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149]
Original Article
Evaluation of chemokines in gingival crevicular fluid
in children with dental caries and stainless steel
crowns: Aclinicobiochemical study
Naveen Kommineni Kumar, Veera Kishore Kasa Reddy, Prathyusha Padakandla, Harshini Togaru,
Swathi Kalagatla, Sarath N Chandra
Department of Pedodontics, C.K.S. Teja Institute of Dental Sciences, Tirupati, AndhraPradesh, India
ABSTRACT
Aims and Objectives: The study was conducted to
detect the presence of macrophage inflammatory
protein1 (MIP1) and MIP1 and estimate
their levels in gingival crevicular fluid (GCF) in
children with dental caries and stainless steel
crowns. Materials and Methods: Atotal of 80
children with primary dentition were selected and
categorized into four groups with twenty in each
group; Group1healthy subjects, Group2dental
caries, Group 3 dental caries involving the pulp,
and Group4stainless steel crowns. GCF samples
were collected by an extracrevicular method with
microcapillary pipettes. The GCF samples were
quantified by ELISA and the levels of MIP1 and
MIP1 were determined. Results: MIP1 and
MIP1 were detected in all the samples. Highest
mean concentration in GCF was obtained for
Group 3 followed by Groups 2 and 4 while the
lowest concentration was seen in Group 1. This
suggests that MIP1 and MIP1 levels in GCF
increased proportionately with the inflammation.
Conclusions: GCF serves as a noninvasive diagnostic
fluid to measure biomarkers released during dental
caries initiation and progression. MIP1 and
MIP1 chemokines can be considered as novel
biomarkers, in biological mechanism underlying
the pathogenesis and inflammation in children with
dental caries and stainless steel crowns.
KEYWORDS: Chemokines, dental caries, gingival
crevicular fluid, macrophage inflammatory protein1,
macrophage inflammatory protein1, pulpal
inflammation, stainless steel crowns
Introduction
Dental caries is an infectious disease with
multifactorial etiology.[1] Bacteria causing dental caries
will colonize the oral cavity and lead to the process
of inflammation. These carious lesions induce both
2016 Journal of Indian Society of Pedodontics and Preventive Dentistry | Published by Wolters Kluwer - Medknow
Address for correspondence:
Dr.Naveen Kommineni Kumar,
Department of Pedodontics, C.K.S. Teja Institute of Dental
Sciences, Tirupati517507,
AndhraPradesh, India.
Email:naveenkommineni@gmail.com
Access this article online
Quick response code Website:
www.jisppd.com
DOI:
10.4103/0970-4388.186754
PMID:
******
innate and adaptive immune responses in the host.[2]
As a response to inflammation, the cytokines are likely
to be released into the systemic circulation. Animal
models indicated that the proinflammatory cytokine
concentrations were higher within the serum in
periapical lesions.[3] The proinflammatory cytokines
concentrations are elevated in serum and gingival
tissues of humans with periodontal inflammation, and
may contribute to a systemic hyperinflammatory state,
which is a risk factor for several systemic diseases. It
is wellknown that in oral cavity disorders, the levels
of cytokines are increased in saliva.[3] It may be argued
that the expression of cytokines in unstimulated
This is an open access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-ShareAlike 3.0 License, which
allows others to remix, tweak, and build upon the work non-commercially,
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For reprints contact: reprints@medknow.com
How to cite this article: Kumar NK, Reddy VK, Padakandla P,
Togaru H, Kalagatla S, Chandra SN. Evaluation of chemokines
in gingival crevicular fluid in children with dental caries and
stainless steel crowns: A clinico-biochemical study. J Indian Soc
Pedod Prev Dent 2016;34:273-9.
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Kumar, etal.: Evaluation of chemokines in children with dental caries and crowns
whole saliva may be due to the leakage of gingival
crevicular fluid (GCF) into the oral cavity.[4] Hence,
it is assumed that the level of cytokines is increased
in GCF of individuals with dental caries. CCL20
expression in human inflamed pulp is observed mostly
in macrophages that have accumulated in the area
adjacent to caries lesions.[5]
Steel crowns are indicated in pediatric dentistry to treat
teeth surfaces when they have been severely affected
by dental caries, development defects, traumatic
dental fractures, and following pulp therapies.[6]
The association between stainless steel crowns and
gingival inflammation has not been fully explained
in the literature. It has been reported that gingivitis
often occurs around primary teeth restored with steel
crowns due to various factors such as poorly adapted,
inadequately contoured margins and subgingivally
located crowns.[7]
The chemokines are considered to be one of the
inflammatory factors which play a crucial role in
mediating the extravasation and accumulation
of selective leukocyte subsets in the process of
inflammation.[8] Chemokines are chemotactic
cytokines that direct the recruitment and subsequent
activation of specific types of leukocytes into inflamed
periodontal tissues.[9] Chemokines play a vital role
in the process of inflammation, physiological and
pathological activities, such as lymphoid trafficking,
Th1/Th2 cell development, and wound healing.[10]
They are secreted by a range of inflammatory cells,
such as neutrophils, monocytes, and lymphocytes
as well as noninflammatory cell types at sites of
inflammation.[10] Cytokines cause selective migration
of human monocytes and lymphocytes. Macrophage
inflammatory protein1 (MIP1) is preferentially
chemotactic for CD8+Tcell subset.[11]
Expression of MIP1 in gingival tissue with chronic
periodontal diseases has been investigated in the
previous studies. Ryu etal.[12] have found that MIP1
expression in gingival epithelial cells is induced
by lipopolysaccharide (LPS), and it is important in
initiating inflammation. Garlet et al.[13] have detected
higher expression of MIP1 in inflamed gingival
tissue of subjects with chronic periodontitis and
aggressive periodontitis. It makes clear that as the
severity of periodontal disease increases, MIP1
levels also increases.[14] MIP1 belongs to the CC
chemokine subfamily. The chemokine MIP1 (also
called CCL4) is considered to be the most abundantly
expressed chemokine in periodontal diseases in
correspondence to MIP1.[15] Both MIP1 and
MIP1 have shown to be potent chemoattractants
for macrophages, lymphocytes, eosinophils, natural
killer cells, and dendritic cells.[10,16,17] Both of these
chemokines exert similar effects on monocytes but
their effects on lymphocytes differ: MIP1 selectively
attracts CD8 lymphocytes and MIP1 selectively
attracts CD4 cells.[10] However, to date, no study has
274 Journal of Indian Society of Pedodontics and Preventive Dentistry | Jul-Sep 2016 | Vol 34 | Issue 3 |
been done to evaluate the GCF levels of MIP1 and
MIP1 in subjects with dental caries and stainless steel
crowns. The present study is thus the first of its kind to
investigate the presence of MIP1 and MIP1 levels
in such subjects.
Materials and Methods
Children were selected from outpatient department,
Department of Pedodontics, C.K.S. Teja Institute of
Dental Sciences, Tirupati, Andhra Pradesh. Healthy
male and female children in the age group of
35 years with healthy gingiva were included in the
study. Children with dental caries and stainless steel
crowns were also included in the study. Subjects with
intraoral and systemic infections, having received
periodontal or antibiotic therapies 6 months before
testing, using mouth rinses containing antimicrobials
in the preceding 2months, with diabetes or with other
orthodontic appliances, were excluded from this study.
All eligible subjects are thoroughly informed as to the
nature, methods, risks, and benefits of the study. Their
participation was obtained by a clear written consent.
The study was carried out after approval of Institutes
Ethical Committees.
Criteria for subject grouping
The selected children were categorized into four
groups(twenty subjects each):
Group 1: (Control group) 35 years of age with
clinically healthy gingiva (gingival index (GI) <1,
plaque index(PI) <1, and probing pocket depth<3)
and with deft score<3
Group 2: 35 years of age with healthy
gingiva(GI<1, PI<1, and probing pocket depth<3)
and deft>3
Group 3: 35 years of age with healthy
gingiva(GI<1, PI, and<1 probing pocket depth<3)
and with deft>3 with pulp involvement
Group4:35years of age with stainless steel crowns
and with deft<3.
GI, PI, probing pocket depth and deft were
assessed. Without touching the marginal gingiva,
supragingival plaque was removed to avoid
contamination and blocking of the microcapillary
pipette. A standard volume of 3 ml GCF was
collected from each site by placing 13 ml calibrated
volumetric microcapillary pipette (SigmaAldrich
Chemical Company, USA: Catalog number p0549)
extracrevicularly (unstimulated) for 520 min. GCF
was collected from the distal sites of primary first
molar and around the gingival sulci of teeth with
stainless steel crown[Figures1 and 2]. The test sites,
which did not express standard volume(3ml) of GCF
and micropipettes contaminated with blood or saliva
were excluded from this study. The collected GCF
was immediately aliquoted and stored at70C until
the time of the assay. An ELISA was performed to
determine the chemokines present in the GCF samples.
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Kumar, etal.: Evaluation of chemokines in children with dental caries and crowns
ELISA employs the quantitative sandwich enzyme
immunoassay technique (R and D Systems: Catalog
numbers DMP300 and DTM100)[Figures3 and 4].
Statistical analysis
Data analysis was carried out using theStatistical Package
for Social Sciences (SPSS version 20, USA). Multiple
comparisons for GI and PI were analyzed by analysis of
variance. Further post hoc comparisons were analyzed
using Tukey honest significant difference test. For other
variables such as probing pocket depth, deft, MIP1,
and MIP1, multiple comparisons were analyzed by
KruskalWallis test. Further pairwise comparisons
were analyzed using MannWhitney Utest.
Results
As shown in Table1, the mean PIPI for Group1 was
0.39 0.21, for Group 2 was 0.41 0.16, for Group 3
was 0.420.14, and for Group4 was 1.610.16. The
mean GI was 0.42 0.19 for Group 1, for Group 2
was 0.42 0.15, for Group 3 was 0.41 0.14, and for
Group4 was 1.630.14[Table1]. The mean probing
pocket depth for Group1 was 1.120.27, for Group2
was 1.13 0.26, for Group 3 was 1.12 0.23, and for
Group4 was 1.190.24[Table2]. The mean deft for
Figure 1: Collection of gingival crevicular fluid from caries tooth
Figure 3: Addition of streptavidin-horseradish peroxidase conjugate
(Cat. no. S100180)
Journal of Indian Society of Pedodontics and Preventive Dentistry | Jul-Sep 2016 | Vol 34 | Issue 3 |
Group1 was 1.500.51, for Group2 was 5.250.96,
for Group 3 was 5.75 0.91, and for Group 4 was
1.400.50[Table2]. The differences in[Tables1 and 2]
were highly significant (P < 0.001). All the samples,
in each group, tested for the presence of MIP1 and
MIP1. The mean total concentration of MIP1 in the
GCF for group 1 was 197.60 pg/l, for Group 2 was
900.40 pg/l, for Group 3 was 1286.55 pg/l, and for
Group 4 was 682.55 pg/l [Table 3 and Graph 1]. The
mean total concentration of MIP1 in the GCF for Group
1 was 287.85 pg/l, for Group 2 was 1048.85 pg/l,
Group 3 was 1208.85 pg/l, and Group 4 was 884.35
pg/l [Table 3 and Graph 2]. Statistically significant
difference existed between these groups (P = 0.001).
Discussion
Cytokines are closely associated with the pathogenesis
of inflammation in soft tissues,[18,19] and evidence
indicates that they contribute to the initiation and
progression of dental caries.[20,21] MIP1 and MIP1
are the most important cytokines in the human immune
system which play a vital role in the antibacterial
defense system. Carious lesions have previously been
shown to be dominated by a variety of infiltrating
immune cells.[22,23] Cytokines and chemokines are
Figure 2: Collection of gingival crevicular fluid from tooth with
stainless steel CROWN
Figure 4: ELISA reader
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Kumar, etal.: Evaluation of chemokines in children with dental caries and crowns

Table1: Mean plaque index and mean gingival index for Groups a range of target cells. Certain cytokines, such as tumor
1, 2, 3, and 4 necrosis factoralpha (TNF), interleukins (ILs), and
Parameters Frequency MeanSD SE F P epithelial cellderived neutrophil attractant 78 are
Plaque index
predominantly proinflammatory. These cytokines
mediate both local and systemic inflammatory
Group1 20 0.390.21 0.048 238.54 0.001*
responses, with increased levels which are associated
Group2 20 0.410.16 0.037
with a variety of diseases.[24] Streptococcus mutans level
Group3 20 0.420.14 0.032
positively correlated with saliva IL1 concentration
Group4 20 1.610.16 0.036
and inversely correlated with saliva IL1 receptor
Gingival index
antagonist concentration.[25] So far, only limited studies
Group1 20 0.420.19 0.044 269.80 0.001* have been performed on the molecular nature of the
Group2 20 0.420.15 0.035 dental tissue immune response, and no study has been
Group3 20 0.410.14 0.033 done to evaluate the presence of MIP1 and MIP1
Group4 20 1.630.14 0.033 in dental caries and stainless steel crowns. Therefore,
ANOVA test: *P<0.05(significant); SD:Standard deviation; SE:Standard the present study is thus the first of its kind to better
error; ANOVA:Analysis of variance
characterize the molecules involved by analyzing the
expression of MIP1 and MIP1.
Table2: Mean probing pocket depth and deft for Groups 1, 2, 3,
and 4 In this study, GI and PI were found to be high in
Parameters Frequency MeanSD Mean 2 P Group 4 when compared with other groups. When
ranks mean concentrations of MIP1 and MIP1 in
Probing pocket GCF were compared between Groups 1 and 4, high
depth concentrations were found in Group 4. The possible
Group1 20 1.120.27 31.20 9.75 0.021* reasons for increase in GCF levels of MIP1 and
Group2 20 1.130.26 38.93 MIP1 in Group 4 could be because of recruitment
Group3 20 1.120.23 40.90 and retention of leukocyte subsets into gingival crevice
Group4 20 1.190.24 50.98 in response to plaque accumulation, periodontal
Deft scores pathogens, and their bacterial components like LPS.
Group1 20 1.500.51 21.50 62.66 0.001* Furthermore, factors like tissue injury contributed to
Group2 20 5.250.96 57.58 the release of chemokines. The variability of MIP1
Group3 20 5.750.91 63.43 and MIP1 concentrations within subjects of each
Group4 20 1.400.50 19.50
group could be due to their role in different stages of
Kruskal-Wallis Test: *P<0.05(significant); SD:Standard deviation
disease process at the time of GCF collection.

The results of this study are in accordance with

Table3: Mean macrophage inflammatory protein1 and Sharaf and Farsi,[7] who stated that the gingival health
macrophage inflammatory protein1 concentrations in for
Groups 1, 2, 3 and 4
is affected due to marginal adaptation of stainless
steel crowns and the level of oral hygiene. This is
Parameters Frequency MeanSD SE 2 P supported by studies carried out by Henderson[26]
(pg/l) and Myers,[27] who reported a high incidence of
MIP1 gingivitis around incorrectly contoured stainless
Group1 20 197.6040.83 10.50 65.32 0.001* steel crowns. These results disagree with those of
Group2 20 900.40209.04 53.28 Checchio etal. study[28] in which improper adaptation
Group3 20 1286.55382.71 65.73 showed no relationship with periodontal problems.
Group4 20 682.5559.97 32.50 Myers[27] showed a significant association between
MIP1 crown defects (34%) and clinical evidence of
Group1 20 287.8542.20 10.50 65.16 0.001* gingivitis(P<0.001), with extension being the most
Group2 20 1048.85212.07 53.40 common error. Many studies supported that there
Group3 20 1208.85235.69 65.58 is a correlation between gingivitis and oral hygiene
Group4 20 884.35125.46 32.53 around stainless steel crowns.[18,29,30] Children with
Kruskal-Wallis test: *P<0.05(significant), MIP:Macrophage inflammatory inadequate oral hygiene showed higher frequency of
protein; SD:Standard deviation, SE:Standard error gingivitis, whereas children with proper oral hygiene
showed a healthy gingiva around steel crowns.[18]
The study by Ramazani etal.[29] showed that gingival
wellstudied in the host response to bacterial infection health is affected by the presence of biofilm around
and are expressed by a variety of immune cells. stainless steel crowns. This is supported by Durr
They are characterized by their pleiotropism and et al.,[30] who reported that there is a correlation
pluripotentiality. They play a key role in mediating the between accumulation of dental biofilm and gingivitis
immune response by exerting their biological effects on in teeth restored with steel crowns.

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Kumar, etal.: Evaluation of chemokines in children with dental caries and crowns
Graph 1: Comparison of macrophage inflammatory protein-1
concentrations in Groups 1, 2, 3 and 4
In this study when all groups were compared for GCF
concentration of MIP1 and MIP1, the differences
were statistically significant with P< 0.001. The mean
concentration of MIP1 and MIP1 in GCF was
found to be higher in Groups2 and 3 when compared
to controls. The possible reasons for increase in GCF
levels of MIP1 and MIP1 in Groups2 and 3 could
be because of systemic inflammatory response to
progressive dental caries. Gornowicz et al.[31] reported
that elevated levels of salivary cytokines such as ILs
and TNF were seen in children with dental caries.
Sotwiska and Zaleska[32] reported that a significant
correlation exists between salivary IL1 concentration
and S. mutans levels in the oral cavity, denoting the
modulation of cytokine concentration by bacterial
antigens in caries pathogenesis. Ruhl etal.[33] measured
the levels of IL1, IL6, IL8, epidermal growth factor,
nerve growth factor, and albumin in salivary glands and
whole saliva. They stated that IL1, IL6, and IL8 were
present in whole saliva at concentrations significantly
higher than in major salivary gland secretions and
concluded that the inflammatory cytokines detected in
whole saliva did not come only from the secretions of
major salivary glands but also from GCF, which was the
likely source of these cytokines.[33] This was supported
by a study done by Wozniak et al., who stated that
whole saliva contains GCF from all periodontal sites
providing an assessment of periodontal disease status.[34]
Miller etal. reported that there is a positive relationship
between clinical parameters of periodontal disease
and the levels of IL1, matrix metalloproteinase8
and osteoprotegerin in whole saliva.[35] Zehnder etal.[36]
analyzed the transcript levels of cytokines in carious
and healthy pulps and their findings indicated that
statistically significant levels of ILs were present in
pulps of teeth with caries.
The results of this study are contrary to Emingil etal.[8]
and Fokkema etal.[37] reported that there is no significant
difference found in MIP1 and MIP1 levels in GCF
samples collected from subjects with periodontitis,
gingivitis, and sound periodontal health. Hence, they
explained that the low MIP1 levels in periodontitis
group could be because of the lack of macrophages as well
Journal of Indian Society of Pedodontics and Preventive Dentistry | Jul-Sep 2016 | Vol 34 | Issue 3 |
Graph 2: Comparison of macrophage inflammatory protein-1
concentrations in Groups 1, 2, 3 and 4
as subsets lymphocytes with specific receptors for MIP1.
Gemmell et al.,[38] who demonstrated that significant
correlation existed between the levels of MIP1 and
degree of inflammation. Antigens within the root canal
are also capable of stimulating a systemic antibody
response. This was first demonstrated by Barnes and
Langeland[39] who showed that there is marked systemic
antibodies against both antigens with the introduction
of bovine serum albumin and sheep erythrocytes into
the root canals of monkeys. Dahln et al.,[40] confirmed
systemic antibody responses to LPS and other bacterial
antigens. Taken together, these observations suggest that
both locally and systemically produced antibodies may
help to protect the periapex against bacterial invasion,
through opsonization or complementmediated lysis.
Conclusions
A better understanding of the molecular mediation of
dental tissue inflammation will ultimately facilitate
improved future diagnosis and treatment. GCF could
be used as a noninvasive diagnostic fluid to measure
biomarkers released during disease initiation and
progression. Molecular diagnosis helps understand
the molecular mechanisms underlying the disease and
plays a pivotal role in the early detection, delivery of
safe and effective therapy for many diseases in the
future. Thus, by determining the disease risk at an
early stage in subjects with dental caries and stainless
steel crowns, preventive measures can be advised and
so the progression and spread of disease are controlled.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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Journal of Indian Society of Pedodontics and Preventive Dentistry | Jul-Sep 2016 | Vol 34 | Issue 3 | 279