Effect of Saliva Contamination on Microleakage of Open Sandwich Restorations.

OBJECTIVES: The purpose of the present study was to evaluate the microleakage of conventional glass-ionomer, resin modified glass-ionomer and glass hybrid ionomer Class II open sandwich restorations with or without saliva contamination.
MATERIALS AND METHODS: Sixty extracted sound human molar teeth were used and 120 class II slot cavities were prepared in mesial and distal surfaces. The gingival margins were located 1 mm below the cementoenamel junction. All specimens were randomly divided in 4 groups (n=15): Group I: High-Viscous Glass Ionomer (Fuji IX GP) Group II: Resin Modified Glass Ionomer (Fuji II LC) Group III: Glass Hybrid Ionomer (Equia-fil Forte), Group IV: Composite Resin (G'aenial Posterior). In open sandwich restoration groups, glass ionomer materials were placed to gingival floor in 1 mm thickness and rest of the cavity was filled with resin composite. After the restorations in mesial surfaces had been performed, distal cavities were restored with the same protocol after saliva contamination. The specimens were thermo-cycled for 10000 cycles at 50C to 550C and immersed in methylene blue dye solution (% 0,5) for 24 hours. Then, they were sectioned vertically through the center of the restorations from mesial to distal surface with a water-cooled diamond saw with 1mm thickness. Subsequently, the dye penetration was evaluated with image analysis software. Data were statistically analyzed (p<0.05).
RESULTS: There was a statistically significant difference between gingival microleakage scores in no contamination groups, between high-viscous glass ionomer, Fuji IX GP and other materials tested (p<0.05). In saliva contaminated groups, there was no statistically significant difference between gingival microleakage scores (p>0.05). Additionally, there was not a statistically significant difference between the no contamination and saliva contaminated groups regardless of dental materials tested (p>0.05).
CONCLUSION: Within the limitations of this study, in open sandwich restorations, saliva contamination did not show an adverse effect on microleakage irrespective of dental materials tested. Glass hybrid ionomers and resin modified glass ionomers showed lower microleakage scores in gingival margins compared to high-viscous glass ionomer material in no contamination groups.

Introduction

Resin composites are the best and most used restorative materials for anterior and posterior teeth due to esthetic and mechanical properties. However, polymerization shrinkage and technique sensitivity are drawbacks of these materials. In some class II restorations, gingival margins could be located below the cementoenamel junction and marginal adaptation would be difficult to achieve (). The open sandwich technique had been demonstrated to overcome these problems (). According to this technique, a glass ionomer material is used to replace the dentin and also fill the cervical part of the box, which results in a part of the glass ionomer being exposed to the oral cavity (). This technique has some limitations due to the fact that the polymerization contraction stress of resin composite might affect the bond between glass ionomer and the dentin at gingival margin in the initial stages of glass ionomer setting reaction (-). Also, there were some concerns about adequate polymerization of resin portion of resin modified glass ionomer materials in deep cavities. Roberts et al. () recommended the use of conventional high powder/liquid ratio glass ionomers for cavities extending to the root surface. In previous studies, Dietrich et al. () and Lougercio et al. () showed that the use of resin modified glass ionomers for the open sandwich technique, improved the marginal adaptation of the restoration. Additionally, it was also stated that bonding of composite to resin-modified glass ionomers could not be a problem due to the HEMA content and these materials could easily adhere to the methacrylate-based resin materials, such as resin composites ().

In the last decade, a hybrid glass ionomer material, Equia-fil Forte (GC, USA), has been introduced to the dental market. Equia-fil Forte was produced by dispersed ultrafine and highly reactive glass particles. The molecular weight of the polyacrylic acid was optimized to give the material excellent mechanical strength and properties. There were numerous studies which evaluated the in vitro and in vivo performance of this glass hybrid ionomer system and successful results were obtained (-).

However, there is no consensus about techniques to minimize microleakage in gingival margins located on dentin in the dental literature. There is also a lack of information about highly viscous glass ionomers, used as a base material in open sandwich restorations and the presence of saliva contamination. Therefore, the aims of the present study are as follows:

To evaluate microleakage scores in gingival margin below the cemento-enamel junction in Class II open sandwich restorations using three different glass ionomer materials and to compare them with a resin composite group; To examine the effect of saliva contamination in microleakage scores of all groups tested.

The null hypothesis of the study was that a glass ionomer material type and saliva contamination have no effect on the microleakage of open sandwich restorations.

Materials and methods

Specimen Preparation

This study was approved by the Kırıkkale University Institutional Review Board (Project no: 2018.10.08). Sixty extracted sound human molar teeth were collected, cleaned and polished with pumice and rubber cups for 10 seconds and kept in 0.1% thymol solution for up to 6 months after extraction until the test procedures were performed.

Class II slot cavities were prepared in mesial and distal surfaces with the gingival cavosurface margins which were located 1 mm below the cementoenamel junction. The overall dimensions of the cavities were standardized: the buccolingual width of the cavities was 1/3 of intercuspal distance; the axiopulpal extension of the cavities was 3 mm. The internal angles were rounded and cavosurface margins were sharp without bevel. The dimensions of the cavities were verified with a periodontal probe. All of the cavities were prepared by the use of a high-speed hand piece with cylindrical diamond bur (# ISO110/012, Hicare Co, Guangzhou, China) under water cooling, which was changed in all 10 cavity preparations. Specimen preparations were performed by one operator who took a part in the study.

All of the materials were used according to the manufacturers’ instructions in this study and shown in Table 1.

Table 1

Material descriptions, lot numbers, manufacturers and chemical composition of the materials used in the study.

Material Description	Material	Lot Number	Manufacturer	ChemicalComposition
High-ViscousGlass Ionomer Cement	Fuji IX GP	1610171	GC Corporation Tokyo, Japan	Powder: %95 aluminum-fluoro-silicate glass, %5 polyacrylic acid powder.Liquid: %50 distilled water, %40 polyacrylic acid, and %10 polybasic carboxylic acid
Resin ModifiedGlass Ionomer Cement	Fuji II LC	1611011	GC Corporation Tokyo, Japan	%25-50 2-hydroxyethyl methacrylate (HEMA), %5-10 polybasic carboxylic acid, %1-5 urethane dimethacrylate (UDMA), %1-5 dimethacrylate
Bulk-fillGlass Hybrid Ionomer	EQUIA-Fil Forte	1707101	GC Corporation Tokyo, Japan	Powder: aluminum-fluoro-silicate glass, polyacrylic acid powder, pigment.Liquid: Distilled water, polyacrylic acid, polybasic carboxylic acid
MicrohybridResin Composite	G-aenial Posterior	1805101	GC Corporation Tokyo, Japan	Pre-polymerized fillers (16–17 μ).Silica, strontium, lanthanide fluoride, fluoroaluminosilicate.
UniversalAdhesive System	G-Premio Bond	1712042	GC Corporation Tokyo, Japan	Functional monomers (4-MET, MDP and MDTP) 10-MDP, acetone, dimethacrylate component, photoinitiator, butylated hydroxytoluene.

Teeth were randomly divided in 4 groups(n=15). In each specimen, mesial slot cavities were identified as no contamination groups and placement procedures of restorative materials in these cavities were described below. Thus, distal slot cavities in all restorative material groups were contaminated with saliva for 5 seconds with a micro brush, dried with cotton pellets and restored using the same protocol. Fresh human saliva was used in this study, collected immediately before application, from a healthy volunteer after the informed consent form had been signed.

Before the restorative material placement, a contoured matrix band with the Tofflemire matrix holder was placed around the teeth for restorative procedures and held by finger pressure against the gingival margin of the cavity.

Group I: High-Viscous Glass Ionomer (Fuji IX GP)

The capsule form of high-viscous glass ionomer cement (Fuji IX GP, GC, Tokyo, Japan) was activated and was placed into an auto-mixer (RotoMix, 3M Espe, St Paul, MN, USA), mixed for 10 seconds. The dentin was conditioned with 20% polyacrylic acid for 10 seconds, rinsed and dried with a cotton pellet. A restorative material was placed to gingival floor in 1 mm thickness with a capsule applier, a plugger was used for condensation procedure, excess material was cleaned with a micro-brush and left undisturbed for 120 seconds until the initial setting time completed. A universal adhesive resin (G-Premio Bond, GC, Tokyo Japan) was applied, air dried for 5 seconds and polymerized with a LED unit with a light intensity of 1200 mW/cm2(Elipar, 3M ESPE, USA) for 10 seconds and a resin composite (G’aenial Posterior, GC, Tokyo Japan) was applied with incremental technique and each increment was light-cured for 20 seconds.

Group II: Resin Modified Glass Ionomer (Fuji II LC)

The capsule form of the resin modified glass ionomer cement (Fuji II LC, GC, Tokyo, Japan) was activated and was placed into an auto-mixer, mixed for 10 seconds. The dentin was conditioned with 20% polyacrylic acid for 10 seconds, rinsed and dried with a cotton pellet. A restorative material was placed to gingival floor in 1 mm thickness with a capsule applier, a plugger was used for condensation procedure and excess material was cleaned with a micro brush. Then, RMGIC layer was polymerized for 10 s. A universal adhesive resin (G-Premio Bond) was applied, air dried for 5 s and polymerized for 10 s and a resin composite (G’aenial Posterior) was applied as described above.

Group III: Glass Hybrid Ionomer (Equia-fil Forte)

The capsule form of a glass hybrid ionomer material (Equia Forte, GC, Tokyo, Japan)was activated and was placed into an auto-mixer (RotoMix), mixed for 10 s. Cavity was rinsed and dried with a cotton pellet. A restorative material was placed to the gingival floor in 1 mm thickness with a capsule applier, a plugger was used for condensation procedure, the excess material was cleaned with a micro-brush and left undisturbed for 150 seconds until the initial setting time was completed. A universal adhesive resin (G-Premio Bond) was applied, air dried for 5 seconds and polymerized with a LED unit (Elipar S10, 3M ESPE, USA) and a resin composite (G’aenial Posterior) was applied as described above.

Group IV: Resin Composite

In this group, a universal adhesive resin (G- Premio Bond) was applied, air dried for 5 seconds and polymerized for 10 seconds. A resin composite material (G’aenial Posterior) was applied with incremental technique as described above.

After the restorative procedures, all teeth were stored in distilled water for 24 hours at 37oC in an incubator. The restorations were finished with ultra-fine grain diamond burs (#ISO 166-016, #ISO 257-018, Hicare Co, Guangzhou, China) and polishing was performed with fine grit silicone polishers (Nais Ltd, Sofia, Bulgaria) and aluminum oxide disks (Super Snap Rainbow Technique Kit, Shofu Dental, Kyoto, Japan) under water cooling.

Thermocycling and Dye Penetration of the Specimens

Specimens were kept in distilled water at 370C for 24 hours and they were thermo-cycled for 10000 cycles at 50C to 550C (MTE 101 Thermocycling Machine, Esetron, Ankara, Turkey). The immersion time of each bath was 30 seconds and internal time was 10 seconds. The apical parts of the teeth were sealed using a resin composite (Filtek Z250, 3M, Espe, St. Paul, MN, USA) then they were coated with nail varnish up to 1 mm to the restoration margins. Then, specimens were immersed in a % 0,5 methylene blue dye solution for 24 hours. After immersion procedure, all teeth were rinsed under running water. They were embedded in autopolymerizing acrylic resin through the 1/3 of the root apices. They were sectioned vertically through the center of the restorations from mesial to distal surface with a water-cooled diamond saw with 1mm thickness (Microcut 151, Metkon, Istanbul, Turkey).

Dye penetration was evaluated at gingival, occlusal margins and interface between glass ionomer and resin composite at 20X magnification using a stereomicroscope (Nexius Zoom, Euromex Microscopen BV, Arnheim, Netherlands). Photographs were saved as TIFF images and processed with a MacBook device. The extent of dye penetration was evaluated quantitatively using the image analysis software (Image J,V.1.42, National Institutes of Health, Bethesda,MD).

Statistical Analysis:

The results were statistically analyzed using the statistical software IBM SPSS for Windows version 22.0 software package (IBM SPSS, New York, ABD). The data were analyzed using One-way ANOVA with the significance level set at 0.05. The Tukey HSD and Student t-tests were performed for multiple comparisons.

Results

The mean microleakage scores of all groups were shown in Table 2 and Figure 1.

Table 2

Mean microleakage scores of groups tested(Mean±SD).

	NO CONTAMINATION		SALIVA CONTAMINATED
	OCCLUSAL	GINGIVAL	OCCLUSAL	GINGIVAL
GROUP I	0.1±0.02a	1.07±0.85b	0.1±0.02a	0.66±0.58a
GROUP II	0.24±0.44a	0.49±0.59a	0.39±0.48a	0.65±0.83a
GROUP III	0.1±0.02a	0.44±0.49a	0.18±0.29a	0.36±0.59a
GROUP IV	0.17±0.21a	0.19±0.18a	0.12±0.07a	0.15±0.13a

*Groups with distinct superscripts exhibit statistically significant differences in column(p<0.05).

Figure 1

Microleakage scores of groups tested.

There was a statistically significant difference between no contamination groups (p<0.05). The High-viscous glass ionomer group, Fuji IX GP demonstrated higher gingival microleakage scores than other groups tested. However, in saliva contaminated groups, gingival microleakage scores indicated similar results and no statistically significant difference was shown (p>0.05). The results of the present study also demonstrated that saliva contamination did not significantly affect the microleakage scores in no contamination and saliva contaminated groups regardless of margin localization and dental materials tested (p>0.05). No dye penetration was observed in the interface of glass ionomer materials and resin composite in open sandwich restoration groups.

Discussion

The present study evaluated the microleakage of class II slot open sandwich restorations performed with a high-viscous glass-ionomer, resin modified glass-ionomer and glass hybrid ionomer materials in case of saliva contamination. According to our results, in no contamination groups, glass ionomer material type affected the gingival microleakage scores. However, saliva contamination did not demonstrate adverse effects in microleakage scores. Therefore, the null hypothesis was partially rejected.

Although different techniques have been used to evaluate microleakage (, ), the dye penetration method was selected for the present study. Although, the semi quantitative dye penetration technique was widely accepted method for in vitro studies, clinical relevance was questioned in a recent systematic review (). However, this technique is easy and fast. Also, it is still a preferred screening method to compare restorative materials (-).

The gingival margin of Class II restorations, extended to the root surface demonstrated microleakage according to isolation problems, polymerization shrinkage of composite materia l () and distance to the tip of the polymerization unit (). These factors could lead to an inadequate marginal adaptation, thus affecting the longevity of a resin restoration (). The difficulties with class II restorations have led to the development of open sandwich technique, as glass ionomer or resin modified glass ionomer material, which is placed to gingival margin and completed with a resin composite in occlusal part of the cavity. Koubi et al. () observed that in open sandwich restorations, resin modified glass ionomer material, Fuji II LC was the best intermediate material. However, Moazzami et al. () evaluated the effect of different materials (resin modified glass ionomer, compomer and self-curing resin, flowable composite resin) on the gingival microleakage of Class II open sandwich restorations. As a result, none of the materials enhanced the microleakage scores. In the present study, in no contamination group, high-viscous glass ionomer group showed the greatest leakage scores, which is in accordance with the results of a previous study conducted by Lougercio et al. (). The authors compared the gingival microleakage of Class II resin restorations and open sandwich restorations and found poor results in a traditional glass ionomer group. Some studies (, , ) found out that the contraction forces which occurred during polymerization of resin composite were sufficiently strong to disrupt the bond between a glass ionomer and the dentin, mainly in the initial stages of the glass-ionomer cement maturation. Hence, they might be blamed for the poor results observed in this group. However, in the present study, there were no statistically significant differences in gingival leakage scores in saliva-contaminated groups. This may be explained by the behavior of conventional glass ionomer at the time of placement. The dentin at the dentin-restorative interface should not be over-dried and saliva could moisturize the surface, which resulted in a good adhesion in our study. It could be also related to the chemical composition and the technique sensitivity of materials tested. Similar to our results, Dietrich et al., in a previous study (), reported that sandwich restorations seem to be less sensitive to contamination with saliva and blood. The results of some studies demonstrated no effect of saliva contamination on bond strength and marginal adaptation (, ). Dursun and Attal () also demonstrated that saliva contamination, regardless of time, did not significantly affect the dentin bond strength when a self-etch adhesive system was used with a resin modified glass ionomer. Saliva contamination time could have an important effect on these results since we contaminated the cavities before acidic primer application for high-viscous and resin modified glass ionomer groups and adhesive application for resin composite groups. Ludlow et al. showed higher microleakage scores when the contamination occurred after the acidic primer application ().

In the present study, among experimental glass ionomers, the best marginal sealing was obtained when a hybrid glass ionomer (Equia-fil Forte) was used and the open sandwich technique was employed. However, a conventional glass ionomer Fuji IX GP showed the worst microleakage results in no saliva contamination groups. Additionally, the best marginal leakage scores were obtained in the resin composite group. In a previous study, Miller et al. () evaluated the marginal microleakage of different materials in sandwich restorations and obtained good results when resin modified glass ionomer and compomers were used as a base material under the resin composites. They obtained good results even in the resin composite group, without any base material in the gingival margin.

One of the limitations of the open sandwich technique was adaptation of two different material increments and bonding the interface of the materials in a restoration. Shafiei and Akbarian () demonstrated no dye penetration at the interface of a nano ionomer and composites specimens, or only a slight dye penetration. Similar to their results, in our study, perfect adhesion was obtained between all glass ionomer materials and the resin composite layer. This result could be associated with the use of universal adhesive, posterior composite and incremental technique, achieving a good bond between materials in the present study. Universal adhesives contain functional monomers such as 10-MDP and can be used in self-etch or etch-and-rinse mode. Simple clinical procedures and ability to bond to various substrates gained popularity to these adhesive systems. Francois et al. () demonstrated similar shear bond strength values of resin composite bonded to Equia-fil Forte using a universal adhesive in self-etch mode and in etch-and-rinse mode. In our study, G-Premio Bond universal was preferred in self-etch mode and it demonstrated good results in control and saliva-contaminated groups in terms of microleakage. In difficult clinical conditions, this technique could be less time-consuming and less sensitive to achieve better marginal adaptation in class II extensive restorations.

Czarnecka et al. () evaluated the adhesion of Fuji IX GP, Fuji II LC and two other glass ionomer materials in slot cavities and noticed problems in establishing a contact between the material and internal walls of the cavity due to handling and the placement of a thin layer of cement. Although the capsule form of all glass ionomers was used in our study, it was not easy to control fluidity of glass ionomer material in the cavity as a base. In some microleakage studies (, , ), glass ionomer materials were applied to the entire cavity using the bulk technique and from clinical point of view, this could be easier than to place a thin layer at the gingival margin. Besides, the application tips of the capsules were not designed to reach narrow spaces such as gingival margins of slot restorations.

Recently, a new material with all advantages of glass ionomers such as fluoride release, hydrophilic nature and enhanced mechanical properties has been introduced to the dental market (-). Francois et al. () speculated that this resin-free and biocompatible characteristics might be useful for the sandwich technique or proximal margin sealing. Moshaverinia et al. reported that Equia-fil Forte is a powerful restorative material with a wide range of clinical applications in dental practice (). In another study, it was reported that Equia Forte was significantly more rigid and less deformed than other materials under functional stress, thus supporting its indication for posterior restorations (). This in vitro result was supported by a clinical follow up study of class II restorations performed by Gürgan et al. () The development of such materials might be promising for the clinicians in terms of restorations extended margins to the root surface. In the present study, Equia-fil Forte demonstrated acceptable microleakage results at gingival margins compared to Fuji II LC and G’aenial Posterior without any conditioner application.

One of the limitations of the present study was only thermocycling which was performed as aging procedure for the restorations. However, mechanical loading could affect the marginal adaptation of restorations in gingival area and the results could mimic clinical conditions in a better way than in the current study. Further studies are needed to clarify this issue in the future. Laboratory tests have also numerous drawbacks. The clinical behavior of restorative materials needs to be simulated. The obtained results need to be supported by long-term clinical trials.

Conclusion

Saliva contamination mostly occurs in posterior restorations with gingival cavosurface margins located in the root dentin. Within the limitations of this study, no adverse effect of saliva contamination has been demonstrated on microleakage of restorations performed with open sandwich technique in gingival and occlusal margins regardless of dental materials tested. Glass hybrid ionomers and resin modified glass ionomers showed lower microleakage scores in gingival margins compared to a high-viscous glass ionomer material in no contamination groups, hence they might have been preferred in cases where isolation was not a problem.