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 Table of Contents  
Year : 2020  |  Volume : 17  |  Issue : 3  |  Page : 97-105

Nanoleakage of different composite restoration systems

1 Ministry of Health, Elgharbia, Tanta University, Tanta, Egypt
2 Department of Restorative Dentistry, Faculty of Dentistry, Tanta University, Tanta, Egypt

Date of Submission10-Dec-2019
Date of Acceptance01-Jun-2020
Date of Web Publication30-Oct-2020

Correspondence Address:
Dina M El-Keredy
Apartment 16, Fourth Floor, Al Firdous Tower, University Teaching Staff Buildings, Hassan Radwan Street, Tanta
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/tdj.tdj_57_19

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This study was carried out to investigate the nanoleakage of different composite restoration systems (composite materials and its recommended adhesives).
Patients and methods
Thirty intact, noncarious human premolars were selected. Root apices of all teeth were sealed with wax. For each tooth, the roots were inserted into self-curing resin mold, with the long axis perpendicular to the acrylic resin base. Standardized class V cavities were prepared above the cemento-enamel junction by 1 mm, on the buccal surfaces of the teeth. The prepared teeth were divided randomly into three equal groups (10 each) according to restorative systems used: group 1: nanohybrid composite system, group 2: bulk-fill composite system, and group 3: flowable composite system. Each group was equally divided into two subgroups (five each) according to the thermal and load cycling treatments (A and B). All specimens were examined at the occlusal margins and cervical margins (divisions 1 and 2). All specimens were coated with two layers of nail varnish except for 1 mm, around the restoration margins and immersed in a 50% (w/v), sectioned in bucco-lingual direction and processed for scanning electron microscopy and energy dispersive analysis radiograph.
The bulk-fill composite system (group 2) recorded the higher silver weight percentage mean value followed by nanohybrid composite system (group 1) while the flowable composite system (group 3) recorded the lowest silver weight percentage mean value in both cases whether the restorations were thermal and load cycled or not. However, a statistically significant difference was recorded in the three tested groups when subgroup A was compared to subgroup B in each group. Moreover, the results indicated that the mean value of nanoleakage at the cervical margins (division 2) in all groups (1, 2, and 3) and subgroups (A and B) recorded a higher significant nanoleakage than the occlusal margins (division 1) except group 2B which recorded no significant difference with a P value of 0.147.
The flowable composite system recorded the lowest silver weight percentage mean values. Thermal and mechanical stresses increase nanoleakage significantly.

Keywords: dental composite, energy dispersive analytical radiograph, nanoleakage, scanning electron microscope

How to cite this article:
El-Keredy DM, Etman WM, Salama MM. Nanoleakage of different composite restoration systems. Tanta Dent J 2020;17:97-105

How to cite this URL:
El-Keredy DM, Etman WM, Salama MM. Nanoleakage of different composite restoration systems. Tanta Dent J [serial online] 2020 [cited 2020 Nov 27];17:97-105. Available from: http://www.tmj.eg.net/text.asp?2020/17/3/97/299633

  Introduction Top

The use of direct resin-based composite has increased primarily due to patient esthetic desires and product improvements[1]. In addition improvements of mechanical properties of the composite have permitted its use in posterior teeth with greater reliability than was the case some of years ago[2].

Different types of composite have been available among these, the flowable composites were found to adapt well to a cavity form preparation and can be used as liners in deep cavities, bulk-fill composites which are more easier in use, saving time and effort and nanohybrid composites that are able to ensure long-term gloss and good adaptation to bonded cavity walls [3],[4],[5]. However, all types of composites are now the materials of choice for most restorations; their polymerization shrinkage remains a problem. The contraction stress associated with this shrinkage can cause cuspal deflection or debonding at the composite-tooth interface [6],[7],[8].

It has been suggested that adding filler particles to adhesive systems can generally improve the mechanical properties of adhesive layer and the bonding to dentin, accompanied by penetration of bonding agents into the dentin tubules where they form resin tags and what is known as the 'hybrid layer'[9].

The formation of an effective hybrid layer is achieved by diffusion of monomers in the collagen fibers, and this is the main bonding mechanism of the total-etch adhesive systems[10]. Due to the technique-complexity and sensitivity, the novelties in the adhesive systems were directed toward a simplified application process. The self-etching adhesive systems were developed to avoid the collapse of the collagen network and to shorten the clinical technique. Thus, while total-etch systems have accumulated more years of data, self-etch adhesives offer a convincing, patient-friendly advantage[11],[12].

Although, gap-free margins at the dentin/restoration interface were detected, however another pattern of leakage called nanoleakage was described by Sano et al.[13], who observed the penetration of silver nitrate along gap-free margins. This might be detected by the aid of scanning electron microscope (SEM)[13].

'Nanoleakage,' is a different pattern of leakage occurring within the hybrid layer in nano-meter scaled spaces, which may be due to the presence of residual water around collagen fibrils, collagen network collapse or imperfect resin infiltration into the exposed collagen network and incomplete polymerization[14].

This pattern may arise within the adhesive layer and likewise within the hybrid layer. Bacterial product or oral fluid penetration across the interface compromised the stability of the resin-dentin bond through hydrolytic breakdown of the adhesive resin or collagen in the hybrid layer[15].

Therefore, nanoleakage assessment could be considered an important indicator of the sealability of a restorative material and the hybrid layer quality, which consequently affect the longevity of the restoration[16]. Usually the silver nitrate solution (AgNO3) 50% (w/v) is used to trace the nanoleakage phenomenon. This solution can penetrate the dentin due to the small diameter of silver ions (0.059 μm)[17].

Researchers concluded that some factors might have an impact effect on the adhesion mechanism, leading to nanoleakage. One of those is demineralized but not fully hybridized dentin layer which might be considered a weak point in the adhesion mechanism that could allow dentinal fluid to slowly permeate the interface, and this is believed to degrade the adhesive resin[13].

Another factor was found recording that during normal function and parafunctional habits, teeth are subjected to stresses[18]. Food bolus can induce vertical loading between antagonistic teeth, which is equally disseminated over the entire occlusal surface to alleviate stresses. Compressive stresses arise on the tooth aspect being bent, while tensile stresses are generated simultaneously on the opposite tooth aspect. The same scenario occurs with restorations placed cervically in teeth when they are subjected to occlusal loading. In theory, the fatigue resulting from the masticatory forces generate stresses in the tooth/restoration interface, increasing the damages in this area [19],[20],[21].

As well as, the temperature changes which induce interfacial stresses due to differences of contraction and expansion in the adhesive interface. These tensions may cause microcracks, which propagate through the interface leading to degradation of adhesive resin quality, and may affect the clinical longevity of restorations[22]. Therefore, the thermo-mechanical simulation might provide data close to the clinical situation. Since it was concluded that the use of thermal and load cycling simulate stresses undertaken by dental restorations and helps to better understanding the dental materials[23],[24]. A confliction whether thermal and load cycling affect nanoleakage or not, led to study the effect of thermal and load cycling on the nanoleakage of three composite restorative systems assuming that all tested types will be affected by different degrees by thermo-loading. However, it was the hypothesis of this research that nanotechnology assumed that composites containing nanofillers will exhibit lesser nanoleakage compared to other types of composites.

  Patients and Methods Top

Thirty intact, noncarious human premolars, extracted for orthodontic reasons were collected from patients aged 18 − 30 years old. These teeth were obtained from Oral Surgery Department in Tanta University. All patients are informed about the purpose of the study and using of their extracted teeth according to Ethics Committee of Faculty of Dentistry, Tanta University. All collected teeth were cleaned from blood, soft tissue and debris, washed thoroughly using distilled water and examined using a light microscope (Kern_11002; Kern and Sohn, Balingen, Germany) to ensure that there were no cracks or enamel defects. The teeth were stored in normal saline at 37°C in the incubator (BJPX-NEWARK; BIOBASE, Jinan, China) 2 months prior to testing.

Root apices of all teeth were sealed with wax (Modelling wax; Cavex, Haarlem, Holland BV). For each tooth, the roots were inserted into self-curing resin (Cold Cure; Acrostone Dental Manufacture, Cairo, Egypt) mold, with the long axis perpendicular to the acrylic resin base. Standardized class V cavities were prepared (3.0 mm in depth, 2.0 mm in width) above the cemento-enamel junction by 1 mm, on the buccal surfaces of the teeth. The cavity depth and width were measured using a caliper (NEIKO, electronic digital caliper; accuracy: 0.001/0.02 mm). Cavities were prepared using #D-07639 carbide round burs in water-cooled and high-speed handpiece. No bevels were made at any of the enamel margins of the prepared cavities[18]. Each bur was used for ten preparations and then replaced.

After storage in distilled water at 37°C for 24 h in the incubator, the prepared teeth were divided randomly into three equal groups (n = 10) according to the type of resin composite and the adhesive used:

  1. Group 1: were restored by Grandio SO Universal nanohybrid composite (VOCO GmbH, Cuxhaven, Germany) in incremental technique (two restorative increments each of 1.5 mm which was cured for 10 s using blue phase n Ivoclar vivadent light curing) with Futurabond M + Universal adhesive (VOCO GmbH).
  2. Group 2: were restored by Tetric N-Ceram Bulk-Fill (Ivoclar Vivadent AG, Schaan, Liechtenstein) (were applied as a bulk and cured for 20 s using the same previous light curing unit) with Tetric N-Bond Universal (Ivoclar Vivadent AG) bond.
  3. Group 3: were restored by flowable composite (Tetric N-Flow) (Ivoclar Vivadent AG) two restorative increments each of 1.5 mm which were cured for 10 s using blue phase n Ivoclar vivadent light curing with Tetric N-Bond and N-Etch (Ivoclar Vivadent AG) bond.

All restorative materials and adhesive systems were applied according to the manufacturer's instructions. After complete restoration of cavities, specimens were stored in distilled water at 37°C for 24 h in the incubator.

Each group was equally divided into two subgroups (n = 5) A and B representing whether specimens were subjected to thermo-cycling (custom made apparatus at Conservative Dentistry Department, Faculty of Dentistry, Alexandria University) and load cycling (Model LRX – Plus; Lloyd Instrument Ltd, Fareham, UK) or not respectively. All specimens were examined at the occlusal margins and cervical margins. These were pointed to as divisions 1 and 2.

Specimens of subgroups 1A, 2A, and 3A were subjected to 600 thermal cycles from 5 to 55°C with 30 s dwell time, 20 s transfer time and then subjected to a maximum vertical load of 5 kg with cyclic frequency of 1.7 Hz for 240 000 cycles which corresponds to 1 year of clinical condition[25].

Specimens of both subgroups were coated with two layers of nail varnish except for 1 mm around the restoration margins, immersed in a 50% (w/v) ammoniacal silver nitrate solution then immersed for 8 h in photo-developing solution while being exposed to fluorescent light. These were sectioned in bucco-lingual direction through the center of the restorations using Isomet diamond saw (Isomet 5000; Buehler Ltd, Lake Bluff, Illinois, USA). Specimens were air dried, mounted on aluminum stubs and sputter-coated with a layer of gold to be subjected to electron microscopy analysis. To analyze the presence of silver particles, energy dispersive analysis radiograph (JEOL, JSM-5300; Akishima, Tokyo, Japan) was used.

Statistical analysis

The collected data were tabulated and analyzed using statistical package of the social science (SPSS) (version 22; SPSS Inc., Chicago, Illinois, USA) for statistical analysis in which data were expressed as mean and SD. One way analysis of variance test was performed to detect difference between all the tested groups. When there was a significant difference found, Tukey's test was performed to detect which group was responsible for this significance. Student t-test was performed to detect significance between subgroups and divisions.

  Results Top

The results revealed that composite systems, thermo-load cycling and location of margin had statistically significant effect on the silver weight percentage mean values, as shown in [Table 1] and [Table 2].
Table 1: Comparison of silver weight penetration % mean values in subgroup A versus B in each tested group regardless location

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Table 2: Effect of margin location on the total values of silver weight penetration percentage (occlusal margin versus cervical margin)

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To confirm the previous results SEM images at × 500 magnification were taken for all samples. All tested groups were examined and represented samples for each group, subgroup and division were presented to show the amount of silver ions penetration to the occlusal and cervical margins. [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5] recorded the randomly selected marginal SEM images for all tested variables.
Figure 1: SEM photomicrograph showing silver penetration to the occlusal margin of class V cavities restored with nanohybrid composite system and treated with thermal and load cycling (group 1AI). SEM, scanning electron microscope. C: Composite, D: Dentin.

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Figure 2: SEM photomicrograph showing silver penetration to the occlusal margin of class V cavities restored with nanohybrid composite system not treated with thermal and load cycling (group 1BI). SEM, scanning electron microscope.

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Figure 3: SEM photomicrograph showing silver penetration to the occlusal margin of class V cavities restored with bulk-fill composite system and treated with thermal and load cycling (group 2AI). SEM, scanning electron microscope..

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Figure 4: SEM photomicrograph showing silver penetration to the occlusal margin of class V cavities restored with flowable composite system and treated with thermal and load cycling (group 3AI). SEM, scanning electron microscope.

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Figure 5: SEM photomicrograph showing silver penetration to the cervical margin of class V cavities restored with flowable composite system and treated with thermal and load cycling (group 3AII). SEM, scanning electron microscope.

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  Discussion Top

The current hypothesis was totally rejected, as it was found that flowable composite restorations exhibited a significant lesser nanoleakage mean values with and without thermal and load cycling compared to nanohybrid and bulk-fill composite restorations. This can be explained by their lower stress induction due to low elastic modulus, compared to the higher elastic modulus, higher viscosity and lower wettability of conventional composites[26],[27].

Our findings are in agreement with many authors. Orlowski et al.[28], stated that statistically significant better marginal integrity of flowable tested materials, SDR, SonicFill, and Filtek bulk-fill compared to the composite TetricEvoCeram bulk-fill and they explained their findings by the flow consistency of flowable composites during application. Moreover, Peutzfeldt and Asmussen[29], showed and reported that the degree of fluidity during application of the composite material influences the marginal adaptation and the increased fluidity of the composite makes it adhere better to the walls of the cavity.

While Zubani et al.[30], did not agree with the current findings. They investigated the cavity margin adaptation of three composites using optical microscope and software evaluations. Materials tested were a new experimental bulk-filling composite (group 1), flowable Bulk-fill Surefil SDR Flow (group 2) and TetricEvoceram (group 3) and reported that no significant differences were found between the tested composite materials, this may be due to the difference in material and evaluation methodology.

On the contrary some authors reported more polymerization shrinkage and lesser mechanical properties of flowable composites compared to conventional types[3]. In addition, Braga et al.[31], and Cadenaro et al.[27], concluded that flowable resin composites have shown shrinkage stress more than conventional resin composites, supporting the hypothesis that the use of flowable materials might increase the risk of debonding at the adhesive interface as a result of polymerization contraction.

The results of the current study also stated that the bulk-fill composite system showed a significant more nanoleakage mean values than nanohybrid composite system applied with incremental technique. This was explained by some authors concluding that using bulk-fill does not eliminate the potential formation of gaps in the inner walls of the cavity[32]. Studies evaluating shrinkage and polymerization stress in bulk-fill composite resins are still scarce. Authors concluded that the success of bulk-fill restoration depends on adequate adaptation, technique of application and composition of the material itself[33].

These current findings came in agreement with Yamazaki et al.[34], who concluded that the use of an incremental technique resulted in significantly less microleakage than use of a bulk technique, regardless of the restorative system employed. They explained this by believing that incremental filling technique reduces stresses in the restoration interface.

On the other hand, Al-Harbi et al.[35], analyzed the cervical marginal integrity of bulk-fill versus incremental and open-sandwich class II resin composite restorations after thermo-mechanical cycling and reported that bulk-fill composites provide similar marginal performance to open-sandwich and incremental composites. Such a variation in result can be related to the difference in cavity configuration and testing methodology.

In addition, Heintze et al.[36], evaluated the marginal quality of composite resin restorations placed in extracted molars either in bulk (4 mm) or three increments. They found that there was no statistically significant difference between the resin restorations placed in bulk and those placed in three increments as they concluded that the adhesive system, rather than the incremental technique, was the determining factor for marginal quality.

Therefore the previous conclusion and the present confliction lighted up the idea of considering the use of a total-etch adhesive with the flowable composite system, compared to self-etch adhesive with the nanohybrid and bulk-fill composite systems a reason for the low nanoleakage mean values of flowable composite, confirming the conclusion that total-etch adhesives results in higher bond strength values compared to self-etch adhesives [37],[38],[39].

It was worthy to mention that the self-etch adhesive does not totally remove the smear layer or open all the tubules since the material has higher pH values than those used with total-etch adhesive systems. In addition self-etching materials are not rinsed away thus, the smear layer or its components are incorporated into the bonded layer[40]. Furthermore, as the self-etch adhesive systems only modify the smear layer, the presence of residual water may lead to incomplete polymerization of the adhesive, limiting resin-dentin bond quality[41].

Another explanation could be related to the fact that self-etching adhesives contain concentrations of water, acidic monomers, and hydroxyethylmethacrylate which makes these polymers very hydrophilic and likely to absorb water into the dentin interface and act even after polymerization as semi-permeable membranes[17]. This means that increased silver uptake into the hybrid and adhesive layers resulting in higher amount of nanoleakage. Furthermore, Sano et al.[13], found that the resin-dentin interfaces bonded by self-etching primers demonstrated the presence of a fine network of silver deposits within thin hybrid layers formed by these systems.

The current findings agreed with Al-Agha and Alagha[39], who studied nanoleakage of two nanofilled adhesive systems by environmental SEM and the energy dispersive analytical radiograph and found that, the self-etch adhesive had statistically significant higher nanoleakage mean values than the total-etch adhesive. Also, this result was in agreement with Kukletová et al.[42], and Gateva and Dikov[43], who studied nanoleakage of adhesive systems by transmission electron microscopy, and reported that, although a certain amount of nanoleakage was observed in all the tested groups, it was more pronounced for the self-etch adhesives when compared to the total-etch adhesives.

Similarly, Kubo et al.[44], found that conventional total-etch adhesive systems tended to show less nanoleakage than the self-etch adhesives when they used the field emission-SEM. This may be due to the specific characteristics of different adhesive systems that determine the degree of smear layer removal, demineralization of the underlying dentin, as well as the ability of the adhesive to wet and penetrate the dentin.

Therefore, it might be concluded that the difference in nanoleakage seemed to be dependent on the dentin adhesive systems used due to variable infiltration ability or difference in resin monomer composition or solvent. The self-etching adhesives are more hydrophilic than the total-etch adhesive systems and more permeable to water originated from dentin and, therefore more susceptible to degradation of resin-dentin bonds[45].

This holds true specifically that other studies performed by Hashimoto et al.[46], and Malekipour et al.[47], considered the type of adhesive system as influential in the leakage of composite restorations as different nanoleakage patterns were observed with different adhesive systems. Furthermore, different parameters of the application technique (e.g. etching time, dentin moisture) could also play a role in this respect[48]. On the other hand, Owens et al.[49], found no statistically significant difference in nanoleakage between dentin bonding agents, this might be due to the difference in evaluation methodology.

However, the current results were not consistent with Durate et al.[50], who studied the nanoleakage pattern in the dentin hybrid layer by using different dentin adhesives. They randomly assigned the specimens to two total-etch and two self-etch dentin adhesives They concluded that self-etch adhesive systems resulted in the least penetration of silver nitrate within the hybrid layer using field emission-SEM and transmission electron microscopy as they have the potential to form a hybrid layer and seal dentin. Also, the collagen fibrils are not completely deprived from hydroxyapatite in contrast to total-etch adhesives. This may be due to the difference in pH of currently used self-etching adhesive with (pH = 2.5 − 3.0) compared to the strong self-etching adhesives (pH = 1) used in the previous research, which resulted in an interfacial ultra-morphology resembling that produced typically by total-etch adhesives with the formation of abundant resin tags.

Also, the results are not harmonizing with Vaysman et al.[51], who observed that the highest reduction in bonded dentin permeability and increase in sealing ability occurred when the self-etching adhesive systems was applied to the roughest dentin surface prepared with the extensively serrated carbide bur. The difference in results may be because they used different bur cutting surface roughness (conventional straight edge bur, cross-cut serrated bur, and extensively serrated bur) which are different than those used currently. They explained their findings by the high surface roughness produced using extensively serrated bur in their samples might have increased dentin surface area, allowing better infiltration of the bonding resins.

Also, Hegde et al.[52], did not agree with the current findings. They held a study to evaluate the resin-dentin interface, quality of the hybrid layer of total-etching and self-etching adhesive systems under SEM. They concluded that the adaptation of self-etch adhesives to the resin-dentin interface was good without voids or separation of phases; showing a thin, continuous hybrid layer. This difference could be related to type of tested bonding agents since they used XP bond, Adper Single Bond II, Adper Easy One, and Xeno V, While currently Futurabond M+, Tetric N-Bond and Tetric N-Bond and N-Etch were used.

This present study assessed the aging of tested materials in terms of thermal and mechanical load cycling. It was found that aging increased nanoleakage significantly in all tested groups. This was in accordance with Kubo et al.[53], and Swathi et al.[54], who stated that nanoleakage of composite restorations occurs because of stress placed along the tooth–restoration interface due to various factors, such as polymerization shrinkage, thermo-cycling in the oral environment and mechanical fatigue through repetitive masticatory loading.

Also, the present findings were in agreement with Bedran-de-Castro et al.[55], and Ameri et al.[18], who concluded that cyclic loading increase nanoleakage of the margins of class V composite restorations. Furthermore, our findings were in agreement with Abo El Naga et al.[56], who concluded that aging of the two tested adhesive systems, as a function of cyclic loading, increased nanoleakage.

On the other hand, the current results did not confirm those of Li et al.[57], who stated that the application of mechanical cyclic loading did not increase nanoleakage. They had more than one explanation for their findings: first, after hybridization of the adhesive and demineralized dentin via micromechanical interlocking, the bond between the adhesive and dentin obtained from the dentin bonding agents used, was believed to be strong enough to resist a moderate amount of occlusal force for some time. Second, the forces and movements during mastication are highly complex. Also, in-vitro test conditions simulate but not duplicate clinical conditions. In addition, the number of load cycles in the laboratory may only represent several days' or months' masticatory forces experienced in the mouth.

In addition, from the results of the present study the lowest marginal adaptation was found in the cervical regions of all groups as compared to the occlusal regions of all groups. This is possibly because class V cavities mostly have gingival margins with thin enamel thickness or in dentin only while the occlusal margin was located in enamel, as a result the adhesion of the restorative materials would be more retentive at occlusal margin as compared to cervical margin[58].

This might also be explained by the high mineral content of enamel which make bonding to it stable and predictable. In contrast, dentin contains a substantial proportion of water and organic material, primarily type I collagen. Dentin also contains a dense network of tubules that connect the pulp with the dentino-enamel junction. Near the amelo-dentinal junctions these tubules may branch. Heterogeneous dentin structure results in different surface chemistries and morphologies. Also, the orientation of dentinal tubules might affect the formation of the hybrid layer. In areas with perpendicular tubule orientation, the hybrid layer was significantly thicker than areas with parallel tubule orientation. Therefore, the dentin surface on the gingival floor of class V preparations might be a surface on which good hybrid layer formation is difficult[59].

These findings came in agreement with the study of Rasheed and Ismael[60], who compared the marginal adaptation of five different surface treatments at four different composite/tooth interface regions (gingival, mesial, distal, occlusal regions) of standardized class V cavity restoration after thermal changes and mechanical load cycling. They found that the lowest marginal adaptation was found in the gingival regions of all tested groups as compared to the occlusal regions.

It was obvious then that more clinical evaluations should be carried out since it was one of the limitations of this in-vitro study the neglection of the effect of pulpal pressure on the used restorative materials.

In addition, it was concluded that bonding durability depends on chemical factors (such as acidic elements in dentinal fluid, saliva and food) and physical factors (such as occlusal chewing forces, repetitive expansion and contraction stresses due to temperature changes within the oral cavity)[17],[61],[62]. All of these factors are difficult to be analyzed in vitro, thus it is recommended to evaluate the nanoleakage of these tested materials in an in-vivo condition, to evaluate their bonding durability.

  Conclusion Top

Under the limitations of this in-vitro study, it was concluded that:

  1. Type of the tested composite systems is an effective variable on the recorded nanoleakage.
  2. Thermal and mechanical stresses increase nanoleakage significantly.
  3. The location of cavity margin is an important factor affecting nanoleakage; composite systems leak cervically more than occlusally.


  1. Nanoleakage evaluation of further composite systems rather than used in the current study is recommended.
  2. Use of different bulk-fill systems either preheated or Sonicfill is recommended.
  3. Use of different flowable systems on experimental animals is recommended.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2]


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