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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
DR. RAKESH.M.P
AND ADDRESS
POST GRADUATE STUDENT
(IN BLOCK LETTERS)
DEPARTMENT OF PERIODONTOLOGY
RAJARAJESWARI DENTAL COLLEGE
AND HOSPITAL, MYSORE ROAD,
BANGALORE – 560060
2
NAME
OF
INSTITUTION
3
AND HOSPITAL, BANGALORE-560060
COURSE OF STUDY AND MASTER OF DENTAL SURGERY
SUBJECT
4
THE RAJARAJESWARI DENTAL COLLEGE
PERIODONTOLOGY
DATE OF ADMISSION TO 26th MAY 2012
THE COLLEGE
5
TITLE OF THE TOPIC
EFFECT OF DIODE LASER ON GINGIVAL CREVICULAR FLUID
STROMAL DERIVED FACTOR – 1 ALPHA LEVELS IN CHRONIC
PERIODONTITIS.
6
BRIEF RESUME OF THE INTENDED WORK:
6.1
NEED FOR THE STUDY
Periodontal diseases are initiated by Gram-negative tooth associated microbial
biofilms that elicit a host response, with resultant osseous and soft tissue destruction.
Mediators produced as a part of host response that contribute to tissue destruction include
proteinases, cytokines and prostaglandins.1
Stromal derived factor-1 (SDF-1α and β or CXC chemokine ligand 12 [CXCL12]) is
a potent chemoattractant for hematopoietic cells, including neutrophils.
This factor
belongs to the C-X-C chemokine family, which was originally isolated from a murine bone
marrow stromal cell line. SDF-1α is selectively expressed by endothelial cells in certain
tissues, perhaps in response to specific signals or tissue damage. This may provide a
mechanism to localize hematopoietic cells to specific tissue compartments.2
A recent study revealed that GCF from patients with chronic periodontal diseases
have significantly higher levels of SDF-1α than subjects who are clinically healthy. SDF1α may be involved in the immune defense pathway activated during periodontal disease.
Upon development of disease, SDF-1α levels increase and may recruit host defensive cells
into sites of inflammation. This suggests that SDF-1α may be a useful biomarker for the
identification of periodontal disease progression.2
A previous study had revealed the effect of diode laser on several cytokine levels in
periodontitis.3
According to our knowledge, there are no data available regarding the effect of
diode laser on SDF-1α levels. Therefore, this study is aimed at quantitative analysis of
SDF-1α levels in GCF of chronic periodontitis patients before and after diode laser therapy.
6.2 REVIEW OF LITERATURE:

A study was done to determine whether SDF-1α secreted by host cells plays a
role in recruiting inflammatory cells into the periodontia during local
inflammation. SDF-1α levels were determined by enzyme-linked immunosorbent
assay in gingival crevicular fluid (GCF) of 24 individuals with periodontitis versus
healthy individuals in tissue biopsies and in a preclinical rat model of
Porphyromonas gingivalis lipopolysaccharide–induced experimental bone loss.
Results have shown that subjects with periodontal disease had higher levels of
SDF-1α in their GCF compared to healthy subjects. Subjects with periodontal
disease who underwent mechanical therapy demonstrated decreased levels of SDF1α. Neutrophil migration was enhanced in the presence of SDF-1α, mimicking
immune cell migration in periodontal lesions. The authors concluded that SDF-1α
may be involved in the immune defense pathway activated during periodontal
disease.2
 A study was done to examined the expression of chemokine SDF-1 receptor
CXCR4 on human PDLSCs and investigated the activation, proliferation,
differentiation, and migratory potential of PDLSCs induced by SDF-1. The
expression of SDF-1 receptor CXCR4 was evaluated by real-time polymerase
chain reaction (PCR) and immunocytochemical staining. PDL tissues were
obtained from clinically healthy premolars extracted for orthodontic reasons and
used to isolate single-cell colonies by the limited-dilution method. SDF-1
significantly increased proliferation and stimulated the migration of this PDLSC
subpopulation at concentrations between 100 and 400 ng/mL. CXCR4 neutralizing
antibody could block cell proliferation and migration, suggesting that SDF-1
exerted its effects on cells through CXCR4. SDF-1 promoted collagen type I level
significantly but had little effect on alkaline phosphatase level. The authors
concluded that SDF-1 may have the potential of promoting periodontal tissue
regeneration by the mechanism of guiding PDLSCs to destructive periodontal
tissue, promoting their activation and proliferation and influencing the
differentiation of these stem cells.4
 A study evaluated and compared the expression and production of macrophage
inflammatory protein (MIP)-1α, stromal-derived factor (SDF)-1, and interleukin
(IL)-6 by human cultured periodontal ligament and gingival fibroblasts challenged
with lipopolysaccharide (LPS) from Porphyromonas gingivalis. Fibroblasts were
cultured from biopsies of gingival tissue and periodontal ligament of the same
donors and used on the fourth passage. After confluence in 24-well plates, the
culture medium alone (control) or with 0.1 to 10 mg/ml of LPS from P. gingivalis
was added to the wells, and after 1, 6, and 24 hours, the supernatant and the cells
were collected and analyzed by enzyme-linked immunosorbent assay and realtime
polymerase chain reaction, respectively. MIP-1α, SDF-1, and IL-6 protein
production was significantly greater in gingival fibroblasts compared to periodontal
ligament fibroblasts. The authors opined that the distinct ability of the gingival and
periodontal ligament fibroblasts to secrete MIP-1α, SDF-1, and IL-6 emphasizes
that these cells may differently contribute to the balance of cytokines in the LPSchallenged periodontium.5
 Another study reported that in the bone marrow,SDF-1 is produced mainly by
immature osteoblasts and endothelial cells and the effect of DNA-damaging agents
such as irradiation, cyclophosphamide (Cy), or 5-fluorouracil (5-FU) on the
expression of SDF-1 in the bone marrow of mice or in cultured stromal cells.
Normal human trabecular bone was obtained from patients undergoing orthopedic
surgery in accordance with the University of Michigan’s Investigational Review
Board. Conditioning
with
DNA
damaging
agents
(ionizing
irradiation,
cyclophosphamide, and 5-fluorouracil) caused an increase in SDF-1 expression and
in CXCR4-dependent homing and repopulation by human stem cells transplanted
into NOD/SCID mice. The authors suggest that immature osteoblasts and
endothelial cells control stem cell homing, retention, and repopulation by secreting
SDF-1, which also participates in host defense responses to DNA damage.6
 A study assessed the regulation of CXCR4 transcription and SDF-1-induced
functional responses in human monocytes during their differentiation in the
presence of granulocyte-macrophage colony-stimulating factor (GM-CSF),
oxidized low-density lipoprotein (Ox-LDL), and unmodified LDL. Human
peripheral blood monocytes from healthy donors were isolated from monocyteenriched leukopacks (Biological Specialty Corp., Colmar, PA) and purified by
standard procedures. Results reveal that the rapid decline of SDF-1-mediated
[Ca2+] influx after monocyte isolation is followed by a gradual functional
restoration and a concomitant reexpression of CXCR4 mRNA over time. A further
three- to fourfold induction of CXCR4 mRNA occurred in macrophage-derived
foam cells on treatment with Ox-LDL. HL-60 cells induced with phorbol myristate
acetate (PMA) showed a rapid fourfold stimulation of CXCR4 mRNA within 1 h,
declining to barely detectable levels at 3 h, with eventual restoration over time,
mirroring the expression pattern in monocytes. Surface expression of CXCR4 is
maintained in HL-60 cells during PMA-induced differentiation, as demonstrated by
flow cytometry. GM-CSF had no effect on CXCR4 mRNA in HL-60 cells and
does not cause its down-regulation in human macrophages. 7

An in vivo study reported that the chemokine stromal-derived factor-1 (SDF-1)
can block human immunodeficiency virus type 1 (HIV-1) infection in vitro by
binding to the CXC chemokine receptor, CXCR-4, which serves as a coreceptor for
T cell tropic HIV-1. The authors reported that SDF-1 is consistently measured in
normal serum (15.4±3.0 ng/ml; mean±SD) and in serum from AIDS patients
(16.6±3.7 ng/ml) but found that the circulating SDF-1 is modified to an inactive
form. When exposed to serum, recombinant SDF-1 is specifically and rapidly
altered to yield an apparently smaller chemokine that does not bind to SDF-1
receptor-expressing cells, does not have chemoattractive or pre-B cell stimulatory
activity, and does not block HIV-1 infection. They concluded that serum
modification and inactivation contribute to the failure of SDF-1 to block HIV-1
infection and spread in man. The inactivation of circulating SDF-1 may be critical
in permitting local gradients to develop and direct cell trafficking.8
 A study investigated the production of stromal cell-derived factor-1 (SDF-1) and
the expression of CXCR4 in human bone marrow endothelial cells (BMECs).
Human BMEC cell line BMEC-1 cells expressed SDF-1 mRNA, and conditioned
medium induced chemoattraction of CD34+ cells. Three-day culture of BMEC-1
and primary human BMEC cells produced 1,710±204 and 1,050±153 pg/mL SDF1 , respectively, which was much less than primary human BM stromal cells
(29,536±532 pg/ mL). By immunohistochemistry, CXCR4 was detected in the
endothelial cells lining sinusoids, arterioles, and venules in the bone marrow.
These results indicate that BMECs produce and release small amounts of SDF-1
and express CXCR4 in vivo only.9
6.3 AIMS AND OBJECTIVES OF THE STUDY:
1. To assess the effect of diode laser on SDF-1α levels in GCF of chronic periodontitis
patients.
2. To quantitate and compare the levels of SDF-1α in GCF samples obtained from chronic
periodontitis patients treated with scaling and root planing and Diode laser.
7
MATERIALS AND METHODS :
7.1 SOURCE OF DATA
Patients visiting to the Department of Periodontology, Rajarajeswari Dental College
and Hospital, Bangalore.
7.2 METHOD OF COLLECTION OF DATA :
A total of 30 patients will be included in the study and will be grouped as follows.
Group I – 15 chronic periodontitis patients to be treated with scaling and root planing
alone.
Group II – 15 chronic periodontitis patients to be treated with scaling and root planing and
Diode laser.
GCF samples will be collected at baseline, 15 days and 21 days after treatment. 45
GCF samples will be collected from each group which forms a total of 90 samples.
INCLUSION CRITERIA :

Patients having minimum of 20 natural teeth.

Sites with a probing depth ≥5mm.

Sites with clinical attachment loss ≥ 3mm.

Radiographic evidence of alveolar bone loss.
EXCLUSION CRITERIA
1. Patients with history of systemic diseases affecting the periodontium.
2. Smokers.
3. Patients on any medication taken within the last 6 months which may alter the
periodontal status.
4. Pregnant and lactating mothers.
5. Patients who have undergone periodontal treatment within a period of 1 year.
SCREENING OF PATIENTS :
All the subjects will undergo a full mouth periodontal probing and charting and will
be screened for suitability, after which an informed consent will be taken. A proforma will
be designed for the present study so as to have a systematic and methodical recording of all
the observations and information. The relevant data will be recorded in the proforma.
Chronic periodontitis patients will be diagnosed based on criteria presented and discussed
at the 1999 International Workshop.
SCREENING EXAMINATION INCLUDE

Gingival index by Loe H & Silness P,1963.

Probing Pocket Depth (PPD) measured using a graduated William’s Periodontal
probe.

Clinical attachment level (CAL) measured from CEJ to base of the pocket.
SAMPLE COLLECTION
The patients will receive instructions regarding the study procedure and written
informed consent will be obtained. GCF samples of approximately 3 μl will be collected
using a graduated micro capillary pipette by an extracrevicular method. The collected GCF
will be immediately transferred to plastic vials, wrapped in tin foil and stored at -70º C
until the time of assay. Micro capillary pipette will be procured from Sigma Aldrich,
Bangalore, India. The storage of GCF samples was done at Rajarajeswari Dental College
and Hospital, Bangalore.
LABORATORY ANALYSIS OF SDF-1α
Quantitative analysis of SDF-1α Levels in the GCF samples will be done using a
Human CXCL/SDF-1 alpha Quantikine Enzyme Linked Immunosorbent Assay(ELISA) kit
before and after treatment.
STATISTICAL ANALYSIS :
Following statistical test will be employed in the present study.

Student Paired t test will be used to compare the level of SDF-1α, before and after
scaling and root planing.

Chi-square test will be used to associate the level of SDF-1α between two groups.

Pearson correlation test will be used to correlate GCF levels of SDF-1α with
clinical parameters.

Fischer’s exact test will be used to access the association between categorical
variables.

Any other statistical methods if required will be used.
Duration of the study: 18 Months.
7.3 DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTION
TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS, ANIMALS? IF SO
PLEASE DESCRIBE BRIEFLY?
Yes.
GCF samples will be obtained from the patients and will be assessed the levels of SDF-1α
by using Diode laser. Patients will be informed and written consent will be obtained.
7.4
HAS
ETHICAL
CLEARANCE
INSTITUTION IN CASE OF 7.3?
Yes.
Ethical clearance letter has been attached.
BEEN
OBTAINED
FROM
YOUR
8
LIST OF REFERENCES:
1. Giannobile WV. Host-response therapeutics for periodontal diseases. J Periodontol
2008;79:1592–1600.
2. Havens AM, Chiu E, Taba M, Wang J, Shiozawa Y, Jung Y et al. Stromal-derived
factor-1alpha (CXCL12) levels increase in periodontal disease. J Periodontol
2008;79(5):845-53.
3. Giannopoulou C, Cappuyns I, Cancela J, Cionca N, Mombelli A. Effect of
Photodynamic Therapy, Diode Laser, and Deep Scaling on Cytokine and AcutePhase Protein Levels in Gingival Crevicular Fluid of Residual Periodontal Pockets.
J Periodontol 2012;83:1018-27
4. Du L, Yang P, Ge S. Stromal cell-derived factor-1 significantly induces
proliferation, migration, and collagen type I expression in a human periodontal
ligament stem cell subpopulation. J Periodontol 2012;83(3):379-88.
5. Morandini AC, Sipert CR, Gasparoto TH, Greghi SL, Passanezi E, Rezende ML et
al. Differential production of macrophage inflammatory protein-1alpha, stromalderived factor-1, and IL-6 by human cultured periodontal ligament and gingival
fibroblasts
challenged
with
lipopolysaccharide
from
P.
gingivalis.
J
Periodontol 2010;81(2):310-7.
6.
Ponomaryov T, Peled A, Petit I, Taichman RS. Induction of the chemokine
stromal-derived factor-1 following DNA damage improves human stem cell
function. J Clin Invest 2000;106:1331–9.
7.
Gupta SK, Pillarisetti K, Lysko PG. Modulation of CXCR4 expression and SDF-1a
functional activity during differentiation of human monocytes and macrophages. J
Leukoc Biol 1999 ;66: 135–43.
8.
Villalba S, Salvucci O, Aoki Y, Sierra MD , Gupta G, Davis D et al. Serum
inactivation contributes to the failure of stromal-derived factor-1 to block HIV-I
infection in vivo. J Leukoc Biol 2003;74:880–8.
9.
Yun HJ, Jo DY. Production of Stromal Cell-Derived Factor-1 (SDF-1) and
Expression of CXCR4 in Human Bone Marrow Endothelial Cells. J Korean Med
Sci 2003; 18: 679-85.
9
SIGNATURE
OF
THE
CANDIDATE
10
REMARKS OF THE GUIDE
11
NAME& DESIGNATION
(in block letters)
11.1 GUIDE
DR. KRISHNA KRIPAL M.D.S
PROFESSOR
DEPARTMENT OF PERIODONTOLOGY
RAJARAJESWARI
DENTAL
COLLEGE
&
HOSPITAL,
&
HOSPITAL,
&
HOSPITAL,
MYSORE ROAD , BANGALORE- 60.
11.2 SIGNATURE
11.3 CO-GUIDE
DR. VINAYA KUMAR.R M.D.S
READER
DEPARTMENT OF PERIODONTOLOGY
RAJARAJESWARI DENTAL COLLEGE
MYSORE ROAD , BANGALORE- 60.
11.4 SIGNATURE
11.5
HEAD
OF
THE DR. SAVITA. S
DEPARTMENT
M.D.S
PROFESSOR AND HEAD
DEPARTMENT OF PERIODONTOLOGY
RAJARAJESWARI
DENTAL
COLLEGE
MYSORE ROAD , BANGALORE- 60.
11.6 SIGNATURE
12
12.1 REMARKS OF THE
PRINCIPAL
12.2 SIGNATURE