Cytokine 107 (2018) 113–117

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Cytokine
journal homepage: www.elsevier.com/locate/cytokine

Proinflammatory cytokines in early childhood caries: Salivary analysis in the T

mother/children pair
⁎
Cecilia Claudia Costa Ribeiroa,b, , Crysthian de Jesus Borges Pachêcob, Elizabeth Lima Costab,
Lorena Lúcia Costa Ladeirab, José Ferreira Costac, Rubenice Amaral da Silvac,
Cadidja Dayanne Sousa Carmob
a
Program Postgraduate in Public Health, Federal University of Maranhão, Brazil
b
Program Postgraduate in Dentistry, Federal University of Maranhão, Brazil
c
Departament of Dentistry II, Federal University of Maranhão, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Hypothesis: Proinflammatory cytokines are increased in saliva of mother/children pairs with caries. Design:
Early childhood caries Case-control study involving caries-free children (n = 20) and children with early childhood caries (ECC)
Cytokines (n = 20), and their mothers (n = 40). The maternal variables analyzed were waist circumference (WC), decayed,
Mother child pairs missing and filled teeth (DMFT) and sugar intake; and in the children were body mass index (BMI), def-t and
sugar intake. Salivary levels of VEGF, IL-6 and TNF-α were analyzed of mother/children pairs. Results: In the
mothers, salivary VEGF levels were correlated with DMFT (r = 0.35; p = .03), WC (r = 0.35; p = .02), and sugar
intake (r = 0.32; p = .04). Higher salivary IL-6 levels were also correlated with maternal DMFT (r = 0.45;
p = .004) and WC (r = 0.32; p = .04). In the children, higher salivary VEGF levels were correlated with higher
def-t scores (r = 0.42; p = .008). Children with caries had a 63% higher median salivary VEGF and twofold
higher mean IL-6 levels compared to caries-free children. Mothers of children with ECC showed higher mean of
salivary IL-6 levels compared to those of children without ECC (p = .03). Conclusion: Salivary proinflammatory
cytokines are correlated with the severity of caries in the mother-children pair. Obesity and excessive sugar
consumption seem to underlie the associations between proinflammatory cytokines and caries in the family
environment.

1. Introduction resistance [9]. The World Health Organization recommends a reduced

Early childhood caries (ECC) is defined as the presence of one or
more carious lesions cavitated or not in children up to 71 months of age
[1]. Mothers play a decisive role in the occurrence of ECC, with a larger
number of carious teeth in the mother being associated with caries in
her children [2,3]. Furthermore, a higher maternal body mass index
(BMI) [3], greater maternal waist circumference (WC) [4] and children
obesity have also been associated with ECC [5,6].
The maternal dietary pattern influences the eating habits of the
child, especially the preference for sweets and sugary drinks [7], sug-
gesting that obesogenic behaviors are perpetuated in the family en-
vironment.
Systematic reviews of the literature have showed an association of
excess intake of sugars with weight gain in children and adults [8], with
an epidemy of obesity, metabolic syndrome [9,10] and also insulin
intake of free sugars not only for the prevention of caries and obesity,
but also to reduce the risk of other chronic noncommunicable diseases
[11]. The American Heart Association published recommendations that
emphasize the limits of sugar intake by children to reduce the cardio-
vascular risk [12].
The adipose tissue is an organ secreting numerous cytokines that
play a role in the regulation of the systemic inflammatory state [13].
These regulatory cytokines are directly involved in sugar metabolism
and obesity. Interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-
α) are adipokines which have been associated to obesity and hyper-
insulinemia [14,15]. These adipokines may regulate the endothelial
function [14]. Therefore, cytokines such as vascular endothelial growth
factor (VEGF) are also overexpressed in obesity and diabetes [16,17].
High serum levels of IL-6 and TNF-α were also associated to an
excessive consumption of added sugars [18]. An increase of salivary

⁎
Corresponding author at: A. dos Portugueses SN, Cidade Universitária Bacanga, São Luís, MA CEP 65080-805, Brazil.
E-mail addresses: cecilia_ribeiro@hotmail.com (C.C.C. Ribeiro), crysthian_bp@hotmail.com (C.d.J.B. Pachêco), bet.lima@terra.com.br (E.L. Costa),
lorenaladeiro0@gmail.com (L.L.C. Ladeira), costa.jf@terra.com.br (J.F. Costa), rubi.sl@hotmail.com (R.A. da Silva), cadidjadayane@yahoo.com.br (C.D.S. Carmo).

https://doi.org/10.1016/j.cyto.2017.12.009
Received 11 April 2017; Received in revised form 7 November 2017; Accepted 6 December 2017
Available online 13 December 2017
1043-4666/ © 2017 Elsevier Ltd. All rights reserved.
C.C.C. Ribeiro et al.
levels of VEGF are observed in sugar metabolism disorders [19,20].
Elevated salivar levels of IL-6 and TNF-α have been evidenced in pre-
sence of high caries severity [21–23].
Based on the premise that the consumption of added sugars in the
family environment is associated with both caries and obesity, the hy-
pothesis of this study is that proinflammatory cytokines linked to high
consumption of sugar would be increased in saliva of mother/children
pairs with severe caries disease.
Therefore, the objective of the present study was to evaluate sali-
vary levels of proinflammatory cytokines (IL-6, TNF-α, and VEGF) and
to correlate them with the presence of caries, obesity and sugar intake
in mothers and their children.
2. Material and methods
2.1. Ethical aspects
The study was approved by the Research Ethics Committee of the
Federal University of Maranhão (UFMA) (Approval No. 23115012534/
2008-41). Prior to the study, the mothers signed a free informed con-
sent form containing clear and objective verbal explanations about their
participation in the study. Children who required dental treatment were
referred to the Pediatric Dental Clinic of UFMA for appropriate therapy.
2.2. Selection of the participant
This was a cross-sectional study nested within a retrospective cohort
(n = 400) that involved children aged 24–71 months enrolled in mu-
nicipal preschools of São Luís, Maranhão, Brazil, and their mothers
participating in the research project “Early childhood caries from the
mother/child pair perspective”.
The sample size was based on two previous studies evaluating caries
disease and inflammatory markers in saliva [21,22]. Children with ECC
and caries-free children were selected from original cohort using the
“Filter” tool of the Excel® 2010 software; this step was to insure ran-
domization of the sample, increasing sample representativeness (in-
ternal validity of the study). Each pair received a numerical identifi-
cation using the RANDBETWEEN function. Twenty children with ECC
and twenty caries-free children and their respective mothers were se-
lected for the analysis of salivary inflammatory markers. Three of the
40 mother/children pairs were lost, including one caries-free child and
two children with caries, since they did not have sufficient amounts of
saliva for the tests, totaling 37 pairs.
2.3. Data collection
The data were collected at the preschools themselves from August
2013 to March 2015. The mothers answered a structured questionnaire
applied by interview that contained socioeconomic and demographic
data, as well as a food frequency questionnaire. The maternal variables
studied were WC, caries experience and sugar intake. For the children,
BMI, ECC and sugar intake were evaluated.
For maternal anthropometric assessment, WC was measured with a
seamless Sanny® tape measure at the midpoint between the last costal
margin and iliac crest, to the nearest 1 mm. Maternal WC was cate-
gorized as follows: < 80 cm (absence of metabolic risk); ≥80–88 cm
(high metabolic risk), and > 88 cm (very high metabolic risk) [24].
The height (cm) of the children was measured with an Alturexata®
portable stadiometer (Belo Horizonte, MG, Brazil) and body weight (kg)
was measured with a Filizola® digital scale (São Paulo, SP, Brazil), with
a maximum variation of 100 g. These measurements were obtained in
duplicate by two evaluators in a blind manner and the mean value of
the two evaluations was recorded. The kappa value was 0.89 ( ± 0.01)
for intraexaminer agreement and 0.91 for interexaminer agreement.
The body mass index (BMI) are expressed as Z-score, which calculates
the BMI for age in percentiles for children aged 0–5 years according to
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Cytokine 107 (2018) 113–117
sex [25]. The WHO Anthro 3.2.2 software was used for children up to
60 months and the WHO Anthro Plus 1.4.0 for children older than
60 months (WHO, Geneva, Switzerland).
Sugar intake by the mothers and children was analyzed using a food
frequency questionnaire [26]. The frequency of added sugar intake was
obtained as the sum of daily intake of the following foods: soft drinks,
processed juices, chocolate drinks, chocolate, cakes, candies, sweets,
and cookies. The sum of these frequencies was used as a continuous
variable.
Clinical oral examination for analysis of caries experience was
performed after tooth brushing using the decayed, missing and filled
teeth (DMFT) index for the mothers and the decayed, tooth indicated
for extraction and filled teeth (def-t) index for the children [27]. The
def-t was modified to include non-cavitated active caries lesions, in-
cluding rough and opaque white spot lesions.
A flat mouth mirror and exploratory probe with a blunt end were
used for clinical examination. The mother and children were examined
under natural light sitting on common chairs in the outside patio of the
school. Clinical examination of the mother and children was performed
at different times to ensure blinding of the evaluator for this variable of
the pair. The data were collected by a single, previously calibrated
examiner (kappa = 0.86).
Stimulated saliva samples were collected from the mother/children
pairs after mastication of Parafilm® for 5 min. The saliva samples were
transferred to a 1.5-mL Falcon tube, stored on ice, and sent to the
Laboratory of Oral Biochemistry of UFMA for processing.
2.4. Salivary analysis of inflammatory markers
The salivar levels of IL-6 pg/mL, TNF-α pg/mL, and VEGF pg/mL
were analyzed saliva using the MILLIPLEX® MAP Human Adipokine
Magnetic Bead Panel 2 Kit – HADKMAG-61K (Merck Millipore, BA,
USA). The standards, controls and samples were processed according to
manufacturer recommendations and the fluorescent beads were read on
a Luminex 200 platform (Gen-Probe, CA, USA). The data obtained were
analyzed with the Milliplex Analyst program (Merck Millipore, BA,
USA).
2.5. Statistical analysis
Continuous variables that showed a normal distribution (maternal
DMFT, maternal sugar intake, def-t and children sugar intake) were
correlated with the salivary inflammatory markers using Pearson’s
correlation test. Categorical variables or non-normally distributed
variables (maternal WC and children BMI Z-score) were correlated with
the salivary inflammatory markers using Spearman’s correlation test.
The correlations were defined as follows: very weak (0 ≤ r < 0.19),
weak (0.20 ≤ r < 0.39), moderate (0.40 ≤ r < 0.69), strong
(0.70 ≤ r < 0.89), and very strong (0.90 ≤ r < 1.0) [28].
In addition, the mean salivary levels of the inflammatory markers
were compared between mothers of children without and with caries.
In this step, the Student t-test was used for normally distributed data
(IL-6 and TNF-α) and the Mann-Whitney test for non-normally dis-
tributed data (VEGF). The salivary levels of the inflammatory markers
were also compared between caries-free children and children with ECC
using the Student t-test for mean IL-6 and TNF-α levels. VEGF levels are
expressed as the median and were compared by the Mann-Whitney test.
All analyses were carried out with the Stata® 14.0 software,
adopting a level of significance of 5%.
3. Results
The characteristics of the sample according to the distribution of
study variables are described in Table 1.
Analysis of the correlation between maternal variables and maternal
proinflammatory cytokines in saliva showed a weak correlation of
C.C.C. Ribeiro et al. Cytokine 107 (2018) 113–117

Table 1 Table 4
Distribution of the variables of the mother–child pairs (mean and standard deviation) Comparison of proinflammatory cytokines in saliva between mothers of children without
according to the presence or absence of early childhood caries (n = 37). and with caries (n = 37).

Mothers of Mothers of Children Children with VEGF (pg/ IL-6 (pg/mL) TNF-α (pg/mL)
children children without ECC ECC (n = 18) mL)
without ECC with ECC (n = 19)
(n = 19) (n = 18) Mothers of children 139.11a 4.3589 ( ± 2.41)b 3.3253 ( ± 1.52)b
without ECC (n = 19)
Age (year) 33.6 ( ± 9.5) 29( ± 3.8) 3.5 ( ± 0.9) 3.8( ± 0.7) Mothers of children with 183.97a 7.0317 ( ± 4.30)b 4.0939 ( ± 1.44)b
Waist 82.1( ± 14.9) 87.4( ± 9.2) – – ECC (n = 18)
Circumferen- p 0.35NS 0.03 0.12NS
ce (cm)
BMI z-score – – 0.3 ( ± 1.37) −0.3( ± 1.1) VEGF: vascular endothelial growth factor; IL-6: interleukin 6; TNF-α: tumor necrosis
def-t – – 0 7.4( ± 3.2) factor alpha; ECC: early childhood caries. p ≤ 0.05: statistically significant. NS: non-
DMFT 4.4( ± 5.9) 8.0( ± 8.8) – – significant.
Sugar intake 2.5( ± 2.6) 3.2( ± 2.4) 3.4 ( ± 2.9) 3.7( ± 1.8) a
Median (Mann-Whitney test).
(times/day) b
Mean ± standard deviation (Student t-test).

Table 5
Table 2
Comparison of proinflammatory cytokines in saliva between children without and with
Correlation analysis of maternal caries index, waist circumference and sugar intake with
caries (mean/median) (n = 37).
maternal salivary levels of VEGF, IL-6 and TNF-α (n = 37).
VEGF (pg/ IL-6 (pg/mL) TNF-α (pg/mL)
VEGF p IL-6 p TNF-α P
mL)

DMFT 0.3504a 0.03 0.4592a 0.004 −0.1997a 0.23NS

Children without ECC 153.83a 4.2411 ( ± 1.68)b 3.7937 ( ± 1.71)b
WC 0.3587b 0.02 0.3255a 0.04 −0.0774b 0.64NS
(n = 19)
Sugar intake 0.3251b 0.04 0.1027a 0.54NS −0.0401a 0.81NS
Children with ECC 245.44a 8.6106 ( ± 7.8442)b 4.0228 ( ± 1.44)b
(n = 18)
VEGF: vascular endothelial growth factor; IL-6: interleukin 6; TNF-α: tumor necrosis p 0.06NS 0.08NS 0.37NS
factor alpha; DMFT: decayed, missing and filled teeth; WC: waist circumference.
p ≤ 0.05: statistically significant. NS: nonsignificant. VEGF: vascular endothelial growth factor; IL-6: interleukin 6; TNF-α: tumor necrosis
a
Pearson’s coefficient (r). factor alpha; ECC: early childhood caries. p ≤ 0.05: statistically significant. NS: non-
b
Spearman’s coefficient (rs). significant.
a
Median (Mann-Whitney test).
DMFT with VEGF (r = 0.35; p = .03) and a moderate correlation with b
Mean ± standard deviation (Student t-test).
IL-6 (r = 0.45; p = .004). Maternal WC was weakly correlated with
VEGF (r = 0.35; p = .02) and IL-6 (r = 0.32; p = .04). There was a mother were also correlated with higher maternal salivary VEGF levels.
weak correlation between maternal sugar intake and VEGF (r = 0.32; Taken together, the correlations of higher caries scores in dyads,
p = .04) (Table 2). higher maternal adiposity and higher maternal sugar intake with in-
Correlation analysis of the children variables with proinflammatory creased salivary VEGF levels suggest that may exist a metabolic risk
cytokines in saliva showed a moderate correlation between def-t and underlying caries experience in the family environment. Supporting this
VEGF (r = 0.42; p = .008) (Table 3). assumption, serum levels of VEGF have been positively associated with
Comparison of mean salivary IL-6 (pg/mL) levels between mothers obesity and hyperinsulinemia [16,17]. The increase of VEGF in saliva is
of children without and with ECC showed higher levels in the latter also linked to sugar metabolism disorders; i.e. increased levels of insulin
(p = .03) (Table 4). and glucose in saliva of obese adolescents [19]. Salivary levels of VEGF
Children with ECC had a 63% higher median VEGF concentration were also increased in diabetic pregnant women associated to higher
than caries- free children, with the p-value approaching borderline caries activity [20]. Although we have not found studies comparing
statistical significance (p = .06). Similarly, children with ECC had serum and salivary levels of VEGF, it would be plausible to presume
means twofold higher in salivary IL-6 levels compared to caries-free that the levels in these both fluids may be increase simultaneously in
children, but the difference was not significant (p = .08) (Table 5). the presence of metabolic alterations.
In the present study, higher caries index in mother were also cor-
4. Discussion related with increased IL-6 levels in saliva. Others studies also showed
elevated levels of IL-6 in children with caries [21,22]; suggesting that a
Our findings show a positive correlation between high caries index dental infection [21] or the severity of caries [22] may increase the
in mother and in their children with salivary levels of VEGF. salivary levels of IL-6, responding to the molecular mechanisms related
Furthermore, higher maternal adiposity and higher sugar intake by the to the caries process [21].
At the meantime, our results also showed that maternal adiposity
Table 3 was also correlated with increased IL-6 levels in maternal saliva; which
Correlation analysis of children caries index, body mass index Z-score and sugar intake may suggest that higher levels of IL-6 in saliva may also be linked to
with children salivary levels of VEGF, IL-6 and TNF-α (n = 37). metabolic disorders. The obesity located in the waist region triggers a
low-grade systemic inflammatory response mediated by the secretion of
VEGF p IL-6 p TNF-α p
adipokynes, such as IL-6 [29]; and also increased serum levels of this
def-t 0.4263 a
0.008 0.1195 a
0.48 NS
0.0355 a
0.83NS cytokine are associated with metabolic syndrome and hyperinsulinemia
BMI Z-score 0.2341b 0.16NS −0.2112a 0.20NS −0.0672a 0.69NS [14].
Sugar intake −0.1613a 0.34NS 0.1521b 0.36NS 0.0381b 0.82NS
The excessive consumption of added sugar could explain the cor-
relation between increase of salivary cytokines, adiposity and caries.
VEGF: vascular endothelial growth factor; IL-6: interleukin 6; TNF-α: tumor necrosis
factor alpha; def-t: decayed, tooth indicated for extraction and filled teeth; BMI: body The consumption of added sugars has a clear role in caries etiology
mass index. p ≤ 0.05: statistically significant. NS: nonsignificant. [11]. An excessive consumption of added sugars may result in high
a
Pearson’s coefficient (r). serum levels of IL-6 [18]. Systematic reviews showed that added sugars,
b
Spearman’s coefficient (rs).

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C.C.C. Ribeiro et al.
especially sugar-sweetened beverages are associated to weight gain [8],
to metabolic syndrome [9,10] and to insulin resistance [9]. In our
findings, mothers of children with caries showed higher mean of sali-
vary IL-6 levels compared to those of children without caries. In this
sense, maternal dietary pattern is determinant in their child's eating
pattern, especially in their preference for sweets [7].
In the children, higher BMI Z-scores or consumption of added sugars
were not correlated with salivary levels of the cytokines studied (IL-6
and TNF-α). A study involving adolescent girls (13–17 years) found no
association between serum IL-6 and TNF-α levels and indicators of
adiposity, suggesting that the inflammatory process is not yet estab-
lished since these cytokines are mainly produced in adipose tissue
which is still scarce in this age group [30]. It is therefore plausible that
the correlation of obesity with IL-6 levels is established later in life, as
demonstrated by the association of greater maternal WC with salivary
IL-6 levels in the present study.
In children with ECC, although the median salivary VEGF con-
centration was higher (63%) and IL-6 levels were twice as high as those
found in children without ECC, comparison of the median/mean levels
of these markers showed a borderline significance. This lack of a sig-
nificant difference between children with ECC and caries-free children
might be explained by the sample size of the present study. However,
the number of participants was similar to that of a study showing ele-
vated IL-6 levels in adolescents with caries (n = 36) [2]. Thus, high IL-6
levels in individuals with caries are plainly established only at older
ages, as demonstrated in adolescentes [21] and in mothers with higher
DMFT scores in the present study. An post estimate calculation to
present study was done comparing IL-6 levels (mean and stand devia-
tion) in children with and without caries, and showed that a sample size
of 20 in each group (n = 40) would have a 80% power; suggesting that
the sample size for the children should be slightly larger than used here
(n = 37) to identify differences between the children groups.
The limitation of the present study was the cross-sectional design;
therefore, the data of inflammatory salivar markers was analyzed at
same time with caries diagnosis, nutritional state and sugar intake in
mother/child pair. However, our intention was not to identify a causal
relationship between sugar intake, salivary cytokines, adiposity and
caries; instead of this, our intention is to warn that there is an increase
in proinflammatory cytokines in saliva occurring simultaneously in the
mother/child pairs with caries.
There are a few factors that were not studied in the present study
that could also explain the present or future children's metabolic risk,
such as genetic or hereditary factors [31] and maternal exposure during
pregnancy [32]. However, the maternal dietary pattern has a de-
terminant role in their children dietary pattern [7]; and the reduction of
added sugars intake is a modifiable risk factor for obesity [11].
In conclusion, salivary inflammatory markers (VEGF and IL-6) are
correlated with the caries severity in the mother-children dyads.
Maternal obesity and excessive sugar consumption seem to underlie the
associations observed here between levels of those proinflammatory
cytokines in saliva and caries in the family environment. In this respect,
controlling the intake of added sugars has been suggested as primary
prevention strategy to reduce caries, obesity and possibly, to reduce risk
of others noncommunicable diseases [11,12].
Acknowledgments
We thank the directors of the community daycare centers and the
parents of the children that participated in this study. Ribeiro CCC
conceived the ideas; Costa EL, Ladeira LLC, Costa JF collected the data;
Carmo CDS, analyzed the data; Ladeira LLC, Costa JF, Carmo CDS, Silva
RA, Pacheco CJB, Costa EL and Ribeiro CCC led the writing.
Funding
This work was supported by the Maranhão Research and Scientific
116
Cytokine 107 (2018) 113–117
and Technological Development Foundation (FAPEMA) and the
National Council for Scientific and Technological Development (CNPq).
Declaration of interest
The authors declare no conflicts of interest.
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