17088240, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jerd.12979 by Cochrane Colombia, Wiley Online Library on [07/03/2023].

See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Received: 24 September 2022 Revised: 16 October 2022 Accepted: 18 October 2022
DOI: 10.1111/jerd.12979

RESEARCH ARTICLE

Clinical in-situ evaluation of the effect of rubber dam isolation

on bond strength to enamel

Rui I. Falacho DMD, PhD 1,2 | Eliana Azevedo Melo DMD 3 |

2,4 2,5
Joana A. Marques DMD | João Carlos Ramos DMD, PhD |
1,2
Fernando Guerra DMD, PhD | Markus B. Blatz DMD, PhD 6

1
Institute of Oral Implantology and
Prosthodontics, Faculty of Medicine, Abstract
University of Coimbra, Coimbra, Portugal
Objective: To evaluate the effect of rubber dam isolation on shear bond strength of
2
Center for Innovation and Research in Oral
Sciences (CIROS), Faculty of Medicine, two different adhesive systems to enamel.
University of Coimbra, Coimbra, Portugal Materials and Methods: The mesial, distal, lingual, and vestibular enamel surfaces of
3
Dentistry Department, Faculty of Medicine,
thirty human third molars were prepared (total n = 120). A custom splint was made
University of Coimbra, Coimbra, Portugal
4 to fit a volunteer's maxilla, holding the specimens in place in the oral cavity. Four
Institute of Endodontics, Faculty of Medicine,
University of Coimbra, Coimbra, Portugal composite resin cylinders were bonded to each tooth with one of two bonding
5
Institute of Operative Dentistry, Faculty of agents (OptiBond FL and Prime&Bond active) with or without rubber dam isolation.
Medicine, University of Coimbra, Coimbra,
Portugal Shear bond strength was tested in a universal testing machine and failure modes
6
Department of Preventive and Restorative were assessed. Significance level for statistical analyses was set at 5%.
Sciences, University of Pennsylvania School of
Results: All pairwise comparisons revealed statistical differences (p < 0.05). The high-
Dental Medicine, Philadelphia,
Pennsylvania, USA est mean shear bond strength values were obtained in rubber dam experimental
groups, regardless of the adhesive system. Group OptiBond FL with rubber dam pre-
Correspondence
Rui I. Falacho, Av. Bissaya Barreto, Bloco de sented the highest mean bond strength values. Fracture modes for specimens
Celas, Faculty of Medicine, University of
bonded without rubber dam isolation were adhesive and cohesive within enamel,
Coimbra, 3000-075 Coimbra, Portugal.
Email: rifalacho@fmed.uc.pt while rubber dam experimental groups revealed only cohesive fractures.
Conclusions: Absolute isolation with rubber dam increases bond strength to enamel,
independent of the adhesive system. The three-step total-etch system OptiBond FL
provided significantly higher bond strength values than Prime&Bond active under
both experimental conditions.
Clinical Significance: Rubber dam isolation has a significant effect on bond strengths
to enamel, independent of the adhesive system. Its application is, therefore, advised
whenever adhesive procedures are performed. A filled three-step etch-and-rinse
adhesive performed superiorly, with or without rubber dam isolation, when bonding
to enamel compared to an isopropanol-based universal adhesive.

KEYWORDS
absolute isolation, adhesion, adhesive system, enamel, humidity, rubber dam, shear bond
strength

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2022 The Authors. Journal of Esthetic and Restorative Dentistry published by Wiley Periodicals LLC.

48 wileyonlinelibrary.com/journal/jerd J Esthet Restor Dent. 2023;35:48–55.


FALACHO ET AL.
1 | I N T RO DU CT I O N
Today, the great majority of restorative procedures requires adhesive
techniques.1 The clinical success of these restorations is directly
related to the bond strength and its durability to prevent marginal gap
formation, bacterial leakage, postoperative sensitivity, recurrent
1,2
caries, and loss of the restoration.
In addition to adequate material selection, proper adhesive proto-
cols and techniques must be followed meticulously to enhance clinical
outcomes and ensure long-term success. Quality of the resin-
adhesive-tooth interface is not only influenced by the chemical
composition of the bonding agents and the applied polymerization
protocols but also by the environment to which they are exposed,
such as temperature and humidity.3–7
Proper moisture control is one of the most challenging steps in
adhesive dentistry. Previous studies have shown that keeping a dry
enamel surface by eliminating remnant moisture prior to adhesive
application is crucial for long-term bond durability.8,9 Bonding sur-
faces are exposed to saliva, blood, and crevicular fluid but also to
water molecules present in exhaled air.9,10
The amount of water saturated in exhaled air is often ignored.
However, it is reported to be about 27 mg/dm3 and its possible
detrimental effects on the bonding interface require thorough
evaluation.11 The average oral temperature and relative humidity are
around 30 C and 80% respectively, with humidity ranging from 74%
to 94%.1,12 Factors that may influence relative humidity include tooth
position within the dental arches, the patient's nose or mouth breath-
ing, and use of protective devices such as rubber dam.12
The rubber dam isolation technique was first proposed in 1864.13
It offers many advantages, such as reduced humidity, lower contami-
nation of the operating field by saliva, blood or crevicular fluid,
decreased risk of cross-infection, and enhanced safety by preventing
injuries to the surrounding soft tissues as well as leakage or aspiration
of dental materials.5,10,14 In spite of the apparent benefits, the major-
ity of dentists never or only rarely use rubber dam isolation during
operative dentistry procedures. Whether absolute isolation with rub-
ber dam or relative isolation, for example with cotton rolls and high-
speed suction, have any effect on clinical outcomes is a common but
15–17
largely unanswered question among clinicians.
Adhesive systems can be divided into etch-and-rinse and
self-etch adhesives.18–20 The effectiveness of etch-and-rinse adhesive
systems on enamel surfaces is well supported by the literature.9
However, to simplify the clinical procedures, universal adhesives have
been introduced. These all-in-one adhesives combine etching, priming,
and bonding into a single bottle and application step, allowing the
operator to select either an etch-and-rinse, self-etch or selective-
enamel-etch adhesive strategy. Such universal adhesive systems are
widely accepted but may not offer the same bond strength values and
durability as their predecessors.20–23
There are only few scientific studies evaluating the effects of oral
humidity on bond strength, and the ones that exist fail to provide valid
experimental models.24,25 In vitro studies can provide valuable infor-
mation about physical and biomechanical properties of materials in a
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49
controlled and calibrated laboratory environment, purposely eliminat-
ing some hard-to-control influencing parameters. On the other hand,
interoperative variations, individual patient factors, and the intraoral
environment may well influence clinical outcomes and contribute to
the differences in bond strength values found between in vivo and
in vitro studies.26,27 Most often, temperature and relative humidity
are simulated in experimental chambers. However, these devices are
not capable of fully replicating oral environment conditions. Testing
adhesive restorations performed in a patient's oral cavity provides
additional and more accurate information on the actual effects of rela-
tive humidity.26
This clinical study evaluated the effect of rubber dam isolation on
shear bond strength of two different adhesive systems, OptiBond FL
(Kerr Corporation, California, USA) and Prime&Bond active (Dentsply
Sirona, Konstanz, Germany), to enamel.
The research hypotheses were:
1. Rubber dam isolation improves shear bond strength to enamel,
independent of the adhesive system used.
2. Highly filled three-step etch-and-rinse adhesive provides higher
bond strength values than an isopropanol-based universal
adhesive.
2 | M A T E R I A L S A N D M ET H O D S
2.1 | Specimen preparation
According to the notification CE-001/2013, the specimen collection
and clinical part of this study were approved by the Ethics Committee
of the Faculty of Medicine of the University of Coimbra. Thirty sound
human third molars, clinically and radiographically free of caries,
cracks, restorations, root canal treatment or other abnormal features
were collected, immediately cleaned with periodontal scalers, and
polished with pumice and water to remove adherent organic material
or calculus. The teeth were stored in distilled water for a maximum of
3 months, followed by immersion in chloramine for 5 weeks at 4 C.
Afterwards, they were placed in a cylindrical mold and embedded in
auto-polymerizing acrylic resin (Schmidt Dental Solutions, Madrid,
Spain; batch number 47975) up to the cementoenamel junction. Each
tooth had the mesial, distal, lingual, and vestibular enamel surfaces
carefully flattened with a conical diamond bur (105–120 μm grit)
under water cooling, attached to a parallelometer. All preparations
were kept in sound enamel. The surfaces were finished with coarse
contouring and polishing discs (Sof-Lex, 3 M, California, USA). Bond-
ing procedures were performed immediately after preparation of the
enamel surfaces.
For the clinical part of the study, a 26-year-old female volunteer
with no history of previous carious lesions and a plaque index lower
than 10% was recruited. All procedures were conveniently explained
without disclosing details regarding the study aims and explicit written
consent was signed by the volunteer patient who agreed and
approved the terms.

50
2.2 | Oral device design
A custom splint was fabricated to fit a volunteer's maxillary arch. The
custom splint was digitally designed (Zirkonzahn Software-Module
CAD/CAM Bite Splints version 9071, Zirkonzahn, Gais, Italy) using an
intraoral scan (Primescan, Dentsply Sirona, Bensheim, Germany) and
3D-printed in NexDent Model 2.0 white resin (Vertex-Dental B.V.,
Soesterberg, Netherlands) with a NextDent 5100 printer
(3D SYSTEMS, Vertex-Dental B.V.). The oral splint featured a palatal
cylindrical slot that fit the acrylic bases of the extracted teeth, expos-
ing only the teeth's crowns while giving full access to all four tooth
surfaces (mesial, distal, vestibular, lingual).
2.3 | Experimental groups
The experimental groups were:
• RD-OFL: OptiBond FL adhesive applied under rubber dam
isolation
• nRD-OFL: OptiBond FL adhesive applied without rubber dam
• RD-PB: Prime&Bond active adhesive applied under rubber dam
isolation
• nRD-PB: Prime&Bond active adhesive applied without rubber dam
Each one of the four prepared enamel surfaces was randomly
assigned to one of the four experimental groups with a research
randomizer (https://www.randomizer.org) for a total of 120 speci-
mens. All four experimental groups were tested on the same
tooth to allow direct comparisons in identical enamel conditions.
Specimen preparation and experimental groups are illustrated in
Figure 1.
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FALACHO ET AL.
F I G U R E 1 Schematic
representation of the specimen
preparation and intraoral set up
during bonding procedures
according to the experimental
groups P (palatal or lingual), D
(distal), V (vestibular), M (mesial),
nRD (no rubber dam), RD (rubber
dam), PB (Prime&Bond active),
OFL (OptiBond FL). Created with
BioRender.com.
2.4 | Bonding procedures
The teeth were attached one by one to the slot in the oral device, with
the area to be restored facing the anterior region, thus simulating the
normal positioning and angle of a central incisor being restored on the
vestibular aspect. A thermo-hygroscope was used to record the dental
office's environmental conditions. The room's relative humidity was
kept at 46% and the temperature was kept at 22 C. One experienced
operator performed all experimental procedures under 10x magnifica-
tion (Leica M320, Leica Microsystems, Heerbrugg, Switzerland).
All procedures prior to the adhesive application were performed
outside of the patient's mouth to avoid any cross-contamination
between fluids that contacted the tooth samples and the patient's oral
cavity. In all experimental groups, each prepared enamel surface was
etched for 30 s with 37.5% phosphoric acid (Gel etchant, Kerr Corpo-
ration, California, USA) followed by a thorough rinse with an air-water
stream for 30 additional seconds and air dried with a strong air flow
until completely dry. Each sample was then placed inside the patient's
mouth according to the specificities of the tested groups.
During the bonding procedure of experimental groups performed
under relative isolation (nRB-OFL, nRB-PB), the patient was instructed
to breathe through the nose and a suction cannula was placed to
remove excess moisture. In both groups, the etched enamel surfaces
were air-dried one more time to ensure total absence of water and
left to rest for 30 s without the presence of any oral fluids to better
mimic clinical conditions. One of the two bonding systems, total-etch
(nRB-OFL) or universal (nRB-PB), was actively applied with a micro-
brush for 20 s, after which the surfaces were airdried with a mild air
flow free of any oil or water residues for 10 s and light-cured for 20 s
with a polywave LED curing light source with a measured intensity
of 1200 mW/cm2 (Bluephase Style 20i, Ivoclar Vivadent, Schaan,
Liechtenstein).
 17088240, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jerd.12979 by Cochrane Colombia, Wiley Online Library on [07/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
FALACHO ET AL. 51

For the bonding procedure of the experimental groups per-
formed under absolute isolation (RB-OFL, RB-PB), a rubber dam
sheet (Nic Tone, MDC Dental, Jalisco, Mexico) was punctured with
a single hole, placed around the tooth's crown, and held in place by
a clamp and a frame holder. Once the enamel surfaces were air
dried with a strong air flow until completely dry, one of the two
adhesive systems, total-etch (RB-OFL) or universal (RB-PB), was
actively applied with a microbrush for 20 s, after which the surfaces
were airdried with a mild air flow free of any oil or water residues
for 10 s and light-cured for 20 s.
The dentin primer of the OptiBond FL system was not applied
to any of the surfaces since all tested samples consisted exclu-
sively of enamel. Once the application of the bonding systems was
concluded, a composite resin (Ceram.X.Spectra ST Low Viscosity,
Dentsply Sirona, Konstanz, Germany) was condensed into a soluble
translucent cylindrical capsule with 4.39 mm height and a 2.54 mm
internal diameter, positioned onto the prepared enamel surface,
and polymerized from all sides with the LED light-curing unit for a
total of 80 s. Materials, manufacturers, chemical composition, and
lot numbers are listed in Table 1. After bonding procedure comple-
tion, each specimen was stored in distilled water at 37 C for
7 days.
2.5 | Shear bond strength testing
The testing sequence was randomly defined for both the teeth and
the groups within each tooth. One blind-to-the-groups, calibrated and
experienced operator performed the shear bond strength tests. All
120 specimens were placed in a universal testing machine (model
AG-I, Shimadzu Corporation, Kyoto, Japan) and a shear load was
applied at the bonding interface until failure with a crosshead speed
of 0.5 mm/min. Failure load was recorded in Newtons (N) and
calculated in Megapascals (MPa) by dividing the failure loads by the
bonding area (N/mm2).
2.6 | Failure mode analysis
The fracture surfaces were independently evaluated by two blinded-
to-the-groups examiners with a dental operating microscope (Leica
M320, Leica Microsystems, Heerbrugg, Switzerland) under 40x magni-
fication. The failure modes were classified as:
(0) - adhesive failure. (1) - cohesive failure within enamel. (2) -
cohesive failure within the composite resin. (3) - mixed failure within
enamel. (4) - mixed failure within the composite resin.
2.7 | Statistical analysis
Statistical analysis was performed with IBM SPSS for Windows ver-
sion 26.0 (SPSS, Chicago, Illinois, USA) and MS Excel (Microsoft Cor-
poration, Redmond, Washington, USA). The significance level was set
at 5% (α = 0.05). The shear bond strength results were described
using mean, standard deviation, minimum and maximum values. After
verifying the normality of data distribution with the Shapiro–Wilk test,
repeated measures ANOVA testing was carried out to detect statisti-
cally significant differences between the means across groups. Post-
hoc multiple pairwise comparisons were performed with the Dunn–Š
idák test.

TABLE 1 Materials specifics

Material (abbreviation) Manufacturer Type Composition Lot number

OptiBond FL (OFL) Kerr Corporation, Three-step total-etch Primer: 2-hydroxyethyl methacrylate, ethanol, 7831887
California, USA adhesive 2-[2(methacryloyloxy) ethoxycarbonyl]
benzoic acid, glycerol phosphate
dimethacrylate
Bond: glass, oxide, chemicals, 2-hydroxyethyl
methacrylate, Ytterbium trifluoride,
3-trimethoxysilylpropyl methacrylate,
2-hydroxy-1,3-propanediyl bismethacrylate,
alkali fluorosilicates (Na)
Prime&Bond active (PB) Dentsply Sirona, Konstanz, Universal adhesive Bi- and multi-functional acrylate, phosphoric 2011000070
Germany acid-modified acrylate resin, initiator,
stabilizer, isopropanol, water
Ceram.X.Spectra ST Low Dentsply Sirona, Konstanz, Composite resin Ethoxylated bisphenol A dimethacrylate, 2008000516
Viscosity Germany urethane modified Bis-GMA dimethacrylate
resin, 2,2-ethylenedioxydiethyl
dimetharcylate, ytterbium trifluoride, 2,6-di-
tert-butyl-p-cresol
Gel etchant 37.5% Kerr Corporation, Etching gel Phosphoric acid 35–40%, cobalt alumina blue 7831887
California, USA spinel
Nic tone dental dam MDC Dental, Jalisco, Rubber dam Latex 11068038
(thick) Mexico
 17088240, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jerd.12979 by Cochrane Colombia, Wiley Online Library on [07/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
52 FALACHO ET AL.

To evaluate the differences between failure modes, McNemar
testing was used between all pairs of groups. The p-value was cor-
rected by the Benjamini-Hochberg method (false discovery rate con-
trolling procedure) for multiple comparisons (false positive rate
of 0.05).
A power analysis was performed in G* Power 3.1.9.2 software
and a repeated measures ANOVA test (α = 0.05) was considered. The
power achieved was 100%.
3 | RESULTS
3.1 | Shear bond strength
Statistically significant differences were observed between the experi-
mental groups regarding shear bond strength (p < 0.001). Multiple
pairwise comparisons revealed statistical differences between all
study groups (p < 0.001), except for the comparison between nRD-PB
and nRD-OFL (p = 0.008), as shown in Figure 2. The highest mean
shear bond strength values were obtained in rubber dam experimental
groups, regardless of the adhesive system (Table 2). OptiBond FL
revealed greater mean shear bond strength values under both condi-
tions (with and without rubber dam isolation).
F I G U R E 2 Box-plot of shear bond strength distribution within
the study groups. Groups indicated by different letters present
statistically significant differences (p < 0.05) according to the Dunn–Š
idák post-hoc test.
3.2 | Failure mode analysis
Cohesive failures within enamel exclusively occurred in rubber dam
experimental groups (Table 2). Failures were mostly adhesive in RD-
PB and RD-OFL groups (56.7% and 66.7%, respectively). All speci-
mens in the two no-rubber dam groups failed adhesively. No mixed or
cohesive failures within the composite resin were observed.
Prime&Bond active and OptiBond FL showed no statistically sig-
nificant differences regarding failure modes when tested under the
same experimental conditions with or without rubber dam isolation
(p > 0.05, Table 2). However, a statistically significant predominance
of cohesive failures was observed in rubber dam compared to no-
rubber dam groups, within the same and between both tested adhe-
sive systems (p < 0.05).
4 | DI SCU SSION
Our findings support the hypothesis that rubber dam placement mini-
mizes the detrimental effects of intraoral humidity, ultimately improv-
ing shear bond strength of both tested adhesive systems, a three-step
total-etch (OptiBond FL) and a universal adhesive (Prime&Bond
active), to enamel under clinical conditions. In addition, OptiBond FL
revealed higher enamel bond strength values than Prime&Bond active,
independent of rubber dam isolation during the bonding procedures.
Therefore, both research hypotheses were accepted.
Of the few bonding studies where relative humidity effects are
considered, most opt to test the bond strength to dentin and overlook
the importance of enamel bonding. While adhesion to dentin may
even benefit from a moist environment, enamel bonding requires dry
conditions without any water or moisture to attain peak bond
strength. Failure to ensure optimal conditions for proper bonding to
enamel will lead to poor marginal sealing and, ultimately, restorative
failure.8 Therefore, bond strength studies to enamel under clinically
relevant conditions are critically important.
Statistically significant differences in bond strength values were
detected among all groups, with higher mean shear bond strength
values in experimental groups with rubber dam isolation. When prop-
erly placed, rubber dam serves as a shield to relative humidity in the
oral cavity, which, as shown, has a negative effect on the adhesive
interface. These findings are in accordance with previous reports that

TABLE 2 Study groups' mean bond strength values (MPa), percentage of failure mode and failure mode proportion comparison

Failure mode
Shear bond strength (MPa)
Study group Mean ± SD* Adhesive Cohesive in enamel Proportion comparison*
a
RD-PB 23.16 ± 4.26 17 (56.7%) 13 (43.3%) a
RD-OFL 30.84 ± 6.31b 20 (66.7%) 10 (33.3%) a
c
nRD-PB 12.57 ± 4.12 30 (100%) 0 (0%) b
nRD-OFL 16.33 ± 6.08d 30 (100%) 0 (0%) b

Note: Different letters within each column indicate statistically significant differences between the study groups regarding shear bond strength and failure
mode proportion (p < 0.05).

FALACHO ET AL.
a dry working field cannot be established in the oral cavity without the
correct application of a rubber dam.12 Similarly, one in vitro study demon-
strated that bond strength declines with increasing temperature and
humidity in an environmental chamber. Significantly worse values were
obtained for the group exposed to 37 C and 90% relative humidity, sup-
porting the recommendation that composite resin restorations should be
3
made under absolute isolation. Authors who studied analogous hypothe-
ses chose simulate the oral environment simulation in a controlled humid-
ity chamber.27 However, there are possible disadvantages to this method:
the chamber's inability to replicate natural inhalation, down time, and
exhalation cycles as they occur in clinical scenarios. In addition, the con-
stant high humidity may impair water evaporation, directly promoting a
12,26
bias towards an adverse outcome.
In another study, the microshear bond strength of a resin com-
posite to enamel was tested with three different adhesive systems
applied at various humidity conditions.27 However, contrary to what
was observed in the present study, no significant influence of humid-
ity on bond strength to enamel was found. The divergence of results
might be explained by the differences in the experimental protocols
and adhesive systems used. Despite that, this same study stated that
total-etch and two-step self-etch adhesive systems exhibited signifi-
cantly higher microshear bond strength values than that of one-step
27
self-etch adhesives to enamel, for all humidity conditions. In the
present study, higher mean shear bond strength values were found
with OptiBond FL when both adhesive systems are tested under the
same isolation conditions. Moreover, when analyzing standard devia-
tions, groups in which rubber dam was used exhibit standard devia-
tions of about 20% of the mean shear bond strength value, whereas
in the experimental groups where absolute isolation was not per-
formed, higher standard deviations of approximately 33% for the uni-
versal system and 42% for the three-step total-etch system were
found, suggesting less variation and greater predictability when rubber
dam isolation is used.
Even though the three-step total-etch system showed higher mean
values of shear bond strength in both experimental conditions, with and
without absolute isolation (30.84 MPa and 16.33 MPa respectively), it
was also the one with greater differences between maximum and mini-
mum bond strength values (25.94 MPa and 3.00 MPa, respectively) in
the group without rubber dam. These findings indicate that OptiBond
FL can be highly susceptible to relative humidity and unpredictable
without proper isolation, which may be due to its chemical composition.
HEMA is a hydrophilic monomer found in OptiBond FL, absent from
Prime&Bond active. If water absorption takes place before polymeriza-
tion, it may lead to a reduction in the degree of polymerization conver-
sion due to dilution of the adhesive system.6,18,21 Increased content of
HEMA in adhesives decreases the degree of conversion and may jeop-
7,19
ardize the polymer mechanical properties.
In this study, failure modes were either adhesive or cohesive in
enamel. Out of the two, there was a higher proportion of overall
adhesive fractures. However, it is worth mentioning that cohesive
enamel fractures were exclusively registered in experimental groups
where rubber dam was used. This suggests that the absence of abso-
lute isolation compromises the bonding interface. When adequate
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53
absolute isolation techniques were used, either adhesive system tested
could provide bond strength values that exceeded the cohesive strength
of enamel itself. Furthermore, failure modes were similar among groups
where the same experimental conditions were tested, meaning that no
significant difference in fracture type between adhesive systems was
found, independent of isolation. Besides bond strength values, failure
patterns are important parameters that must be assessed when evaluat-
ing adhesion, since the cohesive strength of dental substrates sets the
bar for the expected performance of adhesive systems.27
Although the present study was performed in situ, higher relative
humidity values are expected to be found in clinical conditions when
rubber dam is not used or is improperly placed.26 The custom-
designed oral device used in this experimental work may have led to
improved relative isolation compared to the clinical environment
since humidity from surrounding tissues was completely blocked
(i.e., gingival crevicular fluid, saliva, or blood). In addition, the capsule
used to apply the composite resin also shielded the restorative mate-
rial from humidity immediately after being placed. It is also worth not-
ing that restorative procedures in a clinical environment are more
time-consuming and, therefore, increased exposure to humidity and
consequent effects on adhesion are expected. In this study, the best-
case scenario was simulated, meaning that the tooth slot was in a
location equivalent to that of the vestibular surface of an anterior
tooth. Other oral locations, such as mandibular teeth or even posterior
upper teeth, are exposed to a higher degree of humidity and moisture
and may, therefore, suffer from more pronounced effects. A previous
study showed that significantly higher temperatures and relative
humidity values are found at molar sites than those found in the inci-
sor positions (incisor 26.2 C/84.8%RH vs. molar 27.3 C/90.7%RH).26
In the present study, intraoral conditions were standardized by per-
forming procedures in only one patient. However, differences
between patients' oral environments should be considered in further
studies. One study stated that during mouth breathing, temperatures
are significantly higher, and the amount of exhaled water is higher
than during to nose breathing. Thus, even though our volunteer was
instructed to breathe through the nose, differences between patients'
breathing patterns must be taken into consideration.1,11,25 Although
intraoral temperature and relative humidity values were not recorded
in this study, a previous study showed that the placement of different
isolation methods produces significant alterations in intraoral temper-
ature and relative humidity.5 The room's environmental conditions
also influence these values.5 Regarding the use of rubber dam, one
study evaluated how different types, number of exposed teeth and air
vents in the rubber dam sheet influence temperature and relative
humidity. It was concluded that simple moisture exclusion with cotton
rolls is insufficient (100% relative humidity) and that rubber dam isola-
tion lowers relative humidity to levels equivalent to those of the room.
However, the same cannot be said about temperature. Additionally, it
was concluded that there is no difference in moisture exclusion when
a single tooth or multiple teeth are exposed.5
The present study demonstrates that intraoral relative humidity
has a significant effect on bond strength values to enamel. Without
adequate rubber dam isolation, the performance of dental adhesives

54
is compromised, thus potentially compromising the longevity of resto-
rations and with long-term consequences on our patient's oral health.
Further clinical considering different intraoral sites and including mul-
tiple patients with different breathing patterns, followed by sample
aging, are needed to evaluate long-term effects of rubber dam use.
Ultimately, clinical studies should be implemented to correlate resto-
ration survival with humidity levels during bonding procedures.
5 | C O N CL U S I O N S
Within the limitations of this clinical study, the following conclusions
were drawn:
1. Absolute isolation with rubber dam increases bond strength to
enamel, independent of the adhesive system.
2. The three-step total-etch system OptiBond FL provided signifi-
cantly higher bond strength values than Prime&Bond active under
both experimental conditions.
ACKNOWLEDGEMEN T AND DISCLOS URE
The authors thank Professor Francisco Caramelo from the Coimbra
Institute for Clinical and Biomedical Research (iCBR), Laboratory of Bio-
statistics and Medical Informatics (LBIM), Faculty of Medicine, Univer-
sity of Coimbra for promptly and carefully assisting with the statistical
analysis. The authors also thank Dr. Raphaël Gameiro (Dental Techni-
cian) for modeling and manufacturing the custom-designed splint used
to position samples intra-orally. The authors do not have any financial
interest in the companies whose materials are included in this article.
DATA AVAI LAB ILITY S TATEMENT
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
ORCID
Rui I. Falacho https://orcid.org/0000-0001-7099-8871
Eliana Azevedo Melo https://orcid.org/0000-0003-4144-8154
Joana A. Marques https://orcid.org/0000-0001-5210-6155
João Carlos Ramos https://orcid.org/0000-0003-1965-1092
Fernando Guerra https://orcid.org/0000-0001-8780-9379
Markus B. Blatz https://orcid.org/0000-0001-6341-7047
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55
How to cite this article: Falacho RI, Melo EA, Marques JA,
Ramos JC, Guerra F, Blatz MB. Clinical in-situ evaluation of
the effect of rubber dam isolation on bond strength to enamel.
J Esthet Restor Dent. 2023;35(1):48‐55. doi:10.1111/jerd.
12979