Detergents, one of the toothpaste components, are frequently used in removing plaque, due to their antimicrobial properties. However, it is stated that besides these positive properties, they also have the potential to adversely affect the oral mucosa . In this study, when the viability rates of different detergent-containing children's toothpaste solutions on cells were evaluated, it was observed that the lowest viable cell rates were in SLS-containing toothpaste solutions. After the control group, the highest vitality values were determined in the toothpaste without detergent content, followed by the toothpaste containing CAPB.
Clinical intraoral side effects such as mucosal sensitivity, epithelial desquamation and recurrent aphthous ulcerations in vitro studies point to the possible problems of these ingredients used in adult toothpaste [27, 33, 34]. Studies examining the effects of these components in children's toothpastes on intraoral tissues are very few. When looking at the contents of children's toothpaste, it is seen that many paste contents contain SLS as a type of detergent. However, considering the side effects of SLS, the different degree of keratinization and morphology of the gingival of children suggests that these side effects may occur more in children.
Therefore, in this study, the effects of 5 different children's toothpaste with different detergent content on cells were investigated. There are different evaluation methods to investigate the effects on cells, to determine the toxic effects of the materials to be used or to investigate their biocompatibility. These tests can be classified as clinical use tests, in vivo animal experiments and in vitro cell culture tests. Among these alternative methods, cell culture tests are frequently used in cytotoxicity studies due to their ability to mimic the physiological conditions of living tissues. In addition, cell culture studies have many advantages such as rapid application, repeatability, standardization, low cost, easy control of the experimental environment during the experiment and not being affected by different individual factors [35, 36]. Since there are some ethical and legal problems in other test methods, in vitro cell culture tests constitute the starting point of such studies in biocompatibility and cytotoxicity studies. In this study, in vitro cell culture tests were preferred to determine the effects of toothpastes on cells.
The cell type used in cell culture studies should be selected in relation to the area of use of the material whose cytotoxic effects are investigated. Primary cell cultures or continuous cell lines are used in studies as a biological system in biocompatibility tests. It is stated that continuous cell lines such as L929, 3T3, HSC-2, MRC-5 can be used in the cytotoxicity assessment tests of materials used in dentistry, since they can be obtained more easily than primary cell cultures and have rapid reproduction potential. However, since primary cell cultures are more sensitive than continuous cell lines, they are biological systems that best reflect the original physiological state, despite the difficulties that arise during the production phase and the long time to produce [30, 35, 37,38,39,40,41]. For this reason, it was preferred to create a primary cell culture in this study, considering the creation of experimental conditions closer to in vivo conditions. Gingival epithelial cells were used as a biological system in this study, since the majority of oral tissues that toothpastes come into contact with during tooth brushing are gingival tissues.
In studies for the characterization of gingival epithelial cells, the method of determining epithelial cells specific CK13 and Vimentin genes by PCR, analysis of phenotypic properties of cells by transmission electron microscopy, determination of a specific epithelial marker cytokeratin by immunofluorescence method, staining of cells with Papanicolau staining method and analysis under light microscopy. methods were used . In this study, the cells, which are easier and quicker to apply than other methods and are also more cost-effective, were stained with hematoxylin and eosin dyes after fixation with alcohol on the slide, and the cells were analyzed under light microscopy, and the presence of epithelial cells was determined.
Many in vitro tests such as MTT, trypan blue exclusion test, micronucleus are used to determine cell viability [27, 28, 43,44,45,46,47,48]. Flow cytometry analysis is frequently recommended in terms of providing more reliable, faster and more sensitive results than other methods in evaluating cell viability and cytotoxicity . In addition to determining cell viability, information about different properties of cells such as immunophenotypic properties, enzyme activities, and specific markers of the cell can be obtained with this method [49, 50]. In addition, the separation of apoptotic and necrotic cells with this method is important in terms of different biological responses of these two types of death [49, 51]. In this study, since gingival cells are labeled with Annexin V and propidium iodide dyes, since they give faster, more sensitive and reliable results compared to alternative methods used in cell culture studies, it was ensured that live, early apoptotic, late apoptotic and necrotic cells were determined by flow cytometry analysis.
In the literature, changes caused by SLS, which is frequently used in toothpaste, on the oral mucosa have been reported. In addition, in a few studies examining the effects of SLS on cells, it has been stated that they have a negative effect on cell viability [27, 28, 52,53,54,55,56,57]. In this study, SLS, sodium lauryl sarcosinate, sodium C14-16 olefin sulfonate, CAPB containing toothpastes which are reported to be more biocompatible than SLS, toothpaste without detergent and CDMEM were selected as experimental groups. While determining the concentrations of toothpaste solutions in cell viability experiments, similar studies have been examined and optimized as 0.4%, 20%, 50% and 80%. In addition, in this study, the stimulation time of toothpaste solutions with cells was determined as 2 min, since the brushing time was 2 min under normal conditions [27, 28, 43].
When the studies on detergents are examined; Herlofson et al. found a positive relationship between oral desquamation and SLS in their study . Melsen et al. examined the effect of SLS on monoflurophosphate, and it was stated that SLS reduced the amount of fluoride taken up by the enamel . Rantanen et al. reported that toothpastes containing SLS have an irritating effect on the mucosa . Shim et al. investigated the effect of SLS on recurrent aphthous stomatitis and showed that SLS significantly increased the incidence of ulcers, the duration of ulcers in the mouth, and the pain score .
In this study, when the viability rates of different detergent-containing children's toothpaste solutions on human gingival epithelial cells were evaluated, it was seen that the lowest proportion of viable cells was in toothpaste solutions containing SLS. After the control group, the highest vitality values were detected in toothpaste without detergent content, followed by toothpaste containing CAPB. The effects of this study on cell viability Cvikl et al.'s findings in studies examining the effects of adult toothpastes and children's toothpaste on cells [27, 28]. Moore et al. also found that cell viability rates in SLS and betaine containing toothpastes were lower than the control group. These findings are also similar to the findings in our study.
In the literature, the effects of toothpastes on cells have been examined only in terms of living cell proportions [27, 28]. In this study, early apoptotic, late apoptotic and necrotic cell ratios were evaluated as well as the live cell ratios. In the comparisons between the groups, the Colgate group generally shows the highest value in terms of early apoptotic, late apoptotic and necrotic cell ratios, while Splat and the control group generally have similar values in terms of cell death type rates. Considering that SLS increases cellular permeability by causing denaturation of cellular proteins in this study, we think that the opening of the pores between cells may cause the release of apoptosis-inducing proteins into the cytosol and ultimately stimulate apoptosis/necrosis mechanisms. It has been reported that stimulation of apoptosis and necrosis mechanisms in gingival epithelial cells may prevent periodontal wound healing and prolong the healing period [61, 62]. In this study, it is thought that the increase in the ratio of apoptotic and necrotic cells of SLS-containing toothpaste may delay the healing time of periodontal diseases and oral aphthous ulcers and adversely affect wound healing.
This study has some limitations due to the absence of saliva, the protective and immunological properties of tissue barriers. In addition, this study suggests that other ingredients in toothpaste may also have toxic effects, since detergent ingredients cannot be supplied in pure form. However, in order to eliminate this limitation, toothpastes used in similar age groups and having similar contents formed the study groups in our study.