An In-Vitro Evaluation of Microleakage in Resin-Based

Restorative Materials at Different Time Intervals

Abstract: A vital feature of conservative dentistry is the adhesion of the restorative material to the
tooth structure for restoration of the tooth substance lost due to dental decay, trauma, or dental
imperfections. In a perfect world, a restorative material should generate a lasting adhesion by
bonding the restoration with tooth tissues. The ingress of micro-organisms, oral fluids, molecules,
and ions through microscopic spaces due to faulty adhesion between restoration and tooth structure
is known as microleakage. This study is focuses on the evaluation of adhesive failures between the
restorative materials. In the past, studies have focused more on the bonding potential of a restorative
material with the tooth surface. Therefore, there is need to carry out a study that compares the
microleakage between resin-based restorative materials in a sandwich manner with and without the
intermediate bonding layer after immersion in 2% methylene blue dye at different time intervals. The
restorative materials used were composite Ceram X Mono plus (DENTSPLY) and Z350 (3M ESPE),
Vitremer resin modified glass ionomer cement (RMGIC) (3M ESPE), smart dentine replacement SDR
(3M ESPE), Bond NT (DENTSPLY), and Universal Bond (3M ESPE). A light emitting diode (LED)
was used to cure the specimens. Artificial saliva was used as a storage medium for the specimens.
Thermocycling of specimens was carried out at 500 cycles/30 s and 1000 cycles/30 s. The world
health organization (WHO) grading tool for microleakage was used to analyze fluid ingress in the
specimens through disclosing by 2% methylene blue dye. The statistical analysis was carried out with
one-way analysis of variance (ANOVA) and Tukey post hoc test, keeping the level of significance at p _ 0.05. In Grade 0 =
85 samples, Grade 1 = 10 samples, Grade 2 = 7 samples, Grade 3 = 16 samples,
and in Grade 4 = 2 samples were identified. This study describes that no microleakage was observed
in SDR and resin composite groups as compared to Vitremer and resin composite groups.

1. Introduction
Since the invention of dental composites in 1960, they have been modified in the
accomplishment of appearance and durability [1]. An important function of a rtorative
material is to adhere to the dentine when the enamel is lost due to trauma, caries, or dental
treatment [2,3]. The ingress of micro-organisms, oral fluids, molecules, and ions through
microscopic cracks due to faulty adhesion between restoration and tooth structure is known
as microleakage [4,5]. This can cause increased sensitivity, recurrent caries, pulpitis, and
tooth staining [6]. Microleakage in restorative materials could be a consequence of polymerization
shrinkage, thermal contraction, water absorption, and mechanical stresses [7].
The methods to reduce microleakage involve various restorative methods (direct
or indirect) and curing methods such as the incremental technique [8]. The resin-based
restoration shows polymerization shrinkage [9]. Amongst resin restorations, the flowable
composite produces decreased polymerization shrinkage, due to lower filler content [10].
Regardless of all scientific advancements, polymerization shrinkage remains the major
weakness of the composite resins. The contraction produced by it disrupts the bond with
cavity walls and this is a major reason for filling dislodgement, specifically in proximal
Class II deep cavities [11–13]. One of the techniques suggested for overcoming the class II
secondary caries is a “Sandwich Technique” of composite resins and glass ionomer material
[14].
McLean andWilson proposed the concept of open and closed sandwich techniques
for class II cavities to overcome the problem of polymerization shrinkage [15]. In this
procedure, dentine is replaced by GIC and composite resins replace the enamel part of the
tooth. The GIC cement remained uncovered for the release of fluoride at the cervical part of
the cavity to reduce the occurrence of dental caries. The purpose of using GIC as a base was
also to reduce the amount of resin material. The properties of both restorative materials are
combined in a way to decrease caries incidence, enhance chemical adhesion to the tooth
structure, release fluoride, longevity, and aesthetics [16–19]. Other than the conventional
GIC, the resin-modified glass ionomer cement (RMGIC), and smart dentine replacement
(SDR) can also be used as a base material in the sandwich technique. The RMGIC has a
dual ability to bond chemically with the tooth, and also micro mechanically linked to bulk
filling composite restorative materials [20]. Moreover, SDR has an increased depth of cure
of up to 4 mm thickness. Additionally, it possesses less shrinkage stresses, and increased
flowability [21]. This study focuses on analysis of bonding between restorative materials
used in an open sandwich technique of class II model cavities.
The current study will provide insight to observe the problem of microleakage precisely.
In the past, microleakage and total bonding was evaluated between the material
and the tooth structure, but not between the two different materials as in case of sandwich
technique. The addition of this study in scientific literature will aid a deep insight on the
reasons resin-based restorations fail. The null hypothesis of the study states that “there is
no microleakage between the two resin-based restorative materials in a sandwich manner,
with and without the intermediate bonding layer after immersion in 2% methylene blue
dye at different time intervals”.
The purpose of this study was to compare the microleakage between resin-based
restorative materials in a sandwich manner with and without the intermediate bonding
layer after immersion in 2% methylene blue dye at different time intervals.

2. Materials and Methods

2.1. Study Design, Setting, and Duration
This was an in-vitro, experimental study. The samples were made at Dow University
of Health Sciences, OJHA campus, Karachi, Pakistan. Thermocycling (PX2, Massachusetts,
USA) testing was carried out at OJHA campus. The testing under stereo microscope (Motic
DMW 143, PAL System, Hong Kong, China) was done in the Metallurgy department
at Nadirshaw Edulji Dinshaw (NED) University of Engineering & Technology, Karachi,
Pakistan. The study was completed in one year after the approval of project.
2.2. Sampling Technique and Sample Size Estimation
The purposive sampling technique was adopted in this study according to the predefined
sample selection criteria. The sample size was estimated following the study of
Lawrence et al. [22]. The Open-epi software was used for this purpose. Considering the
95% confidence interval with a 5% margin of error, the power of the test was 80%. The total
sample size estimated was 120 specimens, and each group consisted of 10 specimens.
2.3. Materials and Sample Selection Criteria
The following materials were included in the study (Table 1):
1. Composite Z350 (3M ESPE, Minnesota United States).
2. Resin Modified Glass Ionomer (3M ESPE Minnesota United States).
3. Ceram X Mono plus (DENTSPLY, York, Pennsylvania, United States).
4. Smart Dentine Replacement, SDR (DENTSPLY, York, Pennsylvania, United States).
5. Bond NT (DENTSPLY, York, Pennsylvania, United States).
6. Universal Bond (3M ESPE, Minnesota United States).
The curing time recommended by the manufacturer was followed for the polymerization
of samples in the sandwich manner. Only the LED cured specimens were included
in the study. The samples which were broken or contained cracks and air bubbles were
not included. Additionally, the incompletely cured samples were also not included in
the experiments.

2.4. Specification and Fabrication of Samples

A specially fabricated mold of polytetrafluoroethylene (Teflon) was used for the
preparation of samples. Samples were cylindrical (4 mm diameter and 4 mm height) and
followed ISO 4049 for dental composites [22]. Each cylindrical sample consisted of two
materials; each material was 2 mm thick as per ISO 10650 [23] as shown in Figures 1 and 2.
The mold was designed for mimicking class II restoration; it consists of a metallic base and
two Teflon sheets containing nine holes of 4-mm diameter, whereas each sheet was 2 mm
in width. The lining material (Vitremer or SDR) was mixed with plastic instrument and
packed in one Teflon sheet, cured with LED light for 40 s. Second Teflon sheet was placed
on top of the first sheet. The mold was designed in a way that the nine holes of both Teflon
sheets overlay each other. Then, the composite material was filled with or without bond
application, in the second Teflon sheet and cured for 40 s with LED unit. Group-A had a
base material Vitremer or SDR. After curing it was etched for 20 s with 37% phosphoric acid
gel, washed for 10 s, and lastly dried for 5 s. After placement of second Teflon sheet, the
holes were filled with bulk filling material (Z350 or Ceram X). On the contrary, the Group-B
followed the same method with additional application of adhesive material (Vitrebond
plus or Single Bond Universal) between base materials and the bulk materials. The samples
were then removed from the mold.
The specimens were then stored in an incubator at 26 _C/48 h for completion of
the polymerization process. The specimens were placed in dark brown bottles, labeled
for each group containing artificial saliva (pH = 6.7). After incubating, the specimens
were thermocycle in small tubes filled with artificial saliva to prevent drying during the
thermocycling process. It is a method of simulating the temperature variation of the oral
cavity at 5–55 _C as per ISO standard 11405 [24].
After taking out small tubes from thermocycler, specimens were removed, dried
(with tissue paper), and sealed (two-thirds portion) using two coats of nail varnish. The
remaining third portion left uncoated was the dye penetration side (DPS).
Before sectioning, the specimens were dipped in 2% methylene blue dye solution
for 24 h (buffered at pH = 7), then washed properly in running tap water. Ultra-thin
(22.2 mm _ 0.3 mm, ceramic disc) in a micro-motor was used to section the specimens
longitudinally in between the DPS, into two halves. The specimens were observed under
the Stereomicroscope between magnifications of 10–40_ for microleakage observations.

……

Out of the total 34 specimens from the control were classified as grade zero (0.0 mm
leakage), there were 15 specimens of group 1A and 19 specimens of group 1B. Furthermore,
three specimens of the second group had grade zero together with one specimen of
group 2A and two from group 2B. Likewise, all 20 specimens were categorized as grade
zero in the third group, including 10 of group 3A and 10 from group 3B. Nine samples of
4B group were grade zero. Nevertheless, 10 specimens of group 5A and nine specimens of
group 5B from the fifth group were grade zero.
In addition, two specimens of group 1A from the control group were included as grade
one (up to 0.5 mm leakage). Moreover, one specimens of group 2A and five specimens
from group 2B were classified as grade one. In the 4B and 5B groups, only one specimen
from each group was found to be in grade one.
Grade two (up to 1 mm leakage) was associated with one specimen from each
group 1A and 1B from control respectively. One specimen from the 2A group and two
specimens from the 2B group, were grade two, while two specimens from 4A group showed
grade two microleakage.
Likewise, grade three (up to 2mmleakage) categories consisted of two specimens from
group 1A and the control group. Five specimens of group 2A and one sample of group 2B
from the second group, and lastly eight specimens from the 4A group were associated with
grade 3. The grade four microleakage (_2 mm) was found in only two specimens from the
2A group.
3.2. Distribution of Microleakage Values in Control and Experimental Groups after Thermocycling
The mean values of a control group without thermocycling and study groups with
the thermocycling procedure are shown in Table 4 and the pictorial description is given in
Figure 4. The highest mean values in the control group were observed in 2A, 0.82 _ 0.86;
whereas similar microleakage values were found in 2A, 3A, and B, as well as in groups 4
and 5. The mean difference values of specimens were found to be zero in third and fifth
groups with an insignificant p-value > 0.99.

In the second and fourth groups, the mean difference was 0.82 and 0.18 respectively.
There was a significant difference (p = 0.05) in the values of group 2. However, no significant
difference (p = 0.14) was noted in group 4 microleakage values.
The mean difference of second group at 500 cycles per 30 s was 0.75 and fourth group
were 1.16, with a significant p-value of 0.016 and 0.01 respectively. Furthermore, the second, fourth and fifth
group at 1000 cycles per 30 s has shown a mean difference of 0.95, 1.15, and
0.06 with a non-significant p-values of 0.17, 0.17, and 0.32 respectively.
3.3. Proportionality between A Groups
Table 5 is describing the proportionality of A subgroups in control, and after 500 cycles
per 30 s, and 100 cycles per 30 s thermocycling procedures. No difference was found in
microleakage among materials in the control group. Although at 500 cycles per 30 s, a
difference was noted when the second and fourth groups were compared with third and
fifth groups. A difference in values was also noted when the second group was compared
with third and fourth at 1000 cycles per 30 s. The mean values of second and fourth control
group were 0.82 _ 0.86 and 0.18 _ 0.25 respectively. Considering, 500 cycles per 30 s, the
results indicated that in second group the mean value was 1.25 _ 0.13 and in the fourth
group it was 1.22 _ 0.40. Furthermore, at 1000 cycles per 30 s, the second group showed a
mean value of 1.26 _ 1.23 and group four had a mean value of 1.15 _ 0.30. Additionally,
the third and fifth groups had a zero value in the control, and after thermocycling.

3.4. Proportionality among B Groups

Table 6 is showing the proportionality of B subgroups in control, 500 cycles per 30 s
and 1000 cycles per 30 s. After thermocycling at 500 cycles per 30 s, a remarkable difference
was found when second group was compared with all other groups. The second group
revealed a highest mean value of 0.50 _ 0.36, whereas at 1000 cycles per 30 s, the mean
value was 0.31 _ 0.32. Additionally, in the fourth group the mean value was the least
0.07 _ 0.15 at 500 cycles per 30 s, whereas, in the fifth group the mean value was 0.11 _ 0.06
at 500 cycles and 0.24 _ 0.14 at 1000 cycles per 30 s.
Table 6.

4. Discussion
The current study evaluated the success and failure of Class II open sandwiched
technique restoration, by investigating the microleakage at the resin–resin interface. The
stereomicroscope was used to observe eight variable combinations of base and bulk restorative
materials. No significant difference was found in the microleakage values of the third
and fifth groups, both groups showed minimal or zero leakage scores. The SDR material
was used as base material in both the groups where no microleakage was observed, indicating
a better bonding ability of SDR with resin composite material. Whereas a significant
microleakage scores was found in group two and four in this study. The Vitremer and composite
bond was found weaker. Thus, the null hypothesis that “There is no microleakage
between the two resin-based restorative materials in a sandwich manner, with and without the intermediate
bonding layer after immersion in 2% methylene blue dye at different time
intervals” was rejected.
Nicola and Scotti, reported a similar outcome with SDR in their study, the only
difference was in the methodology, they used extracted molar specimens, filled with
restorative materials [27]. Furthermore, the presence of urethane di-methacrylate and dimethacrylate
resin could be the reason for no leakage in SDR, both resins enhance chemical
adhesion with tooth structure. Sadeghi et al. used thermocycle samples at 1500 cycles,
fuchsine dye was applied to identify the microleakage. He applied flowable composite
and SDR in the specimens. An adequate adhesion was found primarily due to the use of
flowable resins which has reduced modulus of elasticity that decrease stresses within the
restoration. A better physical characteristic, easy handling, less polymerization shrinkage,
and improved curing depth of SDR could also be the reason for the favorable outcome [28].
El-Safty et al. [29] and Leprince J.G. et al. [30] found the same results with flowable
composites. They further suggested that ‘swelling’ behavior of particular bulk fill composites
could also be one of the reasons for reducing the microleakage. The authors checked
the properties of the composite by Raman spectroscopy and Vickers hardness testing [30].
In the current study, absolutely no leakage specifies improved bonding ability of the Ceram
X and SDR in groups 1, 3, and 5 which was evaluated after being thermocycled at
500 cycles per 30 s and 1000 cycles per 30 s at room temperature. Schirrmeister et al. found
similar results in an in-vivo study using Ryge’s criteria. It was proposed that Ceram-X
had an improved clinically proven marginal seal [31]. Similarly, Ahmadi et al. [32], used
class V cavities in intact molar to analyze microleakage through fuchsin dye with a similar
methodology and they found no microleakage in nano-ceramic composite at the cervical
margins. The absence of microleakage with Ceram X was also found by Eden et al. [33] with
similar methods. In a clinical study conducted by Schmidt et al. [34], utilizing scanning
electron microscopy showed a similar finding with Ceram X composites. Thus, it is proven
by the literature that a superior sealing ability of Ceram X composites is one of the reasons
for reduced microleakage over time. On the contrary, Sing et al. [35] found that Ceram-X
composite was associated with microleakage. This finding is comparable with our study in
terms of methodology, the difference could be due to the variation in the base materials [35].
Likewise, Bogra et al. [36] used extracted teeth with thermocycling at 2–5 _C for 1500 cycles
and observed leakage at cemento-enamel junction and occlusal margins while using Ceram
X nano-composite in class 2 cavities. Kermanshah et al. [37] encountered microleakage
at the gingival margin with Ceram X nano-ceramic composite comparatively higher than
Filtek silorane composite. They used composite materials in class I and II cavities on
extracted teeth to measure leakage by the same regime as adopted in the present study.
Agrawal et al. [38] also described a considerably low microleakage scores in nano-ceramic
and silorane composite materials with a ribboned fiber base material. In another study
by Owen B et al. [39], an increased microleakage was noted in nano-composite resins in
contrast to micro-hybrid composites while used in class 5 cavities, when sectioned and
immersed in 1% methylene blue dye. Because of the contradictory results related to Ceram
X, this study focused on a combination of materials in a sandwich manner. Nonetheless, in
this study, microleakage was found in Ceram X and Vitremer combination groups 2 and 4.
The etch and rinse protocol is the gold standard in dental adhesive systems to date,
this study followed the same protocol, for an adequate micro-mechanical retention of
composite resin and base materials [40]. The Ceram X and Z350 have proven adhesive
efficacy in past studies of class 2 cavities [41]. The application of SDR as a base material in
third and fifth groups showed no significant difference in this study.
The proportionality of second and fourth groups (Vitremer and Z350; Vitremer, 3M,
and Ceram X) in this study revealed microleakage. It was observed in all subgroups (with
and without bond, under 500 and 1000 cycles), but the extent of leakage was different
among the groups studied. The control groups 2A and 4A also showed microleakage. This
could be due to the internal gaps in the restoration as described Owen B et al. [39] and
Spencer et al. [42].

In this study, the bond between the materials was checked after thermocycling to
reflect the aging effect of the restoration. Gerdolle et al. [43] claimed in a study that bonds
between resin-based materials can be enhanced due to thermal expansion and contraction
processes within matured restorations.
In this study, the SDR and Vitremer comparison as a base material revealed unsatisfactory
sealing with Vitremer, even with an inherent property of chemical adhesion to enamel
and dentin. The brittle nature of RMGIC (Vitremer) could be the cause of this distress by
the formation of cracks with thermal changes [44].
Feilzeret et al. [45] described that the microleakage may result in composite restoration
by water sorption specifically in the unfilled resin type. They observed minimum leakage
in nano-RMGI and nano-composite while used in a sandwich manner. El-Ashiry et al. [46]
used the same material in class V cavities of primary molars with 0.5% fuchsin dye to
identify leakage under stereomicroscope while, AB Malik et al. [47] used in class II cavities.
Both authors found decreased leakage at the occlusal margins compared to the gingival
margin in nano-RMGI, compared to conventional RMGIC. Beznos et al. [48] proposed that
RMGIC decreases volumetric shrinkage of resin-based restorations by 41%. This finding
did not match with the results of the present study. In this study, greater microleakage was
observed in groups having RMGIC—i.e., the second and fourth groups. Polymerization
shrinkage, missing elastic deformation, increased viscosity and porosities could be the
cause of increase microleakage in RMGIC [49]. The other reason of microleakage could be
dual setting characteristics of RMGIC compared to SDR which is light cure [50].
This study adopted the dye penetration method for finding microleakage. Despite visible
flaws in dye penetration, no evidence of errors is reported yet about this method [51–53].
The procedure is validated and easy to perform even in a simple laboratory setup [54].
The 2% methylene blue dye is simple to utilize, it has increased water solubility and quick
diffusion in cracks or imperfections in a substance [55]. The methylene blue is considered
to have a better dye penetration than butyric acid [56,57]. Other methods such as dye
and radioisotope penetration are precise and produce 82% accuracy in microleakage detection
[58,59]. Due to these properties reported in the literature, the methylene blue dye
method was selected to be used in this study [59].
The ingress of fluid or microleakage in restoration is an indicator of bond failure
between them that can occur due to various reasons such as polymerization shrinkage,
materials incompatibility, and thermal changes in a particular material with time [57].
Numerous methods such as matched adhesive use, incremental technique application, and
control of C-factor by balancing the number of bonded and unbonded tooth surfaces are
recommended in the literature to overcome microleakage under restorations [60]. This
study proposes the application of suitable base material (SDR) prior to the filling of bulk
filled resin composite material to achieve adequate bond between materials as well as
with the tooth structure. However, further investigation in the form of clinical trials is
recommended to compare the efficacy of resin-based material combinations in long-term
use, and to better understand the outcome of restorative materials available.
5. Conclusions
This study was carried out to analyze the microleakage between different combinations
of resin-based materials. Within the limitations of the study, following conclusion can
be drawn:
1. SDR with Ceram X and with Z350 exhibited no leakage, indicating that SDR would
have better bonding affinity with resin composites at different time intervals.
2. The bonding of Vitremer with the resin composite was weak.