Assessment of Cytotoxic and Genotoxic Effect of Fissure Sealants in Buccal Epithelial Cells.

AIM: The main purpose of this study was to assess the genotoxic and cytotoxic effect of fissure sealants on buccal epithelial cells.
MATERIAL AND METHODS: The study was conducted on 45 patients (27 girls and 18 boys), seven to 16 years of age (age mean 12.09 ± 2.20). Buccal swabs were collected before (T0), seven (T1), 30 (T2) and 90 days (T3) consequently after fissure sealant placement (Helioseal F®, Equia Fil®, Constic®). Patients or legal guardians filled in the questionnaire regarding the demographic data (age, gender), dietary habits, health status, medication usage, and recent X-ray exposure. DNA damage was analyzed using the micronucleus test.
RESULTS: Statistically significant difference in the number of buccal cells with condensed chromatin was found between T0 (time before fissure sealant placement) and T3 (90 days after fissure sealant placement) period for Helioseal F® (P = 0.025). For the other two analyzed materials, no difference was observed during the tested period. There was no difference between materials in the same sampling time.
CONCLUSION: Apart from an increase in cells with condensed chromatin 90 days after the placement of Helioseal F®, no other nuclear abnormalities were observed for tested fissure sealants. Although these sealants have now largely been used, it is of high importance that their biocompatibility is checked continuously, especially in in vivo clinical studies.

Introduction

The primary assignment of dental medicine is to prevent oral disease and maintain oral health (). Regardless of the current progress of dental medicine and the improvement of living conditions, oral diseases especially caries still cause severe problems and dysfunction of the masticatory system, which impacts the entire human body and the quality of life (). Dental caries is a chronic disease which occurs as the interaction of host, pathogen, environment and time ().

The morphology of the tooth, the composition and quantity of saliva, frequent meals, inadequate nutrition, and poor oral hygiene promote the formation of a carious lesion (). About 80-90% of all caries lesions are localized occlusally, in the fissure system (, ). Food residues can remain in low and narrow fissures, saliva and microorganisms, which creates a biofilm - a complex structure that is both their nutrient substrate as well as protection from external influences (). Acids, as a by-product of carbohydrate metabolism, dissolve the inorganic components of hard tooth tissues, allowing bacteria and their toxins to penetrate deeply through the tooth structure, ultimately infecting the pulp itself, resulting in a pulp and periapical diseases ().

Fissure sealing is an interceptive procedure in which deep and narrow fissure system is sealed with a material that can adhere to the enamel. In this way, the penetration of food and bacteria into the fissure system is prevented (, ). Sealing materials are classified into three groups: materials based on the resin, glass ionomer cement and compomers ().

Biocompatibility is the ability of a material to function in a specific role in the presence of an adequate host response. This definition implies an interaction between the host, the material, and the function that that material is supposed to perform. All three factors must be in harmony before the material can be considered biocompatible. Biocompatibility reflects the physical, mechanical and chemical properties of the material, while the adequate host response implies the ability of the tissue to withstand the presence of foreign material (). Daily, in dental practice, many materials are used, and it is of great importance that those are not harmful or contain substances that would cause local or systemic side effects (, ).

The biocompatibility of all dental materials is essential. Primarily, they come into very close contact with human oral tissues over a long period, and the patient is unable to remove them. Since certain dental materials have already been on the market, many clinicians believe they are safe and should not be reviewed (). But biocompatibility is not in the static state and thus is subject to change since the material is exposed to mechanical, chemical and thermal changes. Furthermore, adverse reactions often appear only after chronic exposure, and scientific studies, especially those on new materials, do not usually last long enough to obtain such data ().

As one of many tests to assess genotoxicity, the micronucleus assay is used to evaluate the damage of genetic material during mitosis due to exposure to potential genotoxic substances (, ). The presence of micronuclei, small extranuclear structures containing fragments of damaged chromosomes in the cell, confirms the existence of damaged genetic content, which proves the genotoxic and cytotoxic effect of the tested material on the cells (). Also, cells with damaged genetic material can pass into cells with condensed chromatin, fragmented nucleus (karyorrhexis), and pyknosis or completely lose nuclear content (karyolysis). In some cases, some cells remain in the binuclear phase (two nuclei of equal size) or have nuclear buds ().

Thus, the aim of this in vivo study was to assess the influence of three sealing materials on the epithelial cells of the buccal mucosa. The null hypothesis was that all tested materials would not cause any genotoxic and cytotoxic effect in buccal epithelial cells.

Material and Methods

The study included 45 subjects, 27 girls and 18 boys; aged seven to 16 (mean age 12.09 ± 2.20) and was conducted in Departments of Pediatric and Preventive Dentistry of the School of Dental Medicine in Zagreb and Study of Dental Medicine, School of Medicine in Split, Croatia. It was approved by the Ethics Committee of the School of Dental Medicine, University of Zagreb. All patients participated voluntarily. The parents and caregivers were informed in details of the study's purpose and design and signed a consent form. Inclusion criteria were healthy children with indications for the sealing procedure on lower molars (), with no other restorations in the oral cavity. Exclusion criteria were special health care children, children with contraindication for fissure sealing, and children with restorations. During the research, only one lower molar was sealed. The subjects were randomly divided into three equal groups, depending on the material used. The material properties and their composition as specified by the manufacturer are listed in Table 1. The respondents, with the help of parents/caregivers, filled a questionnaire, designed for this study, in which they answered the questions related to demographic factors (age, gender), lifestyle habits (diet) and personal factors (health status, use of medications, exposure X-ray radiation).

Table 1

Materials used in the study

Material	Type	Manufacture	Chemical composition
Helioseal F®	Fluoride releasing resin-based	Ivoclar Vivadent, Schaan, Liechtenstein	Bis-GMA (11.8%), TEGDMA (23.4%), UDMA (23.4%), silicon dioxide and fluorosilicate glass (40.5 wt %), titanium dioxide, stabilizers and catalysts (< 1 wt%).
Equia Fil®	Glass ionomer cement	GC, Tokyo, Japan	polyacrylic acid (5%), strontium fluoroaluminosilicate glass (95%)
Constic®	Self-adhesive flowable composite	DMG, Hamburg, Germany	Bis-GMA, EBADMA, UDMA, HEMA, TEGDMA, HDMA, MDP, stabilizers and catalysts
Abbreviations: Bis-GMA, Bis-phenol A diglycidylmethacrylate; TEGDMA, triethylene glycol dimethacrylate; UDMA, urethane dimethacrylate; EBADMA, ethoxylated bisphenol-A dimethacrylate; HEMA, 2-hydroxy ethyl methacrylate; HDMA, 1,6-Hexanediol dimethacrylate; MDP, 10 Methacryloyloxydecyl dihydrogen phosphate.

The sample size was calculated by the G Power software. With a 95% confidence interval, an 80% power, and an effect size of 80% (), a sample size of 45 (15 in each group) was necessary.

Samples of epithelial cells of the buccal mucosa were collected by the brushing technique of each subject immediately before (control, T0), seven (T1), 30 (T2) and 90 (T3) days after the fissure sealing. Participants were asked to abstain from food 1 hour before sampling, and since they are children, it implies that they did not consume alcohol or cigarettes. After the participants rinsed the oral cavity with the tap water, the swab was taken by gently brushing the buccal mucosa areas near the set material. Then, the cells collected with the brush were applied to the encrypted slides pre-heated to 37°C. After the application, the cell samples were air-dried and then fixed with methanol (80% v / v) at four °C for 20 minutes. The samples were stained with 5% Giemsa solution for 10 minutes, washed with distilled water and air-dried. The analysis was performed with an Olympus CX 40 light microscope (Olympus, Tokyo, Japan) at 400x magnification, while some observed chromatin anomalies were additionally tested under magnification 1000x. Two replicate slides were prepared for each sample. On each slide 2000 epithelial cells were analyzed. The occurrence of chromatin abnormalities (micronuclei, binucleated cells, condensed chromatin karyorrhexis, karyolysis, pyknosis, nuclear buds and “broken egg”) was evaluated and qualified, according to Tolbert et al. ().

Statistical analysis

For the statistical analysis of the data, the software package SPSS (IBM Corp., Armonk, New York, USA) was employed. Descriptive statistical analysis was used to determine the basic statistical parameters (mean, standard error, standard deviation, relative standard deviation, median and minimum and maximum values). The difference among the tested groups was assessed by the analysis of variance and the Tukey HSD post hoc test.

Multiple linear regression analysis was used to determine the mentioned chromatin abnormalities as the outcome variable with predictor variables. Predictor variables were: age, gender, use of medication, X-ray exposure, dietary habits such as consumption of sweets, meat, vegetables, fruits, soda drinks and tea, the time elapsed since the placement of sealing material, and also type of material (Constic® was set as the base material for regression analysis). The results were expressed in Pareto diagrams. The level of significance was set at 0.05 for all tests.

Results

Figures 1 and 2 show the basic statistical parameters used to obtain results using a micronucleus test on buccal mucosal cells in three groups of subjects at four times: T0 - immediately before fissure sealing, T1 – seven days, T2 – 30 days and T3 – 90 days after sealing the fissure. A statistically significant difference in the number of cells with condensed chromatin was observed between T0 and T3 time for Helioseal F® (P = 0.025). For other two sealant materials, there was no significant difference. No statistically significant difference was observed between the different materials, comparing the nuclear abnormalities at the same sampling time.

Figure 1

The frequency of binucleated cells, cells with micronuclei, karyolysis and karyorrhexis in 2000 buccal epithelial cells after fissure sealant placement (n = 15/material for each time-point of measurement). Mean values are expressed as columns, error bars represent standard deviations.

Figure 2

The frequency of cells with nuclear buds, “broken egg”, condensed chromatin and pyknotic cells in 2000 buccal epithelial cells after fissure sealant placement (n = 15/material for each time-point of measurement). Mean values are expressed as columns, error bars represent standard deviations.

The dependence of the micronucleus test parameters on all predictor variables was determined by multiple regression analysis and presented in the form of a Pareto diagram (Figures 3 and 4). The appearance of cells with condensed chromatin confirmed a statistically significant effect of the following variables: X-ray exposure (β = -0.386; P = 0.031), the medication intake (β = -1.041; P = 0.031), meat consumption (β = 0.332; P = 0.031), soft drinks consumption (β = 0.673; P < 0.001), tea consumption (β = -0.693; P = 0.001) and consumption of sweets (β = -0.336; P < 0.001). The number of cells with condensed chromatin and karyolysis was statistically significantly affected by the type of material used for fissure sealing. Children who had fissure sealed with Helioseal F® had a significantly lower number of cells with condensed chromatin (β = -0.375; P = 0.043), and a higher number of cells with karyolysis (β = 0.932; P = 0.024) than those children who had fissure sealed with Constic®. The number of binucleated cells was affected by gender (β = -0.200; P = 0.030).

Figure 3

Multiple regression analysis results. Association of measured cytogenetic endpoints (number of cells with micronuclei, karyolysis, karyorrhexis and binucleated cells) and demographic and lifestyle factors exposure duration, type of dental material and the time elapsed since the placement of sealing material.

Figure 4

Results of multiple regression analysis. Association of measured cytogenetic endpoints (number of cells with nuclear buds, “broken egg”, condensed chromatin, and pyknotic cells) and demographic and lifestyle factors exposure duration, type of dental material and the time elapsed since the placement of sealing material.

Discussion

In this study, the potential genotoxic effect of the sealing material on the buccal mucosal epithelial cells was evaluated. The influence of each material on the appearance of micronuclei and other abnormalities of the nucleus (karyorrhexis, condensed chromatin, pyknosis, nuclear buds, “broken egg,” binucleated cells, karyolysis) was observed at three different times: seven (T1), 30 (T2), and 90 (T3) days after placement.

Helioseal F® was the only material in the study, which caused a statistically significant increase of cells with condensed chromatin 90 days after fissure sealing (T3) relative to the baseline values (T0). The reason for this result could be potentially hidden in the phenomenon of the release of free, unbound (residual) monomer from the material, especially if it is improperly polymerized. In the study of Topaloglu-Ak et al. () the polymerization time and the amount of residual monomer released were compared, and the association of increased cytotoxicity with lower polymerization of the material was confirmed. In the in vitro study of Kurt et al. () conducted in 2018, the cytotoxicity and genotoxicity of release monomers of resin-based materials were confirmed with the series of tests. Furthermore, incompletely polymerized materials have lower physical properties, making them more susceptible to degradation in the oral cavity, which is an additional source of free monomer that has been proven to have a cytotoxic and genotoxic effect (, ).

Although the amount of released monomers is detectable and toxic in vitro, it has been concluded that it is still too small to cause long-term in vivo damage (). The analysis of changes in the nucleus within the same period confirmed our hypothesis that there is no difference in genotoxicity between different fissure sealants.

Furthermore, Helioseal F® was the only material containing fluoride. Despite being one of the most important agents for remineralization process, fluoride has potentially genotoxic effect (, ). In addition, the other variables that potentially affect the occurrence of nuclear abnormalities such as gender, age, X-rays exposures medicines intake, the consumption of meat, sweets, soda drinks and tea were analyzed (, , ). Previous studies have confirmed that age is a variable that affects the genetic content (, ). Degenerative changes that occur with ageing are primarily the result of changes at the level of genetic material in the cells. Hence, it is not surprising that the number of recorded nuclear abnormalities detected by the micronucleus test is higher in the elderly population (). In this study, the results obtained by multiple regression analysis that the age had a significant impact on the result were not recorded. That could be explained by the fact that all respondents were children and that the oldest respondent was only 16 years old. Gender was the only predictor variable that showed a statistically significant increase in the number of binuclear cells. Namely, a higher number of binucleated cells was noticed in girls than in boys (β = - 0.200; P = 0.030). Our results coincide with the results obtained in the research of Gajski et al. () in 2013 among healthy children in Croatia, in which larger numbers of binuclear cell changes were observed in girls.

To the best of our knowledge, there are no studies which studied exclusively the biocompatibility of materials for fissure sealing in vivo conditions. Namely, the existing studies were generally based on the analysis of the genotoxicity of other dental materials. However, the results of our study are consistent with previously conducted in vitro studies (-, ).

This study has certain limitations; one of them is a relatively small number of respondents. Therefore, it would be necessary to conduct large-scale research over a more extended time in the future. In this way, an association between material and time with the occurrence of nucleus pathological changes could be observed. Since all dental materials are susceptible to degradation by the enzyme in the mouth, it is necessary to associate clinical durability of fissure sealants with possible genotoxic and cytotoxic changes in surrounding tissues. Also, it could be useful to include fissure sealants with a new monomer 12-methacryloyloxydodecyl pyridinium bromide (MDPB) with antibacterial activity (, ).

We could conclude that a need for more extensive knowledge about the safety of dental materials used in clinical practice is crucial. Sealing materials should be uncompromisingly harmless to the child. The usage of fissure sealing materials has primarily preventive effects, and even at the slightest indication of their toxicity, the ethics of their usage should be fundamentally re-examined.

Conclusion

Based on the results, the following can be concluded:

Over a short period, none of the examined material showed a significant increase in the frequency of evaluated nuclear abnormalities.

Over an extended period (90 days), Helioseal F® caused a statistically significant increase in the incidence of cells with condensed chromatin.