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Table of Contents   
ORIGINAL ARTICLE  
Year : 2020  |  Volume : 23  |  Issue : 5  |  Page : 497-504
Effect of layer thickness on the elution of monomers from two high viscosity bulk-fill composites: A high-performance liquid chromatography analysis


Department of Conservative Dentistry and Endodontics, Annoor Dental College, Ernakulam, Kerala, India

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Date of Submission19-Oct-2020
Date of Acceptance05-Dec-2020
Date of Web Publication10-Feb-2021
 

   Abstract 

Aim: The aim is to evaluate the effect of different layer thickness on the amount of elution of monomers from two high viscosity bulk-fill composites after 24 h and 1 month storage in ethanol using high-performance liquid chromatography (HPLC) analysis.
Materials and Methods: Forty-eight samples prepared from two high viscosity bulk-fill composite resins; Tetric EvoCeram Bulk Fill and x-tra fil were divided into three groups (n = 8) based on their layer thickness, i.e., Group 1 (2 mm), Group 2 (4 mm), and Group 3 (6 mm) and were then subdivided based on their storage period. The analysis of the eluates was performed using HPLC unit. Statistical analysis was performed using One-way ANOVA, independent sample t-test, and paired “t” test at a significance level of 0.05 (P < 0.05).
Results: Increase in layer thickness resulted in increased amount of bisphenol A-glycidyl methacrylate (Bis-GMA) and urethane dimethacrylate (UDMA) elution for both composites, i.e., Group 3 > Group 2 > Group 1 and there was statistically significant difference among all the groups. Tetric EvoCeram Bulk Fill showed a statistically significant increase in amount of Bis-GMA and UDMA elution for all groups compared to x-tra fil. Furthermore, increase in storage period resulted in statistically significant increase in amount of Bis-GMA and UDMA elution for both composites, i.e., Group 1B > Group 1A, Group 2B > Group 2A, and Group 3B > Group 3A. The mean value of UDMA elution was higher when compared to that of Bis-GMA elution for both composites and there was statistically significant difference for all groups.
Conclusion: Within the limitations of this in vitro study, it was concluded that increase in layer thickness resulted in increased amount of Bis-GMA and UDMA elution for both Tetric EvoCeram Bulk Fill and x-tra fil.

Keywords: Bisphenol A-glycidyl methacrylate; bulk fill composites; elution; high-performance liquid chromatography; monomers; Tetric EvoCeram Bulk Fill; x-tra fil

How to cite this article:
Nazar AM, George L, Mathew J. Effect of layer thickness on the elution of monomers from two high viscosity bulk-fill composites: A high-performance liquid chromatography analysis. J Conserv Dent 2020;23:497-504

How to cite this URL:
Nazar AM, George L, Mathew J. Effect of layer thickness on the elution of monomers from two high viscosity bulk-fill composites: A high-performance liquid chromatography analysis. J Conserv Dent [serial online] 2020 [cited 2021 May 17];23:497-504. Available from: https://www.jcd.org.in/text.asp?2020/23/5/497/309030

   Introduction Top


Resin-based composites are now considered as a benchmark for esthetic restorations and their use have increased tremendously over the past decade. Today, they represent one of the most important materials being used in dental practice. Resin-based composites are composed mainly of three major components which include a highly cross-linked polymeric matrix reinforced by a dispersion of glass, mineral, or resin filler particles and/or short fibers bound to the matrix by coupling agents. The polymerizable resin matrix consists of base monomers such as bisphenol A-glycidyl methacrylate (BisGMA) and urethane dimethacrylate (UDMA) along with low-viscosity monomer like triethylene glycol dimethacrylate (TEGDMA).[1]

The remarkable performance of these restorations, even in the masticatory load bearing posterior region, has now been definitely proven in many clinical studies. The major advantage of these composite restorations is the increased conservation of healthy tooth structure attained with these restorations as compared to amalgam restorations. Advances in the resin materials and handling techniques have further allowed resin based composites to render almost all the ideal requisites of a contemporary restorative material.

However, polymerization shrinkage and high residual monomer content are the major drawbacks associated with these composite materials.[2] In addition to these, the polymerization process also causes an increase in viscosity of the resin composites, leading to increased stiffness of the material.[3] Consequently, contraction stress occurs not only within the material but also at the bonded interface. This morphologic alteration has been thought to lead to negative sequelae, including postoperative sensitivity, contraction gaps, microcracks in the tooth structure, cuspal deformation, and recurrent caries.[4]

Incomplete monomer to polymer conversion leads to release of unpolymerized monomers from the dental composites. Numerous in vitro studies have been conducted on the toxicity and biocompatibility of these materials, which have concluded that some of the eluted monomers and additives may even have estrogenic, mutagenic, teratogenic, and genotoxic effects.[5] The elution of residual monomers, oligomers, and other degradation products into the oral environment has also raised concerns regarding these materials. This occurs by diffusion of the resin matrix or by its degradation or erosion over a period of time.

According to Ferracane,[6] the major factors affecting elution are the amount of polymerization reaction, chemistry of the solvent, and also the chemical nature and size of the released components. These unpolymerized monomers can get eluted and directly reach the pulp via dentinal microchannels. BisGMA has been known to have a substantial hemolytic potency which can cause time and concentration-dependent cytotoxicity in human gingival and pulp fibroblasts.[7]

Polymerization of light-cured resin composites is affected by a wide range of factors, including shade, light absorption and dispersion within the resin composite, types of fillers and monomer, and light irradiance conditions. Resin monomers notably have a decisive influence on the characteristics of polymerization. During the course of polymerization, generally, the intermolecular separations between the dimethacrylate monomers are reduced due to the conversion of carbon–carbon double (C = C) bonds, resulting in volumetric shrinkage.[8]

Various clinical approaches have been attempted to counter the effects of polymerization shrinkage and contraction stress such as the sandwich technique using glass-ionomer cements, and the use of low-viscosity flowable resin composites.[9] However, these approaches shows increase in the number of clinical steps required and thereby the technique sensitivity. An incremental placement technique has been used by dentists to minimize the polymerization shrinkage but is generally time-consuming and cumbersome. Hitherto, an increment layering technique in a thickness of 2 mm or less has been the standard to effectively convert the monomers.[10]

Recently, a novel category of resin based composites called “Bulk-fill” composites have been introduced with the main highlight that increments of up to 4 mm can be placed and cured in a single step thereby reducing the chair side time. Furthermore, these composites have a short activation time due to the presence of modified initiation systems and increased translucency owing to increased filler particle size and decreased filler load.[11] Although there is no stated consensus on a particular classification, bulk fill resin composites can be described either as low-viscosity that are similar to flowable resin composites or as high viscosity that resemble conventional resin composites.[12]

Numerous studies have been performed multifariously on the mechanical properties of bulk-fill composites.[13],[14] Consequently, Cuspal deflection, marginal integrity of a restoration as well as depth of cure of bulk-fill composites have shown better results compared to composites which are added using the incremental technique. In spite of all, adverse results have also been found associated with bulk-fill composites when compared to conventional composites such as the conversion rate. A clinically acceptable conversion rate of >55% for bulk-fill composites has been found, but it is howbeit less when compared with that of conventional composites.

It has been shown that monomer elution from bulk-fill composites are comparable to that of conventional materials despite their increased layer thickness of 4 mm. However, the amount of data available on the extent of effect of layer thickness on the amount of monomer elution from high viscosity bulk-fill composites is limited. Therefore, this study aims to evaluate the effect of different layer thickness on the amount of elution of monomers from two high viscosity bulk-fill composites after 24 h and 1 month storage in ethanol using high-performance liquid chromatography (HPLC) analysis.


   Materials and Methods Top


The composites which were tested including their manufacturers' data are listed in [Table 1].
Table 1: Investigated dental materials, manufacturer; composition of each material based on manufacturer's data; curing time recommended by the manufacturer

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Sample preparation

Two high viscosity bulk-fill composite resins Tetric EvoCeram Bulk fill (Ivoclar Vivadent, Schaan, Liechtenstein) and x-tra fil (VOCO, GmbH, Germany) were used [Table 1]. From each of them, 24 samples were prepared with a total of 48 samples. For each composite, samples were divided into three groups of 8 samples each based on different layer thickness of composite resin: Group 1 (2 mm), Group 2 (4 mm), and Group 3 (6 mm). Each group was again divided into two subgroups: Subgroup A and Subgroup B having 4 samples (n = 4) each based on storage periods of 24 h and 1 month in ethanol, respectively.

Three standardized silicone molds having 6 mm diameter and with 2 mm, 4 mm, and 6 mm thickness were used to prepare the specimens for Group 1, 2, and 3, respectively. The silicone molds were filled with uncured dental material, covered with plastic strips to prevent the formation of an oxygen inhibition layer and were finally polymerized with an light emitting diode (LED)-lamp (Bluephase C8, Vivadent, Schaan, Lichenstein), in accordance with the manufacturer's instructions for each specimen [Table 1]. The curing unit was applied directly on the surface of the specimen. An LED curing light intensity meter (Woodpecker LM-1, Woodpecker Medical Instrument Co., Ltd, Guilin, China) was used to verify the irradiance at each use of the light cure unit.

Immediately after curing, the specimens were immersed in 2 mL of ethanol. The storage of samples in Subgroup A was done for a time period of 24 h at room temperature and samples in Subgroup B were stored for 1 month.

Sample evaluation

After 24 h and 1 month, analysis of the eluates from Subgroup A and Subgroup B, respectively, were performed using the high-performance liquid chromatography (HPLC) unit. The separation of monomers took place with a CC 125/4 Nucleodur 100-5 C18ec HPLC Column. The mobile phase used was acetonitrile/water (75/25% v/v) at a flow rate of 1 mL/min, and the detection was performed at a wavelength of 254 nm for 30 min. For the analysis of extracted residual monomers, reference standards of BisGMA (CAS No. 494356, Sigma-Aldrich Chemical Co., USA) and UDMA (CAS No. 436909, Sigma-Aldrich Chemical Co., USA) were purchased and stock solutions were prepared by appropriate quantitative dilution. Twenty microliters from the stock solution was injected into HPLC system and standard chromatograms were obtained for both the monomers individually [Figure 1].
Figure 1: The retention time of high-performance liquid chromatography peaks of bisphenol A-glycidyl methacrylate and urethane dimethacrylate under the experimental conditions

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Statistical analysis

The data were subjected to statistical analysis using one-way ANOVA, independent sample t-test, and paired “t”-test. In all the analysis, significance level was taken to be 0.05 and statistical analysis was carried out using statistical package, SPSS version 24 (IBM Corp, Armonk, USA).


   Results Top


The mean values of amount of elution of monomers from Composite1 (Tetric EvoCeram Bulk Fill) and Composite 2 (x-tra fil) based at different layer thickness and storage periods in ethanol are shown in [Table 2]. The quantification and significant differences are shown in [Figure 2]a and [Figure 2]b.
Table 2: Amount of elution of monomers from composite 1 (Tetric EvoCeram Bulk fill) and composite 2 (x-tra fil) based on different layer thickness and storage periods

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Figure 2: (a) Graphical representation of mean values of bisphenol A-glycidyl methacrylate and urethane dimethacrylate elution of all groups of composite 1 (Tetric EvoCeram Bulk Fill) at different storage periods (24 h and 1 month respectively) (b) Graphical representation of mean values of bisphenol A-glycidyl methacrylate and urethane dimethacrylate elution of all groups of x-tra fil at different storage periods (24 h and 1 month respectively)

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Tetric EvoCeram Bulk Fill

  • The results of the study showed that increase in layer thickness resulted in higher amount of Bis-GMA and UDMA elution for Tetric EvoCeram Bulk Fill (Group 3 > Group 2 > Group 1). The highest amount of Bis-GMA and UDMA elution was recorded for samples having 6 mm thickness stored for 1 month (Group 3B) with mean values of 6.312 μg/ml and 187.688 μg/ml, respectively. One-way ANOVA showed statistically significant difference among all the groups [Table 3]. As there was significant difference, post hoc tests were conducted. The results of post hoc tests suggested that there was significant difference in mean value of Bis-GMA and UDMA elution among all the groups for Tetric EvoCeram Bulk Fill for both 24 h and 1 month storage periods
  • Tetric EvoCeram Bulk Fill showed greater amount of Bis-GMA and UDMA elution for all groups with highest mean values of 6.312 μg/ml and 187.688 μg/ml respectively as compared to x-tra fil with highest mean values of 3.371 μg/ml and 129.530 μg/ml, respectively. The results of independent sample t-test suggested that there was significant difference in mean value of Bis-GMA and UDMA elution among the two composites-TetricEvoCeram Bulk and x-tra fil (Table)
  • Mean value of UDMA elution was higher when compared to that of Bis-GMA elution. The results of the independent sample t-test suggested that there was significant difference in mean value of elution of Bis-GMA and UDMA for all groups (Table)
  • The increase in storage period resulted in increased amount of Bis-GMA and UDMA elution for both composites, i.e., Group 1B > Group 1A, Group 2B > Group 2A, and Group 3B > Group 3A. Amount of Bis-GMA and UDMA elution are higher for 1 month storage period with highest mean values of 6.312 μg/ml and 187.688 μg/ml respectively (Group 3B) as compared to samples stored for 24 h with highest mean values of 2.881 μg/ml and 105.710 μg/ml, respectively (Group 3A). The results of the paired t-test suggested that there was significant difference in mean value of Bis-GMA and UDMA elution of all groups of composite Tetric EvoCeram Bulk Fill at different storage periods.
Table 3: One-way ANOVA comparing mean values of bisphenol A diglycidyl methacrylate and urethane dimethacrylate elution among different groups for composite 1 (Tetric EvoCeram Bulk Fill) after 24 h and 1 month of storage in ethanol

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X-tra fil

  • The results of the study showed that increase in layer thickness and storage period resulted in higher amount of Bis-GMA and UDMA elution for Composite x-tra Fil. Highest amount of Bis-GMA and UDMA elution was recorded for samples having 6 mm thickness stored for 1 month (Group 3B) with mean values of 3.371 μg/ml and 129.530 μg/ml, respectively. One-way ANOVA showed statistically significant difference among all the groups [Table 4]. The results of the ANOVA suggested that there is significant difference in mean value of Bis-GMA and UDMA among the groups for composite 2 (xtra-fil) for both 24 h and 1 month. As there was significant difference, post hoc tests were conducted
  • Mean value of UDMA elution was higher when compared to that of Bis-GMA elution. The results of the independent sample t-test suggested that there was significant difference in mean value of elution of Bis-GMA and UDMA for all groups
  • Amount of Bis-GMA and UDMA elution are higher for 1 month storage period with highest mean values of 3.371 μg/ml and 129.530 respectively (Group 3B) as compared to samples stored for 24 h with highest mean values of 1.572 μg/ml and 65.244 μg/ml respectively (Group 3A). The results of the paired t-test suggested that there was significant difference in mean value of Bis-GMA and UDMA elution of all groups of composite x-tra Fill at different storage periods.
Table 4: One-way ANOVA comparing mean values of bisphenol Aglycidyl methacrylate and urethane dimethacrylate elution among different groups for composite 2 (xtra-fil) after 24 h and 1 month of storage in ethanol

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   Discussion Top


The light-cured resin-based composite restorations have lately become popular, including their use on posterior teeth, owing to its suitable mechanical behavior as well as their superior aesthetic attributes. However, restoring a cavity especially on the posteriors is a complex procedure and is usually carried out using an incremental technique.

The most common drawbacks of all resin-based composites are their incomplete polymerization; not all of the dimethacrylate monomer is converted into a polymer and considerable residual monomers persists within the final product. Numerous in vitro studies have been conducted on the toxicity and biocompatibility of these materials, which have concluded that some of the eluted monomers and additives may have estrogenic, mutagenic, teratogenic, and genotoxic effects.[5] The elution of residual monomers, oligomers, and other degradation products into the oral environment has also raised concerns regarding these materials. This occurs by diffusion of the resin matrix or by its degradation or erosion over a period of time. It has also been reported in previous studies that (co) monomers and additives cause concentration-dependent cytotoxic effects, such as increased cell death, damage of the plasma membrane or an increased content of released lactate dehydrogenase.[15] A study conducted by Reichl et al. concluded that the cytotoxic potency of basic monomers are in the following order: BisGMA > UDMA > TEGDMA > HEMA.[15] Yano et al. have also concluded that BisGMA has cytotoxic effects[16] and Söderholm and Mariotti revealed the estrogenic effects of Bis-GMA.[17]

In the present study, the elution of monomers from samples having layer thickness of 6 mm was also investigated as a worst-case scenario. The results of the current study showed that increase in layer thickness and storage period resulted in higher amount of Bis-GMA and UDMA elution for both composite 1 (Tetric EvoCeram Bulk Fill) and composite 2 (x-tra fil). Thus, the highest amount of Bis-GMA and UDMA elution was recorded for samples having 6 mm thickness stored for 1 month (Group 3B) with mean values of 6.312 μg/ml and 187.688 μg/ml respectively for Tetric EvoCeram Bulk Fill and 3.371 μg/ml and 129.530 μg/ml respectively for x-tra fil. There was statistically significant difference among all the groups for both composites. These results indicated that the elution values of methacrylates increase with increase in surface area which is in accordance with many previous studies.[18],[19] Pelka et al. concluded that the initial release of different components corresponds with the surface area of the composite material and the long-term release additionally depends on the weight and volume of the composite material.[19] Ilie et al. also concluded in their study that polymerization process becomes slower as layer thickness increases. This may be due to the attenuation of light in deeper layers, indicating that less photoinitiator molecules are activated.

It was established in the present study that increase in storage period resulted in higher amount of Bis-GMA and UDMA elution for both composite 1 (Tetric EvoCeram Bulk Fill) and composite 2 (x-tra fil) at all layer thickness with highest concentrations of monomers being found in samples stored for 1 month as compared to those stored for 24 h which is in accordance with the results of study conducted by Manojlovic et al.[20] but in contrast to results obtained by Sajnani and Hegde in which maximum amount of monomer elution was observed during the first 24 h.[21]

The current study employed 99.99% ethanol as a solvent, owing to its maximum ability to extract residual monomers and solubility parameter similar to BisGMA. The rate and extent of elution is greater in organic solvents, as compared to elution into pure water. This difference can be ascribed to the greater ability of the organic solvent to penetrate and swell the polymer network, thus facilitating the liberation of unreacted monomers and promoting a stronger degradative effect. It has been found that the eluted amount is usually proportional to the hydrophobicity and the swelling capacity of the organic solvent.[5]

In this study, the mean value of UDMA elution was higher when compared to that of Bis-GMA elution for both composites. The highest amount of UDMA elution was recorded for Group 3B samples with mean values of 187.688 μg/ml for Tetric EvoCeram Bulk Fill and 129.530 μg/ml for x-tra fil which was greater as compared to amount of Bis-GMA elution having highest mean values of 6.312 μg/ml and 3.371 μg/ml for Tetric EvoCeram Bulk Fill and x-tra fil, respectively. There was statistically significant difference in mean value of elution of Bis-GMA and UDMA for all groups. The size of the molecules is a significant factor affecting the elution of components of dental composites. Smaller molecules are supposed to have increased mobility and might therefore be eluted at a faster rate than larger molecules. The degree of conversion of different monomer systems increases in the following order, i.e., BisGMA < BisEMA < UDMA < TEGDMA.[22] BisGMA is considered to be the most viscous monomer among all due to its strong intramolecular hydrogen bonding, which decreases its reactivity and mobility during the polymerization process. In the present study, the total quantity of eluted residual UDMA was significantly greater than quantity of residual BisGMA. The probable explanation for this is the difference in molecular weight of the monomers, i.e., Bis-GMA > UDMA > TEGDMA. Tanaka et al.[23] discovered that small molecular weight monomers could be extracted in comparatively higher quantities than large molecular weight monomers. Therefore, considering the molecular weight of BisGMA and UDMA, BisGMA has higher weight, thus the release of UDMA is faster and in higher amount at a certain time interval.

The results of present study also showed that Tetric EvoCeram Bulk Fill showed greater amount of Bis-GMA and UDMA elution for all groups with highest mean values of 6.312 μg/ml and 187.688 μg/ml respectively as compared to x-tra fil with highest mean values of 3.371 μg/ml and 129.530 μg/ml, respectively. There was statistically significant difference in mean value of Bis-GMA and UDMA elution among the two composites. This is in accordance with the results obtained in the study conducted by Cebe et al. in which highest eluted monomer amounts were detected in Tetric EvoCeram Bulk Fill as compared to x-tra fil.[24] According to the manufacturer's data, composite 1 (Tetric EvoCeram Bulk Fill) does not contain TEGDMA. It has been reported that TEGDMA increases the degree of conversion of resin owing to its low molecular weight, lower viscosity, and higher reactivity compared to Bis-GMA and UDMA.[25] In addition, Tetric EvoCeram Bulk Fill also contains prepolymer fillers. These fillers are prepolymerized, and any residual unreacted or pendant C = C double bonds increase the final pool of leachable monomers. Hence, the absence of TEGDMA and presence of prepolymers could have contributed to higher elution of monomers from Tetric Evoceram Bulk Fill.

From the results, it was observed that an increase in layer thickness resulted in a higher amount of eluted bulk-fill composite components which in turn may lead to higher uptake in patients. The null hypothesis that the variation in layer thickness of bulk-fill composites has no significant effect on the amount of elution of monomers from the composites at different storage periods in ethanol was thus rejected.


   Conclusion Top


Within the limitations of this in vitro study, the following conclusions were drawn:

  • Increase in layer thickness resulted in increased amount of Bis-GMA and UDMA elution for both Tetric EvoCeram Bulk Fill and x-tra fil
  • Tetric EvoCeram Bulk Fill showed greater amount of Bis-GMA and UDMA elution for all groups when compared to x-tra fil
  • Increase in storage period resulted in increased amount of Bis-GMA and UDMA elution for both the composites, i.e., samples stored for 1 month showed greater amount of elution as compared to those stored for 24 h
  • The amount of UDMA elution was higher when compared to that of Bis-GMA elution for both Tetric EvoCeram Bulk Fill and x-tra fil.


Further considerations and investigations with suitable modifications in the composite materials, curing lights, or curing techniques are needed to reduce monomer elution from composite resins.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Correspondence Address:
Dr. Anciya Mohamed Nazar
Department of Conservative Dentistry and Endodontics, Annoor Dental College, Perumattom, Muvattupuzha, Ernakulam - 686 673, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JCD.JCD_535_20

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