Interfacial microscopic examination and chemical analysis of resin-dentin interface of self-adhering flowable resin composite

Background: The newly introduced self-adhering flowable resin-composites decrease the required time for application by incorporation of an acidic adhesive monomer, thus reducing the number of steps, but its bonding is still uncertain. The aim of this study was to evaluate the interfacial microscopic examination and chemical analysis at the resin-dentin interface of a self-adhering flowable resin composite (Vertise™Flow Self-Adhering Flowable Composite, Kerr Dental, USA) versus a total-etch (Te-Econom Plus) resin composite, using an etching agent (Eco-Etch gel) and bonding agent (Single Bond Universal). Methods: Sixteen freshly extracted sound human posterior teeth were used. The teeth were randomly divided into two groups: 8 specimens per type of composite. Standard-shaped class V cavities were prepared on the buccal surface. One group was restored by Te-Econom Plus resin composite by total-etch technique using Eco-Etch gel, which was applied to dentine for 15 seconds, followed by rinsing, drying and bonding agent application (Single Bond Universal). The other group restored directly with self-adhering resin composite (Vertise-Flow) without application of etch or bond. Curing was done for 20 seconds using a light emitting diode light curing unit. Evaluation of the resin-dentin interface was done microscopically by examination of marginal gap distance in μm using scanning electron microscope (SEM), and chemical analysis of silver particles was observed using SEM with energy-dispersive X-ray spectrometry after 24 hours of specimen storage in ammoniacal silver nitrate. Results: Regarding marginal gap distance (µm) and silver atomic % mean values, teeth restored with self-adhering resin composite (Vertise-Flow) showed significantly higher mean values than the multi-step etch and rinse resin composite group (5.2 vs 0; 12.2 vs 8.2, respectively). Conclusions: Resin-dentin bonding using total-etch resin composite technique was more effective than self-adhering flowable resin composite (Vertise-Flow) regarding marginal gap formation and penetration of silver particles. Further studies for bond strength could be performed.

was more effective than self-adhering flowable resin composite (Vertise-Flow)

Introduction
Adhesive dentistry has seen a paradigm shift from the invasive to be minimally invasive, due to a revolution in bonding systems. There are great demands for simplified restorative materials. A new selfadhering flowable resin composite (Vertise™ Flow Self-Adhering Flowable Composite, Kerr Dental, USA), was recently introduced onto the market. Bonding of flowable composites to tooth structure is achieved by incorporation of an acidic adhesive monomer into the material 1 . It is still a big challenge to seal the resin-dentin interface 2,3 .
The total-etch (etch and rinse) technique is a widely accepted technique to improve bonding of dental resins to tooth structure 2 . The dentin bonding mechanism is based on the micro-mechanical interlocking of the infiltrated resin monomers into porosities created in demineralized inorganic part 4 . Debonding of restorations may arise from gap-formation at the resin-dentin interface and hence recurrent caries, discoloration and tooth pain may follow 5 . Thus sufficient marginal seal should be obtained. Recently, an innovative self-adhesive and flowable resin composite was developed. These materials are claimed to eliminate the need for a separate step of bond-application, finally simplifying the restorative procedure. Therefore, the aim of this study was to evaluate the sealing performance of this new material.

Preparation of specimens
After attaining written informed consent from each patient to use their extracted teeth in research, sixteen sound human molar teeth were extracted in a private dental clinic (Dr. Tamer Hamdy Dental Clinic), which were randomly divided into two groups (eight specimens per group). Standard-shaped class V cavities (3 mm width, 3 mm length, 2 mm of depth) were prepared in the teeth using a #169L carbide bur (KG Sorensen, Brazil) on the buccal surface. One group's (Group A) cavities were filled with Te-Econom Plus ® (Ivoclar Vivadent, Africa) resin composite after etching and bond application. The etching agent, Eco-Etch gel (Ivoclar Vivadent), was applied to dentine for 15 seconds, followed by rinsing and drying. After rinsing, a bonding agent (Single Bond Universal, 3M ESPE, USA) was applied to teeth for 20 seconds, afterwards the teeth were air-dried for 5 seconds and light-cured for 10 seconds. Finally, the Te-Econom Plus resin composite was applied. The other group's (Group B) cavities were filled with self-adhering resin composite (Vertise™Flow Self-Adhering Flowable Composite, Kerr Dental, USA), which was applied without etch or bond. Curing was done for 20 seconds using a light emitting diode (LED) light curing unit (Satelec, Acteon, France).

Interfacial microscopic examination
All teeth were stored in distilled water for 24 hours at 37°C. Subsequently, the specimens were vertically sectioned with a diamond saw (Isomet, Buehler Ltd., USA) under water lubrication into approximately 1mm thick slab composed of tooth structure bonded to resin composite. These were examined for marginal gap distance in μm using scanning electron microscope (SEM; Model Quanta 250 FEG; FEI, Thermo Fisher Scientific, USA): accelerating voltage 30 K.V., magnification 14x up to 1000000 and resolution for Gun.1n, to ensure high brightness and resolution at low accelerating voltage.
Chemical analysis of the interface Specimen slabs were placed in freshly prepared 50 weight % ammoniacal silver nitrate solution for an additional 24 hours at 37°C in the dark. Ammoniacal silver nitrate solution (pH=9.5) was prepared according to Tay et al. (2002) 6 . After 24 hours of storage in silver nitrate solution, the silver impregnated specimens were rinsed thoroughly in distilled water and placed in photo-developing solution for 8 hours under a fluorescent light (200 Watt) 3 .
The specimens were then observed under environmental SEM Model Quanta 250 FEG attached with energy-dispersive X-ray (EDX; Inspect S 50, FEI, Netherlands): accelerating voltage 30 K.V., magnification 4000x and resolution for Gun.1n. The Blackscattered backscattered electron mode was used for elemental analysis of the atomic silver %.

Statistical analysis
Numerical data were explored for normality using Kolmogorov-Smirnov and Shapiro-Wilk tests, followed by Student's t-test to compare between both groups. The significance level was set at P ≤ 0.05. Statistical analysis was performed with IBM ® SPSS ® Version 20 for Windows (SPSS Inc., IBM Corporation, USA).

Results
Regarding marginal gap formation, Group A showed a significantly lower mean gap distance values than Group B (p<0.001), as shown in Table 1

Discussion
A proper marginal seal of restoration is essential to improve the durability of resin composite/bonding systems 7 . Most of the clinical studies assessing the performance of an adhesive system use class V cavities 8 . EDX analysis permits identification of silver particles, thus giving an indication about the chemical analysis of the interface 9 .
Our results revealed better sealing ability of composites treated with multi-step etch and rinse technique, presenting lower marginal gap formation and lower penetration of silver particles compared to Vertise-Flow. The presence of exposed collagen fibers could increase micromechanical interlocking of the bonding agent within the dentin surface 10 . The poorer sealing of Vertise-Flow may be due to included adhesive monomer, the glycerol that etches instead of bonds to hydroxyapatite 11 .

Conclusions
Total-etch resin composite technique was more effective regarding marginal gap formation and penetration of silver particles as compared to a flowable resin composite (Vertise-Flow). Further studies on bond strength should be undertaken.

Grant information
The author(s) declared that no grants were involved in supporting this work.
The following points need to be corrected, otherwise it is approved is based on the micro-mechanical interlocking of the infiltrated resin monomers into porosities created demineralized inorganic part of dentin 4. by created part....

Comment: in demineralized inorganic
Chemical analysis of the interface Specimens slabs were then placed in freshly prepared 50 weight/volume % ammoniacal silver nitrate solution for an additional 24 hours at 37°C in the dark. Specim slab were placed.......

Comment:
en s lower mean gap distance values than Group B (p<0.001), as shown in Table 1 and Figure 1 and Figure 2. and Figur 1 and 2 Comment: es The SEM with EDX analysis results revealed that Group A showed significantly lower mean silver atomic % values than Group B (p<0.001), as shown in Table 2 A. Selected SEM/EDX analysis is shown in Figure 3 and Figure 4. This may be attributed to the fact that phosphoric acid included in acid-etch step demineralized the smear layer, exposing the collagen fibers of superficially demineralized dentin. These could increase micromechanical interlocking of the bonding agent within the dentin surface 10 The presence of exposed collagen fibers could increase....

Comment:
No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The scientific idea of this article to evaluate the adaptability of resin/dentine interface of the most commonly used direct aesthetic restorative material is superb.
The article lacks the name of the company & country of the main investigated material (flowable composite, Vertise-Flow).
Despite the used method is well known for detection of nanoleakage of restorative materials, it is smartly employed for assessment of adaptability of composite at resin/dentine interface.
The suggestions is logical but it is preferable to be separate item not involved in context of conclusion section.

Summary:
The article is presented clearly & accurately The study design is appropriate Sufficient details were provided except the name of the company & country of the investigated material (Vertise-Flow) The used method for detection of marginal gap is smartly employed The data are reproducible And the unique conclusion is obvious The suggestion is promising for further researches

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed. After reviewing the entire manuscript, several modifications were added to different parts of the manuscript.
For the Introduction part, it shortly covered the idea of investigated resin composites describing the aim of the study.

Materials and Methods described steps and method of slab preparation (missing a few details).
Results were objectively presented.
The results were vaguely discussed.
This manuscript is approved for publication after elaborating the changes required.

Specific Comments:
Bonding is achieved by incorporation of an acidic adhesive monomer into the flowable composites . Comment: Bonding of flowable composites to tooth structure is achieved by incorporation of an acidic adhesive monomer into the material.
It is still a big challenge to seal the resin-dentin interface, due to the heterogeneous nature of dentin, the wet tubular structure, composition and surface morphology and or improperly designed adhesives Comment: Too long sentence The total-etch (etch and rinse) technique is a widely accepted technique to improve bonding of dental resins . Comment: improve bonding of dental resins to tooth structure

...is based on the micro-mechanical interlocking of the infiltrated resin monomers into porosities created
demineralized inorganic part of dentin by . Comment: created in demineralized organic part....
Restoration debonding may arise from gap formation at the resin-dentin interface and hence recurrent caries, discoloration and tooth pain Comment: Debonding of restorations may arise from gap-formation ..........., discoloration and tooth pain may follow These materials are claimed to eliminate the need for a separate bonding application step, thus simplifying the restorative procedure Comment: to eliminate the need for a separte step of bond-application, finally simplifying the restorative procedure Thus, the aim of this study was to evaluate the sealing performance of this new material Comment: Therefore, the aim of.....  Table 1 and Figure 1 and Figure 2. Comment: and Figures 1 and 2 The SEM with EDX analysis results revealed that Group A showed significantly lower mean silver atomic % values than Group B (p<0.001), as shown in Table 2 A. Selected SEM/EDX analysis is shown in Figure 3 and

Table 1. Marginal gap formation (μm) between groups of teeth treated with different composites
Comment: groups of teeth restored with different...

Figure 1. Selected scanning electron microscopy shows absence of gap formation in dentin in teeth treated with total etch technique.
Comment: in dentin for teeth treated with....

Figure 2. Selected scanning electron microscopy shows presence of gap formation in dentin in teeth treated with self-adhering resin composite.
Comment: in dentin for teeth........ with a self-adhering ....  This may be attributed to the fact that phosphoric acid included in acid-etch step demineralized the smear layer, exposing the collagen fibers of superficially demineralized dentin. These could increase micromechanical interlocking of the bonding agent within the dentin surface Comment: Presence of exposed collagen fibers could increase....

Dataset 2. Scanning electron microscopy (SEM) showing gap formation (raw values included on the images) and SEM/energy dispersive X-ray analysis (EDX) at the resin-superficial dentin interface in teeth treated with total-etch technique (Group A) and self-adhering resin composite (Group B) (n=8/group/method)
The poorer sealing of Vertise-Flow may be due to the included adhesive monomer, which is called glycerol phosphate dimethacrylate "GPDM", that etches instead of bonds to hydroxyapatite . Comment: included adhesive monomer, the glycerol.........
Further studies for bond strength could be performed. It is important to emphasize that this study ignored the effect of oral condition, Comment: Further studies on bond strength should be undertaken.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? No competing interests were disclosed.

Competing Interests:
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