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Table of Contents   
ORIGINAL RESEARCH  
Year : 2013  |  Volume : 24  |  Issue : 1  |  Page : 81-86
In vitro cytotoxicity of indirect composite resins: Effect of storing in artificial saliva


Department of Prosthodontics, Faculty of Dentistry, Gazi University, Ankara, Turkey

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Date of Submission28-Apr-2010
Date of Decision02-Jun-2010
Date of Acceptance18-Jun-2010
Date of Web Publication12-Jul-2013
 

   Abstract 

Aim: The aim of this study was to compare the cytotoxic effects of two indirect composite resins (Artglass and Solidex) on the viability of L-929 fibroblast cells at different incubation periods by storing them in artificial saliva (AS).
Materials and Methods: Disk-shaped test samples were prepared according to manufacturers' instructions. Test materials were cured with light source (Dentacolor XS, Heraus Kulzer, Germany). The samples were divided into two groups. The first group's samples were transferred into a culture medium for 1 hour, 24 hours, 72 hours, 1 week and 2 weeks. The other group's samples were transferred into a culture medium for 1 hours, 24 hours, 72 hours, 1 week, and 2 weeks after being stored in AS for 48 hours. The eluates were obtained and pipetted for evaluation onto L-929 mouse fibroblast cultures incubated for 24 hours. Measurements were performed by MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The degree of cytotoxicity for each sample was determined according to the reference values represented by the cells with a control group.
Results: Statistical significance was determined by ANOVA. Both groups presented lower cell viability in comparison to the control group at all periods. Storing in artificial saliva reduced cytotoxicity significantly (P < 0.05). Stored Artglass and Solidex showed similar effects on cytotoxicity. Nonstored Solidex samples were found more cytotoxic than Artglass samples. The cell survival rate results of 24-hour incubation period were significantly lower than those of the other experimental periods (P < 0.05).
Conclusion: Storing indirect composite resins in AS may reduce cytotoxic effects on the fibroblast cells. However, resin-based dental materials continue to release sufficient components to cause cytotoxic effects in vitro after 48 hours of storing in AS.

Keywords: Artificial saliva, cytotoxicity, indirect composite

How to cite this article:
Yildirim-Bicer AZ, Ergun G, Egilmez F, Demirkoprulu H. In vitro cytotoxicity of indirect composite resins: Effect of storing in artificial saliva. Indian J Dent Res 2013;24:81-6

How to cite this URL:
Yildirim-Bicer AZ, Ergun G, Egilmez F, Demirkoprulu H. In vitro cytotoxicity of indirect composite resins: Effect of storing in artificial saliva. Indian J Dent Res [serial online] 2013 [cited 2020 Nov 26];24:81-6. Available from: https://www.ijdr.in/text.asp?2013/24/1/81/114962
The second-generation laboratory composite or the "poly-glass" materials have been promoted as a hybridization of composite and ceramic technologies, although they are essentially still a composite resin matrix with differing filler components. [1] Their chemistry is identical to that of the conventional composite resins, although they usually have higher filler contents. [2],[3] The degree of conversion that occurs during polymerization and the type of matrix also influence the properties, especially when aging occurs in the oral environment. [1] They also allow significant postcuring under different laboratory conditions providing combinations of heat, pressure, vacuum, and light to improve their degree of polymerization. [2],[3] However a large proportion of polimerizable groups (residual monomer or short chain polymers) do not react and remain unbonded during curing in set dental composites. [3],[4],[5]

There is still limited information concerning the biological behavior of the second-generation composite resins used for indirect restorations. At the same time, their cytotoxic effects recorded so far vary widely depending on the modes of sample preparation, storing, curing conditions, and preparation of extracts. [6],[7],[8] In most cases, cytotoxicity has been attributed to the release of residual monomers or other substances, derived either from incomplete polymerization or resin degradation. [6],[9] These studies have shown that both the chemistry of the resinous matrix and the degree of polymerization contribute to the cytotoxicity of these composite resins. [6],[10] In an in vivo situation, it can be assumed that saliva, food components, and beverages may effect dental composites. Components may be leached into salivary fluids and/or may diffuse toward the pulp. [4] Several studies have shown the amount of components eluted from dental composites into various solutions. [3],[5],[11],[12],[13],[14] Storage solutions may contain leached soluble components from the resin composite which may provide information about resin degradation. [4],[14],[15],[16],[17] The leaching of components or degradation by products from dental composites has a potential impact on both the structural stability and the biocompatibility of the material. [4]

Unbound monomers and/or additives are eluted by solvents or polymer degradation within the first hours after initial polymerization. The release is due to defective photopolymerization, thermal, mechanical, or chemical factors. Interactions between resin monomers and commercial composite resins with human saliva-derived esterase and pseudocholinesterase occur in the oral cavity and they contribute to the degradation of composite resins. [18] HEMA and TEGDMA are the main monomers released from several resin-based materials. [4],[19],[20] Bis-GMA has also been reported to be released from several types of resin composite and fissure sealants. [20],[21] Released monomers have been found in saliva, dentin, and pulp after placement of resin-based restorative materials. [20],[22],[23] The release of these components into the surrounding tissue may cause an adverse local reaction or even systemic effects. [24]

Cell culture studies are frequently used to assess the cytotoxicity of resin-based materials, their elutes or components (such as monomers). [20],[25],[26] Variable levels of cytotoxicity have been shown to be induced by several resin-based materials and their components. However, a previous study has evaluated the relationship between cytotoxicity and the structures of resin monomers. [20]

The MTT (tetrazolium salt 3- [4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide) test has been used extensively to assess cytotoxicity of dental materials. The MTT assay indicates the effects on cell viability by alterations of mitochondrial dehydrogenase activities. [20],[27] In this test, methylthiazol tetrazolium is metabolically reduced to colored formazan. The factors that inhibit dehydrogenase activity will affect the associated color reaction. It has been shown that activated cells produce more formazan than resting cells; therefore it is possible to measure cell activity or enzyme activities. [20]

The aim of this study was to compare the cytotoxic effects of two second-generation indirect resin composites after being stored in artificial saliva on L-929 mouse fibroblasts.


   Materials and Methods Top


Sample preparation

Two commercially available indirect dental composite systems were investigated [Table 1].
Table 1: Materials used in this study

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A total of 50 disk-shaped samples (10 mm diameter × 1 mm thick) were prepared in the sterile circular polytetrafluoroethylene mould for each test material. The resin material was carefully packed into the mould. The resin was sandwiched between glass slides. Then test materials were polymerized with a photo-curing unit with a xenon stroboscopic light source (Dentacolor XS, Heraus Kulzer, Germany) emitting a total of 4.5 W as usable luminous power, whereas the emission range is between 320 nm and 500 nm. One side of Artglass test materials was cured for 180 seconds and the other side was cured for 90 seconds. Both sides of Solidex test materials were cured for 180 seconds. After the polymerization, both surfaces of the samples were sterilized in ultraviolet sterilizator (BS-4012 NbS Di. Co., Turkey) for 45 minutes. Then the samples were divided into two groups. The samples in the first group were tested without storing in AS (n = 50). The second group (n = 50) was stored in AS for 48 hours before testing of their cytotoxicity. One milliliter per specimen of AS containing 4.1 mM KH 2 PO 4 , 4.0 mM Na 2 HPO 4' 24.8 mM KHCO 3' 16.5 mM NaCl, 0.25 mM CaCl 2 [28] was used. The pH of AS was adjusted to 6.7 and then sterilized by filtration before use. After the samples being stored in AS for 48 hours, they were removed and placed at the bottom of six well plates. In the first group, the freshly prepared samples were placed immediately at the bottom of six well plates (Costar, Cambridge MA, USA.). The ratio of the surface area of the disk samples to the extraction volume was 1.88 cm 2 /ml -1 which was midrange in the ISO-2002 [29] in this study. The samples were placed in DMEM/F12 with 10% FBS and incubated at 37°C in an atmosphere of 5% CO2 in air without agitation for 1 hour, 24 hours, 72 hours, 1 week, and 2 weeks. After the incubation, the extracts were filtered through 0.22-μm cellulose acetate filters (Millipore, Sigma, USA) and then they were used to evaluate cytotoxicity.

Cells

The cells used for the experiments were L-929 mouse fibroblasts (L-929 An 2 HÜKÜK 95030802: Foot and Mouth Disease Institute, Ankara, Turkey). The cells were grown as monolayer cultures in T-25 flasks (Costar, Cambridge, MA, USA), subcultured three times a week at 37°C in an atmosphere of 5% CO 2 in air and 100% relative humidity and maintained at the third passage. The culture medium was Dulbecco's modified Eagle medium (DMEM)/Ham's F12 nutrient mixture (1:1; Sigma, St. Louis, MO, USA) supplemented with 10%(v/v) fetal bovine serum (FBS; Biochrom, Berlin, Germany) without antibiotics. Adherent cells at a logarithmic growth phase were controlled under an inverted tissue culture microscope (Olympus CK40, Tokyo, Japan) and detached with a mixture of 0.025% trypsin (Sigma, St. Louis, MO, USA) and 0.02% ethylenediaminetetraacetic acid (EDTA; Sigma, St. Louis, MO, USA), incubated for 2-5 minutes at 37°C and used for cell inoculation.

Cytotoxicity testing (MTT assay)

The L-929 cell suspension with DMEM/F12 with 10% FBS and 1% antibiotic was prepared at a concentration of 3 × 10 4 cell/ml and inoculated into 96-well cluster cell culture plates (100 μl per well). The multiwell plates were incubated at 37°C, 5% CO 2 in air for 24 hours. After 24 hours, the culture medium was removed from the wells and equal volumes (100 μl) of the extracts (0.44 mol) were added into each well. In control wells 100 μl DMEM/F12 with 10% FBS and 1% antibiotic was added. Then 96-well cluster cell culture plates were incubated for 24 hours at 37°C. After the 1 hour, 24 hours, 72 hours, 1 week, and 2 weeks of incubation period tests extracts were removed. Following removal of the tests extracts, 100 μl per well DMEM/F12 with 10% FBS and 1% antibiotic and 12 μl MTT were added to each well and incubated in a dark environment for 4 hours at 37°C. After incubation 96 wells were checked for formazan crystals with inverted tissue culture microscope. MTT was aspirated and 100 μl per well of isopropanol (Merck, Darmstadt, Germany) was added to each well. Subsequently, the absorbance at 570 nm was measured using a UV-visible spectrophotometer (LPB Pharmacia, Bromma, Sweden).

Then the visible cells were counted under a light microscope and calculated as a percentage of the controls. Triplicate experiments were performed throughout this study.

Statistical analysis

Statistical analysis was performed by Statistical Package for Social Sciences (SPSS) 11.5 software (SPSS Inc., Chicago, IL, USA). Data were expressed as mean ± standard deviation. Proportions of a live cells were evaluated by three-factor ANOVA. When the P value from the interaction terms is statistically significant, to determine which factor causes a difference, one-way ANOVA post hoc Tukey Student's t-test was used, where applicable. Statistical significance was defined at P < 0.05. For all possible multiple comparison tests, the Bonferroni correction was applied for controlling type 1 error.


   Results Top


Results showed that both of the test materials were moderately cytotoxic to L-929 mouse fibroblasts. The level of the cytotoxicity varied according to the tested materials, incubation periods and whether stored or not stored in AS [Table 2].
Table 2: The cell survival rates and standard deviations of each tested material in accordance with the nonstoring/ storing condition


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There was a significant difference between the stored and nonstored materials (F = 51.267, P < 0.05). Storing in AS reduced cytotoxicity significantly (P < 0.05). When the cell survival rate results of stored materials were compared with nonstored materials, stored tested materials demonstrated higher cell survival rates than nonstored ones [Table 3].
Table 3: The cell survival rates and standard deviations of each tested material in accordance with storing/nonstoring condition in AS and incubation period

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There were two-factor interactions between the tested materials and storing/nonstoring condition in AS (F = 4.650, P < 0.05), as well as the tested materials and incubation periods (F = 10.873, P < 0.05). Also a significant three-factor interaction occurred among the tested materials, storing/nonstoring condition in AS and incubation periods (F = 6.839; P < 0.05).

There was no significant difference between the stored materials (P = 0.947). Stored tested samples showed similar effects on cytotoxicity. However, a significant difference was found between the nonstored test materials (P < 0.05). Nonstored Artglass samples showed significantly higher cell survival rates than nonstored Solidex samples.

Also a significant difference was observed in incubation periods. The cell survival rate results of 24-hour incubation period were significantly lower than the other experimental periods (P < 0.05). When the interaction between nonstored test materials and incubation periods were evaluated, Solidex exhibited significantly more cytotoxic effect than Artglass in a 24-hour period (P < 0.05).


   Discussion Top


In vitro cytotoxicity of storing in AS of two indirect resin-based materials on L-929 mouse fibroblasts for 1 hour, 24 hours, 72 hours, 1 week, and 2 weeks of incubation periods was compared. An AS which reacts with the test material in a manner similar to that of natural saliva is the basic requirement of an artificial oral environment [6] such as, in vitro tests that require the chemical conditions pertaining in the mouth. [7] Therefore we decided to use AS containing inorganic components.

In the current study, the first group, the freshly prepared samples, were placed in medium immediately, the other group was put into artificial saliva for 48 hours. It is important to place the materials to be tested in the medium immediately after mixing/curing to avoid the loss of toxic substances released from the tested materials at this initial stage. [30]

Water or other solvents enter the polymer, leading to the release of biodegradation products, namely, oligomers and monomers. This form of erosion leads to weight loss of the polymer. Softening of the bis-GMA matrix allows the solvent to penetrate more easily and expand the polymer network, a process that facilitates the long-term diffusion of unbound monomers. [18],[22] In this study, when the results of stored materials compared to nonstored materials, it was demonstrated that the stored materials had statistically significant higher cell survival rates than nonstored ones [Table 2]. In other words, it was shown that the storing in AS for 48 hours had increased cell survival rates of the tested materials. Wataha et al. [28] reported that aging the resin specimens in artificial saliva significantly reduced the mass released. Also Ferracane and Condon [5] investigated the rate of the elution of molecules from a dental composite and an unfilled resin with time during soaking in either water or an ethanol/water mixture in a previous study and they implied that elution of nearly all of the leachable components was complete within a 24-hour period in either solvent. Our results agree with the work of theirs. [5],[28]

Investigations on the potential toxicity of these materials have therefore focused mainly on the early intervals. [25],[28],[31] Despite this focus, researches done by other investigators show that a substantial portion of resin-based materials remains unconverted to polymer and therefore may be available for longer term release. [28],[32] For this reason, in this study, cytotoxicity of indirect composites evaluated at 1 hour, 24 hours, 72 hours, 1 week, and 2 weeks. At early intervals, resin containing materials are more cytotoxic than at later intervals. However, long-term effects should also be taken into consideration. [18]

The present study revealed that, a significant difference was seen in incubation periods [Table 3]. The cell survival rate results of 24-hour incubation period were significantly more toxic than other experimental periods (P < 0.05). So it was concluded that the majority of the relevant release of substances from composites occur within 24 hours. Our result conformed to that of Ferracane and Condon. [5] They also implied that component release after 24 hours is negligible. Thus, the biological liability of resin materials has often been assumed to be limited to the first 24 hours. However, Wataha et al. [28] demonstrated that several commonly used resin-based restorative materials continue to release biologically relevant amounts of mass into AS in vitro even after storing them for 2 weeks. This result contrasts with those of some authors who have indicated that the extent of mass release from some of these materials is negligible after 24 hours.

Although unbound free monomers released by dental resins during and after polymerization are considered by a majority of authors to be responsible for the cytotoxic effects, additional mechanisms have been also proposed. Short-term release of free monomers occurs during the monomer - polymer conversion. Unbound monomers and/or additives are eluted by solvents or polymer degradation within the first hours after initial polymerization. The release is due to defective photopolymerization, thermal, mechanical, or chemical factors. [18]

Ferracane and Condon [5] reported that approximately 15-50% of the methacrylic groups remain unreacted. Usually composite resins are polymerized by photo-activation and free monomer may be released from resinous materials before and after polymerization. An insufficient photo-activation can contribute to increase in the level of unreacted monomers through a reduced degree of polymerization and cross-linking. [30],[33],[34],[35],[36],[37] A recent series of papers and data demonstrate the importance of the cytotoxicity of composite resins and therefore the importance of the sufficient polymerization with the light sources. [30],[36],[38],[39] The chemical characteristics of leachable substances determine the diffusion through the polymer network. Leachable components are released due to degradation or erosion over time. Chemical degradation is caused by hydrolysis or enzymatic catalysis. [18] The cytotoxic effect of Artglass was indicated in one of with the former studies. [40] In the present study, both nonstored test materials (Artglass and Solidex) had a statistically significant effect on cytotoxicity (P < 0.05) [Table 2]. Solidex was found more cytotoxic than Artglass. Brackett et al. [41] reported that filler load of the composites did not necessarily correlate with the cytotoxic responses, and suggested that the setting chemistry is a more important determinant to the biological response than the filler load. Thus, the difference in cytotoxicity between Artglass and Solidex could be related to the difference in the chemical composition of these materials, since Solidex containing bis-GMA may be responsible for this difference. The use of resin-based dental composites involves in situ polymerization, and because bis-GMA polymerization is never complete, unpolymerized monomers are known to remain in the polymer after the curing process. [37] Various studies have documented that bis-GMA is highly cytotoxic in both primary and immortal cell lines, including human pulp and gingival fibroblasts. [37],[42] Nevertheless, while comparing both aged tested materials, there was no statistically significant difference between their effects of cytotoxicity (P = 0.947) [Table 2]. However Al-Hiyasat et al. [24] demonstrated that the change in the chemical of the composite and variation in the ratio of filler and monomers have a significant effect on the element release and cytotoxicity level of the materials.


   Conclusion Top


The findings of the present study revealed that storing the indirect composite resin samples in artificial saliva for 48 hours had a significant effect on the cytotoxicity. On the other hand, most eluted substances are found to be cytotoxic ex vivo and therefore the materials may not necessarily be cytotoxic in vivo. From the clinical point of view, there are limitations regarding the correlation between ex vivo testing and clinical usage ones. Therefore, it is recommended that dentists soak the resin prostheses in water or artificial saliva at least 48 hours before placing them into the patient's mouth.

 
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Correspondence Address:
Gulfem Ergun
Department of Prosthodontics, Faculty of Dentistry, Gazi University, Ankara
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.114962

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    Tables

  [Table 1], [Table 2], [Table 3]

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Daniela Bastos TUMSCITZ,Laisa Araujo Cortines LAXE,Aislan Cristina Rheder Fagundes PASCOAL,Raphael HIRATA JUNIOR,Renata Ximenes LINS
Revista de Odontologia da UNESP. 2017; 46(4): 203
[Pubmed] | [DOI]
5 Effect of Artificial Saliva with Different pH Levels on the Cytotoxicity of Soft Denture Lining Materials
Canan Akay,Merve . Tanis,Handan Sevim
The International Journal of Artificial Organs. 2017; 40(10): 581
[Pubmed] | [DOI]
6 Effect of Artificial Saliva with Different pH Levels on the Cytotoxicity of Soft Denture Lining Materials
Canan Akay,Merve . Tanis,Handan Sevim
The International Journal of Artificial Organs. 2017; 40(10): 581
[Pubmed] | [DOI]



 

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