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Abstract

Introduction:

The aim of this study was to evaluate the cytotoxic effects of three different light-cured orthodontic composites.

Material and methods:

Light Bond (Reliance orthodontic products), Grengloo (Ormco corporation), and Kurasper F (Kuraray Europe GmbH) were selected for the experiment. Specimens were prepared according to the manufacturers’ instructions, measuring 5 mm in diameter and 2 mm in thickness. Fibroblast cells were obtained from healthy gingival connective tissues. The composite cylinders were incubated in Dulbecco’s modified Eagle’s culture medium for 72 h according to ISO 10993-5 standards. The xCELLigence method was used to evaluate fibroblast cell vitality. After seeding 200 mL of the cell suspensions into the wells (20,000 cells/well) of the E-plate 96, gingival fibroblasts were treated with bioactive components released by the orthodontic composite materials and monitored every 15 min for 121 h.

Results:

There were no significant differences between the human gingival fibroblast (HGF) cell indexes of the control and all testing groups (p > 0.05) at 24 and 48 h. Light Bond demonstrated statistically significant decrease in HGF index (p < 0.05) at 72 h, but there was no significant difference among the Kurasper F, Grengloo, and untreated control groups (p > 0.05). Light Bond (p < 0.001) and Grengloo (p < 0.05) groups had lower HGF cell index values when compared to untreated control group, but Kurasper F demonstrated no significant differences between the control groups at 96 h (p > 0.05).

Conclusion:

Orthodontic composite materials include biologically active components and may change oral tissue. So, biocompatible orthodontic bonding composites should be used.

Keywords

Composite materials biocompatibility toxicity

Introduction

Contemporary fixed orthodontic appliances are largely variations of edgewise systems, such as lingual, Begg, tip-edge techniques, and so on. Generally, in orthodontics, braces are used to align teeth. Bonding of fixed orthodontic appliances such as brackets and tubes has been an important topic in clinical orthodontics, since achieving a stable bond between the enamel and the fixed appliance is necessary.^1,2

Safe bonding has been performed successfully in orthodontic clinics for almost 40 years.³ Light-cured orthodontic resins are especially suitable in clinical settings in which a quick set is essential, such as when re-bonding a loose bracket or placing an attachment on an impacted canine. When extra-long work time is required, light-cured adhesive is preferred. For example, when a difficult premolar bracket or bonded molar tube position must be controlled and second control with the mirror before the bracket placement or meticulously fixing the lingual retainer are considered ideal.

Orthodontic composites for fixed appliances must not only provide good bonding but also reveal excellent biocompatibility. Cytotoxicity of orthodontic composites is a clinical issue when materials are used in the oral cavity. Although biocompatibility or cytotoxicity affects the user’s choice of orthodontic composites, cytotoxicity studies of orthodontic composites are limited.

Little comprehensive data for the biocompatibility of light-cured orthodontic resin composites have been presented in the orthodontic literature. In contrast, manufacturers of orthodontic composite materials possess complete test data. The aim of this study was to evaluate the cytotoxic effects of three light-cured orthodontic composite materials.

Materials and methods

The orthodontic resin composites selected were Light Bond (Reliance Orthodontic Products, Illinois, USA), Grengloo (Ormco Corporation, California, USA), and Kurasper F (Kuraray Europe GmbH, Hattersheim am Main, Germany). Details of the composite materials and their components are shown in Table 1. The specimens were prepared according to the manufacturers’ instructions in standard 2-mm-thick disks made of polytetrafluoroethylene measuring 5 mm in diameter. All test samples were set and manipulated under disinfected conditions to limit the influence of biological contamination on the cell culture tests. The specimens were prepared between polyester film and glass slabs to minimize oxygen inhibition and maximize surface smoothness. Specimens were polymerized using a light-emitting diode-curing unit (Elipar Free Light 2, 3 M ESPE Dental Products, St Paul, Minnesota, USA) according to the manufacturer’s instructions. To maintain maximum sterility, all steps were performed in a laminar hood.

Table 1.

Orthodontic composites used in this study.

Product	Material type	Manufacturer
Light Bond	Bisphenol A diglycidylmethacrylate (3–7%), silica-crystalline, silica, fused (60–99%), amorphous silica (7–13%), and sodium fluoride (<1%)	Reliance Orthodontic Products
Grengloo	Uncured methacrylate ester monomers (20–38%), inert mineral fillers, fumed silica, activators, and preservative	Ormco Corporation
Kurasper F	Bisphenol A diglycidylmethacrylate, triethyleneglycol dimethacrylate, barium glass filler, colloidal silica, dl-camphorquinone, initiators, accelerators, and others	Kuraray Europe GmbH

The test processes for this study were based on ISO standard 10993-5⁴ (surface area-to-volume ratio of the specimen to the culture medium, 3 cm²/mL). ISO 10993-5: Tests for in vitro cytotoxicity specifies procedures for testing devices with direct or indirect contact, extracts of devices, and filter diffusion. Extracts of test devices and materials were tested by exposure to the cell culture system. The presence of cytotoxic leachates is indicated by the loss of cell viability. Thirty cylinders were manufactured with three different orthodontic composites and divided into three groups. Ten samples were prepared for each group for cytotoxicity testing. The composite cylinders were immersed in 7 mL of culture medium for 24 h at 37°C to extract residual monomer or cytotoxic ingredients. The culture medium enclosing the orthodontic composite extracts was sterile and filtered for use on the cell cultures.

Cytotoxicity testing

The xCELLigence system (Roche Applied Science, Mannheim, Germany, and ACEA Biosciences, San Diego, California) consists of an impedance-based real-time cell analyzer (RTCA), a computer, an RTCA single-plate station, and the disposable E-Plate 96. The impedance measured between electrodes in each well depends on the electrode, the ion concentration in the well, and whether the cells are bonded to the electrodes. In the absence of cells, electrode impedance is mainly determined by the ion environment at the electrode–solution interface and in the bulk solution. In the presence of cells, cells attached to the electrode sensor surfaces act as insulators and change the local ion environment at the electrode–solution interface, leading to increased impedance. Thus, the higher the number of cells that grow on the electrodes, the larger the electrode impedance. The data expressed in cell index units can be exported to Excel software (Microsoft, Seattle, Washington) for mathematical analysis.^5,6

Fibroblast cells were obtained from healthy gingival connective tissues of patients who underwent crown-lengthening procedures. In summary, the fresh gingival tissue samples were cut into small sections, rinsed with biopsy medium, placed in culture dishes, and incubated in the biopsy medium in a humidified atmosphere overnight. On the second day, biopsy medium was replaced with culture medium (Dulbecco’s modified eagle medium (DMEM); Biological Industries, Beit Haemek, Israel) with 10% fetal bovine serum. After reaching confluence, the cells were passaged with 0.25% trypsin and 0.1% ethylene diamine tetraacetic acid. Gingival fibroblasts were used between the fourth, five, and sixth passages for all tests.

The composite cylinders were incubated in DMEM culture medium for 72 h according to ISO 10993-5 standards (surface area-to-volume ratio of the specimen to cell-culture medium: 3 cm²/mL). The fibroblasts were maintained with DMEM that contained 10% fetal bovine serum. The xCELLigence method was used to evaluate fibroblast cell vitality. After 200 mL of the cell suspension was seeded in the wells (20,000 cells/well) of the E-Plate 96, gingival fibroblasts were treated with bioactive components released by the orthodontic composite materials and monitored every 15 min for 121 h.

Statistical analysis

All analyses were performed with the RTCA-integrated software of the xCELLigence system. Data are represented as mean (mmol/L) ± SEM. For proliferation experiments, the statistical analyses used were one-way analysis of variance and Tukey–Kramer multiple comparison tests. The data are presented as means and standard deviations. A value of p < 0.05 was considered statistically significant (Table 2).

Table 2.

Cell index by real time cell analysis and comparison of 24, 48, 72, and 96 h with ANOVA and Tukey–Kramer multiple comparison tests.^a

	24 h	48 h	72 h	96 h
Group (n = 10)	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
Control	2.67 ± 0.18	2.91 ± 0.22	3.18 ± 0.33^b	3.55 ± 0.28^b
Light Bond	2.50 ± 0.15	2.15 ± 0.12	2.03 ± 0.12^a,c	1.79 ± 0.09^a,c
Grengloo	2.49 ± 0.29	2.27 ± 0.44	3.40 ± 0.25^b	2.52 ± 0.12^b
Kurasper F	2.68 ± 0.35	2.29 ± 0.39	2.73 ± 0.36^b	2.99 ± 0.12^b

ANOVA: analysis of variance; SD: standard deviation.

^ap < 0.05: means of the same letter are not significantly different at α = 0 .05.

Results

We defined the optimum concentrations for cell proliferation and survival rate measurements. To this end, 40,000, 20,000, 10,000, and 5000 cells per well were seeded in the E-Plate 96, and the impedance was identified.

According to statistical analysis, when the cells were left to elute for 24 and 48 h, there were no significant differences between the human gingival fibroblast (HGF) cell indexes of the control and the testing groups (p > 0.05). While the Light Bond demonstrated a statistically significant decrease in the HGF index at 72 h (p < 0.05), there were no significant differences among the Kurasper F, Grengloo, and untreated control groups (p > 0.05). The Light Bond (p < 0.001) and Grengloo (p < 0.05) groups had lower HGF cell index values when compared to the untreated control group, but Kurasper F was not significantly different from the control group at 96 h (p > 0.05).

Discussion

The present in vitro investigation revealed that Kurasper F was the least cytotoxic orthodontic composite material and the other tested materials, Light Bond and Grengloo, caused cytotoxic effects on the cultured HGF cells, including significantly decreased cell metabolism and remarkable cell death. Furthermore, according to the results of the present study, the most toxic composite tested was Light Bond.

It is essential to assess the biocompatibility of orthodontic composites, since orthodontic composites are placed close to the periodontium and alveolar structure. Toxic elements extracted from orthodontic composites may lead to an inflammatory reaction or necrosis in adjacent tissues, such as the mucosa, gingival tissues, and alveolar crest.³ There are numerous pathways through which orthodontic composites may influence the health of oral mucosa by transporting water-soluble ingredients into the oral liquids and cavity as well as by reacting directly with surrounding tissues.⁷ Another interesting condition of orthodontic dental material toxicity is related to the health of the orthodontic staff. In routine clinical practice, the possibility of unwanted reactions to orthodontic materials is much higher for orthodontic staff than for patients because of continuing exposure to and preparation of these materials while they are being bonded and debonded.

Different designs and several in vitro cytotoxicity study models such as inhibition of cell growth, effects on membrane or cytoplasmic marker cytolysis, mitochondrial dehydrogenase activity of cells, and changes in metabolic activity have been designed over the last 30 years to overcome the disadvantages of in vivo study designs that evaluate the biologic response to different dental materials.^6,8,9 The main advantages of the cell culture test are ethical considerations are not necessary and the test has high reproducibility, adequate sample sizes, and standardization.^3,6,10 Only one test design is appropriate for examining one type of unwanted effect of a great variability of possible reactions. Additionally, specific test methods are usually acceptable only to define a single aspect of a certain type of unwanted reaction.¹⁰ We designed the experiment with a new real-time system that studied the cytotoxicity of the chemical compounds 2-hydroxyethyl methacrylate (HEMA), bis-glycidyl methacrylate (bis-GMA), and other ingredients on HGF cells with real-time and uninterrupted observation of cell growth, proliferation, and vitality.

Real-time continuous monitoring evaluates cell proliferation, vitality, and toxicity, demonstrates the physiologic situation of the cells, and avoids the high cost of expensive reagents used in traditional cell analysis. In this test design, dynamic control of the cellular condition was recorded while the entire test provided nonstop data about cell growth, development, morphologic changes, and cell death. In addition, the real-time cell analysis test design helps calculate time-related physiologic values, which provide more knowledge than the single-value end points of current toxicity testing.^5,6 Compared to classical end point cell-based assays, dynamic monitoring of cell reactions (cell adhesion, increasing, proliferation, growth, and apoptosis) has a great advantage in optimizing the cell concentration and conditions for in vitro assays before and during the experiment. Furthermore, the reaction of live cells to chemical exposure can be observed in real time; this is not possible with current end point assays such as lactate dehydrogenase, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide), water-soluble tetrazolium salt-1, or bromodeoxyuridine viability. The real-time test method was useful for estimating cell densities in small culture volumes. The use of cultivation in small culture volumes and sensitive evaluation with the real-time test lead to the screening and testing of many different substances and fractions can screened and tested to determine cytotoxicity.^5,6 For these reasons, we also preferred the real-time xCELLigence test procedure.

Orthodontic composite resin contains monomers such as Bis-GMA and urethane dimethacrylate (UDMA). Other components are comonomers such as triethylene glycol dimethacrylate (TEGDMA), diethylene glycol dimethacrylate, and ethylene glycol dimethacrylate and various molecules such as photoactivators, co-activators, inhibitors, absorbers, and stabilizers.¹¹ The amount of ingredients that can be extracted from resin composites and cements depends on the extraction medium.¹² Some investigations revealed that between 0.4 wt% and 1.5 wt% Bis-GMA or 4.6–11 wt% of the initial weight of all organic substances is releasable with organic solvents.^12,13 Other comonomers such as TEGDMA were reported very high levels (0.04–2.3 wt%).¹⁴ Animal studies have been concluded concerning biodegradation of HEMA/TEGDMA.³ Water-soluble components are used in different composites and resins, and these elements are extracted from materials. Swallowed HEMA/TEGDMA was almost completely absorbed by the organism. These elements are released from the orthodontic composite and diffused through the oral cavity and are cytotoxic.^15,16 Orthodontic composite cytotoxicity is critical, because a large amount of composite is used for full-mouth orthodontic bonding or fixation of lingual retainers. Clinical examination and the probing depth of orthodontic patients who had good oral hygiene showed that the gingiva close to the orthodontic bracket and composite demonstrated severe to moderate inflammation or allergic reactions. No data are available in the orthodontic literature regarding the systemic toxicity of orthodontic composites and resin-based luting cements for orthodontic bonding and banding as well as fixation of lingual retainers.

Our study results revealed that the tested orthodontic composites were nontoxic and biocompatible except for Light Bond. At the same dilution concentration, the ranked order for cytotoxicity was as follows: Light Bond > Grengloo > Kurasper F. Light Bond was more cytotoxic than Grengloo and Kurasper F. However, there was no significant difference in cytotoxicity between Grengloo and Kurasper F (Figure 1). The present investigation showed that the most intense cytotoxic effects were caused by UDMA and TEGDMA containing an orthodontic composite. In an evaluation of the biocompatibility of 35 resin components, Geurtsen et al. reported that UDMA and TEGDMA were more cytotoxic on human gingival fibroblasts and periodontal ligament cells.¹⁷ This conclusion is in accordance with our results. However, Malkoc et al. reported that Light Bond did not reduce HGF cell survival rates statistically significantly compared to the untreated control group.³ This difference can be explained by the different test methods and study design. In addition, Malkoc et al. evaluated mitochondrial activity with the MTT test with L929 mouse fibroblasts.³

Figure 1.

Dynamic monitoring of cultured HGF adhesion and cell proliferation. HGF: human gingival fibroblast.

The study results demonstrated that light-cured orthodontic composites have adequate high biocompatibility when compared with other restorative resin-based restorative composites and cements.^18,19 This condition may be explained by the combination of orthodontic composite monomers and the high degree of polymerization.^3,20
–22 However, the results of the present investigation remain unclear, and different study designs are needed for future research efforts. Future studies should evaluate the long-term biologic and systemic effects of orthodontic composites. In addition, evaluating the toxicity of clinical materials on microbial models can be useful.

Conclusion

The different cytotoxic effects of orthodontic composites should be evaluated when selecting an appropriate adhesive for orthodontics. Orthodontic composite materials include biologically active components and may change oral tissue. When orthodontic composite materials are used for orthodontic bonding or fixed lingual retainers, biocompatible orthodontic bonding composites are appropriate.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Biocompatibility evaluation of orthodontic composite by real-time cell analysis

Abstract

Introduction:

Material and methods:

Results:

Conclusion:

Keywords

Introduction

Materials and methods

Cytotoxicity testing

Statistical analysis

Results

Discussion

Conclusion

Footnotes

Declaration of Conflicting Interests

Funding

References