Objectives:

To evaluate the cytotoxic effects of five different light-cured orthodontic bonding composites.

Materials and Methods:

The orthodontic composites Heliosit Orthodontic (Ivoclar), Transbond XT (3M Unitek), Bisco ORTHO (Bisco), Light Bond (Reliance), and Quick Cure (Reliance) were prepared, and the samples were extracted in 3 mL of BME (Basal Medium Eagle) with 10% newborn calf serum for 24 hours. The L929 cells were plated (25,000 cells/mL) in a 96-well dish and maintained in a humidified incubator for 24 hours at 37°C, 5% CO2, and 95% air. After 24 hours of incubation of the cells, the incubation medium was replaced by the immersed medium in which the samples were stored. Then, L929 cells were incubated in contact with eluates for 24 hours. The cell mitochondrial activity was evaluated by the methyl tetrazolium (MTT) test. Twelve wells were used for each specimen, and the MTT tests were applied two times. The data were statistically analyzed by one-way analysis of variance (ANOVA) and Tukey HSD tests.

Results:

Results with L929 fibroblasts demonstrated that except for Transbond XT, freshly prepared composite materials did not reduce vital cell numbers (P > .05) compared with the control group. Our data demonstrate that Transbond XT showed significant cytotoxicity compared with the control group.

Conclusion:

Results indicate that tested orthodontic bonding composites are suitable for clinical application, but that further studies using different test methods are needed for Transbond XT.

The introduction of the acid etch bonding technique has led to dramatic changes in clinical practice, and orthodontic bonding has been a critical issue. Today, orthodontists are approaching 40 years of successful, reliable orthodontic bonding in clinics. The desire to cure on demand has caused an increasing number of orthodontic practices to use light-cure adhesives instead of the more traditional paste-paste adhesives requiring in-office mixing.1 The light initiated resins have become the most popular adhesives for a majority of orthodontists. Photo-activated resin composites are the choice of adhesive for orthodontic bonding because of their ease of use and the extended time they allow for bracket placement.2 A number of studies published during the 1990s have investigated the bond strength of dual-cure and light-cure adhesives of transparent ceramic brackets and metallic brackets. The majority of these studies concluded that the light-cured material was not different from that for the chemically cured adhesives, and even these materials are found to have higher bond strength.3 

Although the developments and improvements of the orthodontic light cure adhesive materials are very satisfying and amazing, biocompatibility of the orthodontic composites is still a problem for orthodontists today. In addition, very few data are available in the literature regarding biocompatibility of the commercially available orthodontic composites and the length of time after light polymerization.4 Orthodontists are using a large variety of bonding agents, and newer orthodontic composite materials present new challenges because of the potential for interaction.

No comprehensive data are available in the orthodontic literature regarding the toxicity of light-cured orthodontic resin composites. It should be critically emphasized at this point that the manufacturers of these materials possess comprehensive test data. Therefore, the aim of the present study was to evaluate the cytotoxic effects of five different light-cured orthodontic bonding composites.

Five different orthodontic composites were tested in the experiments: Heliosit Orthodontic (Ivoclar Vivadent AG, Schaan, Liechtenstein), Transbond XT (3M Unitek Ortho Prod, Monrovia, Calif), Bisco ORTHO (Bisco Inc, Schaumburg, Ill), Light Bond (Reliance Orthodontic Products Inc), and Quick Cure (Reliance, Itasca, III). Their components and details are listed in Table 1.

Table 1

The Adhesive Resins Included in the Investigation

The Adhesive Resins Included in the Investigation
The Adhesive Resins Included in the Investigation

Test specimens were prepared according to the manufacturers' instructions in standard Teflon molds of 5 mm in diameter and 2 mm in depth. All specimens were prepared and handled under aseptic conditions to limit the influence of biologic contamination on the cell culture tests. Specimens were prepared between Mylor and glass slabs to minimize the oxygen inhibition and maximize the surface smoothness. Specimens that required light curing were cured using a standard light curing unit (LED, Elipar FreeLight 2, 3M ESPE Dental Products, St Paul, Minn).

Four samples were prepared for each group for cytotoxicity testing. The samples were immersed in 7 mL of culture medium for 24 hours at 37°C to extract residual monomer or cytotoxic substances. The culture medium containing material extracts was sterile filtered for use on the cell cultures.

Cytotoxicity Testing

L929 cells (ATCC CCL 1, Şap Enstitüsü, Ankara, Turkey) were cultured in BME (Basal Medium Eagle) containing 10% newborn calf serum and 100 mg/mL penicillin/streptomycin at 37°C in a humidified atmosphere of 95% air, 5% CO2. Cell cultures between the 12 and 15 passages were used in this study. Confluent cells were detached with 0.25% trypsin and seeded at a density of 5 × 103 into each well of a 96-well plate for 24 hours at 37°C and 5% CO2. After 24 hours of incubation, the culture medium was replaced with 200 µL of culture medium containing material extracts of orthodontic composites. The original culture medium served as control in this study.

Cultures were incubated for 24 hours at 37°C and 5% CO2. The viability of cells exposed to material extracts was assessed using succinic dehydrogenase activity. The succinic dehydrogenase activity has been shown to be reasonably representative of mitochondrial activity in the cells and reflects both cell number and activity.5 The old medium was removed and cell cultures were rinsed with PBS, and 0.5-mL aliquots of freshly prepared MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) solution (0.5 mg/mL in BME) were added to each well.

After a 2-hour incubation period (37°C, 5% CO2), the supernatant was removed and the intracellularly stored MTT formazan was solubilized in 200 µL dimethyl sulfoxide for 30 minutes at room temperature. The absorbance at 540 nm was spectrophotometrically measured.

The worksheets were incorporated into the software, Excel version XP, and then recalculated as follows: cell viability percentage  =  where a is the OD value at 540 nm derived from a well added with a test chemical, b is the mean OD value at 540 nm derived from blank wells, and c is the mean OD value at 540 nm derived from control wells (ie, added culture medium as a test chemical).

Twelve replicate cell cultures were exposed to each concentration of a single material in at least two independent experiments. Cell survival in treated groups was compared with that in the untreated controls. Differences between median values were statistically analyzed using the one-way analysis of variance (ANOVA) and Tukey HSD tests.

Cell survival of L929 cells was evaluated in a methyl tetrazolium test after exposure to orthodontic bracket adhesive materials. Data are expressed as percentage of the control cultures. Cell survival rates were calculated from independent experimental cultures.

Cell Morphology Evaluation

Morphologic alteration of L929 cells was observed directly using an inverted microscope (TS100 Nikon Eclipse, Japan) (10×) and photographed by a camera (Nikon Eclipse, Tokyo, Japan).

Means and standard deviations of cell survival rates for each group are given in Table 2. There were significant differences among the orthodontic composite resins in the cell survival percentage (P < .05).

Table 2

The Cell Viability Percentages by Methyl Tetrazolium (MTT) Assaya

The Cell Viability Percentages by Methyl Tetrazolium (MTT) Assaya
The Cell Viability Percentages by Methyl Tetrazolium (MTT) Assaya

Results demonstrated that all of the orthodontic bracket adhesive materials except Transbond XT did not reduce L929 cell survival (P > .05) when compared with the control group. However, Transbond XT significantly decreased L929 cell survival rates compared to the control group (P < .05; Table 2).

Morphologic Assessment

L929 cells were elongated and spindle-shaped in appearance. While Heliosit Orthodontic led to enlargement of the intercellular space, the cells kept their shape as spindle in appearance. However, the cell densities were decreased when compared with the control. For Bisco ORTHO, Quick Cure, and Light Bond, the cells were retracted, rounded in appearance, and also led to enlargement of the intercellular space, but the cells did not show any differences with the control (Figures 1 through 6). However, in the Transbond XT group the cells were significantly retracted, rounded, and also increased in the intercellular space, while there was no significantly different appearance in the Transbond XT group cell population when compared with the control.

Figure 1

Cultured L929 cells for control group.

Figure 1

Cultured L929 cells for control group.

Close modal
Figure 2

Cultured L929 cells for Heliosit Orthodontic.

Figure 2

Cultured L929 cells for Heliosit Orthodontic.

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Figure 3

Cultured L929 cells for Transbond XT.

Figure 3

Cultured L929 cells for Transbond XT.

Close modal
Figure 4

Cultured L929 cells for Bisco ORTHO.

Figure 4

Cultured L929 cells for Bisco ORTHO.

Close modal
Figure 5

Cultured L929 cells for Light Bond.

Figure 5

Cultured L929 cells for Light Bond.

Close modal
Figure 6

Cultured L929 cells for Quick Cure.

Figure 6

Cultured L929 cells for Quick Cure.

Close modal

It is very important to evaluate the biocompatibility of the orthodontic bonding adhesives, because these materials are located proximate to the periodontal tissue and alveolar bone. Substances released from orthodontic composites may cause a reaction (inflammation or necrosis) in adjacent tissues, such as the oral mucosa and gingiva, or alveolar bone. There are several ways that materials may influence the health of soft tissues—by delivering water-soluble components into the saliva and the oral cavity as well as by interacting directly with adjacent tissues.6 The extraction assay described above is one of the most frequently used methods to assay the mechanism of intraoral cytotoxicity in the study of orthodontic bonding composites.

Another important topic, the area of biocompatibility of materials, is also relevant to the practitioner from the standpoint of the health of the dental team. In many cases, the risk of the adverse effects of biomaterials is much higher for the dental team than for the patients because of chronic exposure of the dental team and manipulation of the materials when they are being placed, set or removed.5 Furthermore, there is the necessity of informing the affected personnel that the dental materials used by orthodontists can pose some risk to the patient and the dental team. It is the orthodontic clinician's problem to conclude whether this evidence is deserved and estimate the risk of these topics in orthodontic practice.

A great variety of different test methods are used to determine the risk of such damage to ensure material compatibility. However, the results of such evaluations are dependent not only on the tested material, but also on the test method used. Evaluation of the biocompatibility of dental materials is complex and comprehensive because unwanted tissue reactions may occur in a great variety of types.7 

Previously, the tissue compatibility of orthodontic bonding agents was studied in animal experiments.8 Ethical considerations, poor reproducibility, and small sample sizes resulted in the development of in vitro cytotoxicity tests and their standardization.9 Any single test method is applicable only for investigating one type of unwanted reaction out of a great variety of possible reactions. Moreover, individual test methods are usually adequate only to describe or document a single aspect of a certain type of unwanted reaction.

Cell culture tests will detect only the influence of a material on isolated cells.7 Isolated cells derived from animal or human tissues are grown in culture plates and then are used for these tests.7 Today, primarily permanently growing cells are used for this purpose because these cells can be easily amplified and their behavior is well known, relatively consistent, and constant.7 Frequently, permanent mouse fibroblast (L929) cells are used. These cell cultures are “incubated” with the materials or their extracts. Subsequently, a series of various parameters will be measured; for example, the number of “surviving” cells, protein synthesis, enzyme activity, or synthesis of inflammatory mediators.7 L929 fibroblasts and gingival fibroblasts have previously been shown to have similar cytotoxicity levels. Consequently, L929 fibroblasts make a useful screening model for in vitro toxicity testing of dental materials. Because of its excellent reproducibility, the L929 cell line was preferred to primary gingival fibroblasts.9 

The advantages of the MTT procedure are simplicity, accuracy, reliability, and the saving of time. The MTT method proved to be useful to estimate cell densities in small culture volumes. The cultivation in small culture volumes and the sensitive evaluation with the MTT assay allow the screening and testing of many different substances and fractions for the determination of cytotoxicity. For these reasons, we also used an MTT assay procedure.

Resin matrixes of the orthodontic composites consist of mainly two monomers: bisphenol A diglycidyl dimethacrylate (bis-GMA) and triethylene glycol dimethacrylate (TEGDMA). In addition, the matrix resin consists of a mixture of various monomers, for example, bis-GMA and/or UDMA, as well as various modifications of these molecules. Other ingredients of the composite matrix are co-monomers (EGDMA, DEGDMA, TEGDMA) and various additives such as photo-initiators (eg, camphorquinone), co-initiators (eg, DMABEE, DEAEMA), inhibitors (eg, BHT), ultraviolet absorbers, photo-stabilizers, and pigments.10,11 Polymerization in products used today is mainly initiated by light; the light-sensitive initiator camphorquinone acts together with an aliphatic amine-type catalyst. TEGDMA has an important function because it decreases the viscosity of the matrix, thus allowing increased filler content. Resin-based composites (ormocers), which are presented recently are based on a Si-O scaffold with methacrylic side chains, which are necessary for polymerization.7 Orthodontic composite resins may be released bis-phenol A, a bis-GMA precursor that exhibits cytotoxic effects concluded potential biologic adverse reactions. Furthermore, various ions are leached out at different times and in different conditions. Recently, data from animal studies have been presented concerning biodegradation of HEMA/TEGDMA.12,13 Both “water-soluble” substances are used in a variety of resin-based composites (TEGDMA) and adhesives (HEMA/TEGDMA), and thus are released from materials. Swallowed HEMA/TEGDMA were almost completely absorbed by the organism. These ions are released from orthodontic resin-composite, diffuse through oral tissues, and are cytotoxic.

In addition, Hansel et al.14 investigated the influence of base monomers (bis-GMA, UDMA) and co-monomers (TEGDMA, EGDMA) on the in vitro proliferation of caries-relevant bacteria. They found that the base monomers had no influence or only a slightly growth-inhibiting effect on these cultures, but that both of the co-monomers tested (TEGDMA, EGDMA) promoted bacterial proliferation. Because these substances usually leach from resin-based composites at higher concentrations than base monomers do, an overall increased bacterial growth may be the consequence in the presence of resin-based composites. Söhoel et al.15 tested two different bis-GMA/TEGDMA containing resins that were used for the bonding of brackets. Both substances generated a sensitization in 50% of the experimental animals, with a subsequent allergic reaction.15 

Usually, mixtures of these monomers are used in orthodontic composites. When evaluating the ingredient of tested materials in the present study, there were significant similarities in resin matrixes. However, Transbond XT also contains bis-EMA. In addition, a bis-EMA monomer showed a cytotoxic effect analogous to that of TEGDMA.16 The mechanism of cytotoxicity induced by TEGDMA in human fibroblasts was recently studied.17 Hence, cytotoxicity of Transbond XT could be explained by the presence of bis-EMA in its matrix.

The result of this study showed that light-cured orthodontic bonding adhesives have acceptable high biocompatibility when compared with other dental adhesives.18,19 This issue may be explained by mixture of orthodontic adhesive monomers and a high degree of cure.20 

However, the results of the present in vitro study remain unclear, and further studies using different test methods are needed for Transbond XT. Research efforts should focus on assessing long-term biologic effects of orthodontic composites.

  • The tested orthodontic bonding composites are suitable for clinical application. However, Transbond XT was cytotoxic (87%), while the other orthodontic composites caused no or only slight cellular alterations (90%, 91%).

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