of the fluoride mouthwash containing fluorapatite or calcium fluoride, as well as to occlude
nanohydroxyapatite would not influence the dentin the dentinal tubules. Moreover, the hydroxyapatite
interface microhardness of direct resin composite crystals are highly stable and more resistant to acidic
resin restorations was rejected. This occurred since challenges when compared to amorphous calcium
the final microhardness values were statistically phosphates, explaining the increased efficiency of
different between the fluoride mouthwash containing the tested formula containing nanohydroxyapatite
nanohydroxyapatite and the placebo fluoride in comparison to the fluoride placebo formula. Thus,
mouthwash containing no nanohydroxyapatite, with the limitations of this crossover in situ study, it
regardless of the adhesive system tested. The was possible to conclude that the fluoride mouthwash
mineralizing agents had been tested to reduce the containing nanohydroxyapatite managed to increase
failure areas of the adhesive interface, hindering the the dentin microhardness of direct resin composite
infiltration of harmful agents. Calcium and fluoride restorations regardless of the adhesive system used
have been described in the literature as being able within one week. On the other hand, the conventional
to fill the microporosity present in the hybrid layer, fluoride mouthwash solution had no influence on
decreasing its permeability [7-10]. As observed dentin microhardness within one week.
in this study, the fluoride mouthwash containing
nanohydroxyapatite managed to influence the
dentin microhardness showing a possible ability to Authors contribution
fill the microporosity present in the dentin interface. DO/LM: Experimental design, crossover in situ
However, due to the similar mean values of the initial study coordinator, statistical analysis, manuscript
and final microhardness, it is presumed that despite writing, manuscript proofreading. LTP: Experimental
the capability of the fluoride to penetrate the hybrid design, blinded in situ study clinical instructor,
layer, it could have been lixiviated from the hybrid manuscript proofreading. EK: Experimental design,
layer during the in situ study, not contributing to filling blinded technical microhardness tester, manuscript
the microporosities present in the dentin interface. proofreading. GP/LMP: Experimental design, in situ
The microporosities in the dentin interface occurs study clinical monitor, manuscript proofreading.
due to the incorrect or incomplete penetration of the
adhesive monomers into the demineralized dentin,
resulting in failure areas within the hybrid layer. These Acknowledgments
failures within the hybrid layer increase the risk of This study thanks FGM for preparing and donating the
micro or nanoleakage through the pores present in mouthwashes used in this study. DO is a Post-Doctoral
the adhesive interface, reducing the longevity of the Researcher at the Sao Paulo Research Foundation
restoration [5-6]. However, the same way oral fluids (FAPESP grant #2016/05823-3 and #2017/22161-7).
can infiltrate through these failures, other substances The authors declare no potential conflict of interest
such as calcium and fluoride, present in the saliva concerning the authorship or the publication of this
or solutions used as mouthwashes [4] could also article.
penetrate and fill the microporosity present in the
hybrid layer, thus decreasing its permeability [8-10],
and possibly increasing the longevity of composite References
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INFLUENCE OF FLUORIDE MOUTHWASH CONTAINING NANOHYDROXYAPATITE ON THE DENTIN INTERFACE OF
DIRECT RESTORATIONS: A RANDOMIZED CONTROLLED CROSSOVER IN SITU STUDY
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Dayane C. R. S. de OLIVEIRA
DDS, MS, PhD, Assistant Professor
Department of Restorative Dentistry Piracicaba Dental School
State University of Campinas
Piracicaba, SP, Brazil
CV
Dayane C. R. S. de Oliveira, DDS, MS, PhD, is a post-doc fellow of the Department of Restorative Dentistry at Piracicaba Dental
School, State University of Campinas (UNICAMP), in Brazil. Dr. Oliveira is a young researcher that contributed to 6 textbook
chapters, authored 7 patents and is the recipient of many awards in her area of expertise. Her areas of interest include esthetic
dentistry, color science and biomaterials development and characterization.
Questions
1. Based on the results in this study, fluoride mouthwashes:
qa. Are capable of increasing microhardness of dentin interface of composite restorations;
qb. Are not capable of increasing microhardness of dentin interface of composite restorations;
qc. Are not necessarily capable of increasing microhardness of dentin interface of composite restorations
depending on composition;
qd. None of the above.
2. Based on the results in this study, fluoride mouthwashes containing
nanohydroxyapatite:
qa. Are capable of increasing microhardness of dentin interface of composite restorations;
qb. Are not capable of increasing microhardness of dentin interface of composite restorations;
qc. Have similar performance compared to conventional fluoride mouthwashes;
qd. None of the above.
3. Based on the discussion section, which statement is true:
qa. Fluoride mouthwashes not containing nanohydroxyapatite might be capable of infiltrating in the hybrid
layer and forming amorphous calcium phosphates; but it is probably dissolved and lixiviated over time;
qb. Fluoride mouthwashes not containing nanohydroxyapatite do not contribute to filling the microporosities
present in the dentin interface over time;
qc. Fluoride mouthwashes containing nanohydroxyapatite are capable of infiltrating in the hybrid layer, and
forming highly stable hydroxyapatite crystals;
qd. All of the above.
4. Based on the discussion section, which statement is not true:
qa. The primary limitation of this study was follow-up time which does not allow the authors to extrapolate the
results in regard the longevity of the composite restorations;
qb. Literature demonstrate the immediate results of fluoride agents, however, this study demonstrated that
these results can be modified over time;
qc. This study demonstrated that increasing the longevity of the composite restorations might be possible
using fluoride agents associated with nanohydroxyapatite, however further studies are necessary;
qd. None of the above.
28 Stoma Edu J. 2019;6(1): 24-28. http://www.stomaeduj.com