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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA.
ANNEXURE – II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1.
NAME OF THE CANDIDATE AND
ADDRESS
DR. SURABHI GIGRAS,
POST GRADUATE STUDENT,
DEPARTMENT OF PERIODONTICS,
K.L.E. SOCIETY’S INSTITUTE OF DENTAL
SCIENCES,
BANGALORE – 560022.
2. NAME OF THE INSTITUTION
K.L.E. SOCIETY’S INSTITUTE OF DENTAL
SCIENCES, BANGALORE – 560022
3. COURSE OF THE STUDY AND
SUBJECT
MASTER OF DENTAL SURGERY (MDS) IN
PERIODONTICS.
4. DATE OF ADMISSION TO
COURSE
29/05/2013
5. TITLE OF THE DISSERTATION
GINGIVAL
CREVICULAR
FLUID
LEVELS OF TRIGGERING RECEPTOR
EXPRESSED ON MYELOID CELLS,
(TREM)-1 IN GINGIVITIS AND CHRONIC
PERIODONTITIS BEFORE AND AFTER
PHASE –I THERAPY- A CASE-CONTROL
STUDY.
,
BRIEF RESUME OF THE INTENDED WORK:
6.1 NEED FOR THE STUDY:
Periodontal diseases are the most common infections in humans caused by complex
polymicrobial biofilms attaching on the tooth surface and causing inflammation of the
periodontal tissues.1 Gingivitis and chronic periodontitis are the two most common forms
of periodontal disease. Gingivitis is a reversible non-destructive periodontal disease that
does not involve the loss of periodontal tissues. Chronic periodontitis is characterized by
impaired immune responses resulting in connective tissue and alveolar bone destruction.
An intermediate mechanism that lies between bacterial stimulation and tissue destruction
is the production of pro-inflammatory cytokines which stimulate the inflammatory
events.2
Triggering receptor expressed on myeloid cells (TREM)-1 is a cell surface receptor of the
immunoglobulin superfamily, which is constitutively expressed on the surface of human
polymorphonuclear neutrophils and monocytes.3 This, along with its adaptor signalling
molecule DAP12, is activated upon bacterial recognition by host cells via toll like
receptors, triggering a number of intracellular signalling events that result in enhanced
pro-inflammatory cytokine production.1 Bacterial or fungal infections can cause upregulation of membrane bound TREM-1, as well as release its soluble form, rendering it
a useful early inflammatory biomarker for systemic infection.1 Since TREM-1 amplifies
the inflammatory response induced by pathogenic bacteria, it may serve as a useful
marker for detecting the progression of periodontal lesions.3
Gingival crevicular fluid (GCF) is a transudate originating from the gingival plexus of
blood vessels in the gingival connective tissue, close to the epithelial lining of the
dento-gingival sulcus.4 It contains an array of measurable inflammatory mediators, which
may result from interaction between the host and microbial challenge. Host response
mediators in GCF are indicators of the processes occurring in the gingival crevice and
adjoining host tissue and may be useful as early risk markers of the disease.5 GCF has
been therefore regarded as a promising medium for the detection of periodontal disease
activity.
The purpose of this study is to estimate GCF levels of TREM-1 in periodontally healthy,
gingivitis and chronic periodontitis subjects and evaluate the effect of scaling and root
planing (SRP) on the same in periodontal disease.
6.2 REVIEW OF LITERATURE:
A study was done to investigate the effect of Porphyromonas gingivalis on the expression
of TREM-1/DAP12 pathway, as well as its engagement in pro-inflammatory cytokine
production. The results showed that Porphyromonas gingivalis enhanced TREM-1 gene
expression by the cells, concomitantly to an increase in soluble TREM-1 (sTREM)-1
secretion.
TREM-1
also
potentiated
pro-inflammatory
cytokine
responses
to
Porphyromonas gingivalis as shown by an enhanced IL-1β and IL-6 secretion.6
A Case control study was done to evaluate the presence of TREM-1 in GCF of patients
with severe periodontitis. The results showed that sTREM-1 was detected in crevicular
fluid and its concentration was higher in pathological sites. They concluded that
sTREM-1 could be a marker of periodontal tissue destruction.3
A study was done to investigate the effect of subantimicrobial doses of doxycycline
(SDD) on Porphyromonas gingivalis induced TREM-1 expression and secretion by the
myelomonocytic cell line MonoMac-6. After 24 hours of challenge, Porphyromonas
gingivalis enhanced TREM-1 gene expression by the cells, with a concomitant increase
in sTREM-1 release. It was concluded that SDD inhibits bacterially induced TREM-1
and this effect may partly account for its generalized anti-inflammatory
properties.7
A cross-sectional study was done to investigate the GCF sTREM-1 levels in chronic
periodontitis (CP) or generalized aggressive periodontitis (G-AgP), and their association
with the levels of key periodontal pathogens in subgingival plaque. Soluble TREM-1
levels in GCF were found to be higher in CP and G-Agp than healthy sites. Also
sTREM-1 levels were positively correlated with the clinical and microbiological
parameters.8
An in vitro study was conducted to investigate the effect of Porphyromonas gingivalis on
TREM-1 expression and production by primary human Polymorphonuclear neutrophils,
and also to evaluate the role of the gingipains in this process. The results showed that
after 4 hours of challenge, Porphyromonas gingivalis enhanced TREM-1 expression as
identified by quantitative real time polymerase chain reaction (PCR). It was concluded
that the differential regulation of TREM-1 by gingipains may present a novel mechanism
by which Porphyromonas gingivalis manipulates the host innate immune response
helping to establish chronic periodontal inflammation.1
A study was conducted to estimate the sTREM-1 concentration in saliva and serum of
patients with chronic or generalized aggressive periodontitis. The results showed that the
sTREM-1 concentration, both in saliva and serum was found to be higher in the
periodontitis group than the control group. It was concluded that sTREM-1 may serve as
a biomarker for periodontitis and may have implications in the association between
periodontal infections and systemic inflammatory response.9
NULL HYPOTHESIS:
GCF levels of TREM-1 has no value as a biomarker in predicting the progression of
periodontal diseases and its response to phase –I therapy.
RESEARCH HYPOTHESIS:
GCF levels of TREM-1 has a significant value as a biomarker in predicting the
progression of periodontal disease and its response to phase –I therapy.
6.3 OBJECTIVES OF THE STUDY:
1. To estimate GCF levels of TREM-1 in periodontal health, Gingivitis and Chronic
periodontitis.
2. To compare GCF levels of TREM-1 in different stages of periodontal disease.
3. To compare GCF levels of TREM-1 before and after scaling and root planing (SRP)
in Gingivitis and Chronic periodontitis.
4. To correlate GCF levels of TREM-1 with Clinical parameters in periodontal health
and disease.
7. MATERIALS AND METHODS:
7.1 Source of data:
The study population will consist of 45 subjects diagnosed as periodontally healthy,
gingivitis or Chronic periodontitis with 15 subjects in each category, belonging to both
genders. All the subjects will be selected from the out-patient division of the department
of Periodontics, K.L.E Society’s institute of Dental Sciences, Bangalore, Karnataka,
India. A written informed consent (in the local language that can be easily understood by
the subject) will be obtained from those who agree to participate voluntarily in the study.
7.2 Method of collection of data:
Study design: An in vivo study.
Sample size: A total of 45 subjects assigned into 3 groups – One control group and
two test groups:
Group I (Control group): 15 subjects with clinically healthy periodontium.
Group II (Test group 1): 15 subjects with gingivitis.
Group III (Test group II): 15 subjects with chronic periodontitis.
Inclusion criteria:

Systemically healthy subjects, of both the gender in the age group of 20-50 years
with at least 20 teeth in the mouth.

Subjects who are willing to participate in the study, cooperative and able to attend
follow up.

Group I(Control group):
 Absence of clinical signs of gingival inflammation (GI= 0).
 Probing depth ≤ 3mm.
 Absence of loss of clinical attachment.

Group II(Test group I):
 Presence of clinical signs of gingival inflammation (GI >1).
 Probing depth ≤ 3mm.
 Absence of loss of clinical attachment.

Group III(Test group II):
 Presence of a minimum of three periodontal pockets with probing pocket
depth of ≥ 5mm along with clinical signs of gingival inflammation.
 Clinical attachment loss of ≥ 3mm.
 Radiographic evidence of alveolar bone loss. (No delineation will be done
within chronic periodontitis based on the extent of alveolar bone loss).
Exclusion criteria:

Subjects with any systemic diseases like diabetes, hypertension, heart disease and
rheumatoid arthritis that can alter the course of periodontal disease.

Subjects
having
taken
antibiotic
therapy/anti-inflammatory
drugs/immunosuppressive drugs in the past 6 months.

Subjects who have undergone any form of periodontal therapy in past 6 month

Pregnant and lactating mothers.

Smokers and subjects using smokeless tobacco.
GCF Sampling:
The study involves collecting gingival crevicular fluid from all the three groups at
baseline, and at 8 weeks following thorough Scaling and Root planing (SRP) in groups II
and III to measure TREM-1 levels.10
The GCF samples will be collected using commercially available 1-5µl calibrated
micropipettes. A total of 75 GCF samples will be collected during the entire study period.
Site Selection:11
Group I: Multiple sites (three to five sites/subject) to ensure collection of an adequate
amount of GCF.
Group II: The site with the greatest clinical signs of gingival inflammation in the
absence of clinical attachment loss.
Group III: The site with the greatest clinical signs of gingival inflammation, highest
probing depth and clinical attachment loss along with radiographic evidence of alveolar
bone loss.
Clinical parameters:
The following clinical parameters will be recorded at baseline for all the three groups and
at follow up visits scheduled at 8 weeks, following SRP in groups II and III:10

Plaque index (Silness and Loe, 1964 ).

Gingival index (Loe and Silness, 1963).

Probing pocket depth (PD).

Clinical attachment level (CAL).
Periodontal probing depth (PD) and Clinical attachment level will be recorded using a
constant pressure probe (Brock probe TM).
Orthopantomogram (OPG) for every patient, showing clinical signs of attachment loss
will be taken at baseline. Radiographic bone loss will be recorded dichotomously to
differentiate subjects with chronic periodontitis from other groups.11
Study Method:
After obtaining informed consent, clinical parameters of each subject will be recorded
and OPG of every subject showing clinical signs of attachment loss will be taken.
Following this, GCF sampling will be done for all the three groups. After drying the test
site gently with air, supragingival plaque will be removed with a sterile curette without
touching the marginal gingiva and isolated with cotton rolls to avoid saliva
contamination. GCF will be collected by placing a microcapillary pipette at the entrance
of the gingival sulcus, gently touching the marginal gingiva .A standard volume of 1µl
will be collected with an extracrevicular approach (unstimulated) from each test site.
Further, micropipettes that will be suspected of being contaminated with blood or saliva
will be discarded. The collected GCF sample will be transferred to airtight plastic vials
containing 0.5ml of phosphate buffered saline (PBS) and stored at -700 C until assayed.
TREM-1 levels in GCF will be determined by ELISA using specific ELISA kit and
reading will be assessed by ELISA microplate reader at 450nm.
Subjects with gingivitis and chronic periodontitis will be subjected to thorough scaling
and root planing (SRP) and will be recalled after 8 weeks for recording the clinical
parameters and collection of GCF samples.
Statistical Analysis :
Normality assumption of data will be tested using Schapiro-Wilks test.
1. If the data will be normal ,
 Mean comparison between two groups will be done using ANOVA.
 If ANOVA results will be significant (p< 0.05 values will be considered as
significant), further comparison between two groups will be done using
Tukey’s test.
 Intra- group comparison will be done using paired student t test.
2. If the data will not be normal,
 Mean comparison between two groups will be done using the Kruskal-Wallis
test.
 If Kruskal-Wallis test will be significant , further comparison between the
two groups will be done using the Mann-Whitney U test.
 Intra-group comparison will be made using Wilcoxon Signed Rank test.
7.3 Does the study require any investigation or interventions to be conducted on
patients or other humans or animals? If so, please describe briefly?
Yes. The study requires collection of gingival crevicular fluid from patients of all the
three groups. Scaling and root planing will be done in test groups I and II and during the
study, OPG will be taken for every patient showing clinical signs of gingival
inflammation. A signed informed consent will be obtained from the patients before the
same.
7.4 Has ethical clearance been obtained from your institute in case of 7.3?
Yes. A copy of the same has been attached.
8. LIST OF REFERENCES:
1.
Bostanci N, Thurnheer T, Aduse-Opoku J, Curtis MA, Zinkernagels AS,
Belibasakis GN. Porphyromonas gingivalis regulates TREM-1 in human
polymorphonuclear neutrophils via its gingipains. PLoS ONE 2013;8:1-8.
2. Graves
D.
Cytokines
that
promote
periodontal
tissue
destruction.
J Periodontol 2008;79:1585-1591.
3. Bisson C, Massin F, Lefevre PA, Thilly N, Miller N, Gibot S. Increased gingival
crevicular fluid levels of soluble triggering receptor expressed on myeloid cells
(sTREM ) -1 in severe periodontitis. J Clin Periodontol 2012;39:1141-1148.
4. Buduneli N, Kinane DF. Host – derived diagnostic markers related to soft tissue
destruction and bone degradation in periodontitis. J Clin Periodontol 2011;3:85105.
5. Lamster IB. Evaluation of components of gingival crevicular fluid as diagnostic
tests.Ann Periodontol 1997;2:123-137.
6. Bostanci N, Thurnheer T, Belibasakis GN. Involvement of the TREM-1/DAP-12
Pathway in the innate immune responses to porphyromonas gingivalis. Mol
Immunol 2011;49:387-394
7. Bostanci N, Belibasakis GN. Doxycycline inhibits TREM-1 induction by
Porphyromonas gingivalis. FEMS Immunol Med Microbiol 2012;66:37-44.
8. Belibasakis GN, Ozturk VO, Emingil G, Bostanci N. Soluble triggering receptor
expressed on myeloid cells (sTREM ) -1 in gingival crevicular fluid: Association
with clinical and microbiological parameters. J periodontol 2013;84:1-11.
9. Bostanci N, Ozturk VO, Emingil G, Belibasakis GN. Elevated oral and systemic
levels of soluble triggering receptor expressed on myeloid cells -1 in
periodontitis. J Dent Res 2013;92:161-165.
10. Segelnick SL, Weinberg MA. Reevaluation of initial therapy:when is the
appropriate time? J Periodontol 2006;77:1598-1601.
11. Pradeep AR, Happy D, Parag H, Garima G, Manojkumar T. Correlation of
gingival crevicular fluid interleukin-18 and monocyte chemoattractant protein-1
levels in periodontal health and disease. J periodontol 2009;80:1454-1461.
9.
SIGNATURE OF THE CANDIDATE
12. REMARKS OF THE GUIDE
13. NAME & DESIGNATION OF (IN
BLOCK LETTERS)
11.1 GUIDE
Dr. SUDHIR R PATIL,
PROFESSOR AND HOD,
DEPARTMENT OF PERIODONTICS,
K.L.E. SOCIETY’S INSTITUTE OF
DENTAL SCIENCES,
BANGALORE-22.
11.2 SIGNATURE
11.3 CO-GUIDE (IF ANY )
Dr. VEENA HR,
READER,
DEPARTMENT OF PERIODONTICS,
K.L.E. SOCIETY’S INSTITUTE OF
DENTAL SCIENCES,
BANGALORE-22.
11.4 SIGNATURE
11.5 HEAD OF THE
DEPARTMENT
1.6 SIGNATURE
DR.SUDHIR R PATIL,
PROFESSOR & HOD,
DEPARTMENT OF PERIODONTICS,
K.L.E. SOCIETY’S INSTITUTE OF
DENTAL SCIENCES,
BANGALORE-560022.
KARNATAKA.
12
12.1 REMARKS OF THE
CHAIRMAN & PRINCIPAL
12.2 SIGNATURE
K.L.E. SOCIETY’S INSTITUTE OF DENTAL SCIENCES,
YESHWANTHPUR SUBURB 2AND STAGE,BANGALORE- 560022
DEPARTMENT OF PERIODONTICS
STUDY TITLE
GINGIVAL
EXPRESSED
CREVICULAR
ON
FLUID
MYELOID
LEVELS
CELLS
-1
OF
IN
TRIGGERRING
GINGIVITIS
AND
RECEPTOR
CHRONIC
PERIODONTITIS BEFORE AND AFTER PHASE –I THERAPY- A CASE CONTROL
STUDY.
INFORMED CONSENT
PATIENT’S NAME-
DATE-
AGE/SEX-
OP NO.-
ADDRESS-
CONTACT NO.-
I ...........................................................................,hereby, authorize Dr.SURABHI GIGRAS at
the department of Periodontics, K.L.E Society’s Institute Of Dental Sciences, Bangalore, to
perform examination/investigation/treatment or any other necessary procedure required for
the research purpose titled as above.
I have been explained the risk and benefits of the treatment procedure and neither the dentist
nor the assistants will be held responsible, if any untoward incident happens.
I have no objection to the data collected being used for research purpose entitiled above.
I also agree to cooperate for the further appointments needed for the scheduled follow up of
the procedure being conducted on me.
PATIENT’S SIGNATURE
DATEPLACE-
TREATING DOCTOR’S SIGNATURE-
DATEPLACE-
NAME OF THE GUIDE AND SIGNATURE-
DATEPLACE-