Clinical and Microbiological Evaluation of Photodynamic Therapy and Diode Laser (960nm) as an Adjunct to Non Surgical Periodontal Treatment: A Comparative study

111  Download (1)

Full text

(1)

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

(2)

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.

(3)

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

(4)

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

(5)

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

(6)

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

(7)

witnesses.

College represented by its Principal PG Student

Witness STUDENT GUIDE

1.

(8)

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,

(9)

– 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.

(10)
(11)

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

(12)

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

(13)

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

(14)

S.NO FIGURES PAGE

1 Changes in clinical parameters for group 1 61

(15)

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

(16)

Page 1 INTRODUCTION

Periodontitis is an inflammatory disease of supporting tissues of the teethwhich 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 19992 and Herrera et al. 20083).

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

(17)

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 20078).Several periodontal pathogens, such as

Porphyromonas gingivalis and A. actinomycetemcomitansare 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

(18)

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.

(19)

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 19992, and Herrera et al. 20083)

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

(20)

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.(

(21)

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 199619reveals

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

(22)

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

(23)

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-gallium-aluminum-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

(24)

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

(25)

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

(26)

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

(27)

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

(28)

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

(29)

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

(30)

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

(31)

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

(32)

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

(33)

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

(34)

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

(35)

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,

(36)

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. gingivaliscould 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 alin200297 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- 106, 108,107 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

(37)

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

(38)

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

(39)

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

(40)

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

(41)

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

(42)

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.

(43)

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

(44)

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

(45)

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

(46)

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

(47)

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

(48)

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

(49)

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

(50)

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

u

e

s

DNA concentration (Log)

(51)

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

(52)

Page 37 Photograph 1: Armamentarium for sample collection

(53)

Page 38 Photograph 3: Sample transport

(54)

Page 39 Photograph 5: Indocyanin green dye

(55)

Page 40 Photograph 7: Pre-operative photograph

(56)

Page 41 Photograph 9: Indocyanin green applied

(57)

Page 42

Photograph 11: Post-operative after 6 months

(58)

Page 43 Photograph 13: Armamentarium for DNA extraction and real time PCR

Figure

TABLE 6: PLAQUE INDEX

TABLE 6:

PLAQUE INDEX p.69
TABLE 7: BLEEDING INDEX

TABLE 7:

BLEEDING INDEX p.69
TABLE 8: PROBING DEPTH

TABLE 8:

PROBING DEPTH p.70
TABLE 9: CLINICAL ATTACHMENT LEVEL

TABLE 9:

CLINICAL ATTACHMENT LEVEL p.70
TABLE 13: PROBING DEPTH

TABLE 13:

PROBING DEPTH p.73
TABLE 14: CLINICAL ATTACHMENT LEVEL

TABLE 14:

CLINICAL ATTACHMENT LEVEL p.73
Figure 1: Changes in clinical parameters for group 1

Figure 1:

Changes in clinical parameters for group 1 p.76
Figure 2: Changes in clinical parameters for group 2

Figure 2:

Changes in clinical parameters for group 2 p.76

References

Updating...