Evaluation of Cytotoxicity and Degree of Conversion of Orthodontic Adhesives over Different Time Periods

As new orthodontic resin adhesives continue to be marketed, rapid and sensitive tests for examining their toxic effects at the ‘ cell and tissue level ’ are needed because patient safety has been identifi ed as a legal concept. The objective of the present study was to evaluate the cytotoxicity and degree of monomer conversion of orthodontic adhesives over different time periods. Seven adhesives: Transbond XT, Transbond Color Change, Quick Cure, EagleBond, Orthobond, Fill Mágic and Biofix were evaluated for their cytotoxicity in L929 fibroblastic cells and for their degree of monomer conversion over different time periods. Three control groups were also analysed: Positive control (C+), consisting of Tween 80 cell detergent; Negative control (C–), consisting of PBS; and cell control (CC), consisting of cells exposed to any material. The dye-uptake technique that involves the absorption of a neutral red dye in viable cells was used for the cytotoxicity evaluation and the degree of conversion was evaluated using spectroscopy with infrared. The results showed the cytotoxicity of the adhesives at 24, 48, 72 and 168 hours. At these times, the viability values presented for these materials were statistically different from the groups CC and C– (p < 0.05). At 168 hours, all the groups showed low cytotoxicity with high cell viability and with no statistical difference with the groups CC and C– (P > 0.05). In the monomer conversions there was a percentage increase of monomer conversion from 24 to 72 hours. A direct correlation could be observed between cytotoxicity and monomer conversions. From this work it can be concluded that all adhesives evaluated are cytotoxic at the times of 24, 48 and 72 hours. Monomers continued conversion even after photopolymerization had stopped.


Introduction
Composite resins or dental composites today are the materials of choice to bond orthodontic accessories to dental enamel 1,2 .These materials have evolved considerably since the 1970's.However, there as yet no materials with ideal properties and therefore there is constant evolution and research.
Dental composites are defined as a three dimensional combination of at least two chemically different materials, with an interface that separates them both 3 .The resinous matrix contains a system of monomers and initiators for polymerization.The inorganic part contains glass, quartz and/or silica and the bonding agent that unites the inorganic particles with the resinous matrix.The resinous component is called the polymeric matrix 4,5 .
The monomer bisphenol A-Bis-glycidyl dimethacrylate (BisGMA) is the most used monomer in dentistry and is notable for its long, rigid molecule with reactive double carbon bonds at both extremities 6 .
One of the critical aspects of these materials is during the polymerization stage, responsible for the majority of the physical, mechanical and biological properties.Incomplete polymerization can produce a composite with high porosity, low hardness, low gloss, high capacity of staining and even cell toxicity provoked by free monomers 7,8 .
The quantity of double carbon bonds (C = C) present in the monomers that are converted into single bonds (C -C) to form the polymeric chain during the process of polymerization, is known as the degree of conversion 9 .The degree to which this conversion of reactive species occurs may affect the compatibility of the resin with the oral tissues.Therefore, a reduction in remaining double bonds to the lowest possible level is considered a desirable feature of polymerization system.
A spectrometer is the most precise way to determined the quantity of residual monomers and evaluate the degree of conversion of composites nowadays 10 .
Based on this premise the aim of this present article was to evaluate the effect of time on the cytotoxicity and degree of conversion after polymerization of different adhesives used in dentistry.

Adhesives evaluated
Seven adhesives were evaluated.Table 1 shows the manufacturer, composition, presentation and lots.

Test sample preparation
For the preparation and standardization of the test samples, 5 mm diameter and 2 mm thick stainless steel bipartite matrices were used (Figure 1).
The metallic matrix was placed on a glass slide and the adhesives were settled in using a plastic spatula.Having filled the matrix, a new glass slide was placed on top for subsequently photo-activation of the materials.To standardize the force of the slide on the adhesives a weight of 300 g was placed on the glass slide.
Photopolymerization lasted 40 seconds.The photopolymerization apparatus was fixed on a rod so the distance between the light and the specimens remained constant.The apparatus used was a Radii (SDI, Victoria, Australia) with a lamp intensity of 1400 mw.cm -2 , calibrated with a radiometer (Demetron, Danburry, CT, USA).
After the photopolymerization the test samples were removed from the matrixes and some were immerged in a culture for post cytotoxic evaluation (n = 30) and the others were for degree of monomer conversion evaluation (n = 5).

Controls
To verify the cell response to extreme situations, three other groups were included in the study: Group CC (cell control), consisting of cells not exposed to any material; Group C+ (positive control), consisting of Tween 80 (Polioxietileno-20-Sorbitan); and Group C-(negative control), consisting of PBS solution (Phosphate-buffered saline) in contact with the cells.

Assessing the cytotoxity of the materials
The materials were previously sterilized by exposing them to ultra-violet light (Labconco, Kansas, Missouri, USA) for 1 hour.Next, thirty samples of each material (n = 30) were placed in 24-wells plates containing Eagles' MEM (Cultilab, Campinas, São Paulo, Brazil).The culture medium was replaced with fresh medium every 24 hours, and the supernatants were collected after 1, 2, 3 and 7 days for toxicity analysis to L929 cells.The supernatants were placed in a 96-well plate containing a single layer of L929 cells and then incubated at 37 °C for 24 hours in a 5% CO 2 environment.After the incubation period, cell viability was determined using the "dye-uptake" technique described by Neyndorff et al. 11 (1990), which was slightly modified.After the 24-hour incubation period, 100 µl of 0.01% neutral-red staining solution (Sigma, St. Louis, Missouri, USA) was added to the medium in each well of the plates, and these were incubated for 3 hours at 37 °C to allow the dye to penetrate into  the living cells.After this period, the cells were fixed using 100 µl of 4% formaldehyde solution (Reagen, Rio de Janeiro, Brazil)) in PBS (130 mM NaCl; 2 mM KCl; 6 mM Na 2 HPO 4 2H 2 O; 1 mM K 2 HPO 4 , pH = 7.2) for 5 minutes.Next, 100 µl of 1% acetic acid solution (Vetec, Rio de Janeiro, Brazil) with 50% methanol (Reagen, Rio de Janeiro, Brazil) was added to the medium to remove the dye.Absorption was measured after 20 minutes by using a spectrophotometer (BioTek, Winooski, Vermont, USA) at a wave length of 492 nm.

Conversion degree analysis
After polymerization the test samples (n = 5) were ground to obtain the ionomer powder, that was forthwith mixed with potassium bromide (KBr), in a ratio of 1/20.This powder was placed in a tablet-maker under an approximate pressure of 8 t.An FT-IR spectrophotometer (Bomen-modelo MB-102, Quebec, Canadá) carried out the infrared spectrum measurements using the Fourier transformation method (FTIR), to determine the percentage degree of monomer conversion (DC%) in the polymer.
The following equation was used for this study (Equation 1): In the double aliphatic carbon-carbon bond (C = C aliphatic) the infrared absorption characteristics are around 1638 cm -1 , while the double carbon-oxygen bond (C = O) has an absorption value at 1720 cm -1 .

Statistical analysis
Statistical analyses were performed by using Statistical Package for the Social Sciences version 13.0 program (SPSS Inc, Chicago, Ilinois, U.S.A) and means and standard deviations were calculated for descriptive statistical analysis.The values for the amount of viable cells and degree of conversion were submitted to analysis of variance (ANOVA) to determine whether statistical differences existed between the groups and Tukey's test was applied thereafter.

Results
The cytotoxity results of the materials evaluated are shown in Table 2.
The degree of conversion values among the adhesives evaluated are shown in Table 3.

Discussion
This work evaluated the cytotoxicity and the degree of conversion of eight adhesives for dental use and the influence of the time factor on these questions.
After 24 hours all the experimental materials were cytotoxic with less cytotoxicity shown by Eagle Bond followed by Fill Mágic ® , Orthobond ® and Quick Cure.On the other hand Transbond ® XT, Transbond ® Color Change and Biofix ® were the most cytotoxic, not presenting differences among them for this evaluation time.The values obtained for the degree of conversion proved exactly what was observed on testing for cytotoxicity.These values agree with those
Area of band C = C (polymer) Area of band C = O (polyme er) x 100 Area of band C = C (monomer) Area of band C C = C (monomer)

Table 1 .
Materials tested with their respective manufacturers, composition and manufacturing lot.

Table 2 .
Average values of cells, standard deviation, cell viability and statistic analysis of the groups studied.Mean = mean values for the amount of viable cells; SD = Standard Deviation; St* = Same letters mean no statistical difference.VB/Cel.=cell viability (%).

Table 3 .
Average values and standard deviation of the degree of conversion of the materials tested at the times of 24, 48, 72 and 168 hours (n = 5).
Mean = mean values for the amount of viable cells; SD = Standard Deviation; St* = Same letters mean no statistical difference.