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Table of Contents   
ORIGINAL RESEARCH  
Year : 2015  |  Volume : 26  |  Issue : 3  |  Page : 284-288
Correlation between the cytotoxicity of self-etching resin cements and the degree of conversion


Department of Restorative Dentistry, Federal University of Minas Gerais, Belo Horizonte, Brazil

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Date of Submission19-Dec-2013
Date of Decision03-Feb-2014
Date of Acceptance29-Jun-2015
Date of Web Publication14-Aug-2015
 

   Abstract 

Aim: The aim of this study was to evaluate the correlation between the cytotoxicity and degree of conversion (DC) of self-etch resin cements with or without photopolymerization. Materials and Methods: Three self-etching resin cements with or without photopolymerization were evaluated. Six test groups and one control group represented by a standardized L929-fibroblast cell culture were formed. The DC was measured by Fourier transform infrared spectrometry and was correlated with cell culture survival. Statistical Analysis: The analysis of variance and Bonferroni–Holm tests were applied (P < 0.05). Results:The results show that, the cytotoxicity of self-etching resin cements is directly related to the DC. With the exception of Unicem, the other cements show some level of cytotoxicity, even with photopolymerization. Conclusion: These results indicate that photopolymerization of dual cure self-etching resin cements decrease toxic effects on cell culture. Adequate photopolymerization should be considered during cementation when using dual polymerization self-etching resin cements.

Keywords: Cell survival, polymerization, resin cement

How to cite this article:
Morgan LF, Teixeira KI, Vasconcellos WA, Albuquerque RC, Cortés ME. Correlation between the cytotoxicity of self-etching resin cements and the degree of conversion. Indian J Dent Res 2015;26:284-8

How to cite this URL:
Morgan LF, Teixeira KI, Vasconcellos WA, Albuquerque RC, Cortés ME. Correlation between the cytotoxicity of self-etching resin cements and the degree of conversion. Indian J Dent Res [serial online] 2015 [cited 2019 Sep 18];26:284-8. Available from: http://www.ijdr.in/text.asp?2015/26/3/284/162878
A large variety of materials is available for cementing indirect restorations. Due to the improved physicochemical properties as compared to conventional cements, that is, zinc phosphate, glass ionomer cement, and resin cements have been used. This choice is supported by the fact that resin cements offer adhesion to dentin and are insoluble in the dental environment; in addition, resin cements fill the gap between the indirect restorations and the remaining tooth structure, which favors frictional retention.[1]

Recently, self-etching resin cements via dual polymerization were developed. These materials emerged to simplify the surgical technique by eliminating the operative steps of etching and the application of the adhesive system. However, these dual polymerization cements depend on light for proper curing. These cements may not achieve an appropriate degree of conversion (DC) because indirect restorations are limited to the transmission of light.[2] Mechanically, incomplete polymerization of these cements results in lower adhesion.[3] The inadequate conversion of monomers into the polymers has a significant influence on the pulpal response of teeth to these materials.[4]

Resin-based adhesive materials have been shown to have potential harmful effects on the pulpal-dentin complex. The cytotoxicity of resin-based agents has been extensively studied,[5][6][7][8][9][10] but reports on the biological safety of self-etch resin cements in relation to their degrees of conversion are still rare.

Invitro cytotoxicity tests using L-929 fibroblast cells have the advantage of having easy control over the experimental factors that are often problematic when performing experiments in vivo. Moreover, these methods are reproducible, relevant, and suitable for the evaluation of basic biological properties of dental materials.[11]

The aim of the present study was to evaluate the correlation between the cytotoxicity of self-etch resin cements with or without photopolymerization and the DC. The hypothesis is all evaluated cements will show high cytotoxicity to fibroblasts independent of photopolymerization.


   Materials and Methods Top


Three self-etching resin cements dual polymerization were used in this study [Table 1]. Each cement represented two groups: One with light curing for 20 s and one without. The light intensity emitted from the curing light unit (Curing Light 3000, 3M ESPE, St. Paul, MN, USA) was continuously measured by a digital power meter (Nova, Ophir, Hicksville, NY, USA) and light intensity was standardized by preheated.[12] Rely-X Unicem without light curing represents Group 1 (G1); with photopolymerization, Group 2 (G2); set without light curing, Group 3 (G3); with the photopolymerization, Group 4 (G4); Bifix SE without light curing, Group 5 (G5); with photopolymerization Group 6 (G6). The fibroblasts without any resin cement representing the control Group 7 (G7). The manipulation of resin cements followed the instructions of each manufacturer. After inserted into the cavity side, with the tip of light cure made pressure and compressed by strips of polyester. Photopolymerization was performed for 2 min after insertion into the glass matrix by 20 s. The containment provided by the polyester strip was kept without reach of light. A sterilization method was used for ethylene oxide that allowed aeration for 7 days before the test. The specimens were tested for cytotoxicity in cultured fibroblasts, and the DC was evaluated by Fourier transform infrared spectrometry (FTIR).
Table 1: Commercially available dental cements investigated in this study

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The mouse fibroblast cell line L929 (American Type Culture Collection, Rockville, MD, CCL-1 NCTC clone 929) was cultured in modified Eagle's medium (LGC, São Paulo, SP, Brazil) with 4.5 g/L glucose, 2 mM L-glutamine, 2.2 M sodium pyruvate, 10 mM N-2-hydroxyethylpiperazine, N-2-ethanesulfonic acid, 2.0 g/L sodium bicarbonate, and 100 U/ml amphotericin–gentamicin containing 10% fetal bovine serum (Sigma, USA). The medium was changed, and the sub-culturing was performed after the cells reached confluence as seen under a phase contrast microscope. Single cell suspension of the L-929 cells was obtained after trypsinization.

Cell viability was determined by a 3-(4.5-dimethyl thiazol-2-yl)-2,5-diphenyl)-tetrazolium bromide (MTT) assay (Molecular Probes, Eugene, Oregon, USA), which is a colorimetric method for determining the number of viable cells in cytotoxicity assays. The dye was reduced by the mitochondrial enzyme succinate dehydrogenase to produce a colored formazan product in live cells as previously described.[13] A single cell suspension of L-929 cells was obtained after trypsinization, and the cells were counted in a hemocytometer (Reichert, Buffalo, NY, USA). The cells were cultured in a 96-well plate (1.5 × 104 cells/well); after 72 h of incubation, the cells were treated for 24 h. The serum-free medium was used as a control. Subsequently, the cells were shifted in 100 μL of fresh medium, and 10 μL of MTT test solution was added to each well. After 4 h of incubation, the optical density (OD) of soluble formazan was measured using a multiwell scanning spectrophotometer (Thermo Scientific Multiskan Spectrum, Vantaa, Finland) at a wavelength of 570 nm. The values are expressed as a mean ± standard deviation and were obtained from three independent experiments conducted in sixplicates.

Similar to Kong et al., 2009, cytotoxicity was rated based on cell viability relative to the controls:[5]





  • Noncytotoxic >90% cell viability
  • Slightly cytotoxic = 60–90% cell viability
  • Moderately cytotoxic = 30–59% cell viability
  • Severely cytotoxic <30% cell viability.


The FTIR spectra of cement samples were recorded in a Perkin Elmer spectrometer model Spectrum GX (Perkin Elmer, Boston, MA, USA) at 4000–400 cm−1 in KBr pellets at a resolution of 4 cm−1 using 32 scans per sample.

For the assessment of chemical activation mode in the percentage of DC (DC%), six specimens of each material were prepared. Specimen preparation was made as mentioned above, except that they were not light-cured; instead, the specimens were placed in dark and dry conditions for 10 min. Ten minutes is a clinically relevant time that corresponds to the setting times given by the manufacturers and is almost twice the proposed setting time for the majority of materials.

The method used for the assessment of DC% was the micro-attenuated total reflectance FTIR (micro-ATR-FTIR), which is a well-established technique in the relevant literature.[14][15][16][17] An FTIR spectrometer Spectrum GX (Perkin Elmer, Boston, MA, USA) was used and operated under the following conditions: 4000–400 cm−1 range, 4 cm−1 resolution, and 32 scans per sample. The specimens were placed one at a time in the sample holder of the device, and spectra were recorded.

The DC% was calculated by the two-frequency technique, using the absorption peak of C═C groups at 1.638 cm−1 (analytical frequency) and the absorption peak of the aromatic C=C groups at 1.608 cm−1 (reference frequency) according to the equation:

% DC = 100 [1−(Aa(C═C) Ab(C─C)/Ab(C═C) Aa(C─C))]

Where Aa (1.638 cm−1) and Ab (1.608 cm−1) the net peak absorption areas after and before polymerization at the specific frequencies.

For the calculation of DC%, the absorption peak of C═O ester groups was used as a reference, and no aromatic peaks were identified. In this case, the DC% was calculated as follows: DC%=100 [1−(Aa(C═C) Ab(C═O)/Ab(C═C) Aa(C═O))]

Descriptive statistics and means of the measurements with 95% confidence interval were used to illustrate the results. Balanced analysis of variance was used for testing the group effect. For the comparison of DC% between self-cured and dual-cured groups for each material, multiple comparisons of pairwise differences were done. The multiple testing was controlled with the method of Bonferroni.


   Results Top


The results showed that the cytotoxicity of the cements is directly related to the DC [Table 2]. There was a significant difference between the group's that using the same cement with or without photopolymerization, that is, G1 versus G2, G3 versus G4, and G5 versus G6. The comparisons between the different groups also presented significant difference (P < 0.05). There was a significant difference among all experiment groups and the control group, except G2.
Table 2: Cell viability and increase degree of conversion after 20 s of polymerization

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The three groups that were tested were significantly more cytotoxic than the other three groups with the same materials after 20 s of photopolymerization (P < 0.05). However, the set and BifixSE are severely cytotoxic, 9.1% and 13.3% cell survival, respectively, and were significantly more cytotoxic than Rely-X Unicem, which was moderately cytotoxic, 30.5% cell survival, without polymerization. In relation to the groups with polymerization, G2 showed 93.9% cell survival and was not significantly different from the control G7. Therefore, Rely-X Unicem, G2, might be considered noncytotoxic in the conditions of this experiment. The groups G6, 67.4% cell survival, and G4, 41.9% cell survival were slightly and moderately cytotoxic, respectively [Figure 1]. Consequently, the null hypothesis was partially rejected because the group G2 was considered to be noncytotoxic.
Figure 1: Cell viability of different groups tested

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   Discussion Top


One important factor to consider when making a clinical decision about dental cements is their potential for adverse biological effects. The cytotoxicity of resin cements has been mainly attributed to the release of monomers such as urethane dimethacrylate (UDMA), 2-hydroxyethyl methacrylate, triethylene glycol dimethacrylate (TEGDMA) or bisphenol glycidyl methacrylate. These monomers are frequently added to the resin cements chemical composition. Several of the released compounds have been found to induce cytotoxic effects such as cell-cycle arrest and apoptosis.[18][19][20][21][22]

Methacrylate monomers may undergoes hydrolysis producing methacrylic acid and capable of rapidly diffusing through dentin that cause cytotoxicity by stimulating the release of tumor necrosis factor-α, apoptosis or by altering the lipid layer of the cell membrane, which affects the membrane permeability. However, these monomers presented different behaviors of water sorption and solubility.[23]

The photoinitiator as camphoroquinone frequently used in dentinal adhesives is another component that may be released after polymer formation. Whether irradiated or not, it may be responsible for part of the cell viability. As camphorquinone is not incorporated into the polymer network, the fraction that is not consumed in the reaction may be completely leached after polymerization and cause oxidative stress, DNA damage, and cytotoxicity.[24],[25]

Curing of resin-based cement is usually not complete; unconverted monomers can be released from the resin in the adjacent aqueous phase and can be diffused through the dentin to the pulp space.[26] These are unconverted monomers such as TEGDMA and UDMA, and photoinitiators;[27] such as camphorquinone results in poor mechanical[28] and chemical[29] properties are cytotoxic for cells.[9]

The present study investigated the cytotoxicity of currently used self-etching dual resin cements. The results show that some dental cements are toxic to the survival of fibroblasts. In the absence of photopolymerization, a significant decrease in cell survival was observed in all of the test groups. The results after 20 s of curing showed a decrease in cytotoxicity for the three tested cements. The results show that polymerization is essential for these self-etching cements dual polymerization in agreement with to Porto et al., 2011[30] that used similar spectroscopic methods were found that an adequate photopolymerization technique is necessary. Without curing, that is, via a self-polymerization reaction, all tested cements showed severe cytotoxicity; the exception to this was Rely-X Unicem, which showed moderate cytotoxicity.

The best results among the groups with light curing G2, G4, and G5 may be explained by the increase in the DC to 12.25%, 9.42% and 16.6%, respectively. The variations in increase the DC between different cements are probably due to quantities of photopolymerizable portions. The term "DC" that was applied to the resin composites refers to the conversion of monomeric carbon-carbon double bonds to polymeric carbon-carbon single bonds.[15]

The mechanisms of cytotoxicity are related to the short-term release of free monomers occurring during the monomer-polymer conversion. However, even after curing, statistically significant differences in cell survival were observed among the cements. These differences may be explained by the different compositions of the cements. The Set and BIFIX SE have the UDMA monomer, which is more cytotoxic than TEGDMA, present in the composition of Rely-X Unicem. The toxicity of these monomers is dependent on their concentration,[9] and this in turn depends on the DC. Kong et al. have been demonstrated that cytotoxic effects of some resin-based cements after polymerization could be attributed to unconverted monomers such of resin-based cements.[5] The set resin cement probably contains more UDMA monomer than the BiFix SE justifying their differences. Regarding the knowledge that the passage of light through the restorative materials is limited,[2] these materials can cause injuries to the dental pulp[31] in addition to a possible involvement of their mechanical properties[32][33][34] if operative care is not adopted.


   Conclusion Top


According to the results, show self-adhesive dual resin cements tested are more toxic with less cell survival of fibroblasts without photopolymerization.







 
   References Top

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Correspondence Address:
Maria E Cortés
Department of Restorative Dentistry, Federal University of Minas Gerais, Belo Horizonte
Brazil
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Source of Support: Nil, Conflict of Interest: None


DOI: 10.4103/0970-9290.162878

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