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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
ANNEXURE – II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION
1.
NAME AND ADDRESS OF THE Dr. SIDDHARTH GAUTAM,
POST GRADUATE STUDENT,
CANDIDATE (in Block letters)
DEPARTMENT OF PERIODONTICS,
V.S. DENTAL COLLEGE & HOSPITAL,
K.R. ROAD, V.V. PURAM,
BENGALURU- 560 004
2.
NAME OF THE INSTITUITION
3.
COURSE
SUBJECT
4.
DATE OF ADMISSION TO THE 31st JULY 2013
COURSE
5.
TITLE OF THE TOPIC
OF
STUDY
VOKKALIGARA SANGHA DENTAL
COLLEGE & HOSPITAL, BENGALURU.
AND MASTER OF DENTAL SURGERY,
PERIODONTICS.
COMPARATIVE ASSESSMENT OF
EXPRESSION OF CYCLOOXYGENASE-2
IN GINGIVAL TISSUES AFTER DIODE
LASER POCKET THERAPY.
6. Brief Resume of intended work:
6.1 Need of the study:
A central feature of periodontitis is loss of bone associated with inflammatory
mediators. Recent data have shown that COX-2 expression is enhanced in inflamed
gingival tissues1, 2, 3 and that COX-2 is responsible for PGE2 production in the cells
stimulated with proinflammatory molecules such as Interleukin-1β & TNF-α.4
Therefore, expression of COX-2 in gingival tissues can be considered a useful
biomarker of inflammation.1
Laser therapy has been proposed as an alternative or adjunct to conventional non
surgical therapy to arrest progression of disease by eliminating bacterial infection &
reducing soft tissue inflammation.5An in vitro study demonstrated that Ga-Al-As
diode low level laser irradiation has a inhibitory effect on Campylobacter rectus
lipopolysaccharide-induced PGE2 in human gingival fibroblasts through a reduction
of COX-2 mRNA levels.6 In contrast, Er:YAG laser has been shown to increase PGE2
production via the induction of COX-2 mRNA in human gingival fibroblasts.7 Laser
therapy in arthritis patients has shown to reduce inflammation and thereby reduction
in pain.8, 9
Laser therapy may provide therapeutic benefit against aggravation of gingivitis or
periodontitis by modulating the expression of COX-2.The present study aims to
assess the response of gingival tissues to diode laser when used as an adjunct to
scaling & root planning in patients with mild to moderate periodontitis.
6.2 Review of literature:
1) A study evaluated 16 patients with moderate to severe chronic periodontitis (CP)
and 8 healthy volunteers in terms of clinical measures, crevicular fluid and gingival
biopsy specimens. IL-1β levels were found to be more in crevicular fluid and COX-2
mRNA protein level was elevated in gingival tissues. These results suggested that
COX-2 is increased at sites of chronic periodontitis and that its measure can be a
useful biomarker of disease severity.1
2) In a study 32 gingival biopsies were taken during routine oral surgical procedures
and were processed histologically to determine degree of inflammation. To explore
mechanism of COX-2 up regulation, gingival connective tissue primary cell culture
were established and challenged with periodontal bacteria or proinflammatory
cytokines in vitro. COX-2 activity was assessed by quantifying PGE2 levels in culture
supernatants by competitive EIA. The results showed that COX-2 expression was
significantly higher in inflamed tissues.2
3)A cross sectional and analytical study was conducted in 108 gingival biopsies from
52 patients with chronic periodontitis (CP), 39 with gingivitis (GV) and 17 controls.
All biopsies were processed for histopathologic examination and
immunohistochemical determination of COX-2 expression. Results showed that
COX-2 expression was higher in patients with gingivitis and chronic periodontitis
than in individuals without periodontal disease.3
4) In a review article, the roles of COX-2 and PGE2 in periodontal disease were
discussed. They concluded that COX-2 plays a crucial role in prostaglandin
production in periodontal disease. Also COX-2 inhibitors may be effective for host
modulatory therapy.4
5) In a study, 50 patients were randomly subdivided into two groups (laser group and
control group). The laser group received diode laser therapy after SRP whereas
control group received SRP with H2O2. The microbiologic samples were collected at
baseline and after 6 months. The results showed that there was significant reduction in
bacterial load with diode laser therapy.5
6) In an in vitro study, human gingival fibroblasts (hGF) were challenged with lipopolysaccharide (LPS) and then Ga-Al-As diode laser was irradiated to hGF cells. The
PGE2 levels were measured by radioimmunoassay (RIA) and COX-2 mRNA level by
RT-PCR. Both showed decrease in their level. These findings suggest that low level
laser irradiation has inhibitory effect on prostaglandin E2 production that could be of
therapeutic benefit against the aggravation of gingivitis and periodontitis by bacterial
infection.6
7) An in vitro study on cultured fibroblasts which were irradiated with low power
Er:YAG laser irradiation. The amount of PGE2 production was measured by ELISA
and COX-2 mRNA level was analyzed by RT-PCR. It showed that Er:YAG laser
irradiation strongly stimulated PGE2 production due to enhanced COX-2 mRNA
expression in human gingival fibroblast cells.7
8) A placebo controlled study of 19 patients with rheumatoid arthritis with carpel
tunnel syndrome who received low level laser therapy (Ga-Al-As diode laser)
concluded that it was effective in pain relief and improved hand function.8
9) A review study (34 cell studies, 54 animal studies and 106 skin incisions) reported
that low level laser therapy (between 633nm -904nm) reduces inflammation
significantly and is equally effective as NSAIDs in reducing pain.9
10) A study compared 980nm diode laser (as adjunctive therapy to SRP) with scaling
and root planning alone in 13 patients. Clinical measurements (PPD, CAL, BOP, GI,
PI) were performed at baseline and after 4th, 8th and 12th weeks and 6 months.
Subgingival plaque samples were taken at baseline and after treatment and were
examined using PCR technique. It showed that the additional treatment with diode
laser may lead to slight improvement of clinical parameters whereas no significant
reduction of periodontopathogens were found.10
6.3 Objectives of the study:
1. To assess & compare the expression of cyclooxygenase-2 in gingival tissue at
baseline & 6 months after laser as an adjunct to SRP& SRP alone.
2. To assess & compare clinical parameters like pocket probing depth (PPD), bleeding
on probing(BOP), clinical attachment level(CAL), plaque index (PI) and gingival
index(GI) at baseline & 3 & 6 months post therapy.
3. To correlate the clinical parameters with expression of cyclooxygenase-2 at various
time intervals
7. MATERIALS AND METHODS:
7.1 Source of Data:
The study will be conducted on the patients reporting to the Department of
Periodontics, VokkaligaraSangha Dental College, Bengaluru.
7.2.1 Method of collection of Data:
Forty patients fulfilling the inclusion criteria will be included in the study. It will be
made clear to all potential subjects that participation will be voluntary and written
informed consent will be obtained from those who agree to participate.
7.2.2 Inclusion Criteria:
- Age in between 18-50 years.
- Systemically healthy (with special regard to disease affecting tissue repair, for
example, Diabetes Mellitus Type II).
- No medications such as NSAIDs and antibiotics in the preceding month.
- No periodontal therapy in the preceding 6 months.
- Non-Smoker
- At least 10-12 teeth per arch.
- Pocket probing depth 5-7mm.
- Co-operative Patients.
7.2.3 Exclusion Criteria:
- Grade III mobile teeth.
- Pregnant or lactating women.
- Patients with immunologic diseases.
7.2.4 Duration of study: 1.5 years.
7.2.5 Study Design: An in vivo comparative parallel design
40 subjects meeting the selection criteria will be randomly allocated to test or control
group of 20 each. The test subjects will be treated with laser as an adjunct to scaling
and root planning. The control subjects will receive scaling and root planning only.
7.2.5 Study Method:
Control Group: SRP will be accomplished using a combination of ultrasonics &
standard Gracey curettes until a hard, smooth calculus free surface is obtained.
Test Group: Ga-Al-As diode laser (AMD Lasers; PICASSO Model) 810nm at a
power output of 2.5 W in pulsed mode (30 Hz, pulse duration 30ms) will be used as
an adjunct to SRP. The optic fiber of 400μm will be used for pocket debridement.
Biopsy Collection: Gingival tissue (1-1.5mm x 2-3mm) will be harvested from the site
with maximum bone loss (mesial or distal or buccal or lingual) pre treatment and 6
weeks post treatment under local anesthesia.The samples will be placed in 10%
buffered formalin solution.
IMMUNOHISTOCHEMISTRY ANALYSIS:
Sample tissues will be subjected to immunohistochemical analysis using anti-COX-2
rabbit monoclonal antibody diluted 1:50 to identify cell expression.
STATISTICAL ANALYSIS: Paired and unpaired student “t” test will be used.
Pearson correlation coefficient test will be done to correlate between clinical and
histopathological parameters.
7.3 Does the study require any investigation or interventions to be conducted on
patients?
YES
7.4
Has ethical clearance been obtained from your institution in case of 7.3?
YES OBTAINED.
8. LIST OF REFERENCES:
1. Zhang F, Engebretson SP, Morton RS, Cavanaugh PF Jr, Subbaramaiah K,
Dannerberg AJ. The overexpression of cyclo-oxygenase-2 in chronic periodontitis.
JADA, Vol.134, July 2003: pg 861-867.
2. Morton RS and Dongari-Bagtzoglou AI. Cyclooxygenase-2 is upregulated in
inflamed gingival tissues. J Periodontol, Vol. 72, number 4, April 2001: pg 461-469.
3. Mesa F, Aguilar M, Galindo-Moreno P, Bravo M and O’Valle F. Cyclooxygenase
– 2 expression in gingival biopsies from periodontal patients is correlated with
connective tissue loss. J Periodontol, Vol 83, number 12, December 2012: pg 15381545.
4. Noguchi K and Ishikawa I. The role of cyclooxygenase-2 and prostaglandin E2 in
periodontal disease.Periodontology 2000, Vol. 43, 2007: pg: 85-101.
5. Moritz A, Schoop U, Goharkhay K, Schauer P, Doertbudak O, Wernisch J and
Sperr W. Treatment of periodontal pockets with a diode laser.Lasers Surg. Med. 22:
1998:pg 302-311.
6. Sakurai Y, Yamaguchi M and Abiko Y. Inhibitory effect of low level laser
irradiation on LPS-stimulated prostaglandin E2 production and cyclo-oxygenase-2 in
human gingival fibroblasts. Eur J Oral Sci2000; 108: pg 29-34.
7. Pourzarandian A, Watanabe H, Ruwanpura SMPM, Aoki A, Noguchi k, Ishikawa I.
Er:YAG laser irradiation increases prostaglandin E2 production via the induction of
cyclooxygenase-2 mRNA in human gingival fibroblasts. J Periodont Res 2005; 40; pg
182-186.
8. Ekim A, Armagan O, Tascioglu F, Oner C, Colak M. Effect of low level laser
therapy in rheumatoid arthritis patients with carpel tunnel syndrome. Swiss Med
Wkly 2007;137: pg 347-352.
9. Bjordal JM, Martins RABL, Joenson J and Iversen VV.The anti-inflammatory
mechanism of low level laser therapy and its relevance for clinical use in
physiotherapy. Physical Therapy Reviews 2010: vol 15, no. 4. pg 286-293.
10. Carsuo U, Nastri L, Piccolomini R, D’Ercole S, Mazza C, Guida L. Use of diode
laser 980nm as adjunctive therapy in the treatment of chronic periodontitits. A
randomized controlled clinical trial. New Microbiologica, 31, 2008: pg 513-518.
ANNEXURES:
V. S. DENTAL COLLEGE AND HOSPITAL, BENGALURU.
CONSENT FORM
STUDY TITLE: COMPARATIVE ASSESSMENT OF EXPRESSION OF
CYCLOOXYGENASE-2 IN GINGIVAL TISSUES AFTER DIODE LASER
POCKET THERAPY.
Conducted By:
Dr. SiddharthGautam
Post Gradyate Student
I………………………………………………………………………son/daughter/wife
of…………………………………………………………aged………………..resident
of…………………………………………………… do hereby give consent to
perform the clinical and radiographical examinations and recommended minor
surgical procedures or treatment. The procedure has been explained to me in my own
language. I hereby give consent to take small piece of gingival (gum) tissue for the
examination.
Any complications arising with it, if any, I agree that no responsibility will be
attached to the surgeon or hospital staff.
Signature of Patient/Parent:Signature of Witness:
Signature of Researcher:
Place:
Date:
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CRITERIA FOR INDICES
PLAQUE INDEX (SILNESS P & LOE H, 1964)
Method: The evaluation or scoring is done on the entire dentition or on selected teeth.
Only plaque of cervical third of the tooth is evaluated. The surfaces examined are the four
gingival areas of the tooth i.e., the distofacial, facial, mesiofacial& lingual surfaces. The
mouth mirror, a light source, a dental explorer and air drying of the teeth and gingiva are
used in the scoring of this index.
SCORING CRITERIA:
Score
Criteria
0
No plaque
1
A film of plaque adhering to the free gingival margin and adjacent area
of the tooth. The plaque may be seen in situ only after application of
disclosing solution or by using the probe on the tooth surface.
2
Moderate accumulation of soft deposits within the gingival pocket, or
the tooth and the gingival margin which can be seen with the naked
eye.
3
Abundance of soft matter within the gingival pocket and / or on the
tooth and gingival margin.
𝐒𝐮𝐦𝐨𝐟𝐬𝐜𝐨𝐫𝐞𝐬𝐨𝐟𝐚𝐥𝐥𝐬𝐮𝐫𝐟𝐚𝐜𝐞𝐬
Plaque score =
𝐍𝐨.𝐨𝐟𝐒𝐮𝐫𝐟𝐚𝐜𝐞𝐬𝐞𝐱𝐚𝐦𝐢𝐧𝐞𝐝
Suggested nominal scale for patient evaluation:
Rating
Excellent
Scores
0
Good
0.1 – 0.9
Fair
1.0 – 1.9
Poor
2.0 – 3.0
GINGIVAL INDEX (GI) (LOE H AND SILNESS P, 1963)
Method: The severity of gingivitis is scored on all teeth. To obtain GI, the tissues
surrounding each tooth are divided into four gingival scoring units: distal-facial papilla,
facial margin, mesial-facial papilla & entire lingual gingival margin. A blunt instrument
such as periodontal probe is used to assess the bleeding potential of the tissues.
SCORING CRITERIA:
Score
Criteria
0
Absence of inflammation / normal gingiva
1
Mild inflammation, slight change in colour, slight edema; no bleeding on
probing
2
Moderate inflammation; moderate glazing, redness, edema & hypertrophy.
Bleeding on probing
3
Severe inflammation; marked redness & hypertrophy ulceration. Tendency
to spontaneous bleeding.
Calculation:
Gingival index score for the area = sum of scores around each tooth
sum of scores around each tooth
Gingival index score for the tooth =
𝟒
sum of scores around each tooth
Gingival index score per person =
No. of teeth examined
The numerical scores of gingival index may be associated with varying degrees of
clinical gingivitis as follows:
GINGIVAL SCORES CONDITION
0.1-1.0
Mild Gingivitis
1.1-2.0
Moderate Gingivitis
2.1-3.0
Severe Gingivitis
CASE RECORD PROFORMA
Name of the patient:
Date:
Age / Sex:
O.P. No. :
Address & Ph. No.:
Occupation:
Chief Complaint:
History of Present Illness:
Past Dental History:
Medical History:
Family History:
Habits:
Clinical Examination:
Oral examination:
PLAQUE INDEX (SILNESS P & LOE H, 1964):
Scoring:
18
17
16
15
14
13
12
11
21
22
23
24
25
26
48
47
46
45
44
43
42
41
31
32
33
34
35
36
37
38
Plaque score =
27
28
𝐒𝐮𝐦𝐨𝐟𝐬𝐜𝐨𝐫𝐞𝐬𝐨𝐟𝐚𝐥𝐥𝐬𝐮𝐫𝐟𝐚𝐜𝐞𝐬
𝐍𝐨.𝐨𝐟𝐒𝐮𝐫𝐟𝐚𝐜𝐞𝐬𝐞𝐱𝐚𝐦𝐢𝐧𝐞𝐝
Rating:
GINGIVAL INDEX (LOE H AND SILNESS P, 1963):
Scoring:
18
17
16
15
14
13
12
11
21
22
23
24
25
26
27
28
48
47
46
45
44
43
42
41
31
32
33
34
35
36
37
38
Gingival Index score =
𝐒𝐮𝐦𝐨𝐟𝐬𝐜𝐨𝐫𝐞𝐬𝐨𝐟𝐚𝐥𝐥𝐬𝐮𝐫𝐟𝐚𝐜𝐞𝐬
𝐍𝐨.𝐨𝐟𝐦𝐚𝐫𝐠𝐢𝐧𝐬𝐞𝐱𝐚𝐦𝐢𝐧𝐞𝐝
Rating:
BLEEDING ON PROBING:
Present or Absent.
PERIODONTAL POCKET DEPTH:
B
P
18
17
16
15
14
13
12
11
21
22
23
24
25
26
27
28
48
47
46
45
44
43
42
41
31
32
33
34
35
36
37
38
L
B
CLINICAL ATTACHMENT LEVEL:
B
P
18
17
16
15
14
13
12
11
21
22
23
24
25
26
27
28
48
47
46
45
44
43
42
41
31
32
33
34
35
36
37
38
L
B
DIAGNOSIS: