Download DR.GOWRI PRIYADARSHINI POSTGRADUATE STUDENT

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
ANNEXURE – II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION
1
NAME AND ADDRESS OF THE
DR.GOWRI PRIYADARSHINI
CANDIDATE (in Block letters)
POSTGRADUATE STUDENT,
DEPARTMENT OF PERIODONTICS,
V.S.DENTALCOLLEGE AND HOSPITAL,
K. R. ROAD, V. V. PURAM,
BANGALORE – 560 004.
2
NAME OF THE INSTITUITION
V.S.DENTALCOLLEGE AND HOSPITAL, BANGALORE
3
COURSE OF STUDY AND
MASTER OF DENTAL SURGERY,
SUBJECT
PERIODONTICS.
DATE OF ADMISSION TO THE
22/05/2010
4
COURSE
5
TITLE OF THE TOPIC
COMPARISON OF GINIGIVAL CREVICULAR FLUID
ALPHA DEFENSIN LEVELS IN HEALTH,
CONTROLLED AND UNCONTROLLED TYPE II
DIABETES MELLITUS WITH CHRONIC
PERIODONTITIS
6
BRIEF RESUME OF THE INTENDED WORK –
6.1 Need for the study–Periodontitis is a multifactorial disease of supporting structures of teeth that
involves microbial dental plaque,genetic and environmental factors. Innate immune response has a
primary role in defense against plaque associated bacteria which is primarily mediated by neutrophils
which are the first line of defense. Neutrophils control bacterial growth by both oxidative and nonoxidative killing mechanisms, but in anaerobic environment as in periodontal pockets, neutrophils
play an important role through non-oxidative microbicidal compounds, such as human neutrophil
peptides[HNP1-3]stored in their azurophillic granules.
Antimicrobial peptides are cationic peptides that are important contributors for maintaining the
balance between periodontal health and disease. Several studies have shown the variations in the
levels of GCF HNP1-3 in periodontal disease and health.
Studies have also shown defective PMN’s functions like chemotaxis, uncontrolled phagocytosis &
killing in diabetic patients. In the present study we would like toevaluate GCF HNP1-3 levels in
controlled and uncontrolled type-2 diabetes patients as there is a paucity of knowledge in this area.
Aim of the study : To investigate the level of HNP1-3(α defensins) in GCF of chronic periodontitis
patients with controlled and uncontrolled diabetes mellitus-type2 and to compare it with the levels
found in systemically healthy patients.
6.2 Review of literature:
 A study was conducted to evaluate the levels of antimicrobial peptides (α and β defensins)
in saliva of patients with oral candidiasis.It was found that oral candidiasis is associated with
gland hypofunction and that decreases of salivary antibacterial proteins induce salivary
Candida overgrowth[1]

During periodontitis, an innate immune response to bacterial challenge is primarily mediated
by neutrophils. A study was conducted to compare neutrophilic content and the level of
neutrophil-derived antimicrobial peptides in gingival crevicular fluid (GCF) in two clinical
forms of severe periodontitis. The amount of HNP1–3 was twofold and fourfold higher in
patients with aggressive and chronic periodontitis, respectively. Those with chronic disease
had significantly elevated amounts of maturecathelicidinLL-37.The increased concentration of
both peptides in chronic periodontitis correlated with the load of Porphyromonasgingivalis,
Tannerella forsythia, and Treponemadenticola.[2]

In a study to investigate the levels of gingival crevicular fluid (GCF) adrenomedullin and
human neutrophil peptides 1 through 3 (HNP1–3) in patients with different periodontal
diseases [patients with chronic periodontitis, generalized aggressive periodontitis, gingivitis,
and healthy subjects, were included in the study] demonstrated that the periodontitis groups
had a significantly higher total amount of GCF adrenomedullin compared to the gingivitis and
healthy control groups. Additionally, GCF adrenomedullin levels were positively correlated
with clinical periodontal parameters of sampling sites. The total amount of GCF HNP1–3 was
not different among the study groups, and there was no correlation between the total amount
of GCF HNP1–3 and clinical periodontal parameters.[3]

In a study conducted to evaluate neutrophil functions in diabetes mellitus patients clearly
demonstrated consistent defects of neutrophil chemotactic, phagocytic and microbicidal
activities along with production of reactive oxygen species and reduced release of cytokines
and prostaglandin by neutrophils[4]

A study was conducted to evaluate polymorphonuclear neutrophil (PMN) cell performance in
61 diabetic patients free of infection , using tests that explore all the functional steps of PMN:
(1) adherence: expression of adhesion molecules, CD 11a, CD 11b, CD 11c; nylon fiber
adherence test; (2) chemotaxis under agarose towards the bacterial oligopeptidefmlp and
complement fractions, used as attracting agents; (3) phagocytosis of opsonized latex
microbeads; (4) bactericidal activity: Results were analysed according to potentially influential
factors: metabolic control (HbA1C, glycaemia), age of patient, type of diabetes, disease
duration, and existence of vascular complications. All steps of PMN functioning like
chemotaxis , adherence, and bactericidal activity were altered in diabetic patients. The type of
diabetes and disease duration did not affect the responses[5]
6.3 Objectives of the study
The objectives of the present study are
1) To compare alpha defensins [HNP 1-3] levels in GCF of clinically healthy indiviuals and
type-II diabetic patients with chronic periodontitis.
2) To evaluate quantitatively, alpha defensins [HNP 1-3] in the gingival crevicular fluid of
individuals with controlled diabetes & uncontrolled diabetes having chronic periodontitis
7. MATERIALS AND METHODS
7.1 Source of the data
The study will be conducted on both male & female patients who are in the age range of 35-60years
reporting to the Department of Periodontics , V.S.Dental college & Hospital.
7.2Method of collection of data
Gingival Crevicular Fluid (GCF) samples will be obtained from 20 healthy controls (C), 20 patients
with controlled diabetics (Do) and 20 patients with uncontrolled diabetics (Dx). All the indiviuals
included in the study should be affected with chronic periodontitis.It will be made clear to all the
potential subjects that participation will be voluntary. Verbal & written informed consent will be
obtained from those who agree to participate.
7.2.2 Inclusion criteria
1) Control Group (Group C)
n = 20
Patients of either sex who clinically healthy but with generalized chronic periodontitis who
haveatleastfour non adjacent teeth with, with CAL≥ 5mm and PD≥ 6mm. BOP should be>50% for
whole mouth in all patients.Clinical findings should commensurate with the amount of local factors
on teeth.
2) Controlled diabetics(Group Do)
n=20
Patients of either sex who are diabetic (type2) but with glycated hemoglobin level less than 7% and
who have generalized chronic periodontitis with atleast four non adjacent teeth with CAL≥ 5mm
and PD≥ 6mm. BOP should be>50% for whole mouth in all patients. Clinical findings should
commensurate with the amount of local factors on teeth.
3)Uncontrolled diabetics(Group Dx)
n = 20
Patients who are diabetic (type2) but with glycated hemoglobin level more than 7% and who have
generalized chronic periodontitis with atleast four non adjacent teeth with CAL≥ 5mm and PD≥
6mm. BOP should be>50% for whole mouth in all patients. Clinical findings should commensurate
with the amount of local factors on teeth.
4) Clinical Periodontal Parameter :
The probing depth (PD), clinical attachment level (CAL), plaque index(PI-Silness and Loe)and
bleeding on probing (BOP at six sites per tooth) will be determined.A manual Williams periodontal
probe will be used for PD(mm) and CAL(mm).
7.2.3
Exclusion criteria
 Patients with immunologic disorders , hepatits, HIV infection
 Pregnant or lactating mothers
 Patients taking oral contraceptives.
 Patients who have received antibiotic therapy within previous 3 months.
 Patients who have undergone periodontal treatment in last 6 months.
 Any other systemic diseases.
7.2.4
Duration of study–
1.5 yrs
7.2.5
Study designComparative Cross-Sectional Study
7.2.6
Study material :
GCF will be collected from the site with CAL≥ 5mm and PD≥6mm from the buccal aspects of mesial/
distal surfaces at the interproximal sites of single rooted teeth. First supragingival plaque will be
removed from interproximal surfaces and the surfaces will be gently dried and isolated before by
cotton rolls before sampling
GCFwill be sampled with caliberated micropipette. The tip of the pipette is inserted carefully in to the
crevice at a level of approximately 1mm below the gingival margin. The crevicular fluid sample will
be then transferred into a microcentrifugetube,immediately frozen and kept at -20 degrees until
analyzed.
7.2.7
Study method :
GCF samples will be analyzed for HNP1-3 by enzyme- linked immunoabsorbent assay (ELISA)
obtained from Hycult Biotechnology, Netherlands according to manufacturer’s instructions.
QUANTITATIVE EVALUATION: The levels of HNP1-3 will be calculated based on dilutions and
the results will be expressed as the total amount and concentration in GCF samples. A calculation of
the concentration data will be performed by dividing the amount of each mediator by the volume of
the sample.
Statistical Test : ANOVA
Sampling : Purposive Sampling
7.3
7.3 Does the study require any investigation or interventions to be conducted on
Patients?
-Yes1)Gingival Crevicular Fluid (GCF) sampling
2)Glycated hemoglobin level
Has et
7.4 Has ethical clearance been obtained from your institution in case of 7.3 ?
YES
8.
1.
LIST OF REFERENCES
ToyohiroTanida ,Tetsuro Okamoto1, Atsuko Okamoto1, Haiyan Wang2, Toshihiro Hamada1,
EisakuUetaTokio Osaki1:Decreased excretion of antimicrobial proteins and peptides in saliva of
patients with oral candidiasis. Journal of Oral Pathology & Medicine2000;32:
586–594
2
M. Puklo, A. Guentsch , P. S. Hiemstra, S. Eick , J. Potemp: Analysis of neutrophil-derived
antimicrobial peptides in gingival crevicular fluid suggests importance of cathelicidin LL-37 in the
innate immune response against periodontogenic bacteria. OralMicrobiology and Immunology Aug
2008 ;23(4):328–335
3
OyaTürkoğlu,GülnurEmingil, NecilKütükçüler,andGülAtilla :Evaluation of Gingival
Crevicular Fluid Adrenomedullin and Human Neutrophil Peptide 1–3 levels of Patients With
Different Periodontal Diseases. J periodontology doi:10.1902/jop.2009.090517
4
Alba-Loureiro TC, Munhoz CD, Martins JO, Cerchiaro GA, Scavone C, Curi R, :
Neutrophil function and metabolism in individuals with diabetes mellitus. Braz J Med Biol Res. Aug
2007;40(8):1037-44.
5
M. Delamaire, D. Maugendre, M. Moreno,M-C. Le Goff ,Allannic ,B. Genetet: Impaired leucocyte
functions in Diabetic Patients. Diabetic MedicineJan1997;14:29–34
DEPARTMENT OF PERIODONTICS
V.S DENTAL COLLEGE AND HOSPITAL, BANGALORE.
CONSENT FORM
I ……………………………………. son/daughter of ……………………………………… aged……………..
resident of …………………………………………………………………. do hereby give consent to the
performance of clinical examination of gingiva, testing of gingival crevicular fluid and evaluation of blood sugar
levels. The procedure has been explained to me in my own language and complications arising, with it if any, I
agree that no responsibility will be attached to the surgeon of hospital staff.
Signature of Patient/Parent:
Signature of Witness:
Place:
Date:
Signature of Researcher:
CASE HISTORY PROFORMA
NAME:
O.P NO:
AGE/SEX:
DATE:
ADDRESS:
OCCUPATION:
CHIEF COMPLAINT:
HISTORY OF PRESENT ILLNESS:
MEDICAL HISTORY: