CLINICAL AND MICROBIOLOGICAL EVALUATION OF PHOTODYNAMIC

THERAPY AND DIODE LASER (960nm) AS AN ADJUNCT TO NON SURGICAL

PERIODONTAL TREATMENT – A COMPARATIVE STUDY

A Dissertation submitted in

Partial fulfillment of the requirements

For the degree of

MASTER OF DENTAL SURGERY

BRANCH – II

PERIODONTICS

THE TAMIL NADU DR. M.G.R. MEDICAL UNIVERSITY

Chennai – 600 032

2014 - 2017

ENDORSEMENT BY HEAD OF THE DEPARTMENT /

HEAD OF THE INSTITUTION

This is to certify that the Dissertation entitled “CLINICAL AND MICROBIOLOGICAL

EVALUATION OF PHOTODYNAMIC THERAPY AND DIODE LASER (960nm) AS

AN ADJUNCT TO NON SURGICAL PERIODONTAL TREATMENT – A

COMPARATIVE STUDY” is a bonafide work done by Dr.NIRMMAL MARIA T Post

Graduate student (2014–2017) in the Department of Periodontics, under the guidance of Dr.

MAHEASWARI RAJENDRAN, Professor, Department of Periodontics, Tamil Nadu

Government Dental College and Hospital, Chennai – 600 003.

Dr. K. MALATHI, M.D.S., Dr. B.SARAVANAN. M.D.S., Ph.D.,

Professor & HOD Principal.

Department of Periodontics.

Tamil Nadu Government Dental College and Hospital

Chennai – 600 003

CERTIFICATE BY THE GUIDE

This is to certify that Dr.NIRMMAL MARIA T, Post Graduate student (2014–2017) in the

Department of Periodontics, Tamil Nadu Government Dental College and Hospital, Chennai –

600 003 has done this dissertation titled “CLINICAL AND MICROBIOLOGICAL

EVALUATION OF PHOTODYNAMIC THERAPY AND DIODE LASER (960nm) AS

AN ADJUNCT TO NON SURGICAL PERIODONTAL TREATMENT – A

COMPARATIVE STUDY” under my direct guidance and supervision in partial fulfillment

of the regulations laid down by Tamil Nadu Dr. M.G.R. Medical University, Chennai – 600

032 for M.D.S., (Branch – II) Periodontics degree examination.

Dr.MAHEASWARI RAJENDRAN.M.D.S.,

Professor and Guide

Department of Periodontics

Tamil Nadu Government Dental College and Hospital

Chennai – 600 003

DECLARATION BY THE CANDIDATE

I hereby declare that this dissertation titled “CLINICAL AND MICROBIOLOGICAL

EVALUATION OF PHOTO DYNAMIC THERAPY AND DIODE LASER (960nm) AS

AN ADJUNCT TO NON SURGICAL PERIODONTAL TREATMENT – A

COMPARATIVE STUDY” is a bonafide and genuine research work carried out by me under

the guidance of Dr.MAHEASWARI RAJENDRAN. M.D.S., Professor and Guide,

Department of Periodontics, Tamil Nadu Government Dental College and Hospital, Chennai -

600003.

Dr.NIRMMAL MARIA T

Signature of the candidate

Tamil Nadu Government Dental College and Hospital

Chennai – 600 003

TRIPARTITE AGREEMENT

This agreement herein after the “Agreement” is entered into on this day ------------

between the Tamil Nadu Government Dental College and Hospital represented by its

Principal having address at Tamil Nadu Government Dental College and Hospital, Chennai –

600 003, (hereafter referred to as, „the college‟)

And

Dr.NIRMMAL MARIA T, aged 28 years currently studying as Post Graduate student in

Department of Periodontics, Tamil Nadu Government Dental College and Hospital, Chennai –

600 003, (hereafter referred to as „the PG student and principal investigator‟)

And

Mrs.Dr.MAHEASWARI RAJENDRAN aged 52 years working as Professor in Department

of Periodontics, Tamil Nadu Government Dental College and Hospital, Chennai (herein after

referred to as the „Co- Investigator‟),

Whereas the PG student as part of his curriculum undertakes this research on “CLINICAL

AND MICROBIOLOGICAL EVALUATION OF PHOTODYNAMIC THERAPY AND

DIODE LASER (960nm) AS AN ADJUNCT TO NON SURGICAL PERIODONTAL

TREATMENT – A COMPARATIVE STUDY” for which purpose the Co-investigator and

the college shall provide the requisite infrastructure based on availability and also provide

facility to the PG student as to the extent possible as a principal investigator.

Whereas the parties, by this agreement have mutually agreed to the various issues including in

particular the copyright and confidentiality issues that arise in this regard.

Now this agreement witnessed as follows

1. The parties agree that all the Research material and ownership therein shall become

the vested right of the college, including in particular all the copyright in the

literature including the study, research and all other related papers.

2. To the extent that the college has the legal right to do go, shall grant to licence or

assign the copyright so vested with it for medical and/or commercial usage of

interested persons/entities subject to a reasonable terms/conditions including

royalty as deemed by the college.

3. The royalty so received by the college shall be shared equally by all the three

parties.

4. The PG student and Co-investigator shall under no circumstances deal with the

copyright, Confidential information and know – how – generated during the course

of research/study in any manner whatsoever, while shall sole west with the college.

5. The PG student and Co-investigator undertake not to divulge (or) cause to be

divulged any of the Confidential information or, know – how to anyone in any

manner whatsoever and for any purpose without the express written consent of the

college.

6. All expenses pertaining to the research shall be decided upon by the principal

investigator/ Co-investigator or borne sole by the PG student.(principal

investigator)

7. The college shall provide all infrastructure and access facilities within and in other

institutes to the extent possible. This includes patient interactions, introductory

letters, recommendation letters and such other acts requires in this regard.

8. The Co-Investigator shall suitably guide the Student Right from selection of the

Research Topic and Area till its completion. However the selection and conduct of

research, topic and area of research by the student researcher under guidance from

the Co-investigator shall be subject to the prior approval, recommendations and

comments of the Ethical Committee of the College constituted for the purpose.

9. It is agreed that as regards other aspects not covered under this agreement, but

which pertain to the research undertaken by the PG student, under the guidance

from the Co-investigator, the decision of the college may be binding and final.

10. If any dispute arises as to the matters related or connected to this agreement

herein, it shall be referred to arbitration in accordance with the provisions of the

Arbitration and Conciliation Act, 1996.

In witness whereof the parties hereinabove mentioned have on this day month and year herein

above mentioned set their hands to this agreement in the presence of the following two

witnesses.

College represented by its Principal PG Student

Witness STUDENT GUIDE

1.

2.

ACKNOWLEDGEMENT

I am privileged to express my deep sense of gratitude to Dr. MAHEASWARI

RAJENDRAN M.D.S., Professor and guide, Department of Periodontics, Tamil

Nadu Government Dental College and Hospital, Chennai – 600 003 for her total

involvement, guidance, encouragement and scrutiny at every step of the dissertation

work and in bringing out a good thesis.

I express my gratitude to Dr. K. MALATHI M.D.S., Professor and Head,

Department of Periodontics, Tamil Nadu Government Dental College and Hospital,

Chennai – 600 003 for her valuable support and continuous encouragement

throughout the study.

I am grateful to Dr. JAISHREE TUKARAM KSHIRSAGAR M.D.S.,

Professor, Department of Periodontics, Tamil Nadu Government Dental College and

Hospital, Chennai – 600 003 for her valuable guidance and support for this study.

I sincerely thank Dr.B.SARAVANAN. M.D.S., Ph.D., Principal, Tamil Nadu

Government Dental College and Hospital, Chennai – 600 003 for his kind permission

and encouragement.

I express my gratitude to Dr.M.JEEVAREKHA. M.D.S., Associate

professor, Department of Periodontics, Tamil Nadu Government Dental College and

Hospital, Chennai – 600003 for her valuable guidance and continuous encouragement

throughout the dissertation preparation.

I am grateful to Dr.P.BHUVANESHWARI,M.D.S., Associate Professor,

Dr.A.MUTHUKUMARASWAMY,M.D.S., Associate Professor, Dr. M.SHABBIR

AHAMED.M.D.S, Dr. P. KAVITHA, M.D.S.,Dr.R.KARTHIKEYAN, M.D.S.,

Dr. A.J. ANAND, M.D.S., Dr. P.R. GANESH, M.D.S., Dr.D.JAYANTHI, M.D.S.,

Assistant Professors, Tamil Nadu Government Dental College and Hospital, Chennai

– 600 003, for helping me with my dissertation and during my study period.

I am thankful to Dr. JOHN KIRUBAHARAN J. M.V.Sc, Ph.D Professor &

Head, Department of Veterinary Microbiology , Madras Veterinary College, Chennai

– 600 007 for providing permission for microbiological evaluation at the laboratory of

microbiology department and for his valuable guidance.

I am also thankful to Miss Madhumathi (microbiologist), Dr.Renjani

(Research student) of Microbiology department, Madras veterinary college for

helping me during the study period.

I thank Dr. MOHAMMED JUNAID.MDS., for helping me with the

statistical analysis in the study.

I would also like to express my gratitude to all my colleagues who have stood

by me always and have been a constant source of encouragement for me during this

period.

I dedicate this work to my parents, siblings and to my husband for without

their encouragement, support and prayers I would not have reached so far.

Last, but not the least, I thank GOD ALMIGHTY for his blessings.

CONTENTS

S.No

TITLE

PAGE NO

1

INTRODUCTION

1

2

AIMS AND OBJECTIVES

4

3

REVIEW OF LITERATURE

5

4

MATERIALS AND METHODS

23

5

STATISTICAL ANALYSIS

45

6

RESULT

46

7

DISCUSSION

62

8

SUMMARY AND CONCLUSION

69

9

BIBLIOGRAPHY

70

10

ANNEXURES

85

LIST OF PHOTOGRAPHS

S.NO PHOTOGRAPHS PAGE

1 Armamentarium for sample collection 37

2 Sample collection 37

3 Sample transport 38

4 Armamentarium for non-surgical periodontal treatment 38

5 Indocyanin green dye 39

6 Diode laser unit 39

7 Pre-operative photograph 40

8 Preoperative photograph 40

9 Indocyanin green applied

41

10 Laser therapy 41

11 Post-operative after 6 months 42

12 Post-operative after 6 months 42

13 Armamentarium for DNA extraction 43

14 Heating bath 43

15 Vortex 44

16 Centrifuge 44

LIST OF TABLES

S.NO TABLES PAGE

1 Baseline measurements of group 1& 2 50

2 Third month measurements of group 1& 2 51

3 Sixth month measurements of group 1& 2 52

4 Group 1 mean and standard deviation 53

5 Group 2 mean and standard deviation 53

6 Group 1 Plaque index intra group analysis 54

7 Group 1 bleeding index intra group analysis 54

8 Group 1 probing depth intra group analysis 55

9 Group 1clinical attachment level intra group analysis 55

10 Wilcoxon signed ranks test 56

11 Group 2 Plaque index intra group analysis 57

12 Group 2 bleeding index intra group analysis 57

13 Group 2 probing depth intra group analysis 58

14 Group 2clinical attachment level intra group analysis 58

15 Wilcoxon signed ranks test 59

16 Mann- whitney test- inter group analysis 60

LIST OF FIGURES

S.NO FIGURES PAGE

1 Changes in clinical parameters for group 1 61

2 Changes in clinical parameters for group 2 61

LIST OF ABBREVIATIONS

SRP Scaling And Root Planning

PDT Photodynamic Therapy

PCR Polymerase Chain Reaction

PI Plaque Index

GBI Gingival Bleeding Index

CAL Clinical Attachment Level

PPD Pocket Probing Depth

ICG Indocyanin Green

BOP Bleeding On Probing

MMP-8 Matrix Metalloproteinases 8

TBO Toluidine Blue O

DL Diode Laser

TNF Tumour Necrosis Factor

RANKL Nuclear Factor Kappa B Ligand

OPG Osteoprotegerin

RT- PCR Real Time Polymerase Chain Reaction

qPCR Quantitative Polymerase Chain Reaction

Introduction

Page 1

INTRODUCTION

Periodontitis is an inflammatory disease of supporting tissues of the teeth which lead to

progressive destruction of periodontal membrane and alveolar bone. This destructive process

is initiated by specific bacteria within the sub gingival biofilm and progresses because of

host’s immune inflammatory mechanisms triggered in responses to these bacteria. Although a

number of Gram-negative anaerobic bacteria have been implicated in this disease process,

Porphyromonas gingivalis is considered as a major etiological agent of periodontitis (van

Winkelhoff et al. 19881, Slots & Ting 1999

2 and Herrera et al. 2008

3).

Treatment procedures for the management of periodontitis aim to reduce and eliminate

the microbial causative factors and to improve the periodontal status. The treatment options

include oral hygiene instructions, non-surgical therapy, surgery and supportive periodontal

maintenance (Carranza) 4

.

The gold standard for non-surgical periodontal treatment is scaling and root planing

(SRP) Hand instruments and ultrasonic scalers were used for the conventional non-surgical

periodontal treatment. Marked changes in the sub gingival microflora and clinical indices were

observed following this non-surgical instrumentation (Haffajee et al. 1997)5.

But these conventional treatments can become less effective, either because of

difficulties in the treatment procedures like inaccessible deep pockets, furcation, concavities or

due to systemic conditions which may compromise host response to the treatment. In these

conditions various adjunctive treatments like systemic antibiotics, local delivery of

antimicrobial agents and lasers are used along with scaling and root planning.

Laser irradiation with its bactericidal and detoxification effect has a significant

potential as an adjunct to traditional nonsurgical therapy (i.e., scaling and root planing). Laser

Introduction

Page 2

improve the effectiveness and efficacy of the removal of sub gingival microorganisms .The

addition of laser may improve periodontal tissue healing, achieving a deeper bacterial

inhibition and prolonging intervals between maintenance visits (Ugo Caruso 2008)6.

Another newer approach in non-surgical management is photodynamic therapy (PDT).

It is a technique combining laser with a photosensitizer dye to produce singlet oxygen

molecules and free radicals which are extremely toxic to certain cells and microorganisms

(Konopka & Goslinski 20077 and Maisch 2007

8).Several periodontal pathogens, such as

Porphyromonas gingivalis and A. actinomycetemcomitans are efficiently eliminated by PDT,

either in the aqueous suspension or as biofilm. In this study indocyanin green is used as the

photosensitizer. The introduction of indocyanin green as photosensitizer is recent and result in

significant reduction of P. gingivalis and A. actinomycetemcomitans, and less than 10% of

bacteria remain viable (Tobias K. 2011)9.

After the non-surgical periodontal treatment there will be improvement in clinical and

microbiological parameters. P. gingivalis count reduces in treated sites but is commonly

encountered in sites that exhibit recurrence of disease (Haffajee AD, 198810

, van Winkelhoff

AJ 19881). Hence microbiological evaluation of P.gingivalis will help to determine the long

term effectiveness of laser and PDT in the management of chronic periodontitis. Real time

PCR is an ideal method to quantify the bacterial species and hence it is used to evaluate the

post treatment P.gingivalis count.

The present study was undertaken to evaluate the effectiveness of photodynamic

therapy and diode laser as an adjunct to scaling and root planning by means of bacterial

reduction and changes in clinical parameters.

Aim and objectives

Page 3

AIM

To compare photodynamic therapy and diode laser as an adjunct to scaling and

root planing in the management of chronic periodontitis.

.

OBJECTIVES

To evaluate the effectiveness of diode laser and photodynamic therapy in the

management of chronic periodontitis patients using

Clinical parameters like plaque index, gingival bleeding index, pocket probing

depth and clinical attachment level.

Microbiological evaluation of Porphyromonas gingivalis.

Review of literature

Page 4

REVIEW OF LITERATURE

Periodontitis is an inflammatory disease of supportive tissue of teeth which

lead to progressive destruction of periodontal membrane and alveolar bone (Caton J

1989)11

. It is a complex interaction between an infection and a susceptible host

(dcna) 12

. It includes the initial microbial challenge, a subsequent host inflammatory

response, and various risk factors that contribute to host susceptibility and progression

of the disease.

Chronic periodontitis is initiated and sustained by microorganisms living in

biofilm communities which are present in supra- and sub gingival plaque in the form

of uncalcified and calcified biofilms. The sub gingival biofilm harbors a variety of

bacterial species; the composition of the biofilm may vary between subjects and sites.

Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia,

Campylobacter rectus, Eikenella corrodens, F. nucleatum, Actinobacillus

actinomycetemcomitans, Peptostreptococcus micros, and Treponema have been most

commonly found to be associated with chronic periodontitis. Although a number of

Gram-negative anaerobic bacteria have been implicated in this disease process,

Porphyromonas gingivalis is considered as a major etiological agent of periodontitis

(van Winkelhoff et al. 19881, Slots & Ting 1999

2, and Herrera et al. 2008

3)

Porphyromonas gingivalis is strongly correlated with chronic periodontitis.

Chronic persistence of P.gingivalis in the periodontium depends on its ability to evade

host immunity without inhibiting the overall inflammatory response, which is

beneficial for both P.gingivalis and other periodontal bacteria. P.gingivalis is found in

40–100% of sites in chronic periodontitis patients.

P.gingivalis comprises a considerably higher proportion of the sub-gingival

microbiota in deep pockets than in shallow pockets (Ali et al. 199613

).

Review of literature

Page 5

High levels of P. gingivalis were observed in samples from periodontitis

patients and low levels from the healthy subjects (Beck et al 199014

). Griffen, A. L. in

199821

reported that P. gingivalis is a causative factor in many cases of periodontitis.

P. gingivalis was found, 11-fold greater in the periodontitis group than in the healthy

group.

Management of chronic periodontitis

Treatment of chronic periodontitis aims to reduce and eliminate the microbial

causative factors and to improve the periodontal status and prognosis of the teeth. The

treatment options include oral hygiene instructions, non-surgical therapy, surgery and

supportive periodontal maintenance (Carranza) 4

.

Non-surgical periodontal treatment

Primary prevention of periodontitis is related to preventing formation and/or

eradication of the microbial biofilm Non-surgical therapy aims to eliminate bacteria in

microbial biofilm and calcified biofilm from tooth surface and adjacent soft tissue

(Lindhe) 16

. Reduction in inflammation of the periodontium due to lesser bacterial

load leads to beneficial clinical changes.

Mechanical debridement and the quality of the patient’s home care are of vital

importance in preventing inflammation that manifests as both gingivitis and

periodontitis. Mechanical debridement includes scaling and root planing (SRP) by

manual instrumentation or with sonic or ultrasonic scalers. SRP has become the gold

standard nonsurgical treatment of periodontitis, with multiple clinical studies

demonstrating that it effectively reduces the microbial load and leads to reductions in

bleeding on probing and probing depths and allows for gains in clinical attachment.(

Maria Emanuel Ryan DCNA 200517

, Doungudomdacha S et al 200118

).

Review of literature

Page 6

P.gingivalis, P.intermedia and A.A comitans were significantly reduced in

both the diseased and healthy sites (86-99%) after the non-surgical treatment.

P.gingivalis is uncommon and in low numbers in health or gingivitis but more

frequently detected in destructive forms of disease. The species has been shown to be

reduced in successfully treated sites but is commonly encountered in sites that exhibit

recurrence of disease post therapy.

A review of nonsurgical mechanical pocket therapy by Cobb in 199619

reveals

mean probing depth reductions and clinical attachment level gains of 1.29 mm and

0.55 mm, respectively, for initial probing depths of 4 to 6 mm before treatment and

2.16 mm and 1.19 mm, respectively, for initial probing depths of 6 mm before

treatment. Conventional nonsurgical periodontal therapy involves performing SRP in

single or multiple quadrants or sextants per visit and is usually completed in 2 to 6

weeks.

Mechanical removal of plaque and calculus are influenced by extent of

disease, anatomic factors, the skill of the operator, and the instruments used (Lindhe)

16. It is also time-consuming, operator and patient dependent, and difficult to master

(Greenstein G.1992)20

. Instrumentation inevitably leaves behind significant numbers

of microorganisms, including putative pathogens. Waerhaug (1978)21

suggested that

in more than 90% of cases, deposits of plaque and calculus remained in sites with

pocket depths (PD) >5 mm following scaling and root planing. Similar conclusions

were reported by Rabbani et al. (1981)22

and Magnusson et al. (1984)23

.

Slots and Ram24

reported that mechanical therapy may fail to eliminate all the

pathogenic bacteria within the gingival tissue and in the tooth structures inaccessible

to periodontal instrumentation. Recolonization of these pathogens can occur within 60

days of SRP, resulting in the need for regular maintenance visits.

Review of literature

Page 7

In these conditions systemic and local administration of various antimicrobial

agents and lasers were used as adjunctive treatment to reduce the pathogenic

microorganisms.

Laser ablation

Laser irradiation with its bactericidal effect has significant potential as

an alternative or adjunct to traditional nonsurgical therapy. The earliest clinical

studies mentioning the application of lasers in the nonsurgical treatment of

periodontitis began in the early 1990s (Trylovich DJ25

).Various laser wavelengths

have been used by clinicians in the treatment of periodontitis; most commonly the

diode lasers (DLs) (809–980 nm), Nd:YAG (1064 nm), Er:YAG and Er,Cr: YSGG

(2940 and 2780 nm respectively) and the CO2 (10,600 nm) (Cobb et al. 201026

).

These lasers reduces the microorganisms in periodontal pocket and remove the

calculus and granulation tissue (Kreisler M 2005)27

.

The high power carbon dioxide (CO2) and neodymium-doped yttrium

aluminum garnet (Nd:YAG) lasers are capable of excellent soft tissue ablation with a

good hemostatic effect and have been generally proposed for periodontal surgery and

oral surgery(Aoki A 200428

). However, these lasers are not suitable for treatment of

root surface or alveolar bone, due to carbonization of these tissues and major thermal

side effects on the target and surrounding tissues. They are mainly indicated for

gingivectomy and frenectomy. Er:YAG laser can be used for periodontal hard tissue

procedures such as dental calculus removal and decontamination of the diseased root

surface.

Diode laser

The DL has been used in dentistry since the early 1980s (Pirnat 200729

, Aoki

et al. 200830

). Over time, the DL has become more popular with clinicians, primarily

Review of literature

Page 8

because of its relatively small size, low cost and flexible fiber delivery system, which

is suitable for pocket insertion (Aoki A 2004)28

. A variety of diode laser systems have

been investigated in numerous in vitro (Kreisler M 200231

,200332

, Moritz A,199733

)

and in vivo studies (Moritz A199834

, Yilmaz S 200235

) Since then, many studies have

been carried out to evaluate the possible advantages of the use of diode lasers as an

adjunct to conventional SRP. They are also effective for soft-tissue applications, such

as incision, hemostasis, and coagulation (Romanos & Nentwig 1999)36

.

The most widely used lasers in the diode family are the gallium-aluminum-

arsenide (GaAlAs) laser (810 nm) and the indium-gallium-arsenide (InGaAs) laser

(980 nm). The shorter wavelength lasers (e.g., 809 nm to 980 nm diodes and 1064 nm

Nd:YAG) are more likely to penetrate deeper into soft tissues (Parker S. 2007)37

. The

extent of tissue penetration by shorter wavelengths is related to their affinity for

pigmented tissues and a low absorption coefficient in water (Parker S 2007)37

. The

potential for undesired tissue penetration can be controlled with proper selection of

parameters, such as power level, pulse repetition rate, pulse width, and energy density

Power output is 2-10 watt and can be either pulsed or continuous mode and has less

thermal effect on deeper tissue (Rasteger 199238

, Wyman 199239

).

According to the literature, potency values already tested varies from 1 up to

2.5 W, either in the continuous or the pulsed mode [Moritz A 199834

, Caruso U 20086,

Borrajo JLL200440

, Kreisler M, 200527

, Kamma JJ, 200941

). A well-designed study by

Harris and Yessik 200442

determined the in vitro ablation threshold for P.g for both

the 810-nm diode and Nd:YAG lasers to be 48 and 96 J/cm2, respectively. Diode

laser is not expected to cause damage to the pulp when operated in pulsed mode and

at an output power of 2.5 W (White et al. 199143

).

Review of literature

Page 9

Diode wavelengths when combined with the appropriate choice of parameters

can result in penetration of soft tissues ranging from about 0.5 to 3 mm (Aoki et al.

2008)30

and exhibits poor energy absorption in mineralized tissues. Thus, the DL is

contraindicated for calculus removal. With the current recommended parameters, the

possibility of inducing collateral damage with the DL, such as root surface alterations,

is not likely to occur (Cobb et al. 2010)26

.

It has bactericidal and detoxification effects and can remove the epithelium

lining and granulation tissue within the periodontal pocket which may potentially

improve healing (Lindhe & Nyman 198544

, Ramfjord et al. 198745

). Romanos et al

(2004)46

in an in vitro histological study on pigs reported the ability of the DL at 2.0

W to completely remove the pocket epithelium. In addition, the application of a DL

has benefits such as promotion of hemostasis, decreased requirement of anaesthesia

during treatment, and less post-operative pain.

Gold standard for successful treatment of chronic periodontitis is gain in

clinical attachment level (Cobb m 199619

, 200647

). Laser irradiation of periodontal

pockets, in which pocket epithelium was eliminated without damaging the collateral

blood supply promote periodontal healing as well as attachment (Romanos GE

2004)46

. The same study also suggested that laser irradiation of periodontal pockets

de-epithelized the pocket and blocked the down growth of epithelium into the healing

periodontal pocket, and so enhanced periodontal reattachment (Romanos GE 2004)46

.

So there will be reduction in probing depth and clinical attachment level. An in vitro

study showed that diode laser irradiation could stimulate the proliferation of

periodontal ligament cells (Kreisler M, 2003)32

The reduction in BOP with laser debridement can be supported by the findings

of a study that reported a decrease in the amounts of matrix metalloproteinase-8

Review of literature

Page 10

(MMP-8) on laser irradiated sites (Qadri T 2005)48

. MMP-8 was used as a surrogate

marker for severity of inflammation since it is stored in the secretory granules of

neutrophil granulocytes and released from the cells to the inflammatory lesion during

migration. Therefore, the decrease in the levels of MMP-8 reduced the inflammation.

Significant reduction of PI was observed when good oral hygiene

instructions were performed (Kreisler M, 200527

, Kamma JJ, 200941

). There is no

evidence that laser therapy can inhibit biofilm formation once a tooth has been

irradiated.

The purported benefits of the DL in periodontal therapy are based on the

premise that sub gingival curettage is an effective treatment and that significant

reduction in sub gingival microbial populations is predictably achieved (Cobb et

al.2010)26

As far as bacterial reduction in periodontal pockets is concerned, the diode

laser is expected to have a disinfecting thermal effect on bacteria that is basically

limited to the root surface. The thermal effect of the laser beam is based on the

absorption of radiation by tissue and subsequent transformation of laser energy into

heat. Tissue absorbs a certain amount of laser radiation per volume and transforms it

into a certain amount of energy, depending on the exposure time used. The amount of

energy absorbed depends on the type of tissue irradiated and the wavelength of the

laser (Andreas Moritz, 1998)34

. Most of the diode laser radiation is absorbed by

superficial layers, thus having a better effect on sites affected by periodontal disease

(Andreas Moritz, 1998)34

.

Sbardone et al in 199049

reported that diseased sites treated with a single

episode of scaling and root planing exhibited a microflora similar to that in healthy

sites at 7 days after treatment. However, the treated sites were repopulating with

Review of literature

Page 11

potentially pathogenic microbes at 21 days after treatment. Lin et al in 199250

indicated that sub gingival treatment with the laser without anesthesia is more

effective in reducing or inhibiting recolonization of bacteria for up to 28 days than is

root planning. Observations at 7 days after laser treatment without scaling and root

planing showed early recolonization by a variety of microbial morphotypes.

High-intensity diode laser has a better penetration and affinity for the

pigments present in some bacteria, which would act as an absorbing chromophorous,

and it would, in turn, intensify its action and thus make it possible to reach black

pigmented anaerobes such as P.gingivalis (Coluzzi DJ 2000)51

. When in vivo it is not

possible to assure that the pigments are produced, and thus that the bacteria are in fact

pigmented. These bacteria need heme (protoporphirin IX) to grow, and this is the

predominant pigment in P. gingivalis and Prevotella intermedia (Smalley JW, 1998)52

.

The amount of free iron on the bacterial surface is photosensitive for some

wavelengths, which would result in the ablation of these bacteria.

(Soukos NS 2005)53

.

Moritz et al in 199834

reported considerable bacterial elimination from

periodontal pocket of greater than or equal to 4mm using irradiation with an 810-nm

diode laser with 2.5 W power settings in pulsed mode (50 Hz, pulse duration 10 ms)

following SRP as compared to SRP alone.. He also reported reduction in pocket depth

and bleeding on probing.

Borrajo et al in 200440

found that there is reduction in bleeding on probing in

laser treated cases when compared to SRP alone. Qadri et al in 200548

reported no

significant difference in reduction of microbial count between laser treated cases and

control group.

Review of literature

Page 12

Kreisler et al (2005)27

did a clinical trial on use of diode laser in chronic

periodontitis patients and reported that there is difference in bleeding on probing in

laser treated group. No difference in other clinical parameters like probing depth and

clinical attachment level.

Kamma et al (2006)54

in his study reported that there is reduction in bleeding

on probing, probing depth and gain in clinical attachment level in laser treated group.

He also reported reduction in microbial count in the same.

Caruso et al in 20086 reported that diode laser may lead to a slight

improvement of clinical parameters when compared to SRP alone.

De Micheli et al in 201155

reported that the high power diode laser adjunct to

the non-surgical periodontal treatment did not promote additional effects to the

conventional periodontal treatment.

Zingale et al. in 201256

and Euzebio Alves et al. in 201357

reported that the

adjunctive use of the diode laser did not significantly differ from SRP alone in

decreasing PD/BOP.

When compared to SRP alone, multiple adjunctive applications of a 980-nm

diode laser with SRP showed PD improvements only in moderate periodontal pockets

of 4–6 mm (Dukic et al. 2013)58

.

Saglam et al. in 201459

reported that diode laser along with SRP provided

significant improvements in clinical parameters.

Photodynamic therapy

. Photodynamic therapy (PDT) is the combination of laser and photosensitizer

application which involves the stimulation of photosensitizer dye molecules by laser

light of particular wavelength. The photosensitizer is generally an organic dye or

similar compound capable of absorbing light of a specific wavelength, after which it

Review of literature

Page 13

is transformed from a ground singlet state to a longer-lived excited triplet state

(Sharman WM 1999)60

.

The longer lifetime of the triplet state enables the interaction of the excited

photosensitizer with the surrounding tissue molecules. It is generally accepted that the

generation of the cytotoxic species produced during PDT occurs while in the triplet

state (Dougherty TJ 199861

, Ochsner M. 199762

). The cytotoxic product, generally O2,

cannot migrate more than 0.02 mm after this formation, thus making it ideal for the

local application of PDT without endangering distant biomolecules, cells, or organs

(Moan J, 1991)63

.

Phenothiazine derivatives (methylene blue and toluidine blue) are most

frequently used as photosensitizers in PDT. Indocyanin green is a tri carbocyanin that

belongs to a large family of cyanine dyes (Mishra a 2000)71

. It can also produce

powerful photosensitized cellular damage (Delaey t 20000)64

. Absorption occurs in

near infra-red wavelength(800-1100). Indocyanine green with laser irradiation results

in significant reduction of P. gingivalis and A. actinomycetemcomitans, as less than

10% of bacteria remain viable (Tobias K Boehm 2011)9.

In general medicine, PDT has been used in the treatment of neoplasms

(Dougherty & Marcus 1992)65

. In dentistry, interest has been in the elimination of

various microorganisms. The major reason for using PDT is to effect reductions in

subgingival microbes. In vitro studies have shown PDT to completely eliminate

Streptococcus sanguis, Fusobacterium nucleatum, Porphyromonas gingivalis and

Aggregatibacter actinomycetemcomitans (Dobson & Wilson 199266

, Pfitzner et al.

200467

). The possibility of the suppression of P. gingivalis was also demonstrated in a

dog study (Sigusch et al. 2005)68

.

Review of literature

Page 14

Photodynamic therapy has been claimed to have a broad spectrum of action,

with efficacy against antibiotic-resistant strains without evidence of development of

photo resistant strains, extensive reduction in the bacterial population with limited

damage to host tissues, the ability to target infected tissues, and overall beneficial

economic factors (Jori G, 2006)69

. Development of resistance to PDT would appear

to be unlikely since its bactericidal activity is due to singlet oxygen and the

photosensitizer needs to be retained in the periodontal pocket for only a short time;

this may be minutes or seconds, depending on the power of the laser light delivery

system used. (N. Ko¨merik 2002)70

It is a new antimicrobial concept with fewer complications (Nagpal S 2012)71

.

It employs a quick and simple protocol that allows the clinician to kill bacteria,

inactivate virulence factors left behind after scaling and root planing. It is used during

initial and maintenance therapy for the treatment of periodontitis. Key virulence

factors like lipopolysaccharide and proteases have been shown to be reduced by

photosensitization (Komerik, N. 200072

, Packer, S., 200073

).

Following exposure of P. gingivalis to low-energy He-Ne laser and TBO (25

um/ ml), the activity of lipopolysaccharide and IL-1 secretion from human peripheral

mononuclear cells exposed to such treatment were significantly reduced (Komerik N

2000)72

. In addition, there was a substantial, light dose dependent decrease in the

proteolytic activity (94 per cent) of P. gingivalis (Packer S, 2000)73

. Such effects may

be of benefit in the treatment of infections due to these organisms. Furthermore, as

stated by Braham, 200974

the inactivation of proteases of P. gingivalis occurs at lower

concentrations of the photosensitizer (methylene blue) than those required for an

effective bacterial killing and more rapidly in the course of irradiation.

Review of literature

Page 15

During inflammation there is venous stagnation and reduced oxygen

consumption by tissues. This decrease in oxygen level and change in pH may enhance

the growth of anaerobic species. In such cases, PDT may improve tissue blood flow in

the microcirculatory system and reduce venous congestion in gingival tissues (Tanaka

M 1998)75

. Furthermore, PDT may increase oxygenation of gingival tissues by 21–47

per cent. This in turn decreases the time and speed of oxygen delivery and utilization,

thus normalizing oxygen metabolism in periodontal tissues (Tanaka M 1998)75

.

Andersen et al in 200776

compared SRP (scaling root planning) alone, PDT

alone and the combination of SRP with PDT. SRP with adjunctive PDT showed

greater CAL gains than did SRP alone or PDT alone, after 3 months (SRP+PDT: 0.86

+/- 0.61, 0.36 +/-0.35 and 0.14 +/- 0.65, respectively). It is evident that the benefits of

PDT alone are negligible from a clinical point of view and hence, PDT should only be

considered adjunctive to SRP.

Superiority of SRP + PDT over SRP alone in the initial periodontal treatment

was also confirmed by Betsy et al.201477

.

Compared to SRP alone, the addition of a single application of PDT resulted

in higher reductions of bleeding scores, but not in additional improvements in pocket

depth reduction and gain of clinical attachment after 6 months (Christodoulides et al.

2008)78

.

Polansky et al 200979

reported no differences in PPD, CAL and the

composition of sub gingival microbiota between the groups (SRP vs SRP + PDT) and

a visibly larger reduction of BOP (bleeding on probing) in the test group, although it

was not significant.

Review of literature

Page 16

Study reported on the clinical effects of a single application of PDT in

maintenance patients and showed a reduction in bleeding on probing (BoP) (Chondros

et al. 2008)80

.

Braun et al. in 200881

reported that the sites treated with adjunctive PDT

shows higher reductions in BoP percentages, PPD and CAL compared with the CSR

therapy alone and were judged to improve non-surgical periodontal therapy.

Polansky et al in 200979

did a study on photodynamic therapy using diode

laser and helbo blue dye and he reported that there were no extra reductions in pocket

depths and bleeding on probing. With regard to eradication of bacteria, there is no

additional effect as compared with conventional treatment alone.

Repeated (five times) applications of PDT improved the clinical outcomes

after 6 months in the treatment of residual pockets (defined as PPDX5 mm) in patients

enrolled in a maintenance care program (Lulic 2009)82

.

In addition, de Oliveira and colleagues (2009)83

compared treatment of

aggressive periodontitis with PDT versus SRP and reported no significant differences

between treatment groups for measures of gingival crevicular fluid levels of tumor

necrosis factor-alpha (TNF-a) or nuclear factor-kappa B ligand (RANKL), both

factors being involved in bone resorption.

Atieh (2010)84

, after a systematic review and meta-analysis concluded that the

combined use of PDT with conventional SRP may provide additional improvements

in CAL, PD and other clinical measures in the treatment of chronic periodontitis.

Campos et al in 201385

reported that sites treated with combined therapy (SRP

+ PDT) presented significantly better clinical parameters (PPD, BoP and CAL) than

control sites (SRP) in a three month observation. Upon the treatment completion, the

percent-age of sites with PPD < 5 mm, and BoP-negative were statistically higher

Review of literature

Page 17

(77.22%) in the test group than in the control group (40.0%), whereas there was no

difference at baseline between the groups.

Franco et al. 201486

applied inclusion criteria similar to Chondros (no active

periodontal treatment in the previous 6 months) and found that the application of PDT

in conjunction to conventional scaling lead to the up-regulation of RANK, OPG and

FGF2, that was statistically significant when compared with sole instrumentation.

Those results suggest that PDT plays a role in controlling osteoclastic activity

(increased expression of RANK and, in particular, OPG) and promotes healing (up-

regulation of FGF2) in inflamed periodontal tissues.

Evaluation of periodontal disease activity and therapeutic efficiency

The determination of disease activity has a direct impact on therapeutic

measures in periodontics. Traditional evaluations like clinical indices and

radiographs, gingival crevicular fluid contents, tissue changes, circulating factors, and

sulcular microbiota are the various methods for evaluation of disease activity

(Hancock E B 1981)87

.

Pocket formation, clinical attachment level and alveolar bone loss provides

only a historical record of the disease condition. Clinical indices like bleeding on

probing have a low predictive value. For the diagnosis of the disease activity of

periodontitis clinical symptoms alone may not be sufficient.

Predictions of recurrence of disease and prognosis for the patient can be

significantly improved when the presence or absence of periodontal pathogens is

monitored as well (Wolff 1994)88

. Microbiological diagnosis is also effective in

evaluating the effects of various periodontal treatments. Examinations of sulcular

microbiota provided evidence suggesting that active periodontal disease was

associated with specific groups of pathogens and with elevated counts of motile

Review of literature

Page 18

organisms. The evaluation of such pathogens and motile organisms currently shows

the most promise for determining periodontal disease activity

Historically culture methods have been widely used for characterization of sub

gingival microflora. Culture methods can grow only live bacteria and it requires strict

sampling and transport conditions. Sensitivity of the culture method is low and the

detection limit is 103

– 104.

Hence low number of specific pathogen in pocket is

undetected.

The development of techniques in molecular biology set the basis for the

development of improved diagnostic techniques which helps in the analysis of DNA,

RNA and the structure or function of the protein. Diagnostic assays employing

molecular biology require specific DNA fragments that recognize complimentary

specific bacterial DNA sequence from target microorganisms. Based on this

technology various diagnostic methods are able to extract bacterial DNA from the

plaque sample and amplify the target periodontal pathogen.

Polymerase chain reaction

The polymerase chain reaction (PCR) is a technology in molecular biology

used to amplify a single copy or a few copies of a piece of DNA across several orders

of magnitude, generating thousands to millions of copies of a particular DNA

sequence. Developed in 1983 by Kary Mullis, PCR is now a common and often

indispensable technique used in medical and biological research labs for a variety of

applications. These include DNA cloning for sequencing, DNA-based phylogeny, or

functional analysis of genes; the diagnosis of hereditary diseases; the identification of

genetic fingerprints (used in forensic sciences and paternity testing); and the detection

and diagnosis of infectious diseases.

Review of literature

Page 19

Bacterial examination methods that detect the presence of bacteria, but not the

amount, are termed qualitative examinations. Owing to the endogenous nature of

periodontal infection, periodontal bacteria often exist in both healthy gingival sulcus

and diseased periodontal pockets, making qualitative methods unsuitable for the

diagnosis of periodontal disease. Most important application of microbiological

examination in periodontal disease is in monitoring changes in bacterial numbers after

periodontal treatment compared with before treatment, providing an assessment of the

effectiveness of periodontal treatment. For this purpose, quantitative bacterial

examinations are required (Akihiro Yoshida 2003)89

.

Quantity is essential because the difference in the microbial species between

periodontal health and disease and between pre- and post-periodontal treatment is

quantitative rather than presence or absence of one or more species of a pathogen.

PCR is a common method capable of detecting low number of cells but it is not able

to provide quantitative data. Real-time PCR overcomes this limitation (Socransky SS,

2005)90

.

Quantitative PCR (q PCR)

It is a variant of the basic PCR technique. Used to measure the quantity of a

target sequence (commonly in real-time). It quantitatively measures starting amounts

of DNA, cDNA, or RNA. Quantitative PCR is commonly used to determine whether a

DNA sequence is present in a sample and the number of its copies in the sample.

Quantitative PCR has a very high degree of precision. Quantitative PCR

methods use fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-

containing DNA probes, such as TaqMan, to measure the amount of amplified

product in real time. It is also sometimes abbreviated to RT-PCR (real-time PCR) but

this abbreviation should be used only for reverse transcription PCR. qPCR is the

Review of literature

Page 20

appropriate contractions for quantitative PCR (real-time PCR). Most real-time PCR

tests are based on the detection of bacterial small-subunit 16S rRNA sequences (Heid

c a 1996)91

. This subunit of DNA is present in multiple copies in all bacterial species

and contains highly conserved species-specific sequences

Real-time PCR assay for periodontal bacteria can be used to determine

bacterial counts for a wide range of purposes in the study of periodontal diseases

(Kawada M, 2004)92

. Real-time PCR with species specific primers can provide a

precise and sensitive method for more accurate quantitation of individual species as

well as total bacteria, and will be a useful tool for studies on the etiology of chronic

periodontitis.

In the clinical conditions when subjects with periodontitis were treated

successfully, the species was eliminated or lowered in counts; treatment failures were

associated with failure to decrease the number of the species in treated sites (Slots J.

1976)93

. During periodontal therapy, factors associated with the etiology of

periodontitis, other than microbiological factors, are relatively stable, whereas the

number of bacteria is variable.

The number of P. gingivalis bacteria increased ten-fold with every millimeter

increase of pocket depth. Furthermore, the number of this organism decreased

significantly after scaling and root planning (Kawada et al., 2004)92

. Real-time PCR

offers the ability to determine the absolute and relative amounts of P. gingivalis in a

mixed sample without culturing the sample. Thus, this method can be used to

quantitatively evaluate the number of periodontopathic bacteria at periodontal sites,

making it applicable for the evaluation of therapeutic efficacy.

Review of literature

Page 21

Using taqman system Lyons, Griffen, and Leys in 200094

determine both the

P.gingivalis count and total no.of bacteria present in plaque sample directly without

culturing.

Real-time quantitative PCR provided a sensitive and accurate method for

measuring the amount of P. gingivalis in plaque samples. In addition, it allowed the

determination of the total number of bacterial cells present in a complex sample so

that the percentage of P. gingivalis could be determined.

Real-time PCR provides precise counts through direct monitoring of the

increasing amount of PCR product throughout the enzymatic assay and is the most

sensitive method with detection limits of 10 genome copies (Kinane DF 200395

,

Kirakodu SS 200896

).

Lyons et al in 200094

and Van Winkelhoff et al in 200297

showed the presence

of P. gingivalis in healthy subjects by PCR assay and concluded that this organism

may also be a normal inhabitant of a periodontally healthy dentition.

Masunaga et al (2010)98

used qPCR to compare the levels of P.gingivalis,

T.forsythia T.denticola and total bacteria detected by different sampling methods. The

no.of total bacteria in samples were 105- 10

6, 10

8,10

7 respectively. The number of

P.gingivalis increase with worsening of clinical status.

Mohammad Taghi and Chitsazi1 (2014)99

conducted a study on clinical and

microbiological effects of photodynamic therapy associated with non-surgical

treatment in aggressive periodontitis. A.a comitans count was evaluated 3 months

after treatment using qPCR method. Bacterial count was significantly reduced after

treatment and mean count of pathogenic microorganism were found to differ

significantly across different probe depth.

Review of literature

Page 22

Milne et al in 2015 evaluate the periodontal pathogen level after using Er

YAG laser in chronic periodontitis patients using qPCR method. He found that

T.denticola and T.forsythia were reduced after treatment.

Materials and methods

Page 23

MATERIALS AND METHODS

The study population was selected from the outpatient sections of the Department of

periodontics, Tamil Nadu Government Dental College & Hospital, Chennai.

CRITERIA FOR SELECTION

Inclusion criteria:

Patients in the age group of 20-50 years.

Good general health.

Presence of at least one tooth with a probing depth of 4–6 mm in each

quadrant.

Patients with established willingness and ability to perform adequate oral

hygiene.

Exclusion criteria:

Patients who are suffering from any known systemic diseases or immune-

compromised patients.

Patients who had received any surgical or non-surgical therapy twelve months

prior to the start of the study.

Patients who had received any antibiotic therapy in the last six month.

Furcation involvement of any tooth

Patients with habit of betel nut, pan masala and tobacco chewing

Patients who are alcoholics.

Pregnant and lactating females.

STUDY DESIGN

Ethical clearances were obtained from the Institution Ethical Committee and

the ethical principles were meticulously followed throughout the study. Subjects for

the study were selected randomly if they are fulfilling inclusion criteria, with no

Materials and methods

Page 24

discrimination on the basis of sex, caste, religion or socioeconomic status as long as

they are ready to follow oral hygiene instruction and other pre-operative and post-

operative instructions. After detailed explanation of the treatment procedure its risk

and advantage, written informed consent was obtained from all the subjects selected

for the study.

Clinical examination was preceded by complete dental and medical history. A

total of 20 patients were selected for the study. The study is a split mouth type and the

study participants were recruited prospectively in this study. Patients suffering from

chronic periodontitis with a probing depth of 4-6mm were selected. Each case was

divided into 2 treatment groups.

Group I: SRP (Scaling and root planing) + laser

Group II: SRP + photosensitizer + laser

STUDY PROTOCOL

1. Institutional Ethical Committee approval

2. Medical history and informed consent

3. Intraoral evaluation and periodontal examination using clinical parameters

namely plaque index, gingival bleeding index, probing depth and clinical

attachment level.

4. Collection of sub gingival plaque for microbiological evaluation.

5. DNA isolation and real time PCR evaluation of P.gingivalis.

6. Clinical photographs

7. Phase I therapy (scaling and root planing)

8. Adjunctive treatments are done using laser and photodynamic therapy

One day after scaling and root planning

Seven days after scaling and root planning

Materials and methods

Page 25

10. Post-operative care

11. Clinical evaluation after 3 months and 6 months.

12. Microbiological evaluation after 6 months.

ARMAMENTARIUM

Clinical examination and sample collection:

Mouth mirror

Williams periodontal probe

Tweezer

Face mask

Head cap

Sterilized disposable gloves

Cotton rolls

Sterile Hufriedy curettes

30 size paper points

1.5 ml micro centrifuge tube

Thermocol box with gel pack

DNA extraction:

QIAGEN DNA extraction kit

Eppendorf tube

Micropipette

Micropipette tips

Vortex

Heat block

Centrifuge

Materials and methods

Page 26

-800C freezer(Preservation of extracted DNA)

Polymerase chain reaction:

Real time plate

Micropipette ,filter tips(10 & 200 micro liter)

Micro centrifuge

Eppendorf tube

Nucleus free water

Primers

Real time PCR machine

For Phase I Therapy:

Mouth Mirror

Explorer

Ultrasonic scaler and Curettes

Gauze pieces

Disposable Gloves, facemask and head cap

Disposable syringe

Local anaesthetic solution (2% LIGNOX)

0.9% normal saline

Laser treatment:

Diode laser unit

Local anaesthetic solution( If needed)

Saline solution

Sterile Gauze pieces

Photodynamic therapy

Indocyanin green dye

Materials and methods

Page 27

Insulin needle

Laser unit

Sterile gauze pieces

Saline solution

Local anaesthesia (If needed)

METHODOLOGY OF CLINICAL EXAMINATION

Patient was examined by mouth mirror and William’s periodontal probe to

assess the overall oral health and periodontal health parameters.

CLINICAL PARAMETERS:

1. Gingival Bleeding Index (AINAMO & BAY)

2. Plaque index (Silness and Loe, 1964) (PI)

3. Pocket Probing depth.

4. Clinical attachment level.

Plaque Index (Sillness and Loe 1964)

All teeth were examined at 4 sites each (disto-facial, facial, mesio-facial,

lingual / palatal) and were scored as follows:

Criteria for Scoring:

Score 0-No plaque

Score 1-Plaque not visible to the naked eye, detected only by running the explorer or

by using a disclosing agent.

Score 2-Thin to moderate accumulation of soft deposits within the gingival pocket or

on tooth and gingival margin, visible to the naked eye.

Score 3-Abundance of soft matter within gingival pocket and / or on tooth surface and

margin, inter-dental area stuffed with soft debris.

Materials and methods

Page 28

Calculation:

Plaque index per tooth = Total score / 4

Plaque index per individual ═ Total PI per tooth/Total number of teeth examined

Interpretation:

Score 0 = Excellent oral hygiene

0.1 to 0.9 = Good oral hygiene

1.0 to 1.9 = Fair oral hygiene

2.0 to 3.0 = Poor oral hygiene

Gingival Bleeding Index (Ainamo & Bay 1975):

Starting distobuccally, the probe was inserted slightly into the sulcus and run

to the buccal and mesial surfaces of every tooth at an angle of about 45°. This was

repeated for all teeth present. Probing was similarly carried out at palatal/lingual sites.

The total number of bleeding sites per tooth was thus recorded for every tooth except

the third molar.

Criteria for Scoring:

Positive score (+) - Presence of bleeding within 10 seconds

Negative score (-) - Absence of bleeding

% of bleeding sites = Total number of positive score x 100

Total number of surfaces of all teeth

Probing pocket depth in mm (PPD):

Probing pocket depth is measured from the gingival margin to the base of the

pocket using Williams periodontal probe. The probe is passed under the gingiva along

the circumference of the tooth. Three measurements are made on the buccal aspect

and three on the lingual aspect of each tooth- total six sites per tooth.

Materials and methods

Page 29

Calculation:

PPD per tooth = sum of all scores per tooth

6

Mean PPD per person = sum of each tooth score

Total number of teeth examined

Clinical Attachment Level:

Clinical attachment level is measured from the cement enamel junction to the

base of the pocket using William’s periodontal probe.

When the gingival margin is located on the anatomic crown, the level of

attachment is determined by subtracting the distance from gingival margin to cemento

enamel junction from the probing pocket depth. If both are same loss of attachment is

zero.

When the gingival margin coincides with the cement enamel junction, loss of

attachment equals to probing pocket depth.

When the gingival margin is located apical to cemento enamel junction, loss

of attachment is greater than the probing pocket depth and therefore the distance

between the cement enamel junction and gingival margin should be added to probing

pocket depth.

Three measurements are made on the buccal aspect and three on the lingual

aspect of each tooth- total six sites per tooth.

Calculation:

CAL per tooth= = sum of all scores per tooth

6

Mean CAL per person = sum of each tooth score

Total number of teeth examined

Materials and methods

Page 30

COLLECTION OF SUBGINGIVAL PLQUE SAMPLE

Experimental site with the deepest probing depth and with no endodontic or

furcation involvement was selected from each patient for microbiological analysis.

Selected sites were localized in two contralateral hemi arches and belong to the same

tooth morphology. Periodontal pocket depth of 4–6 mm was set as experimental site.

Experimental tooth is a tooth with at least one experimental site. At baseline and 6

months after treatment, sub gingival plaque samples were collected from group1 and

group 2. Supragingival plaque and calculus were removed using sterile periodontal

curette. Tooth surface were dried using cotton and plaque sample was taken by the

introduction of two sterile no. 30 paper points into the pocket for 30 s. Paper points

were taken out and kept in a sterile 1.5 ml eppendorf tube. Samples were placed

inside a sealed thermocol box containing gel pack and transported to laboratory for

microbiological analysis.

DNA EXTRACTION

The procedure was done according to the protocol mentioned in QIAGEN DNA

extraction kit. This protocol is for isolation of DNA from paper points.

Methods are:

Add 300 μl Buffer ATL and 10 μl proteinase K, to the mirocentrifuge tube

containing paper points, and mix by pulse vortexing for 10 s. keep it in a

heating block at 560 C for one hour , vortex the tube for 10 s every 10 min

to improve lysis.

Briefly centrifuge the tube to remove drops from the inside of the lid.

Add 150 μl Buffer AL and 5 μl carrier RNA close the lid, and mix by

pulse-vortexing for 10 s.

Materials and methods

Page 31

Place the tube in a heating block of 700C for 10minutes, vortex the tube for

10 s every 3 min to improve lysis.

Briefly centrifuge the 1.5 ml tube to remove drops from the inside of the

lid.

Add 100μl ethanol (96–100%), close the lid, and mix thoroughly by pulse

vortexing for 15 s.

Briefly centrifuge the 1.5 ml tube to remove drops from the inside of the

lid.

Carefully transfer the supernatant to the QIAamp MinElute column (in a 2

ml collection tube) without wetting the rim. Close the lid, and centrifuge at

8000 rpm for 1 min.

Place the QIAamp MinElute column in a clean 2 ml collection tube, and

discard the collection tube containing the flow-through.

Carefully open the QIAamp MinElute column and add 250 μl Buffer AW1

without wetting the rim. Close the lid and centrifuge at 8000 rpm for 1

min.

Place the QIAamp MinElute Column in a clean 2 ml collection tube, and

discard the collection tube containing the flow-through.

Carefully open the QIAamp MinElute column and add 350 μl Buffer AW2

without wetting the rim. Close the lid and centrifuge at 6000 x g (8000

rpm) for 1 min.

Place the QIAamp MinElute column in a clean 2 ml collection tube, and

discard the collection tube containing the flow-through.

Materials and methods

Page 32

Carefully open the QIAamp MinElute column and add 350 μl of ethanol

(96–100%) without wetting the rim. Close the cap and centrifuge at 8000

rpm for 1 min.

Place the QIAamp MinElute column in a clean 2 ml collection tube, and

discard the collection tube containing the flow-through.

Centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min to dry the

membrane completely.

Place the QIAamp MinElute column in a clean 1.5 ml microcentrifuge

tube and discard the collection tube containing the flow-through.

Carefully open the lid of the QIAamp MinElute column, and incubate at

56°C for 3 min.

Apply 20 μl Buffer ATE or distilled water to the center of the membrane.

Close the lid and incubate at room temperature for 5 min. Centrifuge at full

speed 14,000 rpm for 1 min.

Transfer the extracted DNA to a microcentrifuge tube and store at -800c.

QUANTITATIVE POLYMERASE CHAIN REACTION

(QPCR/ REAL TIME PCR)

It is a variant of the basic PCR technique. It is used to measure the quantity

of a target sequence (commonly in real-time). The present study is evaluating the

fimA gene of P.gingivalis.

Materials and methods

Page 33

Primer used in this study:

Sl.no Primer

name

Primer sequence Annealing

temperature(oC)

Fragment

size(bp)

1 P.g (fimA)

forward 5’‑ATA ATG GAG

AAC AGC AGG

AA‑3’

580

C 131 bp

2 P.g (fimA)

reverse 5’‑TCT TGC CAA

CCA GTT CCA

TTG C‑3’

580

C

131 bp

Technique:

Quantitative PCR (qPCR) for fimA gene of P.gingivalis was carried out

following SYBR Green chemistry using SYBR Green dye (Takara, USA, Cat #

RR420) following manufacturer’s instruction in Real time thermal cycler

(LightCycler96, Roche, Switzerland).

Recipe:

Composition Volume

(in µl)

SYBR green master mix : 5.0

Forward primer : 0.5

Reverse primer : 0.5

DNA template : 1.0

Nuclease Free Water (NFW) : 3.0

Total 10.0

The concentration of DNA template was 60ng/10μl reaction.

Materials and methods

Page 34

Reaction condition:

Construction of standard curve:

The standard curve for expression studies was constructed.

i. A representative volume of 5μl cDNA from all the samples was pooled.

ii. The pooled cDNA was diluted serially in five-fold. Six dilutions of the

pooled cDNA sample were done with a known total input RNA

concentration (1000ng, 200ng, 40ng).

iii. 1μl of the serially diluted cDNA was used as PCR template in a 10μl

reaction and the standard curve was constructed.

iv. The curve parameters like slope, intercept and r2 were estimated and the

curve parameters were used for absolute quantification of the gene under

study.

OAT is the Optimal Annealing Temperature

Fluorescence Melting Point

Materials and methods

Page 35

Data analysis

The expression of the gene under study was analyzed by absolute

quantification method using the curve parameters. The post treatment samples were

normalized against the samples before treatment. The difference in expression level of

the gene under study was expressed as fold induction.

TREATMENT PROCEDURE

Following screening, all patients were consented to the planned treatment

strategy. All patients were subjected to a full-mouth periodontal examination at six

sites per tooth. After oral hygiene instructions, all patients received full-mouth scaling

using ultrasonic device. Patients were reviewed after 5 days. Periodontal pocket depth

of 4–6 mm was set as experimental site. Experimental tooth referred to a tooth with at

least one experimental site. Right side of both the arches were assigned as group

1(SRP + laser) and left side as group 2(SRP + photosensitizer+ laser).

Root planing was done under local anaesthesia using Hufriedy universal

curette. One day after SRP diode laser was applied to the experimental tooth. Diode

laser of 960nm at a power output of 2 watt in pulsed mode was used. Optic fiber of

y = -2.8411x + 25.073 R² = 1

15

17

19

21

23

25

27

29

31

33

-3 -2 -1 0 1 2 3 4

Cq

val

ue

s

DNA concentration (Log)

Standard curve

Materials and methods

Page 36

400 micrometer was introduced in the periodontal pocket parallel to the long axis of

the tooth, one millimeter coronal to the base of the pocket, and it is moved coronally

with sweeping movements. Pocket was lased for 30s twice. On the other side of the

arch, indocyanin green (ICG) was meticulously applied using insulin needle at the

bottom of the sulcus in a coronal direction. Three minutes after application of

photosensitizer, removed the excess photosensitizer using gauze piece and irradiate

the area with diode laser at a power output of 1.5 watt in pulsed mode. Both the

treatments were repeated in the same manner after 7days. Post-operative instructions

were given.

Photographs

Page 37

Photograph 1: Armamentarium for sample collection

Photograph 2: Sample collection

Photographs

Page 38

Photograph 3: Sample transport

Photograph 4: Armamentarium for non-surgical periodontal treatment

Photographs

Page 39

Photograph 5: Indocyanin green dye

Photograph 6: Diode laser unit

Photographs

Page 40

Photograph 7: Pre-operative photograph

Photograph 8: Preoperative photograph

Photographs

Page 41

Photograph 9: Indocyanin green applied

Photograph 10: Laser therapy

Photographs

Page 42

Photograph 11: Post-operative after 6 months

Photograph 12: Post-operative after 6 months

Photographs

Page 43

Photograph 13: Armamentarium for DNA extraction and real time PCR

Photograph 14: Heating bath

Photographs

Page 44

Photograph 15: Vortex

Photograph 16: Centrifuge

Statistical analysis

Page 45

STATISTICAL ANALYSIS

Data analysis was performed using the patient as the experimental unit. For all

parameters, the mean values per subject and per visit were calculated. The statistical

analysis was done using the computer software program SPSS version 16.0

(Statistical Package for Social Science, Version 16). Descriptive data are presented as

mean ± SD and range values.

STATISTICAL TEST USED

Intra group analysis:

Multivariate ANOVA with Bonferroni Post hoc test: Statistical test for

clinical evaluation after treatment on the basis of time interval. The mean difference is

significant at the .05 level.

Wilcoxon Signed Ranks Test: Statistical test for microbiological evaluation

after treatment on the basis of time interval.

Inter group analysis:

Mann- Whitney Test: Statistical test for clinical and microbiological

evaluation on the basis of time interval.

Result

Page 46

RESULT

This study was evaluated for a period of 6 months. A total of 20 patients were

selected for the study and 3 patients were excluded late (2 patients didn’t report back

after the treatment and one patient started antibiotic in between the procedure). 17

patients completed the 6 month study period and all the patients were kept under

regular maintenance therapy (Table 1, 2, 3). Clinical and microbiological evaluations

were carried out over a period of 6 months and observations were tabulated as

follows.

INTRA GROUP ANALYSIS

Plaque index

Group 1: Mean plaque score at baseline was 1.8118 ± 0.17640 indicating a fair

to poor oral hygiene with all the subjects (Table 4). The mean plaque score at end of 3

and 6 months after treatment was found to be 0.9412 ± 0.20826 and 0.9047 ± 0.16648

respectively (Table 4). When compared to the baseline, the mean plaque score at 3rd

and 6th

months showed statistically significant decrease (p = 0.000) (Table 6). When

compared to 2nd

month, 3rd

month mean plaque score was reduced but not statistically

significant (p= 0.689) (Table 6).

Group 2: Mean plaque score at base line was 1.7806 ± 0.17134 indicating a

fair to poor oral hygiene with all the subjects (Table 5). The mean plaque score at the

end of 3rd

and 6th

months after treatment was found to be 0.9012 ± 0.16804 and

0.9218 ± 0.15824 respectively (Table 5). When compared to the baseline, the mean

plaque score at 3rd

and 6th

months showed significant decrease (p = 0.000) (Table 11).

When compared to 2nd

month, 3rd

month mean plaque score was slightly increased but

not statistically significant (p= 1.000) (Table 11).

Result

Page 47

Gingival Bleeding index

Group 1: Mean score at baseline was 90.8835 ± 4.90702 (Table 4). The mean

BI score at end of 3 and 6 months after treatment was found to be 16.6000 ± 6.11036

and 14.7924 ± 4.31042 respectively (Table 4). When compared to the baseline, mean

BI score at 3rd

and 6th

months showed statistically significant reduction (p = 0.000)

(Table 7). When compared to 2nd

month, 3rd

month mean score was less but not

statistically significant (p= 0.112) (Table 7).

Group 2: Mean score at baseline was 91.2935 ± 4.83352 (Table 5). The mean

BI score at end of 3 and 6 months after treatment was found to be 15.7253 ± 5.25515

and 14.5035 ± 4.90076 respectively (Table 5). When compared to the baseline, mean

BI score at 3rd

and 6th

months showed statistically significant reduction (p = 0.000)

(Table 12). When compared to 2nd

month, 3rd

month mean also showed significant

decrease in the value (p= 0.05) (Table 12).

Probing depth

Group1: Mean score at baseline was 5.0835 ± 0.19532(Table 4). Mean PD at

the end of 3 and 6 months after treatment was found to be 2.9165 ± 0.39995 and

3.3776 ± 0.34144 respectively (Table 4). When compared to the baseline, mean PD at

3rd

and 6th

months showed statistically significant reduction (p = 0.000) (Table 8).

When compared to 2nd

month, 3rd

month mean was also showed significant decrease

(p= 0.000) (Table 8).

Group 2: Mean score at baseline was 5.1794 ± 0.19772 (Table 5). Mean PD at

the end of 3 and 6 months after treatment was found to be 2.9065 ± 0.35107 and

2.9106 ± 0.40532 respectively (Table 5). When compared to the baseline, mean PD at

3rd

and 6th

months showed statistically significant reduction (p = 0.000) (Table13).

Result

Page 48

When compared to 2nd

month, 3rd

month mean was slightly increased but not

statistically significant (p= 1.000) (Table13).

Clinical attachment level

Group 1: Mean score at baseline was 6.0788 ± 0.31219 (Table 4). Mean CAL

at the end of 3 and 6 months after treatment was found to be 3.3088 ± 0.28926 and

3.2376 ± 0.34525 respectively (Table 4). When compared to the baseline, mean CAL

at 3rd

and 6th

months showed statistically significant reduction (p = 0.000) (Table 9).

When compared to 2nd

month, 3rd

month mean was reduced but not statistically

significant (p= .746) (Table 9).

Group 2: Mean score at baseline was 6.0712 ± 0.31131(Table 5). Mean CAL

at the end of 3 and 6 months after treatment was found to be 3.1776 ± 0.31414 and

3.1818 ± 0.34970 respectively (Table 5). When compared to the baseline, mean CAL

at 3rd

and 6th

months showed statistically significant reduction (p = 0.000) (Table 14).

When compared to 2nd

month, 3rd

month mean was slightly increased but not

statistically significant (p= 1.000) (Table 14).

Microbiological evaluation

Group 1: When compared to baseline, 6th

month P.gingivalis count was less

and statistically significant (p=0.055) (Table 10).

Group 2: When compared to baseline, 6th

month P.gingivalis count was less

and statistically significant (p=0.043) (Table 15).

INTER GROUP ANALYSIS

Clinical parameters

No statistically significant differences were observed in PI, GI and CAL

between the two groups after treatment at 3rd

and 6th

month. When compared to group

Result

Page 49

1, probing depth of group 2 was significantly reduced from 3rd

month to 6th

month and

from baseline to 6th

month (p=0.000) (Table 16).

Microbiological evaluation

Even though there was significant reduction of p.gingivalis in both the groups,

no statistically significant difference was observed between the two groups after 6

months (p=0.808) (Table 16).

Tab

les

Page 5

0

MA

ST

ER

CH

AR

T

TA

BL

E 1

: BA

SE

LIN

E M

EA

SU

RE

ME

NT

S

GR

OU

P I

G

RO

UP

II

S.N

O

AG

E/S

EX

P

I B

I P

PD

C

AL

M

E

PI

BI

PP

D

CA

L

ME

1

28

/M

1.8

6

96

5.0

1

6.1

2

1

1.8

6

96

5.1

5

6.1

2

1

2

39

/M

1.7

8

94

.72

5.3

2

5.3

2

1

1.7

5

94

.72

5.3

2

5.3

2

1

3

38

/F

1.7

9

95

.13

5.1

2

6.4

4

1

1.7

9

95

5.1

2

6.4

1

1

4

35

/M

1.7

1

81

.1

5.0

9

6.0

9

1

1.7

8

2.3

5

.1

6.0

9

1

5

30

/M

1.9

9

2.2

3

5.2

5

6.1

5

1

1.9

9

2.2

3

5.2

4

6.1

2

1

6

38

/F

1.6

9

4.1

2

5.6

1

6.2

1

1

1.6

9

5.1

2

5.5

4

6.2

1

7

27

/M

1.8

1

81

.36

4.9

5

5.8

5

1

1.8

2

82

.3

5.1

2

5.8

5

1

8

49

/M

1.9

8

8.1

2

4.8

6

5.8

6

1

1.9

4

88

.12

4.9

8

5.8

6

1

9

45

/F

1.5

8

91

.9

5.1

3

6.2

3

1

1.5

8

94

.9

5.1

3

6.2

1

10

33

/M

1.6

4

92

.3

4.9

1

6.1

1

1

1.6

4

92

.3

4.9

1

6.1

1

11

32

/M

1.6

9

95

.04

4.8

6

5.9

5

1

1.7

9

6.0

1

5.2

5

6.0

1

1

12

42

/F

1.5

3

96

.23

5.1

3

5.6

7

1

1.5

3

96

.23

5.0

2

5.6

1

1

13

33

/F

1.8

9

4.2

4

.91

6.4

2

1

1.8

9

4.2

5

.05

6.4

1

14

44

/F

2.0

1

92

.11

5.2

6

.71

1

2.0

1

92

.1

5.7

1

6.7

1

1

15

38

/M

2.1

8

8.2

4

.87

6.0

1

1

1.9

7

88

.2

5.2

1

6.0

1

1

16

42

/M

1.9

8

84

.37

5.0

8

6.0

8

1

2.1

2

84

.37

5.0

8

6.0

8

1

17

36

/F

2.1

2

87

.89

5.1

2

6.1

2

1

1.5

6

87

.89

5.1

2

6.1

2

1

Tab

les

Page 5

1

TA

BL

E 2

: TH

IRD

MO

NT

H M

EA

SU

RE

ME

NT

S

G

RO

UP

I G

RO

UP

II

S.N

O

AG

E/S

EX

P

I B

I P

PD

C

AL

P

I B

I P

PD

C

AL

1

28

/M

0.7

8

12

.1

3.0

5

3.6

5

0.7

6

11

.15

3.1

3

.62

2

39

/M

0.8

9

15

.12

3.3

3

.41

0.9

2

15

.1

3.2

5

3.4

1

3

38

/F

0.9

7

28

.05

2.6

5

2.8

1

0.9

6

24

.05

2.6

5

2.8

2

4

35

/M

0.8

2

18

2.9

1

3.3

0

.82

18

.00

2.8

7

3.4

5

5

30

/M

1.0

1

10

.77

3.0

2

3.2

5

1.2

1

10

.00

2.9

8

3.1

5

6

38

/F

0.8

5

24

.33

3.8

3

3.7

5

0.8

4

22

.3

3.4

5

3.6

5

7

27

/M

0.5

2

23

.46

2.6

5

2.8

2

0.4

8

22

.36

2.3

5

2.8

2

8

49

/M

1.0

5

14

.23

3.4

2

3.0

1

1.0

1

14

.23

2.6

5

3.0

1

9

45

/F

0.7

2

30

.01

2.8

4

3.5

0

.93

27

.18

2.9

8

3.5

10

33

/M

1.0

8

13

.5

2.6

5

3.5

2

1.0

8

12

.5

3.1

2

3.5

2

11

32

/M

0.9

2

11

.8

3.5

2

3.3

5

0.8

9

11

.8

3.0

1

3.3

2

12

42

/F

0.7

8

15

.83

2.6

9

2.8

5

0.7

5

15

.83

2.0

1

2.8

5

13

33

/F

0.8

7

11

.56

2.5

4

3.6

5

0.7

2

10

.17

3.1

5

3.6

2

14

44

/F

1.4

2

13

.4

2.5

3

.25

0.9

1

12

.12

2.9

9

3.3

1

15

38

/M

1.2

4

16

.2

2.4

9

3.4

5

0.9

8

15

.17

3.1

2

3.4

16

42

/M

0.9

7

10

.74

2.5

2

3.3

1

0.9

8

11

.25

3.0

8

3.2

8

17

36

/F

1.1

1

13

.1

3.0

0

3.3

7

1.0

8

14

.12

2.6

5

3.3

6

Tab

les

Page 5

2

TA

BL

E 3

: SIX

TH

MO

NT

H M

EA

SU

RE

ME

NT

S

GR

OU

P I

G

RO

UP

II

S.N

O

P

I G

BI

PP

D

CA

L M

E

PI

GB

I P

PD

C

AL

M

E

1

0

.75

9.8

3

.12

3.3

5

0.0

05

0.7

4

11

.01

3.1

1

3.3

7

0.1

68

2

0

.92

12

.1

3.4

5

3.2

5

0.0

25

0.9

5

12

.1

3.2

5

3.2

7

0

3

0

.97

22

.12

3.2

3

2.8

2

0.0

0

0.9

5

22

.01

2.7

5

2.8

7

0

4

0

.87

18

3.1

5

2.4

4

.96

8

0.9

8

17

.18

2.3

2

3.4

5

8.9

67

5

1

.02

11

.5

3.3

4

3.1

0

.00

1

1.2

8

11

.1

2.8

7

3.1

7

0

6

1

.15

22

.1

4.2

8

3.8

0

.00

5

1.0

1

20

.13

3.5

2

3.7

1

0.1

66

7

0

.52

12

.25

3.1

8

2.8

9

1.4

77

0.6

7

17

.36

2.2

5

2.8

7

0.2

22

8

0

.98

16

.24

3.4

6

3.2

5

0.0

01

0.9

5

15

.61

2.7

2

3.2

1

0

9

0

.72

24

.2

3.1

3

.35

0.0

04

0.8

7

27

.21

3.0

1

3.3

4

8.3

88

10

1

.01

12

.28

3.0

5

3.5

7

0.0

01

1.2

3

11

.12

3.2

2

3.5

4

0

11

0

.85

12

.04

3.6

8

3.4

0

.02

3

0.7

9

10

.75

3.1

5

3.3

9

0.8

51

12

0

.79

14

.02

3.8

8

2.8

5

0.0

0

0.8

2

12

.15

2.0

1

2.8

5

0.2

78

13

0

.77

11

.8

3.2

3

.68

4.9

68

0.7

5

11

.01

3.3

5

3.6

8

1.0

91

14

1

.15

12

.23

3.1

3

.25

0.2

22

0.9

5

10

.01

2.8

5

3.3

0

15

1

.12

15

.45

3.3

6

3.4

8

0.0

25

0.9

2

15

.45

3.0

1

3.4

8

0.2

78

16

0

.88

12

.24

3.0

9

3.3

0

.00

4

0.9

6

12

.24

3.1

8

3.3

0

.85

1

17

0

.91

13

.1

3.7

5

3.3

1

.09

1

0.8

5

10

.12

2.9

1

3.3

7

0.0

23

Tab

les

P

age 53

ME

AN

AN

D S

TA

ND

AR

D D

EV

IAT

ION

OF

CL

INIC

AL

PA

RA

ME

TE

RS

TA

BL

E 4

: GR

OU

P 1

PA

RA

ME

TE

RS

PI

B

PI

3rd

M

PI

6th M

BI

B

BI

3rd M

BI

6th M

PD

B

PD

3rd M

PD

6th M

CA

L

B

CA

L

3rd M

CA

L

6th M

ME

AN

1

.81

18

.941

2

.904

7

90

.883

5 1

6.6

00

0 1

4.7

92

4 5

.08

35

2.9

165

3.3

776

6.0

788

3.3

088

3.2

376

SD

.1

76

40

.208

26

.166

48

4.9

070

2 6

.11

03

6 4

.31

04

2 .1

95

32

.399

95

.341

44

.312

19

.289

26

.345

25

N

17

TA

BL

E 5

: GR

OU

P 2

PA

RA

ME

TE

RS

PI

B

PI

3rd

M

PI

6th M

BI

B

BI

3rd

M

BI

6th M

PD

B

PD

3rd

M

PD

6th M

CA

L

B

CA

L

3rd

M

CA

L

6th M

ME

AN

1

.78

06

.90

12

.92

18

91

.293

5

15

.725

3

14

.503

5

5.1

794

2.9

065

2.9

106

6

.07

12

3.1

776

3.1

818

SD

.1

71

34

.16

804

.15

824

4.8

335

2

5.2

551

5

4.9

007

6

.19

772

.35

107

.40

532

.3

11

31

.31

414

.34

970

N

17

Tables

Page 54

INTRA GROUP STATISTICAL ANALYSIS

GROUP 1: PAIRWISE COMPARISONS WITH BONFERRONI POST HOC

TEST

*. The mean difference is significant at the .05 level.

a. Adjustment for multiple comparisons: Bonferroni.

TABLE 6: PLAQUE INDEX

(I)

Time

(J)Time

Mean

difference

(I-J)

Std.

Error

Siga

95% Confidence Interval for

Differencea

Lower Bound Lower Bound

1

2 .871* .042 .000 .758 .758

3 .907* .050 .000 .774 .774

2

1 -.871* .042 .000 -.983 -.983

3 .036 .029 .689 -.042 -.042

3

1 -.907* .050 .000 -1.040 -1.040

2 -.036 .029 .689 -.115 -.115

TABLE 7: BLEEDING INDEX

(I)

time

(J)time

Mean

difference

(I-J)

Std.

Error

Siga

95% Confidence Interval for

Differencea

Lower Bound Lower Bound

1

2 74.284* 1.941 .000 69.095 79.472

3 76.091* 1.583 .000 71.861 80.322

2

1 -74.284* 1.941 .000 -79.472 -69.095

3 1.808 .795 .112 -.318 3.934

3

1 -76.091* 1.583 .000 -80.322 -71.861

2 -1.808 .795 .112 -3.934 .318

Tables

Page 55

TABLE 8: PROBING DEPTH

(I)

Time (J)Time

Mean

Difference

(I-J)

Std.

Error

Siga

95% Confidence Interval

for Differencea

Lower

Bound

Lower

Bound

1

2 2.167* .089 .000 1.929 2.406

3 1.706* .073 .000 1.512 1.900

2

1 -2.167* .089 .000 -2.406 -1.929

3 -.461* .074 .000 -.660 -.263

3

1 -1.706* .073 .000 -1.900 -1.512

2 .461* .074 .000 .263 .660

TABLE 9: CLINICAL ATTACHMENT LEVEL

(I)

Time (J)Time

Mean

Difference

(I-J)

Std.

Error

Siga

95% Confidence Interval for

Differencea

Lower

Bound

Lower

Bound

1

2 2.770* .093 .000 2.522 3.018

3 2.841* .103 .000 2.565 3.117

2

1 -2.770* .093 .000 -3.018 -2.522

3 .071 .059 .746 -.088 .230

3

1 -2.841* .103 .000 -3.117 -2.565

2 -.071 .059 .746 -.230 .088

Tables

Page 56

GROUP 1 MICROBIOLOGICAL ANALYSIS

TABLE 10: WILCOXON SIGNED RANKS TEST

Ranks

N Mean Rank Sum of Ranks

SixthonthME -

baselineME

Negative

Ranks 13

a 9.00 117.00

Positive

Ranks 4

b 9.00 36.00

Ties 0c

Total 17

a. SixthonthME < baselineME

b. SixthonthME > baselineME

c. SixthonthME = baselineME

Test Statisticsb

SixthonthME - baselineME

Z -1.920a

Asymp. Sig. (2-tailed) .055

a. Based on positive ranks.

b. Wilcoxon Signed Ranks Test

Tables

Page 57

GROUP 2: PAIRWISE COMPARISONS WITH BONFERRONI POST HOC

TEST

*. The mean difference is significant at the .05 level.

a. Adjustment for multiple comparisons: Bonferroni.

TABLE 11: PLAQUE INDEX

(I)

Time (J)Time

Mean

Difference

(I-J)

Std.

Error

Sig

95% Confidence Interval

for Differencea

Lower

Bound

Lower

Bound

1

2 .879* .055 .000 .733 1.026

3 .859* .054 .000 .714 1.004

2

1 -.879* .055 .000 -1.026 -.733

3 -.021 .027 1.000 -.092 .051

3

1 -.859* .054 .000 -1.004 -.714

2 .021 .027 1.000 -.051 .092

TABLE 12: BLEEDING INDEX

(I)

Time (J)Time

Mean

Difference

(I-J)

Std.

Error

Siga

95% Confidence Interval

for Differencea

Lower

Bound

Lower

Bound

1

2 75.568* 1.751 .000 70.887 80.249

3 76.790* 1.680 .000 72.299 81.281

2

1 -75.568* 1.751 .000 -80.249 -70.887

3 1.222 .470 .058 -.034 2.478

3

1 -76.790* 1.680 .000 -81.281 -72.299

2 -1.222 .470 .058 -2.478 .034

Tables

Page 58

TABLE 13: PROBING DEPTH

(I)

Time (J)Time

Mean

Difference

(I-J)

Std.

Error

Siga

95% Confidence Interval for

Differencea

Lower

Bound

Lower

Bound

1

2 2.273* .079 .000 2.062 2.484

3 2.269* .097 .000 2.010 2.528

2

1 -2.273* .079 .000 -2.484 -2.062

3 -.004 .044 1.000 -.121 .113

3

1 -2.269* .097 .000 -2.528 -2.010

2 .004 .044 1.000 -.113 .121

TABLE 14: CLINICAL ATTACHMENT LEVEL

(I)

Time (J)Time

Mean

Difference

(I-J)

Std.

Error

Siga

95% Confidence Interval for

Differencea

Lower

Bound

Lower

Bound

1

2 2.894* .094 .000 2.642 3.145

3 2.889* .097 .000 2.631 3.148

2

1 -2.894* .094 .000 -3.145 -2.642

3 -.004 .024 1.000 -.067 .059

3

1 -2.889* .097 .000 -3.148 -2.631

2 .004 .024 1.000 -.059 .067

Tables

Page 59

GROUP 2 MICROBIOLOGICAL ANALYSIS

TABLE 15: WILCOXON SIGNED RANKS TEST

Ranks

N Mean Rank Sum of Ranks

SixthonthME -

baselineME

Negative

Ranks 14

a 8.50 119.00

Positive

Ranks 3

b 11.33 34.00

Ties 0c

Total 17

a. SixthonthME < baselineME

b. SixthonthME > baselineME

c. SixthonthME = baselineME

Test Statisticsb

SixthonthME - baselineME

Z -2.022a

Asymp. Sig. (2-tailed) .043

a. Based on positive ranks.

b. Wilcoxon Signed Ranks Test

Tab

les

P

age 60

INT

ER

GR

OU

P S

TA

TIS

TIC

AL

AN

AL

YS

IS

TA

BL

E 1

6: M

AN

N- W

HIT

NE

Y T

ES

T

Diff P

I

B-3

rd

M

Diff P

I

3rd- 6

th

M

Diff P

I

B –

6th

M

Diff B

I

B-3

rd

M

Diff B

I

3rd- 6

th

M

Diff B

I

B –

6th

M

Diff

PD

B-3

rd

M

Diff P

D

3rd- 6

th

M

Diff P

D

B –

6th

M

Diff

CA

L

B-3

rd M

Diff

CA

L

3rd- 6

th

M

Diff C

AL

B –

6th

M

Diff M

E

B –

6th

M

Ma

nn

-

Wh

itney

U

14

3.5

0

93

.500

12

4.0

00

13

0.0

0

13

7.0

0

12

8.5

0

13

8.0

0

20

.500

31

.500

10

0.0

00

1

06

.50

0

13

3.0

00

13

7.5

00

Wilco

xo

n

W

29

6.5

0

24

6.5

00

27

7.0

00

28

3.0

0

29

0.0

0

28

1.5

0

29

1.0

0

17

3.5

00

18

4.5

00

25

3.0

00

2

59

.50

0

28

6.0

00

29

0.5

00

Z

-.03

4

-1.7

59

-.70

7

-.50

0

-.25

8

-.55

1

-.22

4

-4.2

75

-3.8

93

-1.5

33

-1

.31

4

-.39

6

-.24

3

Asy

mp

.

Sig

. (2-

tailed

)

.97

2

.07

9

.48

0

.61

7

.79

6

.58

1

.82

3

.00

0

.00

0

.12

5

.18

9

.69

2

.80

8

Exa

ct Sig

.

[2*

(1-ta

iled

Sig

.)]

.97

3a

.07

9a

.49

6a

.63

4a

.81

2a

.58

6a

.83

8a

.00

0a

.00

0a

.13

1a

.19

3a

.70

8a

.81

2a

Figures

Page 61

Figure 1: Changes in clinical parameters for group 1

Figure 2: Changes in clinical parameters for group 2

0

10

20

30

40

50

60

70

80

90

100

Baseline 3 months 6 months

PI

BI

PD

CAL

0

10

20

30

40

50

60

70

80

90

100

BASELINE 3 MONTHS 6 MONTHS

PI

BI

PD

CAL

Discussion

Page 62

DISCUSSION

Periodontal diseases are complex, multifactorial, polymicrobial infections

characterized by the destruction of tooth-supporting tissues. It includes initial

microbial challenge, host inflammatory response, and various risk factors that can

contribute to host susceptibility and disease progression.

Periodontitis is initiated and sustained by microorganisms living in biofilm

communities which are present in supra- and sub gingival plaque in the form of

uncalcified and calcified biofilms. The sub gingival biofilm harbors a variety of

bacterial species and the composition of the biofilm may vary between subjects and

sites.

Among major periodontal pathogens, P. gingivalis appears to be one of the

prime etiological agents in the pathogenesis and progression of the inflammatory

events of periodontal disease (Hajishengallis et al., 2012)100

. This periodontopathic

bacterium was found in 85.75% of subgingival plaque samples from patients with

chronic periodontitis (Datta et al 2008)101

. Various immunological studies also

confirmed that serum antibody levels to P. gingivalis are higher in patients diagnosed

with adult periodontitis (Moore et al 1991102

, Schmidt et al 2014103

).

The basic approach for the management of periodontal infections is the

removal of supra and sub gingival bacterial deposits, oral hygiene instructions and

supportive periodontal maintenance (Carranza) 4

. Scaling and root planning (SRP) has

become the gold standard nonsurgical treatment of periodontitis. Various studies

demonstrated that it effectively reduces the microbial load and leads to reductions in

bleeding on probing , probing depths and gain in clinical attachment(Cobb et al

199619

, Maria Emanuel Ryan 200517

, Doungudomdacha S et al 200118

)

Discussion

Page 63

Perio-pathogens cannot be completely eradicated by conventional SRP.

Deeper the pocket, the more incomplete will be the removal of sub gingival plaque

and calculus, and periodontal pathogens like A. actinomycetemcomitans and P.

gingivalis penetrate the periodontal tissues and become difficult to eliminate.

(Rabbani et al. 198122

and Magnusson et al. 198423

).Faster recolonization of

periodontal pathogen can also occur after conventional SRP (Slots and Ram).

Laser has emerged as an adjunctive procedure in enhancing the clinical

outcome of the periodontal treatment. Lasers with its strong bactericidal and

detoxification effects can achieve excellent tissue ablation. Laser can achieve

excellent healing of periodontal tissues by ablating the inflamed lesions and epithelial

lining of the periodontal pockets (Aoki A, 2004)28

. Combination of laser and

photosensitizer can result in significant reduction of sub gingival microorganisms like

P. gingivalis and A. actinomycetemcomitans(Tobias K Boehm 20119, Dobson &

Wilson 199266

, Pfitzner et al. 200467

, Sigusch et al. 200568

).

Present study was undertaken to evaluate the effectiveness of laser and

photodynamic therapy as an adjunct to nonsurgical periodontal treatment in the

management of chronic periodontitis by means of bacterial reduction and clinical

parameters.

In this study laser and PDT was used as adjunctive treatment. Without SRP

calculus may remain on the root surface, which may serve as a niche for

periodontopathogens.

Severe forms of periodontitis are often associated with systemic disorders and

complications which might have a detrimental effect on the periodontium. Hence

cases with mild to moderate periodontitis were selected for the present study (Cabala

A, 2006104

, Seymour GJ2007105

).

Discussion

Page 64

Microbiological sampling, especially when used in conjunction with clinical

trials, complements the diagnosis, and confirms the results of the research. Real-time

PCR with species specific primers can provide a precise and sensitive method for

quantitation of periodontal pathogen. It can detect nonviable bacterial cells and has a

high detection rate.

In this study quantification of fimA gene of p.gingivalis was done using real

time PCR analysis. fimA gene is essential for binding and invasion of host cells and

it promote the progression of periodontal inflammatory reactions and it (Lee et al.

1992106

,Sharma et al 1993107

).

Study is a split mouth type and the main purpose of split-mouth design is to

remove all components related to differences between subjects from the treatment

comparisons. By this error variance of the study can be reduced and can obtain a

higher statistical power along with smaller number of patients required for the trial

(Hujoel &Loesche 1990108

, Hujoel & DeRouen 1992109

).

The selection of high intensity diode laser was based on the studies which

have shown that its wave length has a better penetration and affinity for the pigments

present in some bacteria which would act as an absorbing chromophorous and it

wound in turn intensify its action and thus make it possible to reach black pigmented

anaerobe such as p.gingivalis (coluzzi dj 200051

).

In the present study repeated application of laser and PDT was done at one

and seven days after SRP. Theodoro et al in 2012110

reported that single application

of PAD or TBO did not improve clinical parameters compared with SRP alone.

. Proper oral hygiene maintenance by the patient is an important factor that

determines the success of periodontal treatment. In this study patient is kept under

proper maintenance therapy and evaluated at three and six months. This frequency of

Discussion

Page 65

supportive maintenance care is also necessary to reduce sub gingival proportions of

pathogens (Renvert & Persson 2004111

).

Reduction in clinical parameters was statistically significant in both the

treatment groups at 3 and 6 months from baseline sample. No statistically significant

difference was found between the groups at different time intervals except in probing

depth. When compared to group1 Slight improvement in bleeding on probing and

clinical attachment level also noted in group 2, but not statistically significant.

In group 1 and group 2 probing depth reduced significantly from baseline to 3

months and 6 months. From 3 months to 6 months there was significant increase in

probing depth in group 1 but in group 2 there was only slight increase which was not

significant.

Difference in probing depth from baseline to 6 months and from 3 months to 6

months is more significant in group 2 when compared to group1.

Result of the present study was in accordance with the findings of a previous

study, in which multiple adjunctive applications of a 980-nm diode laser with SRP

showed probing depth improvements in moderate periodontal pockets of 4–6 mm

(Dukic et al. 2013)58

. Study by Saglam et al. in 201459

, reported that diode laser along

with SRP provided significant improvements in clinical parameters. Kamma et al,

200654

and Caruso et al in 20086 reported that there was a slight improvement of

clinical parameters when compared to SRP alone.

The reduction in PD and gain in CAL in group 1 is similar to the findings of

the study that reported laser irradiation of periodontal pockets, in which pocket

epithelium was eliminated without damaging the collateral blood supply that

promoted periodontal healing as well as attachment (Romanos GE, 2004112

).

Systematic review and meta-analysis by Cheng et al in 2016 concluded that there is

Discussion

Page 66

significant reduction in probing depth at 3 and 6 months. No significant improvement

in CAL was observed in 3 and 6 months.

The current results are in line with clinical studies that reported improvement

in clinical outcomes for the photodynamic therapy in combination with scaling and

root debridement (Andersen et al 200776

, Braun et al 2008113

, Lulic et al 200982

,

Sigusch et al. 2010114

). Andersen et al.in 2007 showed that a combination of SRP and

PAD (diode laser 670 nm and methylene blue as photosensitizer) in patients affected

by moderate to advance periodontitis resulted in significant improvements in clinical

outcomes. Similar results were obtained by two randomized clinical studies that

reported statistically better results in sites treated with adjunctive PAD (diode laser

660 nm and phenothiazine chloride as photosensitizer) compared with SRP alone

(Braun et al. 2008113

, Sigusch et al. 2010).

In 10 maintenance patients with residual pockets (PPD > 5 mm), repeated

applications of PAD (diode laser 670 nm, and phenothiazine chloride as

photosensitizer) resulted in significant improvement of clinical outcomes after 6

months. The authors reported that the mean reduction of PPD in PAD treated patients

were significantly higher than that of control group (0.67 mm versus 0.04 mm) (Lulic

et al. 2009).

Alwaeli et al in 2015115

and Berakdar et al in 2012116

reported that PDT had

greater efficacy in decreasing the probing PD 3 months after treatment, which is in

line with the current findings.

In the present study indocyanin green was used as the photosensitizer.

Indocyanine green with laser irradiation results in significant reduction of P.

gingivalis and A. actinomycetemcomitans, as less than 10% of bacteria remain viable

(Tobias K Boehm 2011)9. Study by Abbas Monzavi

in 2016

117 reported that clinical

Discussion

Page 67

parameters like BI and PPD were significantly reduced when indocyanin green is used

as photosensitizer in the management of chronic peiodontits. Reza Fekrazad in

2016118

reported that P.gingivalis count was significantly reduced by the use of

indocyanin green.

The results showed that most of the clinical and microbiological changes

occurred during the first 3 months after treatment in both groups. From 3 months to 6

months there was only slight change in clinical parameters. All patients in this study

maintained low plaque and inflammation scores throughout the duration of the study

.There was significant reduction in PI and BI from baseline to 6months in both the

groups. Thus, the patients’ own infection control and supportive therapy helps to

maintain periodontal stability (Kornman et al. 1994)119

.

Microbiological evaluation showed significant reduction in p.gingivalis count

from baseline to 6 months in both the groups. But there was no significant difference

between the two groups. Reduction in bacterial count was in accordance with the

previous studies by Moritz et al 199834

and Kamma et al, 200654

in which they

reported significant reduction of microbial count in laser treated cases when compared

to SRP alone.

In this study four samples from group 1 and three samples from group 2

showed increase in p.gingivalis count after 6 months. Bleeding on probing and slight

increase in probing depth was observed in those teeth from which samples were taken.

All the remaining samples showed reduction in P.gingivalis count. From these result

we can conclude that the bactericidal effects of the laser and PDT can be maintained

by low plaque and inflammation score. This result was in accordance with the study

by Schmidt et al 2014103

in which he reported that P. gingivalis count increased in

Discussion

Page 68

sites with periodontitis and reduced or non-detectable in sites with good gingival

health.

The present study demonstrated a significant reduction in probing depth in

PDT treated patients when compared to laser alone group. Microbiological parameter

and the remaining clinical parameters did not show any significant difference between

the groups. These findings are in agreement with the study by Martina Lulic in 200982

in which he reported that PPD, mean CAL and mean BoP percentage reduced

significantly in PDT treated patients than in laser treated at 6 month evaluation. But in

2013 Seyed H. Bassir120

did not find any significant clinical improvement in PDT

treated groups than in laser treated patients.

Summary and conclusion

Page 69

SUMMARY AND CONCLUSION

Gold standard for the management of periodontitis is scaling, root planning

and supportive periodontal maintenance. Laser with its excellent bactericidal and

detoxification effect has emerged as an adjunctive procedure in enhancing the clinical

outcome of periodontal treatment.

In the present study effectiveness of diode laser and photodynamic therapy as

an adjunct to scaling and root planning was evaluated . Changes in plaque index,

bleeding index, probing depth, clinical attachment level and P.gingivalis count were

evaluated over a period of 6 months.

In this study additional treatment with laser and PDT showed significant

improvement in clinical parameters like plaque index, probing depth, clinical

attachment level and bleeding index. When evaluated after six month P.gingivalis

count reduced significantly in both laser and PDT treated patients.

When compared to laser assisted non-surgical periodontal treatment,

photodynamic therapy showed significant reduction in probing depth from baseline

to six months. Other clinical parameters like bleeding index, plaque index and

clinical attachment level showed similar result in both laser and PDT treated patients.

P.gingivalis count reduced similarly in both laser and PDT treated patients.

Within the limitations of the study it can be concluded that both laser an PDT

can be used for the successful management of chronic periodontitis patients. When

compared to laser, PDT showed improvement in clinical parameters specifically

probing depth.. Further evaluation on optimization of laser parameters and frequency

of treatments in larger sample size should be carried out in future for confirmation of

result .

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Annexures

Page 85

Annexure 1: Participant Information Sheet in English

PARTICIPANT INFORMATION SHEET

Title of the study: CLINICAL AND MICROBIOLOGICAL EVALUATION OF

PHOTODYNAMIC THERAPY AND DIODE LASER (960nm) AS AN

ADJUNCT TO NONSURGICAL PERIODONTAL TREATMENT- A

COMPARATIVE STUDY

Name of the research institution: Tamil Nadu Government Dental College &

Hospital, Chennai

1. Purpose of the study: Comparative evaluation of photodynamic therapy and

diode laser as an adjunct to scaling and root planing.

2. Procedure: The following examination/investigation will be done for you.

Intra oral examination, Extra oral examination

Routine blood test

X-ray will be taken for the involved teeth.

A test dose of 0.5ml of 2% Lignocaine will be injected below the skin in

your arm

After test dose, local anesthesia will be given and deposits on your teeth will

be removed with ultrasonic scaler and hand instrument. Saline irrigation will be

done at the diseased site. Irradiation of diseased site with diode laser and

placement of indocyanin green dye in the periodontal pocket and their

activation by laser will be done .Clinical evaluation will be performed at

baseline, 6 weeks,3 months and 6 months after the procedure. Sub gingival

plaque sample will be collected at baseline, 6 weeks and 6 months after the

procedure.

Annexures

Page 86

3. Risk of participation: There may be allergic reaction to local anesthetic or to

materials used in the study. Patient may experience pain, swelling and discomfort

after the procedure.

4. Benefits of participation: Patient will be treated for periodontitis.

5. Confidentiality: The identity of the patients participating in the research will be

kept confidential throughout the study. In the event of any publication or

presentation resulting from the research, no personally identifiable information

will be shared.

6. Participant’s rights: Taking part in the study is voluntary. You are free to

decide whether to participate in the study or to withdraw at any time; your

decision will not result in any loss of benefits to which you are otherwise

entitled. The results of this study will be intimated to you at the end of the study

period or during the study if anything is found abnormal which may aid in the

management or treatment.

7. Compensation: Nil

8. Contacts:

For queries related to the study:

Primary Investigator name: XXXXXXXXXX

Contact details: XXXXXXXXX

Phone number: XXXXXXXXX.

Annexures

Page 87

Annexure 2: Informed consent form in English

INFORMED CONSENT FORM

CLINICAL AND MICROBIOLOGICAL EVALUATION OF PHOTODYNAMIC

THERAPY AND DIODE LASER (960nm) AS AN ADJUNCT TO NONSURGICAL

PERIODONTAL TREATMENT – A COMPARATIVE STUDY

Participant ID No: “I have read the foregoing information, or it has been read to me. I have had the opportunity to ask questions about it and any questions I have asked have been answered to my satisfaction. I consent voluntarily to participate as a participant in this study and understand that I have the right to withdraw from the study at any time without in any way it affecting my further medical care.”

Date Name of the participant Signature/thumb impression of the participant

[The literate witness selected by the participant must sign the informed consent form. The witness should not have any relationship with the research team; If the participant doesn’t want to disclose his / her participation details to others, in view of respecting the wishes of the participant,

he / she can be allowed to waive from the witness procedure (This is applicable to literate participant ONLY). This should be documented by the study staff by getting signature from the prospective participant]

________________________________________________________________________________

_________________________ _____________________________________________________

“I have witnessed the accurate reading of the consent form to the potential participant and the individual has had opportunity to ask questions. I confirm that the individual has given consent freely”

Date Name of the witness Signature of the witness

Date Name of the Signature of the interviewer Interviewer

Annexures

Page 88

Annexure 4: Participant Information Sheet

Annexures

Page 89

Annexures

Page 90

Annexure no.5 : Informed consent form in tamil

Annexures

Page 91

Annexure 5: Case sheet proforma

DEPARTMENT OF PERIODONTICS

TAMIL NADU GOVERNMENT DENTAL COLLEGE AND HOSPITAL

CHENNAI- 600003

CLINICAL AND MICROBIOLOGICAL EVALUATION OF

PHOTODYNAMIC THERAPY AND DIODE LASER (960nm) AS AN

ADJUNCT TO NON SURGICAL PERIODONTAL TREATMENT – A

COMPARATIVE STUDY

PROFORMA FOR TREATMENT GROUP:

Date : OP No.:

S.No.:

Name : Age :

Sex:

Occupation : Income:

Address : Phone Number :

CHIEF COMPLAINTS AND DURATION:

HISTORY OF PRESENT ILLNESS:

PAST MEDICAL HISTORY:

PAST DENTAL HISTORY:

FAMILY HISTORY:

PERSONAL HISTORY:

Annexures

Page 92

a) Oral Hygiene Practices :

b) Habits :

c) Menstrual History :

d) Menopause :

e) H/o. Stress Factor :

GENERAL EXAMINATION

a) Extra-Oral Examination:

b) Examination of Lymph nodes:

INTRA-ORAL EXAMINATION WITH CLINICAL FINDINGS:

Buccal mucosa:

Vestibule:

Hard palate:

Soft palate:

Tonsils:

Tongue:

Floor of the mouth:

Teeth:

Decayed

Missing

Filled

Gingiva

Plaque index

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

Calculation Inference

Annexures

Page 93

Bleeding Index

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

Calculation Inference

Probing depth and attachment loss in millimetre

Maxillary:

CAL

PPD

18 17 16 15 14 13 12 11 21 22 23 24 25 26 27 28

PPD

CAL

Mandibular:

CAL

PPD

18 17 16 15 14 13 12 11 21 22 23 24 25 26 27 28

PPD

CAL

Selected site microbiological evaluation:

Investigations:

1. Biochemical / Haematological Investigation :

Hb%

Bleeding time

Clotting time

TLC

DLC

RBS

Annexures

Page 94

2. Others :

Blood Pressure :

Test Dose for L.A:

PROVISIONAL DIAGNOSIS

PROGNOSIS

TREATMENT PLAN

FITNESS FOR TREATMENT

TREATMENT DONE

DATE : PROCEDURE : SIGNATURE :

MAINTENANCE PHASE

EVALUATION AFTER – 3 MONTHS

Plaque index

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

Calculation Inference

Bleeding index

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

Calculation Inference

Annexures

Page 95

Probing depth and attachment loss in millimetre

Maxillary:

CAL

PPD

18 17 16 15 14 13 12 11 21 22 23 24 25 26 27 28

PPD

CAL

Mandibular:

CAL

PPD

48 47 46 45 44 43 42 41 31 32 33 34 35 36 37 38

PPD

CAL

MAINTENANCE PHASE

EVALUATION AFTER - 6 MONTH

Plaque index

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

Calculation Inference

Bleeding index

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

Calculation Inference

Probing depth and attachment loss in millimeter

Maxillary:

CAL

PPD

18 17 16 15 14 13 12 11 21 22 23 24 25 26 27 28

PPD

CAL

Annexures

Page 96

Mandibular:

CAL

PPD

48 47 46 45 44 43 42 41 31 32 33 34 35 36 37 38

PPD

CAL

Selected site microbiological evaluation:

SIGNATURE OF THE PROFESSOR SIGNATURE OF STUDENT