Cytotoxic effects of five dental adhesives

A. Lupi°, G. Gambarini§, G. Bolognini§, L.Testarelli §i, C.Chimenti^, M.Cordaro *, M. Castagnola*, L. Ceccarelli*, G. Nocca*, R. Scatena*, and B. Giardina*.
°CNR Centro Chimica dei Recettori c/o U.C.S.C., § Dental Institute University of Rome “La Sapienza”,. *Dental Schhol & Institute of Biochemical and Clinical Biochemical, U.C.S.C., Rome. ^ University of L’Aquila Dental School,

Summary

The purpose of the study was to evaluate cytotoxicity of five commercially available dental adhesives. Extracts of the tested materials were taken in cell culture medium of freshly-cured and aged sample. Mouse 3T3 fibroblasts were exposed to the extracts for 24 hours and the cytotoxicity was evaluated using neutral red uptake (NRU) assay. Direct toxicity was evaluated also with NRU test.
Results showed that mean values of citotoxicity of freshly cured adhesives were 50%; only one adhesive showed higher values (78%) of toxicity.Aging the samples affected the cytotoxicity of the extracts to a varying degree. Two products showed reduced cytotoxicity in NRU, while three adhesives didn’t show any difference in cytotoxicity respect to freshly extracts.
All the dentin bonding materials were cytotoxic when tested in direct toxicity in NRU. (about 80%). Only one adhesive showed a lower degree (%) of toxicity.
In conclusion, NRU test permitted to rapidly evaluate cytotoxicity of different dental materials, and in same time, to address further biochemical and toxicological studies to identify the molecular mechanisms involved in this toxicity.

Introduction

Dental adhesive systems were introduced in the middle 1960s. The original bonding formulations essentially were chemically identical to the composite resin but contained diluents for more wetting the cavity wall (1). The modern adhesive systems are fourth and fifth generation dentin bonding agents. Generation means of differentiating between various levels of improved handling characteristics and clinical performance. In general sense, the fourth generation dentin adhesives require the use of an adhesive component in addition to the primer to affect bonding of the overlying composite to the prepared dentinal surface. The fifth generation dentin bonding agents permit bonding of the composite restoration without the aid of the traditional adhesive component. Apparently, the wettability of the primer has been so improved that an adhesive agent is not required to effect proper wetting by the restorative agent.

The objective of in vitro biocompatibility tests is to simulate biological reactions to materials when they are placed on or into tissue of the body. Cell culture systems provide convenient, controllable and repeatable means for an initial assessment of biological responses (2).There are a number of cytotoxicity tests available measuring a variety of parameters. The toxicity parameters should ideally be appropriate to the chemical nature of the test material (3). Lipophilic substances are more likely to disrupt membrane integrity, therefore a permeability assay such as neutral red uptake (NRU) is more useful. The NRU assay is so-called vital staining procedure (4; 5). Viable cells incubated in the presence of NR take up and retain the dye compound. On the exposure to substances damaging the plasma or lysosomal membranes, the cell no longer retains NR. Determination of the amount of retained NR in cells exposed to test compounds, comparated with controls, enables the relative toxicity of test chemicals to be assessed. Nru has been applied to ranking cytotoxicity of chemicals according to potency, and to elucidate their structure-toxicity relationships (6). In this study, NRU test has been employed to assay the cytotoxic effects of five adhesives on mouse 3T3 fibroblast cell line, in order to elucidate the cytotoxic potential of these materials.
Clinically an accurate evaluation of cytotoxic properties of different dental materials in general and of adhesives in particular could help to reduce the incidence of early and late complications of different orthodontic implants, moreover could preserve patients from effects related to systemic toxicity.

In general more attention shouold be paid to tolerability and biocompatibility data of dental material.Ameliorating the oral pathophysiology related to dental remodelling could yield positive consequences in clinical and economic terms.

Materials and methods

Materials and reagents. Unless indicated all chemicals and reagents (cell culture grade) were obtained from Sigma Chemical Co., Milan, Italy.
Cells and Treatments. Mouse 3T3-fibroblasts (swiss albino mouse cell line) (Istituto Zooprofilattico, brescia, Italy) were grown in a 5% CO2 atmosphere at 37°C in DMEM (Dulbecco Modified Eagle Medium), 10 mM Hepes, Glucose (1 g/L), NaHCO3 (3,7 g/L), penicillin (100 units/mL) streptomycin (100 mg/mL) and 10% FCS (Fetal Calf Serum) were utilized.
In order to evaluate the cytotoxic effects of the chemicals released in DMEM from the dentin bonding agents of interest to the present study, 1 x 104 fibroblast in 200 mL DMEM were seeded per individual well of 96-well tissue culture plate (Costar, Cambridge, MA), and cultured to subconfluent monolayer for 48 hours. Then, the DMEM–extracts of the adhesives were added to monolayers by medium change. Similar volumes of DMEM without extracted substances were added to control wells. After 24 h of incubation cellular vitality was evaluated by neutral red uptake (NRU) which is a measure of membrane permeability.

Detailed procedures for these measurements have been described by Borenfreund and Puerner (4). In brief: 50 mg/mL of neutral red was add to each well. After incubation for 4 h at 37° C, the supernatant was discarded and the intracellular neutral red were solubilized with 200 ml of 1% acetic acid in ethanol 50%. The absorbance of each 96-well plate was determined using an automatic microplate photometer (Packard SpectracountTM, Packard BioScience Company , Meriden U.S.A.) at 540 nm.
The cell cytotoxicity was calculated according to the equation (7):

% cell mortality
= Control OD – sample OD x 100
Control OD
Each experiment was perfumed in sestuplicate.
Extracts toxicity studies.
Adhesives extract preparation. In order to evaluate the cytopathic effects of five photopolymerized adhesive substances, thirty-five round filter papers (whatman, U.S.A.), 7 mm in diameter, were kept in 70% ethanol for 6 h. After rinsing the papers in PBS, they were dried for 4 hours. On the filter papers, 5 mL of each dentin adhesive materials was applied and light-cured for 20 s (BLUELIGHT pro Mectron medical technology), light source at a standardised distance of 5 mm
Six round filter papers were used for each experimental material (Table 1)
After polymerisation, the adhesive materials were plated on 24 wells plates in for 24 h or for six days (surface to liquid ratio: 11.8 mm2/mL).

Direct toxicity

In order to evaluate the direct cytopathic effects of the photopolymerized adhesive materials, cells of 3T3 cell line were plated at 30.000 cells/cm2 on bottom of every well of two 24-wells plates and maintained for 72 h. Then the round filter papers with and without (control) adhesives were added to monolayer.
After 24 h of incubation the cell viability was evaluated by NRU test.

Statistical Analysis

Each value represents the mean of three experiments, using six replicates of each material per experiment.
All results are expressed as mean SEM. The group means were compared by analysis of variance (ANOVA) followed by a multiple comparison of means by Student-Newman-Keuls. Upon occurence comparison of means by T-Student test was used.
p < 0.05 was considered significant.

Results

24 hours-extract toxicity:
Figure 1 shows cytotoxicity data regarding extracts of freshly cured of five commercially available adhesives, using NRU test. In this experimental conditions, results clearly indicate that all adhesives showed an important cytotoxicity with respect to control. Importantly, extract by adhesive D showed the higher incidence of cytotoxicity (78.3% ± 4.7 SE). However, this cytotoxicity resulted statistically significant different only versus adhesives A and C (cytotoxicity: 49.4% ± 4.9 and 49.5 ± 6.0, respectively).

Six days-extracts toxicity:
Medium conditioned for six days with the five adhesives showed interesting data in term of toxicity using NRU test.
Fig. 2 shows that all adhesive extracts were cytotoxic with respect to control.
Interestingly, cytotoxicity comparison between six days-extracts and 24 hours-extracts permit to realise some considerations:
A, B and E did not show any difference in cytotoxicity with respect to time of incubation of adhesive.
Adhesive D showed a significant reduction in cytotoxicity related to time (% cell mortality 24 hs: 78.3 ± 4.68 versus six days: 52.6 ± 10.64; p < 0.05);
Adhesive C showed a significant reduction in cytotoxicity related to time (% cell mortality: 24 hs: 49.5 ± 5.98 versus 6 days: 15.3 ± 4.63; p < 0.01);
In conclusion, these data seem to show that membrane damage, induced by adhesive C to 3T3 mouse fibroblast cell line in this experimental condition, is less than all other adhesive. However, this difference in cytotoxicity was statistically significant (p <0.05) only with respect to adhesive D.

Direct toxicity:

All of the dentin bonding materials were cytotoxic when tested in direct toxicity in NRU (fig.3) .
A 84.6%, B 76%, C 83%, D 82% and E 53% .
Statistically significant difference was found between the cytotoxicity of E adhesive and all other materials. (E versus A p< 0.01, E versus B p< 0.01, E versus C p< 0.01 and E versus D p< 0.001,).
Interestingly, direct toxicity induced by adhesives A, B and C was statistically higher than one’s by freshly extracts (p< 0.0001, p<0.0001, p<0.05, respectively). Intriguingly, comparison between direct and extract toxicity of adhesives D and E did not show any significant difference.

Discussion

Biocompatibility may be defined as the ability of a material to function in a specific application in the presence of an appropriate host response (8). The need for biocompatible materials implies the necessity of toxicity testing. The toxicity of a dental material can be evaluated by in vitro tests, and by clinical studies in humans. In vitro studies are mainly perfomed to evaluate the cytotoxicity (cell damage) or the genotoxicity (specific DNA damage or chromosomal aberration) of a dental material).
In the present study the biological compatibility of five dental adhesives were examined by in vitro methods. NRU test is considered a fast, valid and reproducible method to evaluate cytotoxicity in vitro, in particular it seems to stress damage to biological membranes in general and plasmatic and lysosomal membranes in particular. In this study, as determined by NRU test adhesives are cytotoxic following application on 3T3 mouse fibroblast cell line. The statistical analysis demonstrated that all extracts reduced cellular viability about 50%, only adhesive Scotchbond 1 ( adhesive D) showed a cytotoxic activity of about 80% (see table 2). Considering six-day extract cytotoxicity, only two samples One Q Bond and Scotchbond 1 (adhesive C e D), showed a decrease in cytotoxicity with respect to corresponding 24 hours-extracts. Interestingly, toxicity induced by Solist, OptiBond Solo Plus and Excite (adhesive A B and E, respectively) did not show any difference respect to 24 hours-extracts.
In direct toxicity all adhesives reduced cellular viability about 80%, only Excite (adhesive E) showed a cytotoxic activity about 50% .

In conclusion, NRU test permitted to rapidly evaluate cytotoxicity of different dental material, and in same time, to address further biochemical and toxicological studies to identify the molecular mechanisms involved in this toxicity.
In particular, comparative studies with others cytotoxicity tests (like the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test) are in program for better clarify the mechanisms of cytopathic effects of dental adhesives.

References

1. Leinfelder K.F. Current status of dentin adhesive systems. Alpha Omegan, 91, 4, 1998.
2. Scuster GS, Lefebvre CA, Wataha JC, White SN. Biocompatibility of posterior restorative materials. J Calif Dent Assoc. 1996; 24: 17-31.
3. Schweikl H, Schmalz G. Toxicity parameters for cytotoxicity testing of dental materials in two different mammalian cell lines. Eur J Oral Sci 1996; 104:292-9.
4. Borenfreund E, Puerner JA. A simple quantitative procedure using monolayer cultures for Cytotoxicity assay (HTD/NR-90) J Tiss Cult Meth 1984; 9:7-9.
5. Borenfreund E, Puerner JA. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicology Lett 1985; 24: 119-24.
6. Babich H, Borenfreund E Applications of the neutral red Cytotoxicity assay to in vitro toxicology. ATLA 1990; 18: 129-44.
7. Hashieh IA, Cosset A. Franquin JC, Camps J In vitro cytotoxicity of one-step dentin bonding systems. Journal of endodontics 25, 2. February 1999.
8. Schmalz G. Use of cell cultures for toxicity testing of dental materials-advantages and limitations. J. Dent. Suppl. 2, 1994; 22: S6-S11.

Table 1: Characteristics of adhesives.

Table 2: Cytotoxicity comparison between six days-extracts and 24 hours-extracts

A, B and E did not show any difference in cytotoxicity with respect to time of incubation of adhesive. Adhesive D showed a significant reduction in cytotoxicity related to time (% cell mortality 24 hs: 78.3 ± 4.68 versus six days: 52.6 ± 10.64; p < 0.05); adhesive C showed a significant reduction in cytotoxicity related to time (% cell mortality: 24 hs: 49.5 ± 5.98 versus 6 days: 15.3 ± 4.63; p < 0.01).



Figures

Figure 1 cytotoxicity of 24 hours-extracts: All adhesives were cytotoxic. Extract by adhesive D showed the higher incidence of cytotoxicity (78.3%±4.7 SEM). this cytotoxicity resulted statistically significant versus adhesives A and C (cytotoxicity: 49.4% ± 4.9 and 49.5 ± 6.0, respectively, p< 0.01).
Figure 2 cytotoxicity of 6 days-extracts: All adhesives were cytotoxic. adhesive C is less toxic than all other adhesive. This difference in cytotoxicity was statistically significant (p <0.05) only with respect to adhesive D.

Figure 3 direct cytotoxicity: All of the dentin bonding materials were cytotoxic. Statistically significant difference was found between the cytotoxicity of E adhesive and all other materials. (E versus A p< 0.01, E versus B p< 0.01, E versus C p< 0.01 and E versus D p< 0.001,).