DOI: 10.1111/ipd.

12270

Inflammatory markers in saliva as indicators of gingival

inflammation in cerebral palsy children with and without
cervical motor control

MARIA TERESA BOTTI RODRIGUES SANTOS1, MICHELE BAFFI DINIZ2, RENATA OLIVEIRA
GUARE
2, MARIA CRISTINA DUARTE FERREIRA3, GABRIELA MANCIA GUTIERREZ1
& RENATA GORJAO~ 4
1
Individuals with Special Needs, Cruzeiro do Sul University, S~ ao Paulo, SP, Brazil, 2Pediatric Dentistry, Cruzeiro do Sul
3
University, S~ao Paulo, SP, Brazil, Pediatric Dentistry, S~
ao Paulo Metodista University, S~ao Bernardo do Campo, SP, Brazil,
and 4Postgraduate Interdisciplinary Program in Health Sciences, Institute of Physical Activity Sciences and Sports, Cruzeiro
do Sul University, S~ao Paulo, SP, Brazil

International Journal of Paediatric Dentistry 2016
Summary
Aim. To evaluate the relation among gingival
inflammation, salivary osmolality, levels of IL-1b,
IL-6, IL-8, TNF-a, and s-IgA concentrations in
children with spastic CP with or without cervical
motor control in a cross-sectional study.
Design. Unstimulated whole saliva and the gingi-
val index were collected in 37 and 34 CP children
with and without cervical motor control, respec-
tively. The data were dichotomized as follows:
(=0) absence of gingival inflammation and (≥0.1)
presence of gingival inflammation.
Results. The group without cervical control pre-
sented statistically higher mean values of salivary
osmolality, s-IgA, and cytokines. In addition,
statistically positive correlation between the gingi-
val index and salivary cytokines was observed in
the group with cervical control. Salivary osmolal-
ity, salivary cytokines, and s-IgA from both groups
presented a significant positive correlation. Signifi-
cant differences (P = 0.00336) in the values of
salivary osmolality were observed between the CP
individuals with (93.9
32.7) and without gingi-
val inflammation (74.4
16.6). ROC analysis was
performed, and values of salivary osmolality >80
indicated a sensitivity of 0.54 and a specificity of
0.79.
Conclusions. Children without cervical motor con-
trol presented a more pronounced oral inflamma-
tory status that was characterized by higher levels
of cytokines.

gradual damage of soft and hard sustaining

Introduction
Gingival health can be described as a condi-
tion characterized by antimicrobial cytokines
with inflammatory properties that are con-
trolled by anti-inflammatory mediators
release to prevent exacerbated inflammation1.
Gingivitis is considered the most frequent
periodontal disease in children, which is
mainly caused by an accumulation of bacteria
in gingival sulcus plaque and their potentially
harmful products, associated with unsatisfac-
tory oral hygiene2. This process promotes the
Correspondence to:
Profa. Dra. M. T. B. R. Santos, Individuals with Special
Needs, Universidade Cruzeiro do Sul Rua Constantino de
Souza, 454, apt 141, S~ao Paulo, SP 04605-001, Brazil.
E-mail: drsantosmt@yahoo.com.br
tissues of the teeth3. In these conditions, local
cytokines and chemokines may be altered
causing a more severe progression of the
inflammatory process4.
Gingivitis is associated with increased medi-
ators such as interleukin (IL)-1b and TNF-a.
IL-6 release in inflamed gingival tissue is also
a significant effect in the development of
periodontal disease5.
These cytokines promote an important
function in immune defense, resulting leuko-
cyte activation in the infection site, such as
macrophages and neutrophils. TNF-alpha
receptor (TNFR) is responsible by macrophage
activation as well other inflammatory cyto-
kine receptors6. TNFR recruitment results in
the NF-jB activation and MAP kinase signal-
ing, promoting the expression of other

© 2016 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 1
2 M. T. B. R. Santos et al.
inflammatory cytokines and chemokines7.
Exacerbated inflammatory cytokine produc-
tion, however, is unfavorable to the tissue
health leading to appearance of inflammatory
disorders8. IL-1b triggers chemotaxis of neu-
trophils and T-cell activation. This cytokine
also stimulates the expression of inflamma-
tory factors that promote extracellular matrix
degradation. Thus, it is involved with several
inflammatory diseases, such as inflammatory
bowel disease, rheumatoid arthritis, and peri-
odontitis9. IL-8 and mucosa-associated epithe-
lial chemokine (CCL28) are also major
chemokines involved in the inflammatory
process related to gingivitis10. One of the
main functions of IL-8 is the stimulation of
polymorphonuclear cell chemotaxis to sites of
tissue damage, leading to neutrophil accumu-
lation, causing tissue damage, and contribut-
ing to the inflammatory process10.
Gingivitis has been described as a common
disease observed in cerebral palsy (CP) sub-
jects11,12 resulting from their incapability to
succeed and preserve adequate oral hygiene
and is dependent on their cervical control13–
15
. Moreover, CP subjects present diminished
salivary flow rates and increased salivary
osmolality16 which contributes to develop-
ment of oral disease17. Due to the central ner-
vous lesion, CP subjects generally present an
inefficient masticatory muscle function
responsible for decreasing fluid intake, which
increase salivary osmolality, accumulation of
pathogenic microorganisms in the biofilm,
and gingival inflammation risk. In a prelimi-
nary evaluation of our group, a salivary
osmolality above 84.5 was observed to
increase the likelihood of the presence of
gingivitis by fivefold18.
After a systematic revision in the published
articles, combining the terms cerebral palsy,
saliva cytokines, and gingivitis, no studies
were achieved. Thus, the purpose of this
study was to evaluate the relation among
gingival inflammation, salivary osmolality,
cytokines (IL-1b, IL-6, IL-8, and TNF-a), and
s-IgA concentrations in children and adoles-
cents with spastic CP with or without cervi-
cal motor control. The null hypothesis was
that children and adolescents with CP with-
out cervical motor control presented higher
© 2016 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
levels of salivary cytokines and s-IgA
compared with children with cervical motor
control.
Material and methods
Ethical statement
This study was approved by the local research
ethics committee (#152/2011). Caregivers
were informed about the purpose of the pre-
sent investigation and signed the written
informed consent for participation of each CP
subject.
Study design
An epidemiological cross-sectional investiga-
tion was performed with CP subjects who
received physical rehabilitation in a reference
center in S~ao Paulo, Brazil.
Subjects
Seventy-one children and adolescents with
spastic CP were consecutively included in
this study, between March and November
2014. CP subjects using anticholinergic, neu-
roleptic, and benzodiazepine drugs, individu-
als with previous history of head and neck
radiation, gastroesophageal reflux disease
(GERD), and salivary gland surgery proce-
dure, or those with non-cooperative behav-
ior were excluded. Moreover, CP subjects
with symptoms of fever, flu, body aches,
and diarrhea, or any inflammatory condition
in the oral mucosa, as a cold sore, were
also excluded. All participants received pre-
ventive oral health care at regular intervals.
Information regarding sex, age, and cervical
control was collected from their medical
reports.
In the present investigation, oral motor effi-
ciency was not assessed because the CP sub-
jects were classified according to the clinical
parameters of CP distribution. The study was
performed in two groups as follows: (A) CP
subjects without cervical motor control (tetra-
paresis – involvement of all four limbs) and
(B) CP subjects with cervical motor control
(diparesis – the legs are more involvement

than the arms or hemiparesis – ipsilateral arm
and leg)19.
Measurements
Clinical examinations were performed in a
dental office under appropriate light-reflector
illumination in a dental chair by a single cali-
brated examiner for the gingival index
(kappa = 0.86).
Saliva collection and assessment
The procedures for unstimulated whole saliva
collection20 and assessment of salivary osmo-
lality18 in individuals with CP were described
in previous studies of our research group.
Salivary cytokine concentrations (IL-1b, IL-
6, IL-8, and TNF-a) were determined on a
Luminex 200 using a MILLIPLEX MAP HCY-
TOMAG-60K-04 kit (Millipore, Billerica, MA,
USA). The assays were performed according
to the manufacturer’s guidelines using Mag-
Pix – Software xPonent/Analyst. A standard
curve was performed for each cytokine using
the standard solutions provided in the kit
according to the manufacturer’s instruc-
tions. The detection limits were 0.4 pg/mL for
IL-8, 0.9 for IL-6, 0.7 for TNF-a, and 0.8 for
IL-1 b.
The s-IgA concentration was determined by
colorimetric immunoenzymatic assay (ELISA)
using an s-IgA saliva kit (DiaMetra, Milano,
Italy) respecting the manufacturer’s guide-
lines. The absorbance was analyzed in a Stat
Fax 2100 (Awareness Technology, Palm City,
FL, USA). The detection limit was 0.5 lg/mL.
Gingival status assessment
The children’s gingival conditions were scored
according to the gingival index21 four-point
scale, varying from 0 (absence of inflamma-
tion) to 3 (severe inflammation), using plastic
periodontal probes18.
Gingival inflammation extension was deter-
mined by the gingival index for the area,
based on six index teeth18. The index of each
CP subject was the average score for the
areas examined as follows: 0.1–1.0 (mild),
1.1–2.0 (moderate), and 2.1–3.0 (severe)
© 2016 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Salivary cytokines in cerebral palsy 3
inflammation21. Then, the data were dichoto-
mized as absence of gingival inflammation (0)
and presence of gingival inflammation (≥0.1).
Statistical analysis
Statistical tests were performed using the
SPSS software program for Windows. The
mean and standard deviation values were cal-
culated for dependent variables (gingival
inflammation and salivary osmolality) and
independent variables (s-IgA and cytokines).
The scores for gingival index (0–3) were
calculated for a general description of the
degree of gingival inflammation in each
group. Then, the dichotomized classification
(absence/presence) was used to perform the
inferential statistical analysis (ROC curve).
Student’s t-test and chi-square test were
used to compare the two groups regarding
age and sex, respectively. The Mann–Whitney
U-test was applied to compare the variables
between the groups.
The correlation between the different vari-
ables was performed using Spearman0 s corre-
lation coefficient (rho). Receiver operating
characteristic (ROC) was performed, and the
area under the ROC curve (AUC) was calcu-
lated for the groups merged. The greatest cut-
off point for salivary osmolality and the
presence of gingival inflammation were
obtained through ROC analyses. The signifi-
cance level was established at P < 0.05.
Power calculation for the difference
between two means of gingival index was cal-
culated using OpenEpi online (www.openepi.
com), at a 95% confidence interval.
Results
Seventy-one children and adolescents with
spastic CP were evaluated, including 37 males
(52.1%) and 34 females (47.9%) ranging
from 6 to 17 years old (mean
standard
deviation, SD = 10.2
3.4 years old).
Table 1 shows the descriptive statistics
regarding gender, age, gingival index and its
scores, salivary osmolality, salivary cytokines,
and s-IgA for both groups. The sample
included 37 (52.1%) and 34 (47.9 %) CP
children and adolescents with and without
4 M. T. B. R. Santos et al.

Table 1. Descriptive statistics for all variables analyzed in compared with the group with cervical control
both groups.
(P < 0.05). Considering the difference of
Without With means between both groups for the gingival
cervical cervical index (1.5 – 0.8 = 0.7), the test provided a sta-
control control tistical power of 98.7% of this study.
Variable (n = 34) (n = 37) P-value
Spearman0 s rank correlation coefficients
Gender – n (%) among gingival index, salivary osmolality,
Female 13 (38.2) 21 (56.7) 0.1186† salivary cytokines, and s-IgA are shown in
Male 21 (61.8) 16 (43.3)
Age years 9.8
2.6 8.6
2.7 0.0658*
Tables 2 and 3. A positive significant correla-
(mean
SD) tion was observed between gingival index
Gingival Index 1.5
0.6 0.8
0.8 0.0005* and salivary cytokines in the group with cer-
(mean
SD) vical control. With respect to the salivary
Score 0 – n (%) 2 (5.9) 12 (32.4) 0.0064†
Score 1 – n (%) 6 (17.7) 11 (29.7) osmolality and salivary cytokines and s-IgA,
Score 2 – n (%) 25 (73.5) 13 (35.1) both groups presented a significant positive
Score 3 – n (%) 1 (2.9) 1 (2.8) correlation (P < 0.05).
Salivary osmolality 99.1
35.5 81.7
24 0.0175*
(mean
SD)
Salivary osmolality values (P = 0.00336)
IL-1b (mean
SD) 22.5
21.4 12.5
22.4 0.0001* were statistically different for CP individuals
IL-6 (mean
SD) 14.3
20.1 5.9
13 0.0012* with gingival inflammation (n = 57; salivary
IL-8 (mean
SD) 46.6
27.0 26.2
21.2 <0.0001*
TNF-a (mean
SD) 13.4
16.6 3.8
7.1 0.0001*
osmolality = 93.9
32.7 and median = 84.0;
IgA (mean
SD) 63.4
77.2 41.1
35.1 0.0235* gingival index = 1.4
0.6) compared to indi-
viduals without gingival inflammation
*Mann–Whitney U-test (P < 0.05). (n = 14; salivary osmolality = 74.4
16.6 and
†Chi-square test (P < 0.05).
median = 73.5; gingival index = 0.0) (Fig. 1).
Consequently, it was determined a cutoff
cervical motor control, respectively. The limit from the ROC analysis (maximizing sen-
group without cervical motor control was sitivity and specificity) for salivary osmolality
composed of 13 (38.2%) females and 21 for CP subjects with gingival inflammation.
(61.8%) males, and the group with cervical The AUC was 0.684 (0.563–0.789)
motor control of 21 (56.7%) females and 16 (P = 0.0147) (Fig. 2). Values of salivary osmo-
(43.3%) males. Regarding age, it ranged from lality >80 indicated a sensitivity of 0.54 (0.41–
6 to 14 years (9.8
2.6) for the group with- 0.68) and a specificity of 0.79 (0.49–0.95).
out cervical motor control, and from 6 to
17 years (8.7
2.7) for the group with cervi-
Discussion
cal motor control. The groups did not differ
regarding sex (P = 0.1186) and age This is the first study to evaluate salivary IL-
(P = 0.0658). 1b, IL-6, IL-8, TNF-a, and s-IgA as inflamma-
According to the individual score of the gin- tory markers of gingivitis in children with CP.
gival index, the group without cervical motor The levels of salivary cytokines could indicate
control presented two individuals (5.9%) with an initial gingival inflammatory process,
no gingival inflammation, six (17.7%) with requiring a preventive management.
mild inflammation, 25 (73.5%) with moder- The secretion of cytokines in tissues as a
ate inflammation, and one (2.9%) with severe response to periodontal bacteria and their
inflammation. The group with cervical motor products promotes higher serum concentra-
control presented 12 (32.4%) with no gingival tions of pro-inflammatory biomarkers22. The
inflammation, 11 (29.7%) with mild inflam- comparison between CP subjects with and
mation, 13 (35.1%) with moderate inflamma- without cervical motor control demonstrated
tion, and 1 (2.8%) with severe inflammation. that the values of the gingival index, salivary
The groups were statistically different regard- osmolality, salivary cytokines, and IgA were
ing the gingival index (P = 0.0064). significantly higher for the group without cer-
The group without cervical control presented vical motor control. Higher expression of IL-6
statistically higher mean values for all variables and IL-1b was also described in children with

© 2016 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
 Salivary cytokines in cerebral palsy 5

Table 2. Spearman0 s rank correlation coefficients (rho) between gingival index (GI) and salivary cytokines and IgA in both
groups.

Without cervical control (n = 34) With cervical control (n = 37)

Variable rho 95% Confidence Interval P-value rho 95% Confidence Interval P-value

GI versus IL-1b 0.183 0.165–0.491 0.2992 0.417 0.107–0.653 0.0102*

GI versus IL-6 0.307 0.034–0.585 0.0769 0.460 0.160–0.683 0.0041*
GI versus IL-8 0.279 0.065–0.564 0.1105 0.347 0.0253–0.603 0.0356*
GI versus TNF-a 0.179 0.017–0.149 0.3101 0.354 0.0334–0.608 0.0318*
GI versus IgA 0.135 0.258–0.490 0.5017 0.0584 0.285 to 0.389 0.7430

*Variables statistically correlated (P < 0.05).

Table 3. Spearman0 s rank correlation coefficients (rho) between salivary osmolality (SO) and salivary cytokines and IgA in
both groups.

Without cervical control (n = 34) With cervical control (n = 37)

Variable rho 95% Confidence Interval P-value rho 95% Confidence Interval P-value

SO versus IL-1b 0.651 0.401–0.811 <0.0001* 0.632 0.387–0.793 <0.0001*

SO versus IL-6 0.602 0.332–0.781 0.0002* 0.509 0.221–0.715 0.0013*
SO versus IL-8 0.752 0.554–0.869 <0.0001* 0.569 0.301–0.754 0.0002*
SO versus TNF-a 0.442 0.122–0.679 0.0089* 0.348 0.027–0.604 0.0346*
SO versus IgA 0.767 0.547–0.888 <0.0001* 0.562 0.276–0.756 0.0005*

*Variables statistically correlated (P < 0.05).

240
100

220

200
80
180
Salivary osmolality

160

140 60
Sensitivity

120

100
40
80

60

40
Absence Presence
20
Gingival inflammation

Fig. 1. Box plots regarding salivary osmolality according to

0
the presence or absence of gingival inflammation.
0 20 40 60 80 100
23 100-Specificity
oral health issues . A previous study by our
group also observed higher levels of s-IgA Fig. 2. ROC curve for salivary osmolality and the presence of
concentration in cerebral palsy individuals24. gingival inflammation.
Elevated saliva concentrations of inflamma-
tory cytokines observed in children and ado- indicative of systemic immune function and
lescents without cervical control indicated cannot replace serum and plasma evaluations
activation of the immune system, resulting in of immunity25.
an increase in immunoglobulin secretion. In this study, even though all participants
Importantly, salivary profiles may not be received the same preventive oral health care

© 2016 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
6 M. T. B. R. Santos et al.
at regular intervals, the results observed in CP
subjects without cervical motor control may
be related to more severe neurological dam-
age; consequently, these individuals are more
prone to cumulative and irreversible biologi-
cal changes26. These alterations have a signifi-
cant effect on their oral health status, due to
difficulty with mouth opening, biting reflex,
and maintenance of adequate oral hygiene,
thereby increasing the risk for oral dis-
eases27,28.
Importantly, IL-8 values were approxi-
mately twice more expressive for the group
without cervical motor control, indicating its
local presence due to the gingival inflamma-
tory process. Indeed, Ertugrul, Sahin, Dikili-
tas, Alpaslan, & Bozoglan10 demonstrated that
IL-8 expression is correlated with the degree
of periodontitis severity. IL-8 is an important
chemokine that promotes recruitment of neu-
trophils during healing, and its levels can be
significant associated with an increased sus-
ceptibility to periodontitis22.
It is accepted that saliva is an important
fluid for the maintenance of oral health29.
The higher the values of salivary osmolality,
the higher bacterial accumulation of bio-
film30, thereby increasing inflammatory pro-
cess18. Furthermore, the positive correlation
among salivary osmolality, cytokines, and
IgA was strong, demonstrating that salivary
osmolality is strongly correlated with gingivi-
tis in CP subjects without cervical motor
control. Salivary osmolality values >80 were
indicative of mild gingival inflammation pro-
cess (gingival index cutoff >0.1). In the liter-
ature, one investigation demonstrated that
salivary osmolality values >84.5 can predict
moderate gingivitis (gingival index cutoff
≥2.0) in CP subjects18, showing that salivary
osmolality can be used as an adjunct to
determine the degree of gingival inflamma-
tion in CP individuals. Moreover, the group
without cervical motor control presented
more moderate gingival inflammation, proba-
bly due to difficulty in mouth opening,
drinking liquids, and oral hygiene perfor-
mance, whereas the group with cervical
motor control presented more mild gingival
inflammation, which can facilitate oral
hygiene maintenance.
© 2016 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Cytokine concentrations (IL-6, IL-1b, and
TNF-a) stimulate the expression of adhesion
molecules and chemokines, matrix metallo-
proteinase, apoptosis of matrix-producing
cells, and increased osteoclast activity31. Thus,
it can be suggested that the chronic elevation
of these cytokines may interfere in gingival
tissue repair and teeth bone sustentation31. In
the present study, children without cervical
control have higher levels of these inflamma-
tory cytokines compared with children with
cervical control. In a previous study, it was
suggested that higher levels of salivary IL-6
could be related to the presence of intellec-
tual disabilities, which could also be related
to our findings32.
The limitation of the present investigation
could be attributed to a convenient sampling
of subjects with CP receiving preventive oral
health care at regular intervals in a referred
specialized physical rehabilitation center in
Brazil. The number of CP children and ado-
lescents, however, was sufficient to infer an
internal validity to the results. The informa-
tion generated in the present study should be
taken into consideration to provide support to
the development of innovative studies and
guidelines and specific recommendations to
control gingival inflammation in this popula-
tion.
The levels of salivary cytokines could indi-
cate an initial gingival inflammatory process,
which can progress to gingival bleeding and
tissue damage, demonstrating the possibility
of periodontal disease progression. The deter-
mination of salivary cytokines could con-
tribute to a better treatment decision in
individuals with cerebral palsy, considering
that the clinical examination is subjective and
the periodontal assessment and probing is a
hard task due to their oral motor impairment.
Individuals with cerebral palsy without cervi-
cal control are more prone to present gingivi-
tis, requiring procedures for preventing
periodontal disease, such as short interval
recall, professional oral hygiene maintenance,
and constant re-orientation for brushing and
flossing teeth with adapted techniques at
home by the caregivers.
In conclusion, children and adolescents
without cervical motor control present a more

pronounced oral inflammatory status charac-
terized by higher levels of TNF-a, IL-1b, IL-6,
and IL-8. These elevated levels of inflamma-
tory mediators are correlated with increased
salivary osmolality.
Why this paper is important for paediatric dentists
 Individuals with cerebral palsy without cervical motor
control are more prone to present gingival inflamma-
tion.
 Higher values of salivary osmolality can be indicative
of the presence of gingivitis in subjects with cerebral
palsy.
 Elevated levels of proinflammatory salivary cytokines
and salivary immunoglobulin A (IgA) are correlated
with increased salivary osmolality in children and
adolescents with cerebral palsy.
Acknowledgements
This study was supported by the S~ao Paulo
Research Foundation (Fundacß~
ao de Amparo a
Pesquisa do Estado de S~ao Paulo, FAPESP
#2011/12475-8).
Ethics Committee Approval
The research was approved by the Research
Ethics Committee of Cruzeiro do Sul Univer-
sity Institutional Review Board, under proto-
col no 152/2011.
Study Population
The study population consisted of children
with cerebral palsy who were referred to a
specialized rehabilitation center in the city of
S~
ao Paulo, SP, Brazil.
Conflict of Interest
The authors declare no conflict of interest.
Author Contributions
M.T.B.R. Santos involved in concepts, design,
literature search, definition of intellectual
content, experimental study, manuscript
preparation, manuscript editing, and manu-
script review; M.B. Diniz involved in concepts,
statistical analysis, manuscript preparation,
© 2016 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Salivary cytokines in cerebral palsy 7
manuscript editing, and manuscript review;
R.O. Guar
e involved in concepts, manuscript
preparation, and manuscript review; M.C.D
Ferreira involved in concepts and experimen-
tal study; G.M. Gutierrez reviewed the manu-
script; and R. Gorj~ao involved in concepts,
statistical analysis, manuscript preparation,
and manuscript review.
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