Tissue and Cell 72 (2021) 101538

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Tissue and Cell

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Effects of different detergent-containing children’s toothpastes on the

viability, osteogenic and chondrogenic differentiation of human dental
periodontal ligament stem cells and gingival stem cells in vitro
a, b b b c
Sinem Birant *, Yazgul Duran , Muazzez Gokalp , Tunc Akkoc , Figen Seymen
a
Istanbul University-Cerrahpas¸a, Faculty of Dentistry, Department of Pedodontics, Istanbul, Turkey
b
Marmara University, Faculty of Medicine, Department of Pediatric Allergy-Immunology, Istanbul, Turkey
c
Istanbul University, Faculty of Dentistry, Department of Pedodontics, Istanbul, Turkey

ARTICLE INFO mesenchymal stem cells exposed to toothpaste solutions were examined morphologically.
Datas were analyzed with IBM SPSS V23. One way ANOVA test was used to determine
Keywords:
the differences between the groups for multiple comparisons, while the Tukey post-hoc
Toothpaste
test was used for pair wise comparisons in determining which groups differed.
Stem cell
Anneksin V Results: A higher percentage of cell viability was detected in Control group at 20 %, 50 %
Detergent and 80 % (p = 0.000) on hGMSCs. After the Control group, the highest cell viability
SLS ratios were observed in the detergent-free Splat group (p = 0.000) followed by the
ABSTRACT Sensodyne experimental group containing CABP (p = 0.000). While the cell viability
rates in Nenedent group was found significantly higher than the Perlodent group at other
Background: Detergents are the most commonly used compounds in toothpastes due to
concentrations except for 20 % concentration (p = 0.000). Colgate group had the lowest
their foaming and cleaning peoperties. This study aimed to investigate the effects of
percentage of cell viability among the experimental groups at all concentrations on
children’s toothpastes with different detergent content on the viability, the osteogenic
hPDMSCs (p = 0.000). The highest live cell ratios was detected in Control group (p =
and chondrogenic differentiation potentials of human mesenchymal stem cells.
0.000), followed by Splat and Sensodyne groups (p = 0.000). The cell viability ratios at
Methods: The necessary tissues for human periodontal ligament mesenchymal stem cells
50 % concentration were higher in Perlodent group than Nenedent group (p = 0.000).
(hPDLMSCs) and human gingival mesenchymal stem cells (hGMSCs) isolation were
The highest osteogenic and chondrogenic differentiation potential of mesenchymal stem
obtained during extraction of 10 impacted third molar teeth. The viability of the cells
cells stimulated with different toothpaste was determined in Control and Splat group.
stimulated with different concentratiaons of Colgate, Sensodyne, Splat, Nenedent,
Conclusions: As a result of the findings, it was observed that toothpaste containing SLS
Perlodent toothpaste solutions and complete Dulbocco’s modified eagle medium (control
had a more negative effect on the viability of the cells and the differentiation potentials
group) were evaluated by using the flow cytometer. In addition, the osteogenic and
than the other groups.
chondrogenic differentiation potential of human gingival and periodontal ligament

Abbreviations: MSCs, mesenchymal stem cells; hGMSCs, human gingival mesenchymal stem cells; hPDLMSCs, human periodontal mesenchymal stem cells; GEC,
gingival epithelial cells; LC50, half lethal dose concentration; SLS, sodium lauryl sulfate; PDL, periodontal ligament; CDMEM, complete dulbecco ’s modified eagles
medium; CABP, cocoamidopropyl betaine; DPBS, dulbecco’s phosphate buffered saline.
* Corresponding author at: Istanbul University-Cerrahpas¸a, Faculty of Dentistry, Department of Pedodontics, Istanbul, 34093, Turkey. E-mail addresses:
sinembirant@istanbul.edu.tr (S. Birant), yazgul.duran@gmail.com (Y. Duran), muazzez.gklp@gmail.com (M. Gokalp), tuncakkoc@marmara. edu.tr (T. Akkoc),
fseymen@istanbul.edu.tr (F. Seymen).

https://doi.org/10.1016/j.tice.2021.101538
Received 4 February 2021; Received in revised form 22 March 2021; Accepted 23 March 2021

Available online 26 March 2021

 0040-8166/© 2021 Elsevier Ltd. All rights reserved.
S. Birant et al.
1. Introduction
Toothpastes are ideal personal care products for removing plaque from tooth
surfaces and for maintaining oral hygiene. The many com ponents in the
formulation of toothpaste each play a different role. Detergents are the most
commonly used compounds in toothpaste due to their foaming and cleaning
properties (Forward et al., 2000; Petersen et al., 2006).
The anionic and amphoteric detergent classes are frequently used in
toothpastes. In addition to foaming, they are routinely added to tooth pastes for
their antibacterial and plaque inhibition properties. (Buma et al., 2006). The
compound most often used in toothpastes is sodium lauryl sulfate (SLS) from the
anionic detergent group. Other anionic detergents used in toothpastes are
sodium lauryl sarcosinate and sodium C14− 16 olefin sulfanate (Healy et al.,
2000).
SLS inhibits the growth of a number of microorganisms. The anti microbial
effect of SLS is related to its adsorption and penetration through the porous cell
wall, followed by its interaction with the cell membrane, lipids and proteins
components. Penetration by SLS into the cell membrane causes an increase in
the cell permeability of bacteria, which may lead to leakage of intracellular
components and cell lysis (Law and Seow, 2006; Nordstrom et al., 2009).
Despite these positive characteristics, some toxic effects of SLS have been
reported. In clinical studies, oral epithelial destruction, ulceration
inflammation have been observed to be caused by SLS (Macdonald et al., 2015).
Studies show that SLS in toothpaste may lead to the deterioration in the barrier
function of the oral mucosa by denaturing glycoproteins of the mucin layer,
leading to the gum and buccal mucosa being more sensitive to irritants such as
exogenous antigens (Neppelberg et al., 2007; Siegel and Gordon, 1986). It is
also reported that SLS may be responsible for a decrease in the keratinization of
the human oral epithelium. It has been shown that SLS can penetrate deeper
layers of the mucosa, breaking down the mucin layer, causing denaturation of
proteins in epithelial cells, increasing the solubility of the structural lipids of
cells and interfering with the function of living tissue (Mac donald et al., 2015;
Siegel and Gordon, 1986; Healy et al., 1999). Altough SLS is the most common
surfactant in toothpaste, surfactants with fewer side effects such as the betaines,
are also used. Cocoamido
propyl betaine (CAPB) has been shown to cause less mucosal irritation than
sodium lauryl sulfate (Cvikl et al., 2017; Cvikl et al., 2015). Cocoamidopropyl
betaine, due to its milder nature and low level of skin irritation, is more
common in children’s toothpastes than in adult toothpastes (Danov et al., 2004;
Herrwerth et al., 2008; McLachlan and Marangoni, 2006).
In addition to the reactions that these toothpaste detergents may have with
the tissues of the oral mucosa, their effects on cells are also important.
Cytotoxicity tests used to determine the biocompatibility of a material are
known as first-line tests. These cytotoxicity tests are methods by which cell
numbers and viability are evaluated, and they are the preliminary tests used to
determine the biological properties of a material (Turkcan and Nalbant, 2016;
Rodriguez-Lozano et al., 2013).
When studies on deterjents are considered, it becomes evident that very few
studies have investigated the toxic effects of toothpastes con taining different
detergent on cells and cell lines such as epithelium and keratinocytes (Cvikl et
al., 2015, 2017).
However, it is also very important to know their effects on the mesenchymal
stem cells (MSCs). Studies have shown that the MSCs contribute to tissue
regeneration by replacing damaged tissue cells, they support the production of
many factors with anti-inflammatory func
tions and differentiate into epithelial-like cells in mucosal infections ( Hermann
et al., 2006; Kuroda et al., 2010; Roddy et al., 2011; Prockop and Oh, 2012).
In addition, periodontal ligament (PDL) MSCs contribute to the regeneration
of the periodontium by playing a role in the formation of cementum and PDL
tissues in vivo (Seo et al., 2004; Wang et al., 2011; Zhou et al., 2008). Gingival
mesenchymal stem cells (GMSCs) are also
Tissue and Cell 72 (2021) 101538
great importance for stem cell studies since they are easily accessible and can be
obtained with minimally invasive cell isolation techniques, while they also have
regenerative and immunodulatory properties (Marynka-Kalmani et al., 2010;
Zhang et al., 2009; Thang et al., 2011; Zhang et al., 2012a; Venkatesh et al.,
2017).
Therefore, if the type of detergent used in a toothpaste does not damage the
MSCs during brushing, it is a significant advantage in relation to maintaining
oral mucosal health or supporting a successful treatment process. Taking these
factors into consideration, this study was the first to include cytotoxic effects on
gingival mesenchymal stem cells and periodontal ligament mesenchmal stem
cells. The viability of MSCs, affecting their ability to contribute to wound repair
and the healing and regeneration of periodontal tissues, will depend on their
differentiation potential. They help in the healing and repair processes by
migrating to different tissues and by differentiating into different cell types
and (Gronthos et al., 2000). Therefore, the effects of specific toothpaste detergents
on stem cells and on osteogenic and chondrogenic differen tiation were also
investigated in this study in addition to the viability tests. The study also aimed
to investigate the effect of children’s toothpastes with a different detergent
content on the viability and osteogenic and chondrogenic differentiation
potential of GMSCs and PDLMSCs. This study is the first to examine the effects
of toothpastes containing different detergents on primary stem cells.
2. Materials and methods
The study was approved by the ethics committee of Istanbul Uni versity,
Faculty of Dentistry (170/2017) following Helsinki Declaration guidelines.
2.1. Isolation of primary mesenchymal stem cells (MSCs)
10 human third molars, which were removed for impact resason from
systemically healthy patients (aged 18–25 years) were used for tissue biopsy
and periodontal ligament (PDL) cell isolation. Meanwhile, gingival tisues
surrounding the tooth sockets were collected after tooth extraction. The PDL
tissues were seperated from the surface of the middle third of the root under a
3
laminar hood. The PDL and gingival tissues were divided into 1 mm pieces by
applying the mechanical seperation method with the scalpel. After
micromechanical digestion, colllagenase solution was prepared in 1 mL of DPBS
(Dulbecco’s phos
phate buffered saline) (Gibco, Grand Island, NY, USA) to 0.003 g Type 1
collagenase (Gibco) and 2 mL was added to micromechanically decomposed
tissues and enzymatic digestion was started. The mechan ically disintegrated
tissues taken into 15 mL sterile tubes were incubated in the incubator for 45
min-1 h at 37 ◦C, 5% CO2. Tubes connected to the rotator were vortexed at
frequent intervals until the cell suspension. After the tubes were removed from
the incubator, the cells were centrifuged at 1500 rpm for 5 min and the cells
2
were transferered to T-25 cm flask with 4 mL culture medium [45 mL DMEM
(Dulbecco’s modified eagle medium); Gibco, 5 mL PBS; Gibco, 500 μL penicillin;
 Gibco]. The culture medium was changed every 2 days and the prolif Phenotype of isolated hPDLMSCs and hGMSCs was determined by flow
eration and spreading of the cells on the flask was monitored at regular intervals cytometry analysis for expression of CD146-FITC, CD45-FITC, CD29-APC, CD25-
by invert miscroscıpe (EVOS-AMG, Thermo Fisher Scientific, Waltham, MA, APC, CD105-PE, CD90-PE, CD73-PE, CD34-PE, CD28- PE and CD14-PE (BD
5
USA). Cells from 3rd passage were used for viability and differentation Biosciences, CA, USA) markers. The cells (5 × 10 cells for each cell type) were
experiments. incubated with antibodies specific for human markers associated with
mesenchymal and hematopoietic line
2.2. Characterization of stem cells ages at room temperature in the dark. Flow cytometry outcomes were

2
Tissue and Cell 72 (2021) 101538
S. Birant et al. toothpaste solutions were obtained at a concentration of 80 % from each
toothpaste. Then, 80 % concentration of toothpaste solutions were diluted with
examined using a flow cytometer (BD, FACS Calibur, San Jose, CA, USA).
DMEM (Gibco) and 50 %, 20 % and 0.4 % concentrations were prepared. The
The ostogenic, chondrogenic and adipogenic differentiation poten tials of the
prepared solutions were vortexed to obtain a ho
cells were analyzed using the differentiation kits [Stem Pro Osteogenesis
mogeneous toothpaste mixture. The homogenized toothpaste solutions were
Differentiation Kit (Thermo Fisher Scientific), StemPro Chondrogenesis
incubated for 24 h at 37 ◦C in a humidified 5% CO2 environment. to obtain
Differentiation Kit (Thermo Fisher Scientific), Stem Pro Adipogenesis
Differentiation Kit (Thermo Fisher Scientific)]. The cells were plated in 6 well extraction fluids in accordance with ISO (International Orga nization for
5 Standardization) 10993− 12 standards (2007). Subse quently, each tube was
plates (5 × 10 ) cell/well) and the differentiations mediums were applied on
centrifuged at 4200 rpm for 10 min. After centrifugation the solid particle were
cells according to the manufacturer’s in structions. The differentiation mediums
collected at the bottom of the tube and the toothpaste solutions were passed
were changed 3 times per week. After 14 days the chondroctes and adipoctes
through the 0.22 μm filter 2 times to remove the remaining small particles. The
were stained with Alcian Blue (Sigma-Aldrich, St. Louis, MO, USA)) and Oil Red
material extracts were obtained for the cell viability tests (ISO 10993-5, 2009).
(Sigma-Aldrich) respectively. After 28 days, Alizarin Red staining (Sigma-
Aldrich) was used for characterization of the osteocytes. The staining cells were 2.4. Evaluation of cell viability by flow cytometry
evaluated using a binocular microscope (Olympus, BH2-RFCA, Olympus, Tokyo,
5
Japan). GMSCs and PDLMSCs (5 × 10 cells for each toothpaste solution) were plated
into 48-well plates. Then the cells exposed for 2 min to different concentrations
of toothpaste, washed with PBS and suspended in serum-free medium. 4 μL of
2.3. Preparation of toothpaste solutions
Annexin V (BD Biosciences) was added to the tubes and kept in a dark place for

The toothpastes used in this study were Colgate6+ (type of deter gent: SLS), 10 min. Then 200 μL of binding buffer was added and centrifuged at 1500 rpm

Sensodyne Pronamel 6+ (type of detergent: CAPB), Nenedent (4− 9 aged) for 5 min to remove the supernatant. Tubes were vortexed with 200 μL binding

(detergent type: Sodium Lauryl Sarcosinate), Perlodent Ju nior 6+ (type of buffer and added to 10 μL propidium iodide to read the effects of toothpaste

detergent: Sodium C14-C16 Olefin Sulfanate), Splat Juicy (detergent free). Their solutions on live, necrosis, early and late apoptotic cell ratios. Viability

complete specifications can be read in Table 1. Four different toothpaste experiments performed with flow cytometry were repeated 5 times, and the
average of the values obtained was calculated and the proportions of viable,
solutions were prepared from the children’s toothpastes used in this study. For
early apototic, late apoptotic and necrotic cells were determined.
this process, the amount of toothpaste was weighed in the sensitive balance and
diluted in ster ileconical plastic tubes in serum free culture medium. First of all,
2.5. Evaluating of the differentiation potentials of stem cells

Table 1
For examination the differentiation potentials of GMSCs and PDLMSCs cells
Composition of materials evaluated. 5
were seeded with 5 × 10 cells per well by inserting type I
Materials Composition Manufacturer

Colgate 6þ Sorbitol, aqua, hydrated silica, PEG-12, Sodium Lauryl Colgate Palmolive Company, Belgium performed; calcification densities and cell
Sulfate, cellulose gum, collagen coated coverslips into each of the 6 well morphologies were evaluated at the binocular reserach
sodium saccharin, sodium fluoride (1450
−
plates. Cells in each well were stimulated with 2 mL microscopy (Olympus, BH2-RFCA) for examination of
ppm F ), aroma,hydroxypropyl
methylcellulose, menthol, glycerin,
differentiations mediums after stimu lated with % 0,4 the osteogenic differentiation potential. At the end of
cinnamal, eugenol, limonene, CI 77891, CI concentration of toothpaste solutions for 2 min. At the the 14th day, Alcian Blue staining
42,090 end of the 21 st day, Alizarin Red staining was
Nenedent Kids (4¡9 aged) 6þ Aqua, sorbitol, hydrated silica, glycerin, PEG-6, Glaxo Smith Kline, ABD
Aqua, hydrated silica, glycerin, xylitol, propylene Cocamidopropyl Betaine, xanthan gum, aroma,
glycol, xanthan gum, titanium dioxide, aroma, sodium fluoride(1450 ppm F− ), sodium saccharin, was performed; presence of proteoglycan
Sodium Lauryl sucralose, titanium dioxide, sodium hydroxide, and cell morphologies were evaluated at
Sarcosinate, disodium EDTA, limonene
Perlodent the binocular reserach microscopy
Junior 6þ sodiummonofluorophophate (500 ppm F− ), sodium Dentinox, Berlin, Germany
chloride
(Olympus, BH2-RFCA) for examination
Aqua, sorbitol, hydrated silica, propylene glycol, of the chondrogenic differentiation
tetrapotassium pyrophosphate, xanthan gum, potential. At the end of the 14th day, Oil
Sodium C14− 16 Olefin Sulfonate, aroma, titaniumRossmann,
Red O staining was performed; presence
dioxide, sodium fluoride (1450 ppm F− ), sodium
Sensodyne Germany
saccharin, phenoxyethanol, ethylhexyl glycerin of oil droplets and cell morphologies
Pronamel
were evaluated at the Binocular Reser
ach Microscopy (Olympus, BH2-RFCA,
Splat Juicy Aqua*, dicalcium phosphate dihydrate*, hydrogenated
starch hydrolsate*,
glycerin*, hydroxyapatite, cellulose gum*,
aroma, xanthan gum*, potassium
thiocyanate, lactoferrin*,
lactoperoxidase*, glucose oxidase*,
glucose pentaacetate, aloe barbadensis
leaf extract*, sodium mthylparaben,
Complete DMEM
(CDMEM)
%10 oranında FBS (Fotal ¨ sıgır ˘ serumu), % 1
S. Birant et al.
Fig. 1. Morphological appearance of hGMSCs (x10). (A) hGMSCs on 0 passage 0 (B) hGMSCs on 1 passage (C) hGMSCs on 2
Tokyo, Japan). 2.6. Statistical analysis One way ANOVA test was used to the Tukey post-hoc test was used for pair
determine the differences between the wise comparisons in determining which
Datas were analyzed with IBM SPSS V23. groups for multiple comparisons, while groups differed.
hydrolyzed casein*, glycyrrhiza glabra (hGMSC and hPDLMSC) showed spindle-shaped
root extract* (*natural origins)
fibroblast-like morphology (Fig. 1, Fig. 2Figs. 1,2 ). As
SIA Splat Trading, Okulovka, Russia
a result of analysis of surface markers by using flow
3. Results
cytometry, both GMSCs and PDLMSCs showed strong
3.1. Isolation and characterization of cells positivity for mesenchymal stem cell markers such as
CD105, CD146, CD90, CD73
The cells isolated and reaching the third passage
penisilin/ streptomycin ilave edilmis¸ DMEM and CD29, while the negative expression (CD45, CD34, CD25, CD28 and CD14)
(Dulbecco’s Modified Eagles Medium)
of surface markers related to (Fig. 3,Fig. 4Figs. 3,4 ).
Gibco, Grand Island, USA
hematopoietic stem cells was detected
3
Tissue and Cell 72 (2021) 101538
th st nd rd
passage (D) hGMSCs on 3 passage.
 th st nd
Fig. 2. Morphological appearance of hPDLMSCs (x10). (A) hPDLMSCs on 0 passage 0 (B) hPDLMSCs on 1 passage (C) hPDLMSCs on 2 passage (D) hPDLMSCs on
rd
3 passage.

In the osteogenic differentiation test of hGMSCs and hPDLMSCs, mineralized
nodule formation and differentiation into osteocyte-like cells were detected. The
presence of calcium deposits stained with red color was observed. In the
adipogenic differentiation experiment, dif
ferentiation into adipocyte-like cells and the formation of oil droplets in the
cells was detected. The presence of bright red-pinkish intracellular
oil droplets was observed. In the chondrogenic differentiation experi ment,
differentiation into chondrocyte-like cells was observed with the formation of
proteoglycans stained in blue-turquoise color in the cells (Fig. 5,Fig. 6Figs. 5,6 ).
As a result of the morphological examination, determination of trilineage
differentiation potentials and marker anal ysis by using the flow cytometry, it
was proved that the cells isolated

4
S. Birant et al. Fig. 4. Determination of the immunophenotypic characteristics of hPDLMSCs by flow
cytometry.
Tissue and Cell 72 (2021) 101538

Fig. 3. Determination of the immunophenotypic characteristics of hGMSCs by flow

cytometry.
 5
S. Birant et al. Tissue and Cell 72 (2021) 101538

Fig. 5. Differentiation analysis in hGMSCs. A, Differentiation of hGMSCs into osteoblasts was confirmed by Alizarin red stain (x40). B, Differentiation of hGMSCs into
chondroytes was confirmed by Alcian blue stain (x40). C, Differentiation of hGMSCs into adipocytes was confirmed by Oil red o stain (x40).

Fig. 6. Differentiation analysis in hPDLMSCs. A, Differentiation of PDLMSCs into osteoblasts was confirmed by Alizarin red stain (x40). B, Differentiation of PDLMSCs
into chondroytes was confirmed by Alcian blue stain (x40). C, Differentiation of PDLMSCs into adipocytes was confirmed by Oil red o stain (x40).

showed mesenchymal stem cell characteristics (Figs. 3, 4, 5, 6). Table 3

Comparison of viability of PDLMCHs between groups.

3.2. Cell viability in cells cultured exposured to the children’s toothpaste containing the Concentration

different detergent content 0,4% 20 % 50 % 80 %

The percentages of cell viability GMCHs had lower values than all higher percentage of cell < 42.96 ± 2.15a 78.98 ± 2.36b
Colgate 6þ 64.74 ± 1.33a 20.96 ± 2.54a 75.38 ± 1.98b
on hGMSCs is represented in groups at 20 %, 50 % and 80 % viability was detected in Control
Splat Juicy 80.54 ± 1.65b 13.05 ± 2.07a 67.41 ± 1.58b
Table 2. Colgate group with concentrations (p < 0.05). A group at 20 %, 50 % and 80 % (p

0.05). After the Control group, the highest cell viability ratios were Sensodyne 75.20 ± 1.87c 57.83 ± 1.05c
Pronamel 6þ 71.68 ± 2.11c 52.31 ± 1.89c

observed in the detergent-free Sensodyne experimental group There was no signifi 50.42 ± 1.48d 24.4 ± 1.5d

Nenedent Kids 68.06 ± 1.63d 34.6 ± 1.41d

Splat group followed by the containing CABP (p < 0.05).

cant difference between the groups in terms of viablity viability rates in Nenedent Perlodent Junior 6þ 53.82 ± 2.16d 25.6 ± 1.67d
70.47 ± 1.89d 41.13 ± 2.07e
Nenedent and Perlodent rates of 20 %, while the cell group

was found significantly higher than the Perlodent group at othercon CDMEM 84.11 ± 1.08e 84.11 ± 1.08f
84.11 ± 1.08e 84.11 ± 1.08e

centrations except for 20 % concentration (p < 0.05) (Table 2). The percentages a-b-c-D-e-f: There is no difference between the groups with the same. One
of cell viability on hPDLMSCs is represented in Way ANOVA Test ; Tukey post–hoc Test.
*
Significant p value at 0.05 level.
Table 2
Comparison of viability of GMCHs between groups. Table 3. Colgate group had the lowest percentage of cell viability among the

Concentration experimental groups at all concentrations (p < 0.05). The highest live cell ratios
was detected in Control group, followed by Splat and
0,40 % 20 % 50 % 80 %
p 0.000* 0.000* 0.000* 0.000*
Colgate 6þ 73.86 ± 2.6ab 15.05 ± 3.07a viability ratios at 50 % concen tration Nenedent group (p < 0.05).
33 ± 2.87a 20.55 ± 2.51a Sensodyne groups (p < 0.05). The cell were higher in Perlodent group than
Splat Juicy 88.11 ± 0.9d 79.81 ± 1.06b The percentages of dead cell rates on hPDLMSCs is represented in
82.93 ± 1.23b 75.52 ± 1.15b

Sensodyne 80.55 ± 1.19c 62.8 ± 1.21c Table 4. In the evaluation the lowest early apoptosis were found in Splat group
Pronamel 6þ 72.73 ± 1.57c 51.92 ± 1.28c of early apoptotic cell rates, rates among toothpastes at all
Nenedent Kids 72.21 ± 0.92a 49.74 ± 1.21d concentrations (p < 0.05).In the rates, a statistically significant the groups at all
60.73 ± 1.09d 33.53 ± 1.41d
evaluation of late apoptotic cell difference was found between
Perlodent Junior 6þ 60.4 ± 0.95d 26.72 ± 0.98e concentrations. While the highest rates of late apoptotic cells at 0.4 %
76.34 ± 1.3b 45.25 ± 1.21e

CDMEM 89.54 ± 1.27d 89.54 ± 1.27f and 80 % concentrations were < 0.05) and the Nenedent group no significant
89.54 ± 1.27e 89.54 ± 1.27f
detected in the Colgate group (p at 20 % concentration (p < 0.05),
p 0.000* 0.000* 0.000* 0.000* difference was found between the Colgate and Nenedent groups at 50 %

a-b-c-D-e-f: There is no difference between the groups with the same. One concentration. In the evaluation of necrosis rates on hPDLMSCs, the statistically
Way ANOVA Test ; Tukey post–hoc Test. highest rates of necrotic cells were found in the Colgate
*
Significant p value at 0.05 level.

6
S. Birant et al. Table 4 Table 5 Tissue and Cell 72 (2021) 101538

Comparison of dead cells rates on hPDLMSCs between groups. Early Comparison of dead cells rates on hGMSCs between groups. Early

apoptosis Concentration apoptosis Concentration

0,4% 20 % 50 % 80 % 0,4% 20 % 50 % 80 %

Colgate 6þ 16.29 ± 2.05c 42.46 ± 6.05c Colgate 6þ 6.55 ± 1.29 22.17 ± 1.8d 21.64 ± 4.56c
26.93 ± 3.07 cd 40.85 ± 1.18d cd 24.61 ± 2.28d
Splat Juicy 7.18 ± 2.85a 17.41 ± 1.52b 0.92a 6.22 ± 0.69a
7.82 ± 0.3a 10.65 ± 0.6a Splat Juicy 2.36 ± 1.01a 3.71 ± 3.06 ± 1.02a
Sensodyne Pronamel 6þ 15.06 ± 2.37b 27.18 ± 1.74b 33.79 ± 2.25c Sensodyne Pronamel 6þ 8.96 ± 0.42c 14.08 ± 0.91b 15.6 ± 0.91b
11.19 ± 2.03b 4.14 ± 1.02abc
Nenedent Kids 20.26 ± 26.34 ± 2c 38.9 ± 1.56c Nenedent Kids 7.12 ± 1.67d 12.99 ± 1.06b
0.83d 41.91 ± 2.52d 10.17 ± 1.36c 21.04 ± 2.14c
Perlodent Junior 17.36 ± 1.72 cd 36.76 ± 3.49c Perlodent Junior 5.11 ± 1.66bcd 20.15 ± 1.17c
6þ 30.3 ± 1.41d 30.72 ± 2.96c 6þ 6.44 ± 1.09b 26.37 ± 2.09d
CDMEM 6.91 ± 1.13a 6.91 ± 1.13a 6.91 ± 1.13a 6.91 ± 1.13a CDMEM 2.62 ± 1.26ab 2.62 ± 1.26a 2.62 ± 1.26a 2.62 ± 1.26a
p 0.000* 0.000* 0.000* 0.000* Late apoptosis Concentration p 0.000* 0.000* 0.000* 0.000* Late apoptosis Concentration

0,4% 20 % 50 % 80 % 0,4% 20 % 50 % 80 %

Colgate 6þ 9.88 ± 2.03c 12.64 ± 2.01d 18.77 ± 2.24d 28.02 ± 2.58d Colgate 6þ 7.81 ± 1.28c 21.14 ± 2.18d 31.13 ± 1.91d 42.64 ± 2.18a
Splat Juicy 5.63 ± 1.12b 6.51 ± 1.28bc 6.1 ± 0.97b 5.39 ± 0.69ab Splat Juicy 2.57 ± 0.45a 4.25 ± 0.95a 5.78 ± 0.59b 6.32 ± 0.92b
Sensodyne Pronamel 6þ 5.36 ± 0.94ab 6.83 ± 0.53b 6 ± 0.89b Pronamel 6þ 7.1 ± 1.13b 7.66 ± 1.09b 10.83 ± 1.12c
7.2 ± 0.95b Sensodyne 4.79 ± 1.13b
Nenedent Kids 5.38 ± 15.98 ± 1.02e 20.65 ± 1.83c 0.66c 17.69 ± 0.95c
1.4ab 18.2 ± 1.53 cd Nenedent Kids 7.75 ± 10.54 ± 1.71c 21.57 ± 0.87d
Perlodent Junior 6.42 ± 1.18b 15.47 ± 2.01c Perlodent Junior 7.13 ± 0.83c 16.51 ± 0.62c
6þ 9.02 ± 1.12c 19.4 ± 1.3c 6þ 12.59 ± 1.09c 21.04 ± 1.05d
CDMEM 2.81 ± 1.21a 2.81 ± 1.21a 2.81 ± 1.21a 2.81 ± 1.21a CDMEM 1.58 ± 0.73a 1.58 ± 0.73a 1.58 ± 0.73a 1.58 ± 0.73e
p 0.001* 0.000* 0.000* 0.000* Necrosis Concentration p 0.000* 0.000* 0.000* 0.000* Necrosis Concentration

0,4% 20 % 50 % 80 % 0,4% 20 % 50 % 80 %

Colgate 6þ 9.1 ± 0.81a 17.47 ± 1.52a 17.81 ± 1.96a 18.09 ± 1.87d Colgate 6þ 11.78 ± 1.63b 23.69 ± 1.69e 21.73 ± 2.15e 20.66 ± 2.77c
Splat Juicy 6.65 ± 1.42b 6.69 ± 1.22b 7.87 ± 0.95b 9.78 ± 1.11b Splat Juicy 6.97 ± 0.91a 9.1 ± 0.9b 11.35 ± 0.59b 11.94 ± 0.78b
Sensodyne Pronamel 6þ 7.9 ± 1.03b 8.17 ± 7.91 ± 0.56ab Pronamel 6þ 11.44 ± 1.27c 19.65 ± 1.51c
6.41 ± 1.5b 0.69b Sensodyne 10.91 ± 1.27b 15.43 ± 1.38c
Nenedent Kids 6.3 ± 7.26 ± 0.87b 13.03 ± 1.03c 0.77b 19.57 ± 1.58de
0.83b 7.91 ± 1.15b Nenedent Kids 12.92 ± 18.55 ± 1.57d 23.86 ± 1.02d
Perlodent Junior 5.75 ± 1.45b 6.62 ± 1.61b Perlodent Junior 11.43 ± 1.61b 18.09 ± 0.95 cd
6þ 6.85 ± 0.93b 24.26 ± 1.13e 6þ 20.57 ± 1.36d 25.87 ± 1.8d
CDMEM 6.17 ± 0.97b 6.17 ± 0.97b 6.17 ± 0.97b 6.17 ± 0.97a CDMEM 6.26 ± 1.11a 6.26 ± 1.11a 6.26 ± 1.11a 6.26 ± 1.11a
p 0.003* 0.000* 0.000* 0.000* Sensodyne group after these groups (p < 0.05). The highest rates of late

a-b-c-D-e-f: There is no difference between the groups with the same. One
Way ANOVA Test ; Tukey post–hoc Test.
*
Significant p value at 0.05 level.
group at 0.4 %, 20 % and 50 % concentrations (p < 0.05). The percentages of
dead cell rates on hGMSCs is represented in Table 5. While the lowest rates of
early apoptotic cells were detected in the Control group and Splat group at all
apoptosis at all con centrations were found in the Colgate group (p < 0.05). No
significant difference was found between Colgate, Nenedent and perlodent
groups at 0.4 % concentrations.In the evaluation of necrotic cell ratios, the
lowest necrotic cell ratios at all concentrations were found in the Splat group
after the Control group (p < 0.05).
3.3. Evaluation of osteogenic differentiation potential in MSCs

concentrations, the lowest rates of early apoptosis were generally found in the
The osteogenic differentiation potentials of hGMSCs and hPDLMSCs exposured
 different toothpaste solutions were qualitatively assessed by intense staining for calcium concentration was slightly less than the Control group but when
Alizarin Red staining and the presence of extracel lular calcium deposits dyed compared to the other experimantal groups the most intense calcium nodules
bright orange red. Images of hGMSCs and hPDLMSCs that stimulated with the were found in this group. In Sensodyne group, the potential of osteogenic
osteogenic differentiaiton medium differentiation was slightly lower than CDMEM and Splat group, but it was
p 0.000* 0.000* 0.000* 0.000* found to be quite high in Nenedent, Perlodent and Colgate groups. While

a-b-c-D-e-f: There is no difference between the groups with the same. One Perlodent group contained much less calcium nodules than CDMEM, Splat and
Way ANOVA Test ; Tukey post–hoc Test. Sensodyne groups in terms of the presence of calcium deposits, it was examined
*
Significant p value at 0.05 level. on microscope images that contained a slightly more mineralized nodule than
Nene dent and Colgate groups. The presence of calcium nodules in Perlodent
after incubating in different toothpaste solutions for 0.4 % toothpaste and Nenedent groups was close to each other but less calcium deposits were
concentration are shown in Fig. 7,Fig. 9Figs. 7 and 9 respectively. In the GMSCs detected in Nenedent group. The lowest amount of intracellular calcium
and PDLMSCs, the highest amount of osteoblast-like cells and mineralized deposition and presence of intense apoptotic cells were observed in Colgate
calcium accumulation were observed in Control group. In Splat group, the group. (Figs. 7 and 9).

7
S. Birant et al. Tissue and Cell 72 (2021) 101538

Fig. 7. Alizarin red staining images of GMSCs that were transferred to the osteogenic differentiation environment after incubation in CDMEM and 4% toothpaste
solutions. *Black arrows indicate intercellular spaces. *Yellow arrows indicate differentiated cells.
 Fig. 9. Alizarin red staining images of PDLMSCs that were transferred to the osteogenic differentiation environment after incubation in CDMEM and 4% toothpaste
solutions. *Black arrows indicate intercellular spaces. *Yellow arrows indicate differentiated cells.

3.4. Evaluation of chondrogenic differentiation potential in MSCs
The chondrogenic differentiation potentials of hGMSCs and hPDLMSCs
exposured different toothpaste solutions were qualitatively assessed by intense
staining for Alcian Blue staining and the presence of blue/turquoise dyed
glycoaminoglycans. Images of hGMSCs and hPDLMSCs that stimulated with the
chondrogenic differentiaiton me
dium after incubating in different toothpaste solutions for 0.4 %
toothpaste concentration are shown in Fig. 8,Fig. 10Figs. 8 and 10 respectively.
In the GMSCs and PDLMSCs, the most cells differed morphologically to the
chondrocyte like cells and the most intense proteoglycan accumulations were
observed in Control group as a result of the chondrogenic differentiation test.
After the Control group, Splat group had the highest the amount of proteoglycan
accumulations and the concentration of glycosaminogly cans. When the
Sensodyne group was compared to the CDMEM and

8
S. Birant et al. Tissue and Cell 72 (2021) 101538

Fig. 8. Alcian Blue staining images of GMSCs that were transferred to the chonrogenic differentiation environment after incubation in CDMEM and 4% toothpaste
solutions. * Arrows indicate differentiated cells.
 Fig. 10. Alcian Blue staining images of PDLMSCs that were transferred to the chondrogenic differentiation environment after incubation in CDMEM and 4% toothpaste
solutions. *Arrows indicate differentiated cells.
Splat groups, it was observed that the confluent structures of the cells were
disturbed in some regions and the breaks in the intercellular con nections
occured. The potential for chondrogenic differentiation was slightly lower in this
experimental group than in the CDMEM and Splat group, but it was found to be
significantly higher in Nenedent, Perlodent and Colgate experimental groups.
It was determined that the cells stimulated with Perlodent, Nenedent and
Colgate toothpaste did not differ morphologically to the chonroblast like cells as
a result of the chondrogenic differentiation test. It was
S. Birant et al.
to its foaming effect, pleasant taste and low cost. Some manufacturers
started to use alternative detergents in toothpastes due to the irritant side-effects
of SLS (Salzer et al., 2016). It is reported that toothpastes containing SLS may
reduce oral mucosal resistance, causing negative effects in individuals with
recurrent oral aphthous ulcers (Siegel and Gordon, 1986; Healy et al., 1999;
Salzer et al., 2016; Bark
voll et al., 1989). In addition, a relationship has been identified between the use
of toothpastes containing SLS and increased oral desquamation ( Stec, 1972;
Allen et al., 1975). Shim et al. show that the healing time for patients with oral
aphthous ulcers, and their pain scores, decreased significantly in individuals
with who used SLS-free toothpaste compared to individuals who continued to
use toothpaste containing SLS (Shim et al., 2012).
Children and adults differ in their gingival thickness, morphology
degree of keratinization. The type and amount of detergents, sweetener and
fluoride in toothpastes for children and adults therefore differ. However, when
the contents of children’s toothpaste are exam
ined, it can be seen that most children’s toothpastes do contain SLS. It is
suggested that the side-effects of SLS may be greater in children due to their
lower degree of gingival keratinization (Cvikl et al., 2015, 2017). It is thought
that the negative effects of SLS may occur more frequently in children whose
gingival problems are due to systemic disease. SLS may increase inflammatory
factors, such as IL-6, and IL-8, in the gingival crevicular fluid, causing gingival
bleeding and prolonging the healing time for gingival diseases.
Herlofson and Barkvoll investigated the effects of different tooth pastes on
oral mucosa and clinically evaluated zones of desquamation. From their results,
they concluded that most of the desquamation was related to toothpaste
containing SLS. (Herlofson and Barkvoll, 1996). Shim et al. studied the effects of
SLS on recurrent aphthous stomatitis, considering clinical parameters such as
the number of oral ulcers, the frequency of their recurrence, their duration and
pain sensation. They stated that scores for the pain experienced by the patients
during tooth-brushing was statistically significantly lower with SLS- free
toothpaste than with toothpastes containing SLS. In addition, they observed that
the frequency of ulcers increased with toothpastes con
taining SLS, as did the duration of the ulcers and the pain score (Shim et al.,
2012). Pretty et al. compared the antimicrobial and plaque inhi bition properties
of a detergent-free toothpaste with those of a tooth paste containing detergent.
Their results showed, statistically, that the detergent-free toothpaste
significantly increased plaque inhibition relative to the toothpaste containing
detergent (Pretty et al., 2003). Rantanen et al. examined the effects of different
toothpastes on human oral mucosa and stated that the toothpastes containing
SLS had an irritant effect on the mucosa while the SLS-free toothpaste
observed that cells lost their confluent structures due to apoptosis and the
intercellular connections disappeared (Figure 8 and 10).
4. Discussion
Detergents, also known as surfactans, are among the ingredients added to
the toothpastes due to their cleaning and antimicrobial effects on tooth surfaces,
resulting from their hydrophilic and hydrophobic properties. SLS is the
detergent most commonly used in toothpastes due
9
(containing betaine only) showed no significant signs of irritation on the mucosa
(Rantanen et al., 2003). Melsen et al. showed that SLS may reduce the
have
cariostatic effect of fluoride when used in combination with sodium
monofluorophosphate during topical application (Melsen and Rolla, 1983).
Altough there are many studies in the literature that examine the effects of SLS
on the antimicrobial and clinical parameters, there are very few studies that
have examined the effects of SLS on the cells of the oral tissues.
Cvikl et al. examined the effects of toothpastes containing different
detergents on the gingival fibroblasts, on oral squamous cell carcinoma HSC-2
cells and on L929 cells in vitro. They stated that toothpastes containing SLS and
amine fluoride inhibited cell viability at a 1% toothpaste solution. In addition,
they reported that the half lethal dose concentration (LC50) of the toothpastes
containing cocamidopropyl betaine and streath-20 was higher than that of
toothpastes containing SLS and amine fluoride (Cvikl et al., 2015). Cvikl et al.
and
also evaluated the cytotoxicity of different children’s toothpaste on gingival
fibroblasts, oral squamous cell carsinoma HSC-2 cells and L929 mouse
fibroblasts. In their study, toothpastes containing SLS showed LC50 values
below 5%, which were consistent with the results of their study with adult tooth
pastes (Cvikl et al., 2017). In these cell studies, only the cytotoxic effects
Tissue and Cell 72 (2021) 101538
on thye gingival epithelium and fibroblast cells were investigated. The current
study will be the first to examine the effects of SLS on primary stem cells.
Primary cell cultures are the biological systems that can best reflect the original
physiological state compared to continuous cell lines. In this study, primary cell
cultures were preferred, considering the creation of the experimental condition
to be closer to the in vivo con
dition (Illeperuma et al., 2012).
Studies report that MSCs contribute to tissue regeneration by replacing
damaged tissue cells, by supporting the production of factors with anti-
inflammatory properties and by differentiating into epithelial like cells in
mucosal infections. In vivo and in vitro experiments show that hPDLSCs play a
major role in protecting against infectious diseases (such as periodontal disease)
related to their immunomodulatory properties and regulating osteogenic
differentiation. It has been stated that in cases of bone loss, they can stimulate
the bone regeneration process and induce differentiation of host cells. These
cells have also been reported to contribute to tissue regeneration in the
treatment of periodontal diseases. Gingiva-derived MSCs (GMSCs), on the other
hand, have the potential to differentiate into several cell lines and have
immunodulatory capacity. In addition, GMSCs can be isolated easily and show
high proliferative activity. Furthermore, it has been reported that the detergents
in toothpaste can penetrate deeper tissues such as the lamina propria in the oral
mucosa. This shows that toothpastes are effective on PDL cells as well as gingiva
 cells (Hermann et al., 2006; Kuroda et al., 2010; Prockop, 2009; Roddy et al.,
2011; Prockop and Oh, 2012; Zhang et al., 2012b; Trubiani et al., 2019;
Marconi et al., 2021). For this reason, it would be seen as advantageous for the
protection of oral-dental health and for the success of any treatment process if
the type of detergent used in a toothpaste did not damage the mesenchymal
stem cells. Therefore, in our study, the viability rates for hGMSCs and
hPDLMSCs, which are of great importance in the repair and regeneration of
periodontal tissues, were evaluated after the cells had been incubated in
toothpastes solutions containing different detergents.
Studies vary in the methods they use for cell viability analyses. In this study,
flow cytometry analysis was used because it gives faster, more sensitive and
more reliable results than the alternative methods often used in cell culture
studies. The flow cytometry ensured that live, early apoptotic, late apoptotic
and necrotic cells were all determined in the analysis. This study evaluated the
viability rates for cells where the children’s toothpaste solutions contained
different detergents: it became evident that the lowest live cell rate was in the
toothpaste solution containing SLS. After the control group, the highest live cell
rates were detected in toothpaste solutions without detergent, followed by tooth
paste solutions containing CAPB. These results are similar to findings of Cvikl et
al. in studies that examined the effects of adults’ and children’s toothpastes on
S. Birant et al.
of the mesenchymal stem cells were also evaluated in this study since the stem
cells repair small defects in the gingival and periodontal tissues.
Tissue and Cell 72 (2021) 101538
References
other ingredients in the toothpastes may also have a toxic effect. However, to
The literature review revealed no studies that investigated the effect of eliminate this limitation, the toothpastes used in the study groups had similar
toothpastes on the differentiation potential of human mesenchymal stem cells. content and were used in similar age groups. In vitro cell studies do have other
This study is the first to examine the effects of children’s toothpastes on the limitations due to the absence of saliva and the lack of the protective and
differentiation potential of hMSCs. When the osteo immunological properties of the
genic differentiation potentials of hGMSCs and hPDLMSCs were evalu ated, it was conclusions of this study need to be supported by in vivo studies where the in
seen that the Colgate group inhibited the osteogenic differentiation of cells. The
inhibition of osteogenic differentiation in this group can be explained by a
deterioration in adhesion between the cells and a decrease of live cell ratios
during the differentiation experi ment, which lasted for three weeks. It was also
suggested that the excessive foaming property of SLS has a negative effect on the
plasticity of the stem cells. When the chondrogenic differentiation potential of
hGMSCs and hPDLMSCs were being evaluated in the Colgate, Nenedent and
Perlodent experimental groups, microscopic examination did not reveal the
presence of any chondroblast-like cells or proteoglycans. It was thought that
these three experimental groups, all of which have high anionic detergent
content, caused deterioration of cell adhesion due to their foaming property. It
was observed that the rate of dead cells increased due to destruction of the cell
connections.
These results show that the toothpastes containig SLS had a greater cytotoxic
effect on oral tissue cells than the other toothpastes. It was shown that
toothpastes with no detergent or CAPB were more biocom patible with oral tissue
cells. While the highest rates of dead cells were detected in toothpaste groups
containing SLS, after the control group, the lowest rates of dead cells were
observed with the detergent-free toothpaste and with that containing CAPB. It
was determined that the experimental group in which the differentiation
potential of the cells was most negatively affected was that where the toothpaste
contained SLS. It is suggested that toothpastes containing SLS have a negative ef
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cells. (Cvikl et al., 2015, 2017).
In the literature, the effect of toothpaste on cells is reported only in terms of
living cell proportions. In this study, early apoptotic, late apoptotic and necrotic
cell ratios were evaluated as well as the live cell ratio. In a comparison between
groups, the toohpaste containing SLS generally showed the highest value in
terms of early apoptotic, late apoptotic and necrotic cell ratios, while Splat and
the CDMEM generally had similar rates in terms of cell death type. These results
suggest that SLS increases cellular permeability by causing denaturation in the
cellular proteins, while opening the pores between the cells causes apoptosis-
inducing proteins to be released into the cytosol, ultimately to stimulate the
apoptosis/necrosis mechanisms.
It is reported that stimulation of the apoptotic pathway in epithelial cells
inhibits epithelial and mesenchymal cell proliferation and can inhibit cell
migration and wound healing (Semlali et al., 2011; Weindl et al., 2010). In this
study, the fact that toothpaste containing SLS caused an increase in apoptotic
and necrotic cell ratios suggests that the healing time for periodontal diseases
and oral aphthous ulcers may be delayed and wound healing negatively affected
by SLS.
In addition to the cell viability, the effects of different toothpaste solutions
on the osteogenic and chondrogenic differentiation potential
10
the healing of periodontal tissue disease and may lead to pro longation of the
healing process. In addition, we think that the increased rate of dead cells where
the toothpastes contained SLS may support the hypothesis mentioned in the
literature that toothpastes with an SLS content can cause oral apthous ulcers.
A limitation of this study is its use of toothpaste solutions, since the detergent
ingredients are not supplied in pure form. This means that it is possible that
normal tissue barriers. Therefore, the
vitro experiments will constitute only the initial steps.
Author statement
Designing the study by Figen Seymen, Sinem Birant; generating the data by
Sinem Birant; analyses the data by Yazgul Duran, Muazzez Gokalp, Tunc Akkoc;
writing the paper by Sinem Birant; approved the final version of this paper by
Sinem Birant, Yazgul Duran, Muazzez Gokalp, Tunc Akkoc, Figen Seymen.
Declaration of Competing Interest
The authors do not have any conflict of interest.
Acknowledgements
This study was supported by the Research Fund of Istanbul Univer sity,
project no:25696.
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