comparing effectiveness of three different …repository-tnmgrmu.ac.in/5257/1/240502017kakkad... ·...

Dissertation submitted to

THE TAMILNADU DR. M.G.R. MEDICAL

UNIVERSITY

For Partial fulfilment of the requirements for the degree of

MASTER OF DENTAL SURGERY

BRANCH - V

ORTHODONTICS AND DENTOFACIAL ORTHOPEDICS

THE TAMILNADU DR. M.G.R. MEDICAL UNIVERSITY

CHENNAI – 600 032

2014 – 2017

COMPARING EFFECTIVENESS OF THREE DIFFERENT

PROBIOTICS IN INHIBITION OF STREPTOCOCCUS

MUTANS IN PLAQUE AROUND ORTHODONTIC

BRACKETS: A RANDOMIZED CONTROLLED TRIAL

CERTIFICATE

This is to certify that Dr.KAKKAD KRUPALI NITINBHAI Post graduate student

(2014-2017) in the Department of Orthodontics and Dentofacial Orthopaedics, Tamil Nadu

Government Dental College and Hospital, Chennai – 600003 has done this dissertation titled

“Comparing effectiveness of three different probiotics in inhibition of streptococcus

mutans in plaque around orthodontic brackets: a randomized controlled trial” under my

direct guidance and supervision for partial fulfilment of the M.D.S. degree examination in

April 2017 as per the regulations laid down by The Tamil Nadu Dr. M.G.R. Medical

University, Chennai – 600032 for M.D.S., Orthodontics and Dentofacial Orthopaedics

(Branch – V) degree examination.

Guided by

Dr. B. Balashanmugam, M.D.S.

Professor of Orthodontics and Dentofacial Orthopaedics,

Tamil Nadu Government Dental College &

Hospital, Chennai - 600003

Dr. G. Vimala, M.D.S.,

Professor and Head,

Dept. of orthodontics and Dentofacial Orthopedics



Dr. B. Saravanan, M.D.S., Ph. D.,

Principal,



DECLARATION

I, Dr KAKKAD KRUPALI NITINBHAI, do hereby declare that the dissertation

titled “Comparing effectiveness of three different probiotics in inhibition of streptococcus

mutans in plaque around orthodontic brackets: a randomized controlled trial” was done in

the Department of Orthodontics, Tamil Nadu Government Dental College & Hospital,

Chennai 600 003. I have utilized the facilities provided in the Government Dental College for

the study in partial fulfilment of the requirements for the degree of Master of Dental Surgery

in the specialty of Orthodontics and Dentofacial Orthopaedics (Branch V) during the course

period 2014-2017 under the conceptualization and guidance of my dissertation guide,

Professor Dr. B. BALASHANMUGAM, MDS.,

I declare that no part of the dissertation will be utilized for gaining financial assistance

for research or other promotions without obtaining prior permission from The Tamil Nadu

Government Dental College & Hospital.

I also declare that no part of this work will be published either in the print or

electronic media except with those who have been actively involved in this dissertation work

and I firmly affirm that the right to preserve or publish this work rests solely with the prior

permission of the Principal, Tamil Nadu Government Dental College & Hospital, Chennai

600 003, but with the vested right that I shall be cited as the author(s).

Signature of the PG student Signature of the HOD

Signature of the Head of the Institution

ACKNOWLEDGEMENT

I seek the blessings of the ALMIGHTYGOD without whose benevolence this study

would not have been possible.

My sincere and heartfelt thanks to Dr. B.SARAVANAN, M.D.S., Ph.D., our

Principal and Professor of Orthodontics, Tamil Nadu Government Dental College and

Hospital, Chennai – 3,for his continuous and enormous support in allowing me to conduct

this study and for his constant encouragement and advice during my tough phases in

curriculum.

With my heartfelt respect, immeasurable gratitude and honour, I thank my benevolent

guide, Dr. B. BALASHANMUGAM, M.D.S., Professor, Department of Orthodontics and

Dentofacial orthopedics, Tamil Nadu Government Dental College and Hospital, Chennai – 3,

for his astute guidance, support and encouragement throughout my post graduate course and

to bring this dissertation to a successful completion.

I owe my thanks and great honour to Dr. G. VIMALA, M.D.S., Professor & HOD,

Department of Orthodontics and Dentofacial Orthopaedics, Tamilnadu Govt. Dental College

and Hospital, Chennai - 3, for helping me with her valuable and timely suggestions and

encouragement.

I express my deep sense of gratitude and honour to my respected Professor,

Dr. SRIDHAR PREMKUMAR, MDS., Department of Orthodontics and Dentofacial

Orthopaedics, Tamilnadu Govt. Dental College and Hospital, Chennai – 3, for his constant

inspiration and encouragement throughout the entire course.

I sincerely thank Associate professors Dr. G. USHA RAO,

Dr. M. VIJJAYKANTH, Dr. M. D. SOFITHA and Senior Assistant professors

Dr. K. USHA, Dr. M.S. JAYANTHI, Dr. D. NAGARAJAN, Dr. MOHAMMED IQBAL,

and Dr. R. SELVARANI for their continuous support and encouragement.

I thank Dr. DINTA KAKKAD for helping me in statistical analysis.

I thank my god “LORD SHRINATHJI”, my father NITINBHAI P. KAKKAD,

my mother MANJU N. KAKKAD my sister Dr. DINTA KAKKAD for their blessings,

unconditional love, affection, care and prayers. Without them, nothing would have been made

possible.

I thank all my patients for their co-operation.

I also thank my post graduate colleagues for their help and constant support.

TRIPARTITE AGREEMENT

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

December 2016 between the Tamil Nadu Government Dental College and Hospital

represented by its Principal having address at Tamil Nadu Government Dental College and

Hospital, Chennai-03, (hereafter referred to as, “the college”)

And

Dr. B. BALASHANMUGAM aged 45 years working as professor at the college, having

residence address at 8-B,Crescent road, Shenoy nagar,Chennai-600030, Tamil Nadu (Herein

after referred to as the „Principal investigator‟)

And

Dr.KAKKAD KRUPALI NITINBHAIaged 28 years currently studying as postgraduate

student in Department of Orthodontics in Tamil Nadu Government Dental College and

Hospital (Herein after referred to as the „PG/Research student and co- investigator‟).

Whereas the „PG/Research student as part of his curriculum undertakes to research

“Comparing effectiveness of three different probiotics in inhibition of streptococcus

mutans in plaque around orthodontic brackets: a randomized controlled trial” for which

purpose the PG/Principal investigator shall act as principal investigator and the college shall

provide the requisite infrastructure based on availability and also provide facility to the

PG/Research student as to the extent possible as a Co-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 witnesseth 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 legal right to do go, shall grant to license or assign

the copyright do 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 parties.

4. The PG/Research student and PG/Principal 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 vest with the

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

college.

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

investigator/Co-investigator or borne sole by the PG/research student (co-

investigator).

6. 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 required in this regard.

7. The principal investigator shall suitably guide the student Research right from

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

and conduct of research, topic and area research by the student researcher under

guidance from the principal investigator shall be subject to the prior approval,

recommendations and comments of the Ethical Committee of the college constituted

for this purpose.

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

pertain to the research undertaken by the student Researcher, under guidance from the

Principal Investigator, the decision of the college shall be binding and final.

9. 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.

10. In witness where of the parties herein above mentioned have on this the day month

and year herein above mentioned set their hands to this agreement in the presence of

the following two witnesses.

Principal PG Student

Witnesses Student Guide

1.

2.

CONTENTS

SL. NO. TITLE PAGE NO.

1. INTRODUCTION 1

2. AIMS AND OBJECTIVES 5

3. REVIEW OF LITERATURE 6

4. MATERIALS AND METHOD 36

5. RESULTS 44

6. DISCUSSION 48

7. SUMMARY AND CONCLUSION 54

8. BIBLIOGRAPHY I

9. ANNEXURE i

LIST OF TABLES

SL. NO. TOPIC PAGE NO.

1 Demographic Distribution of study subjects according to

Groups, Age and Gender 45

2

Comparison of S. mutans CFU/ml before and after 30 days of

administration of probiotics in each study groups and without

use of probiotics in control group.

46

3

Comparison of S. mutans CFU between different groups at

baseline and the variation of S. mutans CFU between different

groups after 30 days

47

LIST OF CHART

SL. NO. TOPIC

1 Groups and no of participants in each group

2 Distribution of study participants according to Gender

3 Comparison of S. mutans CFU/ml before and after 30 days in

all four groups

LIST OF FIGURES

FIGURE NO.

TITLE

1 Potential mechanisms by which probiotic bacteria could affect

oral health

2 Armamentarium

3 Test tube and Glass rod

4 Thioglycolate broth

5 Blood Agar

6 Incubator

7 Candle jar

8 Digital colony counter

9 Prebiotic lozenges (BIFILAC lozenges)

10 Probiotic sachets (DAROLAC- Z Sachets)

11 Probiotic drink (YAKULT)

12 Collection of plaque samples from the labial surfaces surrounding

the orthodontic brackets of the maxillary lateral incisors

13 Mixed growth of sample

14 Growth of sample in controls

15 Pure isolate of Streptococcus Mutans

16 Colony counting method

17 Colony counting

LIST OF ANNEXURES

SL. NO. TOPIC PAGE NO.

1 Patient information sheet in Tamil i

2 Patient information sheet in English Iii

3 Informed consent form in Tamil v

4 Informed consent form in English vi

5 Ethical committee approval certificate vii

LIST OF ABBREVIATIONS

FAO Food Agriculture Organization

WHO World Health Organization

et al And others

WSLs White spot lesions

S. mutans Streptococcus Mutans

CFU Colony Forming Units

df Degree of freedom

Introduction

Page 1

INTRODUCTION

Orthodontics is the branch of dentistry concerned with prevention,

interception and correction of malocclusion and other abnormalities of dentofacial

region. In recent years orthodontics has played an important role in dentistry and

this trend is likely to continue in the future. Fixed orthodontic appliances have

been contemplated for more efficient and precise orthodontic tooth movement to

achieve ideal results. A survey of the long-term effects of orthodontic treatment

revealed that the majority of individuals who had undergone orthodontic treatment

felt that they were benefitted and were satisfied with the result1. Although many

patients experience dramatic improvement in their dental and facial aesthetics,

occlusal function and oral health, treatment with fixed orthodontic appliances

have disadvantages because it may be associated with gingival inflammation and

enamel demineralisation.2 Gingivitis is reversible while enamel demineralisation

is usually irreversible.3 Though the quality of appliances and treatment protocols

have been improved, it creates the areas of plaque stagnation, especially around

brackets, bands, wires and other attachments. It causes specific changes in the oral

environment, such as decreased PH,

leading to increased pathogenic plaque

formation and enamel demineralisation.4Levels of acidogenic bacteria, present in

the plaque, notably Streptococcus mutans, have been implicated as the main

causative organisms of dental caries, are higher in orthodontic patients as

compared to the non-orthodontic patients.5,6

Among the many orthodontic

appliances, brackets can play a key role in enamel demineralisation because their

complex design increases the retention of food particles and dental plaque by

impeding access to the tooth surface for cleaning.4 Metal brackets are known to

have the highest critical surface tension and have increased risk for enamel

Introduction

Page 2

demineralisation;7,8

leading to the formation of white spot lesion,9 which is the

precursor of enamel caries; this is a grave concern to orthodontists and patients.

White spot lesions are the first clinical signs of enamel demineralisation

and dental caries.10

It is one of the most common, undesirable and potentially

avoidable sequelae of orthodontic treatment. Pre-existing demineralisation and

white spot lesions may be present in orthodontic patients; however,

demineralisation occurring during orthodontic treatment is an indicator of a

current bacterial infection which must be addressed promptly. White spot lesions

are the direct result of an uncoupled demineralisation-remineralisation process

favouring increased demineralisation.11

The overall prevalence of white spot

lesions among orthodontic patients has been reported to be between 4.9%12

and

84%.10

They are most prevalent around the cervical region of bands in the

posterior region, where as in the anterior region, the lateral incisors, followed by

canines, and are most commonly affected.13

In the anterior region the lateral

incisors are the most susceptible because of decreased salivary clearance and also

less space between the brackets and the gingiva. This creates less accessibility for

oral hygiene techniques. White spot lesions in the anterior region are clearly

visible and can cause aesthetic problem after orthodontic treatment.6 Maintenance

of proper oral hygiene6, fluoride delivery systems

14, casein phosphopeptide

15,

amorphous calcium phosphate16

, antimicrobials6, Micro abrasion

17, 18 and enamel

surface attenuation with an argon laser16

have proved to be useful to prevent or to

treat enamel demineralisation but, they have their own disadvantages.13

Probiotics are emerging as a fascinating field in the today’s antibiotic

dominated field. At the turn of the 20th

century, Elie Metchinchkoff, a Nobel

Introduction

Page 3

Prize-winning Russian, made the revolutionary discovery of probiotics and stated

that Probiotics could provide a health benefit. The term probiotics was first used

by Lilly and Stillwell in 1965. In 2001, FAO/WHO defined Probiotics as “live

microorganisms which when administered in adequate amounts confer a health

benefit on host.” First probiotic species to be introduced in research was

Lactobacillus acidophilus by Hull et al in 1984, followed by Bifidobacterium

bifidum by Holcombh et al in 1991.4The general mechanisms of probiotics can be

divided into three categories: normalization of the intestinal microbiota,

modulation of the immune response, and metabolic effect.19

Researchers are now

investigating the use of probiotics for preventing enamel demineralization leading

to white spot lesions in patients undergoing orthodontic therapy. Although only a

few studies have been conducted so far, the results of these studies have identified

the positive role of probiotics in preventing and treating oral infections like dental

caries, periodontal diseases and halitosis. The probiotic or good bacteria found in

the oral cavity are Lactobacillus acidophilus, Lactobacillus fermentum,

Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus salivarius.

Possible ways that probiotic might affect oral health are summarized in figure 1.

Studies have established that the level of s. mutans is reduced after the use

of probiotics.20

Few studies were done to study the effect of local administration

of probiotics agent such as mouthwashes, lozenges, cheese, milk, chewing gums

and yoghurt.21

Hence we conducted this study to evaluate the efficacy of 3 Different

probiotics in inhibition of streptococcus mutans in plaque around orthodontic

brackets.

Introduction

Page 4

Figure 1: Potential mechanisms by which probiotic bacteria could affect oral

health

BINDING IN ORAL CAVITY

1. Compete with pathogens for

adhesion site

2. Compete with pathogens for

nutrients

MODIFY ORAL CONDITION

1. Modification of oxidation and reduction potential

PRODUCTION OF ANTI MICRIBIAL SUBSTANCES

1. Hydrogen peroxide

2. Bacteriocins

IMMUNOMODULATORY

1. Simulate non specific immunity

2. Modulate humoral and cellular immune response

PROBIOTICS

Aim and Objectives

Page 5

AIM AND OBJECTIVES

AIM:

To compare the efficacy of three different probiotics in inhibition of

streptococcus mutans in plaque around orthodontic brackets.

OBJECTIVES:

To evaluate effects of probiotic lozenges on streptococcus mutans

levels in the plaque surrounding orthodontic brackets.

To evaluate effects of probiotic sachets on streptococcus mutans levels

in the plaque surrounding orthodontic brackets.

To evaluate effects of probiotic drink on streptococcus mutans levels in

the plaque surrounding orthodontic brackets.

To compare the efficacy of above listed three different probiotics in

reducing streptococcus mutans levels in the plaque surrounding

orthodontic brackets.

Review of Literature

Page 6

REVIEW OF LITERATURE

Demineralization of enamel adjacent to orthodontic appliances frequently

occurs, commonly due to insufficient oral hygiene. White spot lesions (WSLs) are

an undesirable side effect of fixed orthodontic appliance therapy and are reported

to occur in 2–96 % of orthodontic patients. Equal susceptibility to white spot

formation has been reported whether teeth are banded or bonded. There has been

general agreement that the development of white spots seems to be related to (1)

the retention of plaque on the gingival side of brackets or bands, (2) oral hygiene

efficiency, and (3) the inherent resistance of the individual.

Oral infections constitute some of the most common and costly forms of

infections in humans. Numerous studies have shown that the levels of

streptococcus mutans were increased significantly in patients undergoing

orthodontic treatment with fixed appliance which increase the microbiological risk

of dental caries than in patients being treated with removable appliance or

individuals receiving no orthodontic treatment at all. The concept of microbial

ecological change as a mechanism for preventing dental disease is an important

one while altered microbial ecology may lead to dental disease. New methods

such as probiotic approaches (i.e. whole bacteria replacement therapy) to

eliminate pathogenic members of the microbiota can be investigated.

Bacteriotherapy is an alternative and promising way to combat infections by using

harmless bacteria to displace pathogenic microorganisms. Probiotics are one of

these new agents which are widely used for their therapeutic action. Limited

research is available showing that some probiotic cultures may help dental

improvement.


Page 7

STUDIES RELATED TO PLAQUE AND DENTAL CARIES

BURTON ROSAN, RICHARD J. LAMONT (2000)22

stated that dental plaque

is a complex biofilm that accumulates on the hard tissues (teeth) in the oral cavity.

Although over 500 bacterial species comprise plaque, colonization follows a

regimented pattern with adhesion of initial colonizers to the enamel salivary

pellicle followed by secondary colonization through interbacterial adhesion. A

variety of adhesions and molecular interactions underlie these adhesive

interactions and contribute to plaque development and ultimately to diseases such

as caries and periodontal disease.

STUDIES RELATED TO S.MUTANS LEVEL IN PLAQUE AND SALIVA

& ORTHODONTIC TREATMENT

SCHEIE, ARNEBERG AND KROGSTAD (1984)23

investigated the effect of

orthodontic treatment on the prevalence of Streptococcus mutans in 14 subjects.

They stated that the insertion of appliances tended to give a transient decrease in

S.mutans levels, in both plaque and saliva, possibly due to elimination of

S.mutans reservoirs by the banding procedure. After 3 months of extensive

banding, the S.mutans proportions surpassed pre-treatment levels in saliva and on

banded teeth, whereas un-banded surfaces only showed a slight increase. It was

thus concluded that creation of new retentive areas favours the local growth of

S.mutans, which in turn increases the general infection level of this organism.

ROSENBLOOM et al (1991)24

did a study to evaluate salivary Streptococcus

mutans levels in patients before, during, and after orthodontic treatment.

S.mutans levels were significantly elevated during active treatment. However,


Page 8

when sampled 6 to 15 weeks into the retention phase of treatment, the microbial

levels decreased significantly to levels comparable to age-matched untreated

controls. In addition, patients who were no longer wearing any retention

appliances had S. mutans levels similar to those subjects sampled in the retention

phase of treatment as well as to subjects in age-matched control groups. The

findings of the study suggest that orthodontic treatment does not result in any

long-term elevations of S.mutans levels.

ARI KUPIETZKY et al (2006)25

conducted a study to compare the levels of

Lactobacilli (LB) and Streptococcus mutans (SM) colony forming units (CFU) in

the saliva of subjects before and after orthodontic appliance placement. This was a

controlled, prospective two-group, two-measurement, clinical trial performed on

64 study patients, 12-15 years old. Subjects in the experimental group were

sampled for LB and SM in stimulated saliva collected on the same day but prior to

band and bracket placement. The subjects in the control group were sampled on

their first screening appointment two months prior to band and bracket placement.

The second samples of LB and SM were taken from the experimental and control

groups after two months. Saliva was transferred to a selective agar carrier and

incubated for 48 hours at 37°C / 99 °F. LB and SM colonies forming units were

compared with standard densities. The experimental group wearing orthodontic

appliances had significantly higher mean LB CFU counts than the control group at

the 2 month follow- up (3.25 vs. 2.57, p =0.0036). The two groups, however, did

not show any difference in mean SM CFU counts at the 2 month follow-up (3.0

vs. 3.1, p = 0.66). The results of this study showed that a higher number of CFUs

of LB were associated with the group wearing orthodontic appliances after two

months and may play a role in the increased levels.


Page 9

STUDIES RELATED TO CARIES INCIDENCE AND ORTHODONTIC

TREATMENT

BJORN U. ZACHRISSON AND SIGRUN ZACHRISSON (1971)26

examined

one hundred and seventy-three individuals receiving orthodontic full band

treatment in one or both dental arches, in order to assess the relationship between

caries incidence and oral hygiene during treatment. Topical application of sodium

fluoride was used regularly throughout the experimental period. The average

period of treatment was 19 months. Monthly assessments of oral hygiene were

performed through partial recordings utilizing the Plaque (PlI) and Gingival Index

(GI) systems. Smooth surface carious lesions on vestibular and lingual surfaces of

banded teeth were assessed according to a proposed Caries Index (CI) at the time

of removal of the orthodontic appliances. The results demonstrated a definite

correlation between oral health and caries incidence. With increasing mean PI1

and GI scores, there were concomitant, almost linear increases in mean C1 scores.

CARLOS ALBERTO FELDENSA et al (2000)27

conducted a study to

investigate the association between malocclusion/dentofacial anomalies and dental

caries among adolescents. A cross-sectional study was conducted with 509

adolescents aged 11 to 14 years enrolled at public schools in southern Brazil.

Parents/caregivers answered a structured questionnaire on demographic and

socioeconomic variables. A trained examiner recorded the presence of

malocclusion (Dental Aesthetic Index [DAI]), traumatic dental injury, and dental

caries. Results showed a total of 44.8% of the adolescents had dental caries (mean

DFMT 5 1.33 6 1.84). The DAI index ranged from 15 to 77 (mean 5 29.0 6 7.9);

43.6% of the sample had severe malocclusion and 11.6% had traumatic dental


Page 10

injury. The prevalence and severity of dental caries were significantly greater

among adolescents with severe malocclusion. It was concluded as handicapping

malocclusion, maxillary irregularity, and abnormal molar relationship were

associated with the occurrence and severity of dental caries. The findings suggest

that the prevention and treatment of these conditions can contribute to a reduction

in dental caries among adolescents.

BOERSMA et al (2005)28

did a study to determine caries prevalence on the

buccal surface of teeth in orthodontic patients with QLF and visual examination

immediately after removal of fixed appliances. 97% of all subjects and on

average, in male 40% of surfaces and in female 22% of surfaces showed white

spots (P < 0.01). Caries prevalence was lower (P < 0.01) in incisors and cupids

than in molars and premolars. A positive correlation with caries prevalence was

found for the bleeding scores 6 weeks after debonding and lactobacillus counts

before debonding. Mutans streptococci counts, age, treatment duration,

socioeconomic status and dietary habits showed no correlation with caries

prevalence.

STEFAN BAUMGARTNER, GIORGIO MENGHINI, THOMAS IMFELD

(2013)29

conducted a retrospective, cross-sectional study to investigate the

prevalence of a proximal carious lesions in patients after fixed multi-bracket

therapy and in subjects without orthodontics on bitewing radiographs. Bitewing

radiographs of 104 orthodontically treated patients with fixed multi-bracket

appliances were compared to those of 111 untreated subjects. Results showed the

average number of enamel lesions in the test group after fixed orthodontic

treatment was lower than in the control group (0.57 vs. 1.85, p < 0.001). The same


Page 11

was found for dentin lesions (0.06 vs. 0.49, p < 0.001). The distribution of lesions

was similar in both groups. It was concluded that a proximal carious lesions were

detected in the test group after fixed multibracket appliances than in the age-

matched control group without orthodontic treatment.

WEITING CHEN, YU ZHOU (2015)30

conducted a study to assess the

relationship between orthodontic and the development of dental caries in the same

patients who received single jaw orthodontic treatment. A consecutive sample

consisted of 60 subjects was taken. The dental examinations were routinely

carried out by one dentists at the following stages: pre-treatment (T1); post-

treatment (T2); more than 7 years after T1 (T3). The DMFS count which reflect

the caries experience was recorded. Results showed there was no significant

difference between the treated groups and untreated groups for the DMFS before

received fixed orthodontic treatment. The same result was found after orthodontic

treatment. However, the average number of DMFS in the treatment jaw after fixed

orthodontic treatment was lower than in the without treatment jaw after long-term

follow-up period. Hence it was concluded as fixed orthodontic appliances

significant decrease the patient’s caries risk after orthodontic treatment.

STUDIES RELATED TO WHITE SPOT LESION AND ORTHODONTIC

TREATMENT

ELIAKIM MIZRAHI (1982)10

carried out a cross-sectional study to determine

the prevalence and severity of enamel opacities in patients before and after

orthodontic treatment. The sample consisted of 527 patients examined prior to and

269 patients examined after completion of multi-banded orthodontic treatment.

The results showed that there was a significant increase in both the prevalence


Page 12

(before, 72.3 per cent; after, 84.0 per cent) and severity (Opacity Index: before,

0.125; after, 0.200) following completion of orthodontic treatment. Male patients

experienced a significantly higher increase in the severity of enamel opacities

following orthodontic treatment. This study showed that orthodontic treatment

with multibanded appliances contributed to the development of new areas of

enamel demineralization and to an increase in the severity of enamel opacities as

measured by the Opacity Index.

SAMIR E. BISHARA, ADAM W. OSTBY (2008)6 stated that as oral hygiene

becomes more difficult in patients with fixed orthodontic appliances, the

decalcification of the enamel surface adjacent to these appliances is prevalent.

Decalcification is manifested as a white spot lesion (WSL), and orthodontic

patients develop significantly more WSLs than non-orthodontic patients. If WSLs

are left untreated, they may progress to produce carious cavitations.

DERRICK WILLMOT (2008)13

examined the prevalence and distribution of

white spots after orthodontic treatment. They stated that in the first few weeks

after removal of appliances there is typically an exponential reduction of white

spot lesion size by remineralization, and about half of the original lesion has

remineralized after 6 months with no specific treatment.

JOSHUA A. CHAPMAN et al (2010)31

stated that the development of incipient

caries, or white spot lesions (WSLs), is a significant clinical problem in

orthodontics. Hence the purpose of their study was to retrospectively determine

the incidence and severity of WSLs by examining pre-treatment and post

treatment digital photographs. A total of 332 consecutive finished patients were

evaluated. Initial and final digital images were compared to assess WSLs. Results


Page 13

showed Agreement between direct clinical examination and digital photo data was

excellent, with an intra-class correlation coefficient 0.88 and a 0.3% average

difference between methods. The study Concluded as Risk factors for the

development of incipient caries during orthodontic treatment at the start of

treatment.

ESER TUFEKCI et al (2011)32

conducted a study to determine the prevalence of

white spot lesions (WSLs) in orthodontic patients at 6 and 12 months into

treatment using the visual examination methods. Patients 6 and 12 months into

treatment were examined for the presence of WSLs. Results showed the

percentages of individuals having at least one WSL were 38%, 46%, and 11% for

the 6-month, 12-month, and control groups, respectively. The 6-month (P = .021)

and 12-month groups (P = .005) were significantly different from the control

group but were not significantly different from each other (P = .50). Of subjects in

the study who had at least one visible WSL, 76% were males and 24% were

females (P = .009). It was concluded that this clinical study showed a sharp

increase in the number of WSLs during the first 6 months of treatment that

continued to rise at a slower rate to 12 months.

ALESSANDRA LUCCHESE, ENRICO GHERLONE (2013)33

did a study to

determine the prevalence of white-spot lesions (WSLs) in patients with fixed

orthodontic appliances. The cross-sectional study sample consisted of three groups

of patients: group I, 59 patients treated orthodontically for 6 months; group II, 64

patients treated for 12 months; group 0 (control), 68 patients examined

immediately before appliance placement. The presence of WSLs was evaluated by

visual examination using the scoring system proposed by Gorelick. The


Page 14

mandibular first molars and maxillary lateral incisors were the most affected teeth,

in both the treated and untreated groups. The study revealed significant

decalcification at 6 months after orthodontic bonding. Considering how quickly

these lesions can develop and become irreversible, early diagnosis is of critical

importance.

STUDIES RELATED TO PREVENTION OF WHITE SPOT LESION

M. M. O’RELLLY AND J. D. B. FEATHERSTONE (1987)34

conducted a

study with aim to determine quantitatively (1) the amount of demineralization and

(2) the ability of commercially available products to inhibit or reverse

orthodontically related demineralization. Twenty orthodontic patients scheduled to

have premolars extracted were randomly divided into four groups-one control and

three test groups. The extracted premolars (numbering 58) were bracketed using

an acid-etch composite system; each patient was given precise oral hygiene

instructions and supplied with a sodium fluoride (1,100 ppm fluoride) dentifrice

and an orthodontic toothbrush. The control group brushed only with the supplied

dentifrice. In addition to brushing with the dentifrice, those in test group I rinsed

once each night with a sodium fluoride (0.05%) mouth rinse; group II received a

weekly topical APF treatment (1.2% fluoride); and Group Ill received a weekly

topical APF treatment and rinsed once each night with the sodium fluoride mouth

rinse. The study demonstrated that (1) measurable demineralization occurred

around orthodontic appliances after only 1 month and (2) this demineralization

can be completely inhibited and/or reversed by the use of commercially available

fluoride products.


Page 15

ARNOLD M. GEIGER ET AL (1988)14

conducted an experimental preventive

fluoride program in their orthodontic office. The study was designed to provide

answers to the following questions. 1. Was the preventive fluoride program

beneficial in reducing the incidence and severity of white spots after full-term

bonded orthodontic treatment? 2. Will such preventive efforts reduce the white

spot formation for all teeth that have been bonded? 3. What degree of cooperation

(compliance) can be expected from patients? 4. How does the degree of

compliance with the fluoride program affect white spot formation? In the office,

acidulated phosphate gel was applied about 3 minutes after brackets were bonded.

This was followed by a spray or rinse with a0.05% solution of sodium fluoride.

The home care procedure consisted of the use of a 0.05% fluoride solution as a

rinse after brushing with a fluoridated toothpaste. This was to be done just before

retiring at night and was not diluted by subsequent eating or rinsing. It was

concluded as 1. Decalcification of the labial (buccal) surfaces of teeth during

orthodontic therapy can be significantly reduced by the consistent use of a 0.05%

sodium fluoride rinse during treatment. 2. The incidence and severity of white

spot formation are related to the length of time teeth are bracketed. This suggests

the need for a preventive fluoride rinse used continuously during treatment. 3.

Despite efforts to educate patients and parents, poor compliance with a preventive

fluoride rinse program occurred in 50% of patients. This suggests the need for

more effective methods to change behaviour patterns. 4. The one-time topical

application of acidulated phosphate fluoride gel immediately after bonding

appears to be ‘of little benefit in reducing the incidence of white spots.


Page 16

GRAY, M. M. FERGUSON (1996)35

conducted a clinical study to determine the

acceptability of a sugar-free, low-tack chewing gum by orthodontic patients

undergoing fixed appliance treatment. Twenty-five orthodontic and 25 non

orthodontic control subjects were questioned on their preference between regular-

tack and low-tack chewing gum. The orthodontic subjects showed a strong

preference for the low-tack gum compared with the regular-tack gum. It was

concluded that low-tack, sugar-free chewing gum can be used by orthodontic

patients to increase saliva flow, with the potential to promote remineralization and

help reduce white spot lesion formation related to fixed orthodontic appliances.

This gum should also be of value in patients being treated for xerostomia who are

wearing a partial denture.

PERNILLA LIF HOLGERSON et al (2007)36

did a study to investigate the

effect of a fixed daily dose of xylitol on mutans streptococci in saliva and the

amount of visible dental plaque. A second aim was to explore if the possible

effects differed between children with and without caries experience. The study

was designed as a double-blind randomized controlled trial with two parallel arms.

All pupils (n = 149) in grades 1–6 in a comprehensive school in northern Sweden

were invited, and 128 children (mean age = 12.7 years) consented to participate.

The children were stratified as having caries experience (DMFS/dmfs ≥ 1) or not

before the random allocation to a test or control group. The control group (A) was

given two pellets containing sorbitol and maltitol three times daily for 4 weeks,

and the test group (B) received corresponding pellets with xylitol as single

sweetener (total dose = 6.18 g day). Clinical scoring and saliva samples were

collected at baseline and immediately after the test period. The outcome measures

were visible plaque index, salivary mutans streptococci counts and salivary lactic


Page 17

acid production. Results showed the amount of visible plaque was significantly

reduced in both groups after 4 weeks (P< 0.05). Likewise, the sucrose-induced

lactic acid formation in saliva diminished in both groups (P< 0.05). The

proportion of mutans streptococci decreased significantly in the test group

compared to baseline but not in the control group (P< 0.05). The alterations in the

test group seemed most prominent among children without previous caries

experience. The results suggest that chewing gum with xylitol or sorbitol/maltitol

can reduce the amount of dental plaque and acid production in saliva in

schoolchildren, but only the xylitol-containing gum may also interfere with the

microbial composition.

GF FERRAZZANO et al (2008)37

stated that the casein phosphopeptides (CPPs)

are phosphorylated casein-derived peptides produced synthetically by proteolytic

digestion of alpha (S1)1-, alpha (S2) - and beta-casein. The anticariogenic activity

of CPPs is due to their ability to stabilize high levels of amorphous calcium

phosphate (ACP) on tooth surface, preventing demineralization and enhancing

remineralization of enamel caries. Hence they did an in vitro study to test the

ability of natural CPPs (contained in yogurt) to prevent demineralization and

promote remineralization of dental enamel. Eighty human molars were used. After

standardizing an in vitro demineralization procedure for producing artificial caries

(Group 1: pH 4.8; Group 2: pH 3.97), this procedure was used on teeth, but with

the addition of natural CPPs (Group 3: pH 4.8; Group 4: pH 3.97). Statistical

analysis showed significant differences in weight changes between the groups

with and without natural CPPs. The results demonstrated that CPPs contained in

yogurt have an inhibitory effect on demineralization and promote the

remineralization of dental enamel.


Page 18

BEERENS MW et al (2010)15

conducted a double-blind prospective randomized

clinical Trial to find out the effects of casein phosphopeptide amorphous calcium

fluoride phosphate (CPPACFP) paste vs. control paste on the remineralization of

white spot caries lesions and on plaque composition. Fifty-four orthodontic

patients, with multiple white spot lesions observed upon the removal of fixed

appliances, were followed up for 3 months. A significant decrease in fluorescence

loss was found with respect to baseline for both groups and no difference was

found between groups. The size of the lesion area did not change significantly

over time or between the groups. The percentages of aciduric bacteria and of S.

mutans decreased from 47.4 to 38.1% and from 9.6 to 6.6%, respectively. They

observed no clinical advantage for use of the CPP-ACFP paste supplementary to

normal oral hygiene over the time span of 12 weeks.

ANN BRÖCHNER et al (2011)38

did a study to investigate the effect of topical

applications of 10% casein phosphopeptide–amorphous calcium phosphate (CPP–

ACP) on white spot lesions (WSL) detected after treatment with fixed orthodontic

appliances. Sixty healthy adolescents with ≥1 clinically visible WSL at debonding

were recruited and randomly allocated to a randomised controlled trial with two

parallel groups. The intervention group was instructed to topically apply a CPP–

ACP -containing agent (Tooth Mousse, GC Europe) once daily and the subjects of

the control group brushed their teeth with standard fluoride toothpaste. The

intervention period was 4 weeks and the endpoints were quantitative light-induced

fluorescence (QLF) on buccal surfaces of the upper incisors, cuspids and first

premolars and visual scoring from digital photos. A statistically significant

(p<0.05) regression of the WSL was disclosed in both study groups compared to

baseline, but there was no difference between the groups. The mean area of the


Page 19

lesions decreased by 58% in the CPP–ACP group and 26% in the fluoride group

(p=0.06). The QLF findings were largely reflected by the clinical scores. No side

effects were reported. Topical treatment of white spot lesions after debonding of

orthodontic appliances with a casein phosphopeptide-stabilised amorphous

calcium phosphate agent resulted in significantly reduced fluorescence and a

reduced area of the lesions after 4 weeks as assessed by QLF.

FREDRIK BERGSTRAND AND SVANTETWETMAN (2011)39

published a

research paper to update the evidence for primary and secondary prevention

(treatment) of white spot lesions (WSL) adjacent to fixed orthodontic appliances.

They concluded their research as the use of topical fluorides in addition to fluoride

toothpaste as the best evidence-based way to avoid WSL. The mean prevented

fraction based on 6 trials was 42.5% with a range from -4% to 73%. The recent

papers provided the strongest support for regular professional applications of

fluoride varnish around the bracket base during the course of orthodontic

treatment. For the treatment of post-orthodontic WSL, home-care applications of a

remineralizing cream, based on casein phosphopeptide-stabilized amorphous

calcium phosphate, as adjunct to fluoride toothpaste could be beneficial but the

findings were equivocal.

BENJAMIN T. PLISKA et al (2012)40

did a study to examine the effects of

application of casein phosphopeptide amorphous calcium phosphate (CPP-ACP)

paste and microabrasion treatment on the regression of white spot lesions (WSLs).

Artificially-induced WSLs in bovine enamel were randomly assigned to one of

four treatment groups: CPP-ACP paste only, microabrasion only, microabrasion

and CPPACP, and a control. Samples were treated with each regimen twice daily


Page 20

for 2 weeks and stored in remineralizing solution between the treatments.

Quantitative light-induced fluorescence was used to measure changes in

fluorescence, which indicate changes in mineral content of WSLs immediately

before (T1) and 2 weeks after treatment (T2). A two-within-subject factor analysis

of variance was used to analyze the significance of any changes in mineral content

of the lesions from T1 to T2. Results showed there was a statistically significant

(P < .05) gain in fluorescence associated with the microabrasion only, as well as

the microabrasion and CPP-ACP treatments. The changes in fluorescence for the

CPP-ACP treatment alone were not statistically significant. Hence it was

concluded that CPP-ACP paste alone does not significantly improve the

fluorescence value (ie, the mineral content) of WSLs.

MEHMET AKIN; FARUK AYHAN BASCIFTCI (2012)41

conducted a study

to compare the effects of sodium fluoride mouth rinse, casein phosphopeptide

amorphous calcium phosphate (CPP-ACP), and the microabrasion technique in

treating white spot lesions. The study population consisted of 80 patients who had

developed multiple decalcified enamel lesions after fixed orthodontic therapy. The

study population was divided into four groups of 20 patients each. The control

group (group I) participants were to just brush their teeth, the fluoride group

(group II) participants were instructed to use 20 ml of neutral 0.025% sodium

fluoride rinse, the participants in the CPP-ACP group (group III) were instructed

to use tooth mousse twice a day in addition to fluoride toothpaste for 6 months,

and the participants in the microabrasion group (group IV) were to undergo

treatment by the microabrasion technique, which is a commonly used mixture of

18% hydrochloric acid. Results showed the area of the white spot lesions

decreased significantly in all groups. The highest success rate was observed for


Page 21

group IV (97%). The success rate of group III (58%) was significantly higher than

that of groups II (48%) and I (45%). It was concluded that the use of CPP-ACP

can be more beneficial than fluoride rinse for post orthodontic remineralization.

MIKAEL SONESSON, SVANTE TWETMAN AND LARS BONDEMARK

(2013)42

conducted a study to evaluate the effectiveness of daily tooth brushing

with high-fluoride toothpaste on white spot lesion (WSL) formation in adolescents

during treatment with fixed orthodontic appliances (FOA). Four hundred and

twenty-four healthy patients were randomized to use either toothpaste containing

5000 ppm fluoride or regular toothpaste with 1450 ppm fluoride. The primary and

secondary outcome measures were prevalence and incidence of WSL, as

registered from digital photos of the maxillary incisors, canines, and premolars

taken before onset and immediately after debonding. The photos were evaluated

separately by two blinded and calibrated clinicians using a 4-step score. A random

sample of 50 cases was reassessed to check intra- and inter examiner reliability.

Results showed the use of high-fluoride toothpaste resulted in fewer WSL with a

prevented fraction of 32%. The lateral incisor was most commonly affected in

both groups. Study concluded as to prevent WSL during treatment of FOA, daily

use of high-fluoride toothpaste may be recommended.

CARLO FORNAINI et al (2014)43

did an in-vitro study to test using a universal

testing machine whether sub-ablative Er:YAG laser irradiation prior to acid

etching is effective in orthodontic bracket bonding and secondly using micro-

hardness measurements and Scanning Electron Microscopy (SEM) observations to

investigate the effectiveness of de-mineralization reduction in enamel treated with

sub-ablative Er:YAG laser irradiation followed by fluoride varnish application.


Page 22

One hundred and eighty bovine permanent maxillary incisors were selected for

shear bond strength testing and micro hardness measurements. Sub-ablative

Er:YAG laser irradiation was set at a power density of 2.5 J/cm², a frequency of 7

Hz and air/water spray. Brackets were bonded with an auto-curing resin paste. The

shear bond strength was measured comparing laser irradiated and non-irradiated

enamel surface, followed by SEM observation of the bracket-resin-enamel

interface. Micro hardness measurements were made on enamel samples before

treatment, after samples preparation, and after demineralization. Results showed

While the adhesion of orthodontic brackets to bovine enamel after sub-ablative

Er:YAG laser irradiation and acid etching is comparable to that obtained after

conventional acid etching, the effect of laser irradiation associated with topical

application of fluoride varnish increases the micro hardness of enamel. It was

concluded that Sub-ablative Er:YAG laser irradiation before the acid etching

doesn’t reduce the shear bond whereas when associated with fluoride application

it may play a role in caries prevention.

SRUTHI SUNIL et al (2015)44

stated that the modern dentistry aims to manage

non cavitated carious lesions through remineralization in order to prevent

progression of carious lesion, so as to improve, function, strength and aesthetics of

teeth. The emphasis for enamel remineralizationcurrently is being laid upon new

technologies. Nowadays many remineralization components are available in the

market, out of which one such material is Caries Prevention through Casein

Phosphopeptide–Amorphous Calcium Phosphate (CPP–ACP).

AREZOO JAHANBIN et al (2015)45

did a study to compare two microabrasion

techniques on improving the white spot lesions as well as subsequent enamel


Page 23

discoloration. Sixty extracted premolar teeth without caries and hypoplasia were

selected for this study. White spot lesions were artificially induced on the buccal

surface of each tooth. Teeth were randomly assigned to three treatment groups,

each treated with pumice powder as the control, microabrasion with 18% HCl, and

microabrasion with 37% H3PO4. Subsequently, the three groups were daily

immersed for five minutes in a tea-coffee solution for a period of one week.

Colorimetric evaluation was done before and after formation of white spot lesions,

after microabrasion, and after immersion in the colored solution; then the color

differences ∆E were calculated. This study showed that ∆E between the stages of

white spot formation and microabrasion for H3PO4 was more than other groups

and for the pumice powder group it was less than the others. it was concluded that

Pumice powder alone had similar effects as 18% HCl on removing the white spot

lesions. Nevertheless, 18% HCl makes the enamel susceptible for subsequent

colour staining more than the other microabrasion methods.

SHAZA M. HAMMAD, MICHAEL KNO¨SEL (2016)46

conducted a study to

evaluate the efficacy of a new sealant to prevent WSLs during fixed orthodontic

treatment was compared to a control group that did not receive sealant. Patients

and methods for this 2-arm parallel-group randomized trial, 50 subjects were

recruited. Subjects were randomized in a 1:1 ratio to one of the two arms prior to

undergoing fixed orthodontic treatment, namely a single application of SeLECT

DefenseTM

sealant during the bracketing appointment or no sealant (control arm).

Instructions and dentifrices for local home fluoridation regimen were identical in

both groups. Oral hygiene was assessed using the Approximal Plaque Index (API)

at specified time intervals. Results showed only excellent or good oral hygiene

were independent prognostic factors for preventing severe WSLs (p = 0.035). No


Page 24

significant effect on caries incidence was observed for the sealant. It was

concluded that in combination with adequate oral hygiene SeLECT DefenseTM

helps to reduce the frequency of WSLs.

MATTHEW J. MILLER et al (2016)47

stated that White spot lesions (WSLs)

are an all too common negative outcome of orthodontic treatment. WSLs are areas

of enamel demineralization 100–150-mm deep, with an intact porous surface

layer, which can progress until a complete inward collapse of the surface occurs.

Their un-aesthetic opaque appearance is potentially reversible, but irreversible

once cavitated. Clinically detectable WSLs can occur as early as1month after

fixed appliance placement. It is estimated that 50% of patients develop WSLs in at

least one tooth by the end of orthodontic treatment. An immediate application of

fluoride to a white spot lesion will cause a rapid surface remineralization, leaving

deeper layers demineralized, so prevention of lesion progression is necessary for

an ideal aesthetic outcome. Aside from excellent oral hygiene, fluoride varnish,

MI Paste, and smooth surface sealants are currently the primary methods of WSL

prevention. Research has shown no improvement in WSLs when comparing non-

invasive treatment methods such as MI Paste to routine oral hygiene practice.

Success has been shown in treating arrested WSLs with a resin infiltration

technique, but this is most useful on a small scale.

STUDIES RELATED TO PROBIOTICS

H.J. BUSSCHER et al (1999)48

conducted a research to investigate the in vitro

adhesion of two lactobacillus strains, isolated from a bio-yoghurt with active

bacteria, to enamel with and without a salivary conditioning film and to determine

whether daily consumption of such a bio-yoghurt might lead to the installation of


Page 25

lactobacilli in a group of test persons authentically devoid of oral lactobacilli. The

results suggested that both isolates have an ability to adhere to enamel chips

without and with a salivary conditioning film. L. acidophilus strain could fully

withstand this high shear. It was concluded that lactobacilli cannot installed by the

consumption of bio-yoghurts containing active lactobacilli on tooth surfaces of

test persons, selected on the basis of unfavourable ecological conditions for

lactobacilli to flourish, despite the ability of these strains to adhere to enamel.

H. NIKAWA et al (2004)49

studied the effect of Lactobacillus reuteri against one

of the major cariogenic organism, Streptococcus mutans. They stated that Yogurt

products showed a significant growth inhibitory effect against S. mutans, yoghurts

with lactobacilli other than L. reuteri did not show such inhibition. Further,

double-blind, placebo-controlled trial demonstrated that consuming yogurt with L.

reuteri significantly reduced the oral carriage of mutans streptococci, compared

with the placebo yogurt.

ESBER C¸ AGLAR et al (2006)50

did a study to examine the impact of two

different vehicles of probiotic bacterium Lactobacillus reuteri ATCC 55730 on the

levels of salivary mutans streptococci and lactobacilli in young adults. 120 healthy

young adults were included in the study. A placebo-controlled study design with

parallel arms was utilized. The subjects were randomly assigned to four equally

sized groups: group A drank 200 ml of water through a prepared straw containing

L. reuteri ATCC 55730 once daily for 3 weeks, while group B took 200 ml water

through a placebo straw during the same period. Group C was given one tablet

containing L. reuteri ATCC 55730 once daily for 3 weeks, while group D received

placebo tablets without bacteria. Salivary mutans streptococci and lactobacilli


Page 26

were enumerated with chair-side kits at baseline and 1 day after the final

ingestion. They concluded that a short-term daily ingestion of lactobacilli-derived

probiotics delivered by prepared straws or lozenges reduced the levels of salivary

mutans streptococci in young adults.

E. ÇAGLAR et al (2007)51

conducted a study to evaluate the effect of xylitol and

Probiotic chewing gums on salivary mutans streptococci (MS) and lactobacilli

(LB). It was concluded that daily chewing on gums containing probiotic bacteria

or xylitol reduced the levels of salivary MS in a significant way. No alterations of

salivary LB was demonstrated in any group. However, a combination of probiotic

and xylitol gums did not seem to enhance this effect.

ESBER C¸ AGLAR et al (2008)20

conducted a double-blind, randomized

crossover study to examine whether short-term consumption of ice-cream

containing bifidobacteria can affect the salivary levels of mutans streptococci and

lactobacilli in young adults. They included 24 healthy subjects were followed over

4 periods. During periods 2 and 4 (10 days each), they ingested 100 ml (53 g) ice-

cream containing Bifidobacteriumlactis Bb-12 once daily or a control ice-cream

without viable bacteria. Results showed statistically significant reduction (pB0.05)

of salivary mutans streptococci after consumption of the probiotic ice-cream.

Hence it was concluded that Daily consumption of ice-cream containing probiotic

bifidobacteria may reduce the salivary levels of mutans streptococci in young

adults.

HAUKIOJA A, LOIMARANTA V, TENOVUO J. (2008)52

conducted an in

vitro study to investigate whether the probiotic bacteria used in commercial

products affect the protein composition of the salivary pellicle and the adherence


Page 27

of other oral bacteria. Salivary pellicle on hydroxyapatite and the adhesion of

Streptococcus mutans. Results showed Probiotic bacteria that bound to saliva-

coated hydroxyapatite reduced the adhesion of S. mutans. Salivary pellicle protein

composition was modified by all the strains tested. The modifications in the

pellicle affected the adherence of S. mutans. This in vitro study showed that

probiotic strains used in commercial products may affect the oral ecology by

specifically preventing the adherence of other bacteria and by modifying the

protein composition of the salivary pellicle.

SULE KAVALOGLU CILDIR et al (2009)53

did a study to examine whether

short-term consumption of fruit yogurt containing probiotic bifidobacteria would

affect the levels of salivary mutans streptococci and lactobacilli in patients with

fixed orthodontic appliances. A double-blind, randomized crossover study was

performed and 24 healthy adolescents (12 – 16 years) undergoing orthodontic

treatment were followed over four periods. During periods 2 and 4 (2 weeks

each), the subjects ingested 200 g fruit yogurt containing Bifidobacteriumanimalis

subsp. Lactis DN- 173010 (2 × 10 8

colony forming units/g) once daily or a

control yogurt without viable bacteria. Periods 1 and 3 were run-in and wash-out

periods of 1 and 6 weeks, respectively. Salivary mutans streptococci and

lactobacilli were enumerated with chair-side kits before and after the yogurt

consumption periods. Pre and Post-treatment values within each regimen were

compared with a two-tailed marginal homogeneity test for categorical data.

Results showed a statistically significant reduction of salivary mutans streptococci

after probiotic yogurt consumption (P< 0.05), which was in contrast to the control

yogurt. No significant alterations of the salivary lactobacilli counts were observed.

Hence concluded that Short-term daily consumption of fruit yogurt containing


Page 28

Bifidobacteriumanimalis subsp. lactis DN- 173010 may reduce the levels of

mutans streptococci in saliva during orthodontic treatment with fixed appliances.

ANTONIO-JUAN FLICHY-FERNÁNDEZ et al (2010)54

reviewed published

studies regarding probiotics and their effects on the oral cavity. Most authors

concluded that the use of oral probiotics was associated with an improvement in

oral health, including a significantly reduced level of cariogenic and periodontal

pathogens and a lower crevicular fluid volume and cytokine concentration.

BERNARD BIZZINI1 et al (2012)55

stated that Probiotic bacteria belonging to

the genus of Lactobacillus, Bifidobacterium and Streptococcus have been proven

effective for preventing caries by reducing the number of cariogenic bacteria in

saliva after a short period of consuming the probiotic. In contrast, the effect of

probiotics on improving oral malodour, gingivitis and periodontitis has been less

investigated.

PRANAY JAIN AND PRIYANKA SHARMA (2012)56

stated that Probiotics or

the foods with ‘live cultures’ have come up as one of the most promising alternate

to traditional disease management. Probiotics are those viable microorganisms

which are constituents of natural microflora of human body. Probiotic therapy

decreases the risk of colonization by oral pathogens without depleting the friendly

microflora. Probiotics resembles the human body microbiota and are readily

incorporated in the natural microflora of human body. They are harmless and easy

to consume in many edible forms. The inability of the antibiotics to discriminate

good bacteria from the disease causing bacteria, the development of antimicrobial

resistant mutants and the increasing rate of antibiotic associated side effects and

complications suggests an urgent need to switch our therapeutic approach from


Page 29

traditional antibiotics to the probiotic therapy for oral care. The use of probiotics

in routine life is likely to improve the oral health. This review demonstrates the

action of Probiotics on oral health and disease.

R. SUDHIR et al (2012)57

conducted study to compare the effect of short‑term

consumption of probiotic curd containing Lactobacillus acidophilus and normal

curd on salivary Streptococcus mutans counts, as well as salivary pH. Forty,

caries‑free, 10‑12 years old children were selected and randomly allocated to two

groups. Test Group consisted of 20 children who consumed 200ml of probiotic

curd daily for 30 days. Control Group consisted of 20 children who were given

200ml of regular curd for 30 days. Salivary pH and salivary Streptococcus mutans

counts were recorded at baseline and after 30 days. Results showed consumption

of probiotic curd resulted in a statistically significant reduction in S. mutans

colony counts (P<0.001) as compared to regular curd. However, there was a slight

reduction in pH (P>0.05) in both the groups. Hence it was concluded the

short-term consumption of probiotic curds can reduce oral S. mutans counts.

JUBIN EASO JOSE, SRIDEVI PADMANABHAN, AND ARUN B.

CHITHARANJAN (2013)58

conducted a study to evaluate and compare the

effects of the systemic consumption of probiotic curd and the topical application

of probiotic toothpaste on the Streptococcus mutans levels in the plaque of

orthodontic patients. The study consisted of 60 orthodontic patients divided into 3

groups of 20 each. Group 1 was the control group. The patients in group 2 were

given probiotic curd, and those in group 3 were asked to brush twice daily with

probiotic toothpaste (GD toothpaste; Dental Asia Manufacturing, Shah Alam,

Selangor, Malaysia). Samples were collected at 2 times: before the study began


Page 30

and after 30 days. Plaque specimens were collected from the labial surfaces

immediately surrounding the orthodontic brackets of the maxillary lateral incisors

using a 4-pass technique. Results showed at the end of the study, there were

reductions in S mutans concentration in groups 2 and 3 that were statistically

significant compared with group 1, but there was no statistically significant

difference between groups 2 and 3. The study Concluded as the consumption of

probiotic curd and the use of probiotic toothpaste cause a significant decrease in

the S mutans levels in the plaque around bracketsin orthodontic patients.

ISABELLE LALEMAN et al (2014)59

systematically reviewed the available

literature regarding the caries-preventive effect of probiotics. The outcomes had to

be presented as the effect of probiotics on the incidence of caries or on the levels

of mutans streptococci and/or Lactobacillus species. Human studies, with at least

15 participants, comparing a probiotic product with a placebo/no probiotic were

included where possible, a meta-analysis was performed to obtain quantitative

data. The Meta analysis showed that when the probiotic and control group are

compared after treatment, significantly more patients in the probiotic group had

low mutans streptococci (<105 CFU/ml) counts and significantly less patients had

high (>106 CFU/ml) counts. Regarding the Lactobacillus counts, comparing the

probiotic and control group at the end of the probiotic use, no significant

differences could be observed, neither in low (<104 CFU/ml) nor in high

Lactobacillus (>106 CFU/ml) counts. Within the limitations of the available data,

they have concluded that probiotics decrease the mutans streptococci counts. This

suggests that probiotics could have a positive effect in the prevention of caries.

They further stated that there is insufficient evidence that probiotics can prevent

caries, but they can reduce the mutans streptococci counts.


Page 31

WIPAPUN RITTHAGOL, CHONTIRA SAETANG (2014)60

studied the effect

of powdered milk containing Lactobacillus paracasei on salivary mutans

streptococci in the orthodontically treated Cleft patients. A total of 30

orthodontically treated non-syndromic cleft lip and palate patients (aged 19.22 ±

3.66 years): 15 in the intervention group (A) and 15 in the control group (B).

Average daily consumption of milk in both groups was 50 mL for 4 weeks.

Salivary mutans streptococci and lactobacilli were enumerated using a

quantitative differential culture at baseline and once a week after the end of the

administration period for 4 weeks. A statistically significant reduction in mutans

streptococci counts occurred in group A, in contrast to group B (P < .001). A

significant increase of lactobacilli numbers was found in group A (P < .001),

and L. paracasei SD1 could be detected up to 4 weeks following cessation of

dosing. The probiotic milk powder containing L. paracasei SD1 could reduce

mutans streptococci counts and was apparently able to colonize the oral cavity of

the orthodontically treated cleft lip and palate patients. However, the potentially

beneficial influence of the probiotic milk on the complex oral microflora justifies

further studies with a larger group of volunteers.

KOUR S et al (2015)19

stated that Enamel demineralization leading to white spot

lesions is the most common problem faced during or after orthodontic treatment.

Various methods have been suggested to prevent enamel demineralization among

which fluorides are the most common, but they also have some disadvantages.

Probiotics could be a viable option in preventing and treating the enamel

demineralization by reducing the levels of pathogenic bacteria i.e streptococcus

mutans.


Page 32

SAURAV CHATURVEDI, UPENDRA JAIN (2015)4stated that White spot

lesion development of the enamel surface is by far the most important iatrogenic

effect of fixed orthodontic appliance therapy and can have lasting negative effects

on dental aesthetics. Probiotics are live microorganisms which when administered

in adequate amounts confer a health benefit on host.

SOTIRIA GIZANI et al (2015)61

conducted a research to evaluate the effect of

daily intake of lozenges containing probiotic bacteria on white spot lesion (WSL)

formation as well as on salivary lactobacilli (LB) and mutans streptococci (MS)

counts, in patients undergoing orthodontic treatment with fixed appliances. A

randomized double-blind placebo-controlled study design with two parallel arms

was employed. Patients (n = 85, mean age 15.9 years) with maxillary braces on at

least eight anterior teeth and a remaining treatment period of 7–24 months were

finally enrolled and randomly allocated to a test or placebo group. Subjects in the

test group were instructed to take one probiotic lozenge containing two strains of

Lactobacillus reuteri once daily. An identical lozenge without active bacteria was

used in the placebo group. Dental plaque, WSL, and salivary MS and LB levels

were recorded at baseline and immediately after debonding. Results showed the

groups were balanced at baseline. The mean duration of the intervention was 17

months and the total dropout rate was 10 per cent. There were no differences in

the incidence of WSL between the groups at debonding. The patients had

generally a neglected oral hygiene, both at baseline and at the follow-up. The

levels of salivary LB were significantly reduced in both groups (P < 0.05) at the

time of debonding compared with baseline, while no alterations of the MS counts

were unveiled. It was concluded as daily intake of probiotic lozenges did not seem


Page 33

to affect the development of WSL during orthodontic treatment with fixed

appliances.

RAJAN DHAWAN, SHIVANI DHAWAN (2016)62

did a study to investigate

commercially available combined probiotic formulation for its effect on plaque,

gingivitis, and salivary Streptococcus mutans levels in subjects with chronic

gingivitis. A total of 36 subjects were finally enrolled in a double‑blind,

placebo‑controlled, randomized trial. Selected subjects were randomly divided

into two groups: Group A (control group) with 17 subjects receiving placebo

tablets twice daily and Group B (probiotic group) with 19 subjects receiving

probiotic tablets twice daily. The study consisted of two 2‑week periods: a

2‑week intervention period (T1–T2) and a 2‑week post treatment period (T2–T3).

Clinical parameters and bacterial counts of salivary Streptococcus Mutans were

evaluated at baseline (T1), at the completion (T2) of medication, and 2 weeks

after the medication (T3).On comparative evaluation between the two groups,

results indicated that Group B (probiotic group) exhibited statistically significant

reduction in Plaque Index, Gingival Index, Calculus Index and Streptococcus.

Mutans level than Group A (control group) over the entire span of the study.

Hence concluded as probiotic could be useful in the improvement/maintenance of

oral health.

SHIVANGI SRIVASTAVA et al (2016)63

stated that dairy products like curd

seem to be the most natural way to ingest probiotics which can reduce

Streptococcus mutans level and also increase salivary pH thereby reducing the

dental caries risk. The double blind parallel randomized clinical trial was carried

out with 60 caries free volunteers belonging to the age group of 20-25 years who


Page 34

were randomly allocated into two groups. Test Group consisted of 30 subjects

who consumed 100ml of probiotic curd daily for seven days while an equal

numbered Control Group were given 100ml of regular curd for seven days. Saliva

samples were assessed at baseline, after ½ hour 1 hour and 7 days of intervention

period using pH meter and Mitis Salivarius Bacitracin agar to estimate salivary pH

and S. mutans count. The study revealed a reduction in salivary pH after ½ hour

and 1 hour in both the groups. However after 7 days, normal curd showed a

statistically significant (p< 0.05) reduction in salivary pH while probiotic curd

showed a statistically significant (p< 0.05) increase in salivary pH. Similarly with

regard to S. mutans colony counts probiotic curd showed statistically significant

reduction (p< 0.05) as compared to normal curd. It was concluded as short-term

consumption of probiotic curds showed marked salivary pH elevation and

reduction of salivary S. mutans counts and thus can be exploited for the

prevention of enamel demineralization as a long-term remedy keeping in mind its

cost effectiveness.

SAURAV CHATURVEDI et al (2016)64

conducted a study to evaluate the effect

of the application of probiotic lozenge on the Streptococcus mutans levels in the

plaque of orthodontic patients. A sample of 30 randomly selected patients (14

females and 16 males) having orthodontic treatment were selected for the study.

They were divided into two groups of 15 each. One group of patients (Group 2)

was given the probiotic (Lactobacillus brevis CD2) lozenges, and the other group

(Group 1) was given the placebo lozenges. The lozenges were administered to the

patients from day 1 after the first plaque sample had been assessed and continued

until day 30. Plaque samples were again taken and evaluated at the end of day 30.

The samples were placed into individual microcentrifuge tubes with anonymous


Page 35

coding and sealed for transport for real-time polymerase chain reaction to the 3B

Blackbio Biotech India Ltd, Bhopal, India. Results showed, after the use of the

probiotic lozenges, 14 of 15 subjects in Group 2 showed reduction in the S.

mutans levels; in two subjects, there was no detectable S. mutans after 30 days. In

Group 1 after use of placebo, 3 out of 15 patients showed a decrease in S. mutans

levels. Hence concluded as daily short-term ingestion of a Lactobacilli

brevis derived probiotic through a lozenge tablet could reduce the levels of S.

mutans in plaque around orthodontic brackets.

Materials and Method

Page 36

MATERIALS AND METHOD

Materials:

Brackets : “0.022”-inch slot stainless steel MBT prescription (3M

Gemini series, USA)

Transbond XT adhesive

Sterile scalar (Figure – 2)

Test tube (Figure -3)

Petri plates

Glass rods (Figure - 3)

Culture media

o Thioglycolate Broth/Agar(Figure – 4)

o Blood Agar(Figure – 5)

o Peptone

Saline

Incubator (Figure – 6)

Candle jar (Figure -7)

Digital colony counter (Figure – 8)

Pipette


Page 37

PRODUCTS:

BIFILAC lozenges:

Each BIFILAC lozenges (Figure – 9)contains:

Bacillus Mesentericus (1 Million Spores)

Clostridium Butyricum (2 Million Spores)

Lactobacillus Sporogens (50 Million Spores)

Streptococcus Thermophilus (30 Million Spores)

DAROLAC- Z sachets:

Each DAROLAC –Z sachets (Figure – 10) contains:

Lactobacillus rhamnosus (1 billion cells)

Saccharomyces boulardii (125 mg)

Zinc enriched yeast

Equivalent to elemental zinc (20 mg)

YAKULT 65 ml:

Each bottle of Yakult (Figure – 11) contains:

Sugar (sucrose, dextrose)

Skimmed milk powder

Natural flavours

Live lactobacillus caseiShitora strain, 6.5 billion per 65 ml bottle

(concentration of 108

CFU/ml)


Page 38

PATIENT SELECTION:

Inclusion criteria:

Patient undergoing orthodontic treatment with the PEA appliance (MBT),

“.022” inch slot; (3M Gemini series, USA) for a minimum period of 9

months and maximum of 12 months.

Age between 14 - 29 years of both the genders.

Patient with permanent dentition.

Patient with good general health.

Habit of brushing twice daily.

Patients who volunteered for participation and have signed informed

consent.

Exclusion criteria:

Patient with significant medical history of chronic systemic illness, immune

disorders.

Patients on any other medication during or previous month of study.

Patient who have had topical fluoride treatment within 4 weeks.

Patient with poor oral health or active untreated carious lesions or gingival

inflammation.

Patient with habit of chewing gum or using mouthwash during previous

month and during study.

Elimination criteria:

If patients included in the study would have developed any medical

condition which would have required antibiotics to be taken within the

study duration, would have been referred to general physician and

excluded from the study.


Page 39

METHODOLOGY:

Source of data:

The study population for the present study was selected from the

outpatient Section of Department of orthodontics and dentofacial Orthopaedics at

Tamil Nadu Government Dental College & Hospital, Chennai, Tamil Nadu, India,

who were undergoing orthodontic treatment with fixed appliance mechanotherapy

based on inclusion and exclusion criteria.

Ethical clearance was obtained from Institutional Review Board (IRB) of

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

Informed consent from each subject was obtained after explaining the nature of

the study. Study participants were divided into four groups

Study groups:

o Group 1: Study group receiving probiotic lozenges

o Group 2: Study group receiving probiotic sachets

o Group 3: Study group receiving probiotic drink

Control group:

o Group 4: Group receiving no probiotics

Type of study:

Randomized controlled trial

Study sample:

I. Sampling Technique

A simple random technique was used to select participants for each group.

II. Sample Size

The sample size of 80 Subjects was determined empirically.


Page 40

Pilot Study:

Pilot study was conducted before the main study to check the feasibility

and validity of the study. By standardizing all the materials and method, the study

was conducted by considering a total of 12 participants, 3 in each group. Pilot

study assessments were utilized for proper planning and execution of the main

study.

Method of collection of data:

80 subjects of age group between 14 to 29 years undergoing orthodontic

treatment with pre adjusted orthodontic appliance, bonded with Transbond XT

adhesive, were selected from post graduate clinic of Department of orthodontics

and dentofacial orthopaedics, Tamil Nadu Government Dental College And

Hospital, Chennai.

Randomisation:

A total of 80 subjects were included in this study those who fulfilled the

above mentioned criteria and randomly allocated in four groups using lottery

method with 20 subjects in each group.

Study groups:

o Group 1: Study group receiving probiotic lozenges

o Group 2: Study group receiving probiotic sachets

o Group 3: Study group receiving probiotic drink

Control group:

o Group 4: Group receiving no probiotics


Page 41

Chart 1: Group distribution chart

Group 1: Subjects were asked to chew the tablet for 2-3 minutes and then

swallow twice a day for one month and were instructed not to eat or drink for at

least 30 minutes after application.

Group 2: Participants were asked to mix onesachet powder in 10 ml of water in a

measuring cup and consume twice a day (once in a morning and once at night) for

one month.

Group 3: Subjects were asked to have 1 bottle of probiotic drink with their

breakfast every morning for a month.

Group 4: Control group participants were not taking probiotics in any form.

The participants were instructed to avoid chewing gums, mouthwashes,

and antibiotics during the study. Samples were collected at 2 times: before the

study began and after 30 days.

Groups

Study group

Group-B

Probiotic

yogurt

Group-C

Probiotic lozenges

Group-D

Probiotic Sachets

Group-A

Control group

No probiotics


Page 42

Blinding:

The products were administered by investigator and instructions were

given to participants for use of it. Hence single blinding was ensured by blinding

lab technician.

Collection of samples

At each time interval, the elastomeric modules were carefully removed to

disengage the arch wires by the same operator. Plaque specimens were collected

from labial surfaces immediately surrounding the orthodontic brackets of the

maxillary lateral incisors with a sterilised scalar using a 4-pass technique as

suggested by Pellengrini et al. four passes each along the tooth at the bracket

interface at the gingival, mesial, distal, and occlusal aspects, were used to prevent

overloading the instrument tip. (Figure – 12)

Inoculation of samples:

Plaque samples were taken in a screw cap vials containing 10ml of 10%

sterile thioglycollate broth which was a transport medium. The samples were then

inoculated on culture media to evaluate for streptococcus mutans, in the

Microbiology lab, Hi-tech lab, Chennai, Tamil Nadu.

Samples were inoculated on anaerobic blood agar to estimate the colony

count of streptococci. One percent Potassium tellurite solution was added to make

the solution selective for streptococci and 0.2u/ml of sterile bacitracin solution

was added to the solution to make the medium highly selective for streptococcus

mutans.100 micro litre of broth was transferred under sterile conditions onto the

sterile anaerobic blood agar plates. It was then uniformly spread over the surface

of medium using a sterile L spreader. After 10 minutes, the Anaerobic blood agar


Page 43

plates were incubated at 370c in anaerobic jar with anaerobic pack with additional

five percent carbondioxide for 48 hours. Colonies were counted under digital

colony counter.

The data were suitably compiled and analyzed with appropriate statistical

method using Statistical Package for the Social Sciences (SPSS) software

version 21.

Results

Page 44

RESULTS

In the present study, eighty orthodontic patients were selected and divided

into four groups, each group consisting of 20 patients; Probiotic lozenges,

Probiotic sachets, Probiotic Drink and Control groups respectively. The results

were evaluated and compared for S.mutans CFU before and after thirty days of

use of probiotics in study groups and without use of probiotics in control group.

STATISTICAL ANALYSIS:

Data collected in the present study was compiled and analysed using the

Statistical Package for Social Sciences (SPSS) version 21.0. Data comparison was

done by applying specific statistical tests to find out the statistical significance of

the results.

Since the data were continuous and quantitative type, parametric tests were

used for analysis. The mean and standard deviation of S.mutans CFU at baseline

(0 day) and after 30 days of intervention were calculated. Paired T test was used

for pairwise comparison of S.mutans CFU before and after administration of

Probiotic formulation in study groups and without use of probiotics in control

group. The variation of S.mutans CFU between different groups at baseline and

the variation of S. mutans CFU between different groups after 30 days was

analysed using one way Analysis of Variance (ANOVA) followed by Tukey’s

Post hoc test. P value less than 0.05 was considered for statistical significance.

Results

Page 45

Table 1: Demographic Distribution of study subjects according to Groups, Age

and Gender

Groups

No of

subjects

Age

Gender

Males Females

Group 1 Probiotic Lozenges 20 14-29 years 12 08

Group 2 Probiotic Sachets 20 14-29 years 14 06

Group 3 Probiotic Drink 20 14-29 years 09 11

Group 4 Control 20 14-29 years 13 07

20 subjects were assigned to each of Probiotic lozenges, Probiotic sachets,

Probiotic Drink and Control group. All subjects were aged between 14-29 years.

In group 1 out of 20 subjects, 12 males and 8 females, in group 2; 14 males and 6

females, in group 3; 9 males and 11 females, in group 4; 13 males and 7 females

participated in present study.

Results

Page 46

Table 2: Comparison of S.mutans CFU/ml before and after 30 days of

administration of probiotics in each study groups and without use of probiotics in

control group.

Groups CFU/ml Mean + SD t p value

Probiotic Lozenges

n=20

Baseline CFU/ml (103) 247.6 + 38.82

2.852 0.010*

After CFU/ml (103) 46 + 09

Probiotic Sachets

n=20

Baseline CFU/ml (103) 210.25 + 40.56

2.317 0.032*

After CFU/ml (103) 104.5 + 21.55

Probiotic Drink

n=20

Baseline CFU/ml (103) 224.65+ 39.94

2.514 0.021*

After CFU/ml (103) 64.0 + 27.63

Control

n=20

Baseline CFU/ml(103) 243 + 22.5

0.416 0.682

After CFU/ml (103) 221 + 40.6

*Statistically Significant(Significance level p < 0.05)

In group 1, at baseline S. mutans CFU counts were 247.6 x 103 which were

reduced to 46 x 103 after 30 days of consumption of Lozenges. In group 2, at

baseline S. mutans CFU counts were 210.25 x 103 which were reduced to 104.5 x

103 after 30 days of consumption of Probiotic Sachets. In group 3, at baseline S.

mutans CFU counts were 224.65 x 103 which were reduced to 64.0 x 10

3 after 30

days of consumption of Probiotic Drink. There was reduction in s mutants CFU

counts after 30 days of consumption of probiotic formulations in group 1 Probiotic

Lozenges, group 2 Probiotic sachet and group 3 Probiotic drink compare to

baseline. The difference was statistically significant. Whereas, there was no

statistically significant difference found in S. mutans CFU after 30 days compare

to baseline in control group.

Results

Page 47

Table 3: Comparison of S.mutans CFU between different groups at baseline and

the variation of S.mutans CFU between different groups after 30 days.

*Statistically Significant (Significance level p < 0.05)

1= Probiotic Lozenges, 2=Probiotic Sachet, 3=Probiotic Drink, 4=Control Group

While Comparing S.mutans CFU between different groups at baseline,

there was no statistically significant difference found. Whereas after 30 days while

comparing S.mutans CFU between different groups, there was statistically

significant difference found. Tukey Post hoc Test analysis confirmed the

maximum reduction of S. mutans CFU in 1Probiotic lozenges group followed by

3 Probiotic Drink group followed by 2 Probiotic Sachet group.

Sum of

Squares

df

Mean

Square

F P value

Tukey

Post hoc

Test

Baseline

CFU

Between

Groups

1636.443 3 5454.810

0.325

0.807

1=2=3=4 Within

Groups

127373.228 76 1675.963

Total 1290096.720 79

After

CFU

Between

Groups

13164.333 3 43881.110

10.014

0.001*

1>3>2>4 Within

Groups

333036.767 76 43820.627

Total 464680.098 79

Discussion

Page 48

DISCUSSION

Oral infection constitutes one of the most common and recurrent forms of

infections in humans. Orthodontic treatment with fixed appliance increases plaque

retention and produces a greater difficulty in optimal oral hygiene maintenance

which predisposes to enamel demineralization and white spot formation. Fixed

and removable orthodontic appliances facilitate the colonization of cariogenic

bacteria resulting in increased susceptibility to caries when undergoing

orthodontic treatment.65

Among the cariogenic microorganisms, the initiation of carious lesions is

mainly due to streptococcus mutans and streptococcus sobrinus, they are the first

organisms to colonize the initial carious lesion. They are also the most cariogenic

among the mutans streptococci species because of their greater adhesive

properties and acid release, their presence increases the risk for enamel

demineralization. Therefore, adhesion of these bacteria to fixed appliances might

influence the formation of pathogenic plaque and enamel demineralization during

orthodontic treatment.66

Although good oral hygiene is a prerequisite for fixed

appliance therapy, plaque control can vary during this time among individuals.

Various studies have shown a difference between the Streptococcus

mutans counts in saliva and the Streptococcus mutans counts in plaque. Since the

anterior region, the lateral incisors are the most susceptible because of decreased

salivary clearance and also less space between the bracket and gingiva12

, plaque

accumulation around orthodontic brackets is a more specific region that harbours

the Streptococcus mutans and can be considered more reliable in regard to white

spot lesions.

Discussion

Page 49

White spot lesions have been prevented over the years using fluoride

delivery systems, casein phosphor peptide amorphous calcium phosphate, and

enamel surface attenuation with an argon laser have proved to be useful.

Continuous fluoride release from fluoride-containing sealants, elastomeric chains,

primers and adhesives in bonding brackets is also useful. They reduce the number

and size of white spot lesions.58

Many other methods, including antimicrobial and

antibiotic therapies have been tried, but their efficiency lasts only as long as they

are supplied at regular intervals.67

The concept of microbial ecological change as a mechanism for preventing

dental disease is an important one while altered microbial ecology may lead to

dental disease. Bacteriotherapy is an alternative and promising way to combat

infections by using harmless bacteria to displace pathogenic microorganisms.

Probiotics are one of these new agents which are widely used for their therapeutic

action (i.e. whole bacteria replacement therapy) to eliminate pathogenic members

of the microbiota. Limited research is available showing that some probiotic

cultures may help dental improvement during orthodontic treatment.

The use of probiotics has taken giant leaps since the 20th

century.

Probiotics can create a biofilm, acting as a protective lining for oral tissues against

oral diseases by keeping the bacterial pathogens off oral tissues by filling the

spaces where the pathogens would invade. The probiotic organisms Lactobacillus,

Streptococci, and Bifidobacterium species, are genetically designed to have

greater adhesion and hence competitively inhibit Streptococcus mutans.58

Discussion

Page 50

During the last few years, several authors have suggested the use of

probiotic bacteria for oral health. Inefficiency of the antibiotics to discriminate

between good bacteria and disease causing bacteria, the development of antibiotic

resistant mutans and side effects of antibiotic use changed our therapeutic

approach to the probiotic therapy for oral care.

To be able to display a probiotic effect against caries, a bacterium must

first be able to adhere to the tooth surface, where cariogenic bacteria reside.

Secondly, it has to become part of the biofilm that develops on teeth. Finally, it

must compete with cariogenic bacteria, thus reducing the level of their

colonization.68

The present study aims at comparing the effectiveness of three different

probiotics in reducing streptococcal mutans colony count. For the study, we have

randomly selected 80 patients with different types of malocclusion comprising

both males and females within the age group of 14-29 years, and divided into four

groups each 20; probiotic lozenges, probiotic sachets, probiotic drink and control

group. Products were administered for 30 days so that there was ample time for us

to observe the action of probiotics. There were almost same levels of S. mutans in

all four groups before the study started. After 30 days, there was a statistically

significant reduction in S. mutans colony count in all the three study groups. In

group 1, at baseline S. mutans CFU counts were 247.6 x 103 which were reduced

to 46 x 103 after 30 days of consumption of Lozenges. In group 2, it was210.25 x

103 which has been reduced to 104.5 x 10

3 after 30 days of consumption of

Probiotic Sachets. In group 3, at baseline S. mutans CFU counts were 224.65 x

103 which were reduced to 64.0 x 10

3 after 30 days of consumption of Probiotic

Discussion

Page 51

Drink. Whereas, there was no statistically significant difference found in S.

mutans CFU after 30 days compare to baseline in control group. Comparison

between all three study groups showed, group 1 Probiotic lozenges showed

maximum reduction of S. mutans followed by 3 Probiotic Drink group followed

by 2 Probiotic Sachet group.

The probiotic strains used in probiotic lozenges group, Each contains

Bacillus Mesentericus (1 Million Spores), Clostridium Butyricum (2 Million

Spores), Lactobacillus Sporogens (50 Million Spores), Streptococcus

Thermophilus (30 Million Spores) and instructed to take twice a day, has shown

similar results such as other studies with the consumption of probiotic lozenges on

the plaque around orthodontic brackets, done by Saurav chaturvedi et al (2016)64

where they have used probiotic lozenge contained not <1 billion colony-forming

units (CFU) of L. brevis and each subjects were instructed to take two lozenges by

sucking in the morning and two lozenges at night (2 lozenges b.i.d) and a study by

Esber Caglar et al (2008)20

where they have used medical device containing the

probiotic lozenge with L. reuteri ATCC 55730/ L. reuteri ATCC PTA 5289

(1.1×108CFU) once daily for 10 days. This reduction can be due to the formation

of a protective biofilm that prevents adhesion of pathogens69,

inhibiting

pathogenic bacteria, or due to antimicrobial properties of the bacteriocins.50

In

contrast to our study some previous studies could not confirm the efficiency of

short term use of probiotic lozenges on reduction of S. mutans. A study by

Keller et al. (2012)70

concluded that daily oral administration of probiotic

lozenges containing Lactobacillus reuteri did not affect the regrowth of salivary S.

mutans levels. Another study by Sotiria Gizani et al (2015)61

also concluded that

Discussion

Page 52

Daily intake of probiotic lozenges did not seem to affect the development of WSL

and do not alter MS count during orthodontic treatment with fixed appliances.

The probiotic strains used in probiotic drink group, each drink contains

6.5×108 viable lactobacillus casei Shitora strain per 65 ml bottle instructed to take

with their breakfast every morning for a month shown similar results such as other

similar studies: a study by Maryam Siddiqui et al (2016)71

concluded that there is

a statistically significant reduction of salivary S. mutans after consumption of the

probiotic drink. Another study by Yadav M et al (2014)72

also concluded that

daily consumption of drink containing probiotic bacteria can reduce the levels of

mutans streptococci and may contribute to the prevention of dental caries. In

contrast a study by Sutula et al. (2012)73

failed to find any overall effect of a

probiotic drink (Yakult) in healthy denture wearers.

The probiotic strains used in probiotic sachets group, each 2g sachets

contains Lactobacillus rhamnosus (1 billion cells), Saccharomyces boulardii (125

mg), Zinc enriched yeast, Equivalent to elemental zinc (20 mg). Patients asked to

mix one sachet powder in 10 ml of water in a measuring cup and consume twice a

day (once in a morning and once at night) for one month.The results showed that

the daily consumption of probiotics sachets for 30 days reduced the Streptococcus

mutans colony counts which were similar with a study by Jindal et al (2011)74

in

which they have mixed one sachet powder in 20 ml of water in a measuring cup

and each subject was instructed to swish the mixture in the mouth for one minute.

Science is providing us the tools to diagnose and prevent infection before

it causes damage. The application of probiotic strategies may, in the near future,

provide the end of new cavities in treated populations. Bacteriotherapy in the form

Discussion

Page 53

of probiotics seems to be a new alternative for oral health giving a new research

field for dental science to proceed, economically viable bacterial product for

maintaining oral health.

This study showed that probiotics can strongly be considered an option for

the control of white spot lesions in orthodontic patients as an alternative to

fluorides. Orthodontists would probably find introducing an oral hygiene regimen

involving a probiotics for their patients more practical during treatment.

Limitations:

1. The next predominant bacteria which induces white spot lesions i.e.,

lactobacillus and other microorganisms were not included in the present

study.

2. Shorter duration. Further research with the longer time period is required.

Summary and Conclusion

Page 54

SUMMARY AND CONCLUSION

SUMMARY

The present study was done in the department of orthodontics and

Dentofacial Orthopaedics, Tamil Nadu government dental college and hospital,

Chennai in collaboration with Hi-Tech lab, Chennai. A total of 80 patients in the

age range of 14- 29 years of both gender who were undergoing orthodontic

treatment with fixed appliance mechanotherapy for a minimum period of 9

months and maximum period of 12 months were included in this study based on

inclusion and exclusion criteria. Plaque specimens were collected from labial

surfaces immediately surrounding the orthodontic brackets of the maxillary lateral

incisors with a sterilised scalar using a 4-pass technique and assessed for the

levels of mutans streptococci using anaerobic blood agar.

From the findings observed in this present study it can be concluded that

1. There were almost same levels of S. mutans in all four groups before the

study started.

2. After 30 days, showed there is a statistically significant reduction in the

streptococcus mutans count in each group

Probiotic lozenges is more effective than probiotic drink followed by

sachets and control in reducing streptococcus mutans count in plaque around

orthodontic brackets.

Summary and Conclusion

Page 55

CONCLUSION

Probiotics used for the management of oral disease may reduce the cost of

conventional therapy and prevention programs. The idea of replacing harmful

microorganisms with non‑harmful, inactivated, or genetically modified bacteria is

attractive. With the focus on disease prevention and optimal health for all ages,

the potential for probiotics’ use is enormous. Efforts should be made to increase

the awareness of the general dental practitioners about this aspect of oral disease

therapy. The existence of probiotics in the indigenous oral microflora of humans

warrants exploration because these bacteria offer the advantage of being perfectly

adapted to the oral ecosystem. Much more scientific developments are needed to

have a better understanding of these tiny forms of lives in order to broaden their

potential applications.

Bibliography

Page I

BIBLIOGRAPHY

1. Al-Omiri, M. K., & Abu Alhaija, E. S. (2006). Factors affecting patient

satisfaction after orthodontic treatment. The Angle Orthodontist, 76(3),

422-431.

2. Morrow, D., Wood, D. P., & Speechley, M. (1992). Clinical effect of

subgingival chlorhexidine irrigation on gingivitis in adolescent orthodontic

patients. American Journal of Orthodontics and Dentofacial

Orthopedics, 101(5), 408-413.

3. Øgaard, B., Rølla, G., &Arends, J. (1988). Orthodontic appliances and

enamel demineralization: Part 1. Lesion development. American Journal

of Orthodontics and Dentofacial Orthopedics, 94(1), 68-73.

4. Chaturvedi S, Jain U. (2015) Importance of Probiotics in Orthodontics. J

Orofac Res 2; 5(3):99-103.

5. Øgaard, B. (2008, September). White spot lesions during orthodontic

treatment: mechanisms and fluoride preventive aspects. In Seminars in

orthodontics (Vol. 14, No. 3, pp. 183-193). WB Saunders.

6. Bishara, S. E., &Ostby, A. W. (2008, September). White spot lesions:

formation, prevention, and treatment. In Seminars in Orthodontics (Vol.

14, No. 3, pp. 174-182). WB Saunders.

7. Eliades, T., Eliades, G., & Brantley, W. A. (1995). Microbial attachment

on orthodontic appliances: I. Wettability and early pellicle formation on

bracket materials. American Journal of Orthodontics and Dentofacial

Orthopedics, 108(4), 351-360.

Bibliography

Page II

8. Ahn, S. J., Kho, H. S., Lee, S. W., &Nahm, D. S. (2002). Roles of salivary

proteins in the adherence of oral streptococci to various orthodontic

brackets. Journal of dental research, 81(6), 411-415.

9. Mitchell, L. (1992). Decalcification during orthodontic treatment with

fixed appliances--an overview. British Journal of Orthodontics, 19(3),

199.

10. Mizrahi, E. (1982). Enamel demineralization following orthodontic

treatment. American journal of orthodontics, 82(1), 62-67.

11. Featherstone, J. D. (2000). The science and practice of caries

prevention. The Journal of the American dental association, 131(7),

887-899.

12. Gorelick, L., Geiger, A. M., &Gwinnett, A. J. (1982). Incidence of white

spot formation after bonding and banding. American journal of

orthodontics, 81(2), 93-98.

13. Willmot, D. (2008, September). White spot lesions after orthodontic

treatment. In Seminars in Orthodontics (Vol. 14, No. 3, pp. 209-219). WB

Saunders.

14. Geiger, A. M., Gorelick, L., Gwinnett, A. J., & Griswold, P. G. (1988).

The effect of a fluoride program on white spot formation during

orthodontic treatment. American Journal of Orthodontics and Dentofacial

Orthopedics, 93(1), 29-37.

15. Beerens, M. W., Van Der Veen, M. H., Van Beek, H., & Ten Cate, J. M.

(2010). Effects of casein phosphopeptide amorphous calcium fluoride

phosphate paste on white spot lesions and dental plaque after orthodontic

Bibliography

Page III

treatment: a 3‐month follow‐up. European journal of oral

sciences, 118(6), 610-617.

16. Sudjalim, T. R., Woods, M. G., & Manton, D. J. (2006). Prevention of

white spot lesions in orthodontic practice: a contemporary

review. Australian dental journal, 51(4), 284-289.

17. Welbury, R. R., & Carter, N. E. (1993). The hydrochloric acid-pumice

microabrasion technique in the treatment of post-orthodontic

decalcification. British journal of orthodontics, 20(3), 181-185.

18. Croll, T. P., & Cavanaugh, R. R. (1986). Enamel color modification by

controlled hydrochloric acid-pumice abrasion. I. technique and

examples. Quintessence international (Berlin, Germany: 1985), 17(2),

81-87.

19. Kour S, Verma VK, Sachan A, Singh K, Arora A, Kaur G. (2015) Role of

Probiotics in Orthodontics. Rama Univ J Dent Sci.;2(3):26-31.

20. Çaglar, E., Kuscu, O. O., Cildir, S. K., Kuvvetli, S. S., &Sandalli, N.

(2008). A probiotic lozenge administered medical device and its effect on

salivary mutans streptococci and lactobacilli. International Journal of

Paediatric Dentistry, 18(1), 35-39.

21. Meurman, J. H., &Stamatova, I. (2007). Probiotics: contributions to oral

health. Oral diseases, 13(5), 443-451.

22. Rosan, B., & Lamont, R. J. (2000). Dental plaque formation. Microbes and

infection, 2(13), 1599-1607.

23. Scheie, A. A., Arneberg, P., & Krogstad, O. (1984). Effect of orthodontic

treatment on prevalence of Streptococcus mutans in plaque and

saliva. european Journal of oral Sciences, 92(3), 211-217.

Bibliography

Page IV

24. Rosenbloom, R. G., &Tinanoff, N. (1991). Salivary Streptococcus mutans

levels in patients before, during, and after orthodontic treatment. American

Journal of Orthodontics and DentofacialOrthopedics, 100(1), 35-37.

25. Kupietzky, A., Majumdar, A., Shey, Z., Binder, R., & Matheson, P.

(2006). Colony forming unit levels of salivary Lactobacilli and

Streptococcus mutans in orthodontic patients. Journal of Clinical Pediatric

Dentistry, 30(1), 51-53.

26. Zachrisson, B. U., & Zachrisson, S. (1971). Caries incidence and

orthodontic treatment with fixed appliances. European Journal of Oral

Sciences, 79(2), 183-192.

27. Feldens, C. A., Nakamura, E. K., Tessarollo, F. R., &Closs, L. Q. (2014).

Desire for orthodontic treatment and associated factors among adolescents

in southern Brazil. The Angle Orthodontist, 85(2), 224-232.

28. Boersma, J. G., Van der Veen, M. H., Lagerweij, M. D., Bokhout, B.,

&Prahl-Andersen, B. (2004). Caries prevalence measured with QLF after

treatment with fixed orthodontic appliances: influencing factors. Caries

research, 39(1), 41-47.

29. Baumgartner, S., Menghini, G., &Imfeld, T. (2013). The prevalence of

approximal caries in patients after fixed orthodontic treatment and in

untreated subjects. Journal of OrofacialOrthopedics/Fortschritte der

Kieferorthopädie, 74(1), 64-72.

30. Chen, W., & Zhou, Y. (2015). Caries outcomes after orthodontic treatment

with fixed appliances: a longitudinal prospective study. International

journal of clinical and experimental medicine, 8(2), 2815

Bibliography

Page V

31. Chapman, J. A., Roberts, W. E., Eckert, G. J., Kula, K. S., & González-

Cabezas, C. (2010). Risk factors for incidence and severity of white spot

lesions during treatment with fixed orthodontic appliances. American

Journal of Orthodontics and Dentofacial Orthopedics, 138(2), 188-194.

32. Tufekci, E., Dixon, J. S., Gunsolley, J. C., &Lindauer, S. J. (2011).

Prevalence of white spot lesions during orthodontic treatment with fixed

appliances. The Angle orthodontist, 81(2), 206-210.

33. Lucchese, A., &Gherlone, E. (2012). Prevalence of white-spot lesions

before and during orthodontic treatment with fixed appliances. The

European Journal of Orthodontics, cjs070.

34. O'reilly, M. M., & Featherstone, J. D. B. (1987). Demineralization and

remineralization around orthodontic appliances: an in vivo

study. American Journal of Orthodontics and Dentofacial

Orthopedics, 92(1), 33-40.

35. Gray, A., & Ferguson, M. M. (1996). The use of low‐tack chewing gum

for individuals wearing orthodontic appliances. Australian dental

journal, 41(6), 373-376.

36. Holgerson, P. L., Sjöström, I., Stecksén‐Blicks, C. H. R. I. S. T. I. N. A., &

Twetman, S. (2007). Dental plaque formation and salivary mutans

streptococci in schoolchildren after use of xylitol‐containing chewing

gum. International Journal of Paediatric Dentistry, 17(2), 79-85.

37. Ferrazzano, G. F., Cantile, T., Quarto, M., Ingenito, A., Chianese, L.,

&Addeo, F. (2008). Protective effect of yogurt extract on dental enamel

demineralization in vitro. Australian dental journal, 53(4), 314-319.

Bibliography

Page VI

38. Bröchner, A., Christensen, C., Kristensen, B., Tranæus, S., Karlsson, L.,

Sonnesen, L., &Twetman, S. (2011). Treatment of post-orthodontic white

spot lesions with casein phosphopeptide-stabilised amorphous calcium

phosphate. Clinical oral investigations, 15(3), 369-373.

39. Bergstrand, F., & Twetman, S. (2011). A review on prevention and

treatment of post-orthodontic white spot lesions–evidence-based methods

and emerging technologies. The open dentistry journal, 5(1).

40. Pliska, B. T., Warner, G. A., Tantbirojn, D., & Larson, B. E. (2012).

Treatment of white spot lesions with ACP paste and microabrasion. The

Angle orthodontist, 82(5), 765-769.

41. Akin, M., &Basciftci, F. A. (2012). Can white spot lesions be treated

effectively?. The Angle orthodontist, 82(5), 770-775.

42. Sonesson, M., Twetman, S., &Bondemark, L. (2014). Effectiveness of

high-fluoride toothpaste on enamel demineralization during orthodontic

treatment—a multicenter randomized controlled trial. The European

Journal of Orthodontics, 36(6), 678-682.

43. Fornaini, C., Brulat, N., Milia, G., Rockl, A., &Rocca, J. P. (2014). The

use of sub-ablative Er: YAG laser irradiation in prevention of dental caries

during orthodontic treatment. Laser therapy, 23(3), 173-181.

44. Sunil, S., Panchmal, G. S., Shenoy, R. P., Jodalli, P., &Sonde, L. (2015)

Caries Prevention through Casein Phosphopeptide–Amorphous Calcium.

Int J Oral Health Med Res;2(4):70-73.

45. Jahanbin, A., Ameri, H., Shahabi, M., & Ghazi, A. (2015). Management of

Post-orthodontic White Spot Lesions and Subsequent Enamel

Bibliography

Page VII

Discoloration with Two Microabrasion Techniques. Journal of

Dentistry, 16(1 Suppl), 56.

46. Hammad, S. M., &Knösel, M. (2016). Efficacy of a new sealant to prevent

white spot lesions during fixed orthodontic treatment. Journal of

OrofacialOrthopedics/Fortschritte der Kieferorthopädie, 77(6), 439-445.

47. Miller, M. J., Bernstein, S., Colaiacovo, S. L., Nicolay, O., & Cisneros, G.

J. (2016, June). Demineralized white spot lesions: An unmet challenge for

orthodontists. In Seminars in Orthodontics. WB Saunders.

48. Busscher, H. J., Mulder, A. F. J. M., & Van der Mei, H. C. (1999). In vitro

adhesion to enamel and in vivo colonization of tooth surfaces by

lactobacilli from a Bio–Yoghurt. Caries research, 33(5), 403-404

49. Nikawa, H., Makihira, S., Fukushima, H., Nishimura, H., Ozaki, Y.,

Ishida, K., & Takemoto, T. (2004). Lactobacillus reuteri in bovine milk

fermented decreases the oral carriage of mutans streptococci. International

journal of food microbiology, 95(2), 219-223.

50. Çaglar, E., KavalogluCildir, S., Ergeneli, S., Sandalli, N., &Twetman, S.

(2006). Salivary mutans streptococci and lactobacilli levels after ingestion

of the probiotic bacterium Lactobacillus reuteri ATCC 55730 by straws or

tablets. ActaOdontologica Scandinavica, 64(5), 314-318.

51. Caglar, E., Kavaloglu, S. C., Kuscu, O. O., Sandalli, N., Holgerson, P. L.,

& Twetman, S. (2007). Effect of chewing gums containing xylitol or

probiotic bacteria on salivary mutans streptococci and lactobacilli. Clinical

oral investigations, 11(4), 425-429.

Bibliography

Page VIII

52. Haukioja, A., Loimaranta, V., &Tenovuo, J. (2008). Probiotic bacteria

affect the composition of salivary pellicle and streptococcal adhesion in

vitro. Oral microbiology and immunology, 23(4), 336-343.

53. Cildir, S. K., Germec, D., Sandalli, N., Ozdemir, F. I., Arun, T., Twetman,

S., & Caglar, E. (2009). Reduction of salivary mutans streptococci in

orthodontic patients during daily consumption of yoghurt containing

probiotic bacteria. The European Journal of Orthodontics, 31(4), 407-411.

54. Flichy-Fernández, A. J., Alegre-Domingo, T., Peñarrocha-Oltra, D., &

Peñarrocha-Diago, M. (2010). Probiotic treatment in the oral cavity: An

update. Med Oral Patol Oral Cir Bucal, 15(5), e677-80.

55. Bizzini, B., Pizzo, G., Scapagnini, G., Nuzzo, D., & Vasto, S. (2012).

Probiotics and oral health. Current pharmaceutical design, 18(34), 5522-

5531.

56. Jain, P., & Sharma, P. (2012). Probiotics and Their Efficacy in Improving

Oral Health: A Review. J App Pharm Sci; 2 (11):151-163

57. Sudhir, R., Praveen, P., Anantharaj, A., & Venkataraghavan, K. (2012).

Assessment of the effect of probiotic curd consumption on salivary pH and

streptococcus mutans counts. Nigerian medical journal: journal of the

Nigeria Medical Association, 53(3), 135.

58. Jose, J. E., Padmanabhan, S., &Chitharanjan, A. B. (2013). Systemic

consumption of probiotic curd and use of probiotic toothpaste to reduce

Streptococcus mutans in plaque around orthodontic brackets. American

Journal of Orthodontics and Dentofacial Orthopedics, 144(1), 67-72.

59. Laleman, I., Detailleur, V., Slot, D. E., Slomka, V., Quirynen, M., &

Teughels, W. (2014). Probiotics reduce mutans streptococci counts in

Bibliography

Page IX

humans: a systematic review and meta-analysis. Clinical oral

investigations, 18(6), 1539-1552.

60. Ritthagol, W., Saetang, C., &Teanpaisan, R. (2014). Effect of probiotics

containing Lactobacillus paracasei SD1 on salivary mutans streptococci

and lactobacilli in orthodontic cleft patients: A double-blinded,

randomized, placebo-controlled study. The Cleft Palate-Craniofacial

Journal, 51(3), 257-263.

61. Gizani, S., Petsi, G., Twetman, S., Caroni, C., Makou, M., &

Papagianoulis, L. (2016). Effect of the probiotic bacterium Lactobacillus

reuteri on white spot lesion development in orthodontic patients. The

European Journal of Orthodontics, 38(1), 85-89.

62. Dhawan, R., & Dhawan, S. (2013). Role of probiotics on oral health: A

randomized, double-blind, placebo-controlled study. Journal of

Interdisciplinary Dentistry, 3(2), 71.

63. Srivastava, S., Saha, S., &MintiKumari, S. M. (2016). Effect of Probiotic

Curd on Salivary pH and Streptococcus mutans: A Double Blind Parallel

Randomized Controlled Trial. Journal of clinical and diagnostic research:

JCDR, 10(2), ZC13.

64. Chaturvedi, S., Jain, U., Prakash, A., Sharma, A., Shukla, C., & Chhajed,

R. (2016). Efficacy of probiotic lozenges to reduce Streptococcus mutans

in plaque around orthodontic brackets. Journal of Indian Orthodontic

Society, 50(4), 222.

65. Andrucioli, M. C. D., Nelson-Filho, P., Matsumoto, M. A. N., Saraiva, M.

C. P., Feres, M., De Figueiredo, L. C., & Martins, L. P. (2012). Molecular

detection of in-vivo microbial contamination of metallic orthodontic

Bibliography

Page X

brackets by checkerboard DNA-DNA hybridization. American Journal of

Orthodontics and DentofacialOrthopedics, 141(1), 24-29.

66. Ahn, S. J., Lee, S. J., Lim, B. S., &Nahm, D. S. (2007). Quantitative

determination of adhesion patterns of cariogenic streptococci to various

orthodontic brackets. American Journal of Orthodontics and Dentofacial

Orthopedics, 132(6), 815-821.

67. Anderson, M. H., & Shi, W. (2006). A probiotic approach to caries

management. Pediatric dentistry, 28(2), 151-153.

68. Comelli, E. M., Guggenheim, B., Stingele, F., &Neeser, J. R. (2002).

Selection of dairy bacterial strains as probiotics for oral health. European

journal of oral sciences, 110(3), 218-224.

69. Söderling, E. M., Marttinen, A. M., &Haukioja, A. L. (2011). Probiotic

lactobacilli interfere with Streptococcus mutans biofilm formation in

vitro. Current microbiology, 62(2), 618-622.

70. Keller, M. K., Hasslöf, P., Dahlén, G., Stecksén-Blicks, C., &Twetman, S.

(2012). Probiotic supplements (Lactobacillus reuteri DSM 17938 and

ATCC PTA 5289) do not affect regrowth of mutans streptococci after full-

mouth disinfection with chlorhexidine: a randomized controlled

multicenter trial. Caries research, 46(2), 140-146.

71. Siddiqui, M., Singh, C., Masih, U., Chaudhry, K., Hegde, D. Y.,

&Gojanur, S. (2016). Evaluation of Streptococcus mutans Levels in Saliva

before and after Consumption of Probiotic Milk: A Clinical Study. Journal

of International Oral Health, 8(2), 195.

72. Yadav, M., Poornima, P., Roshan, N. M., Prachi, N., Veena, M., &Neena,

I. E. (2014). Evaluation of probiotic milk on salivary mutans streptococci

Bibliography

Page XI

count: an in vivo microbiological study. Journal of Clinical Pediatric

Dentistry, 39(1), 23-26.

73. Sutula, J., Coulthwaite, L., Thomas, L., &Verran, J. (2012). The effect of a

commercial probiotic drink on oral microbiota in healthy complete denture

wearers. Microbial ecology in health and disease, 23.

74. Jindal, G., Pandey, R. K., Agarwal, J., & Singh, M. (2011). A comparative

evaluation of probiotics on salivary mutans streptococci counts in Indian

children. European Archives of Paediatric Dentistry, 12(4), 211-215.

Annexure

Page i

ANNEXURE - I

Muha;r;rpgw;wpajfty; gotk;

kU/ff;fl; fpUghypepjpd; gha; Mfpa ehd; kU/ghyrz;Kfk; MDS.,

mth;fspd; tHpelj;Jjypd; fPH; @gy;rPuikg;g[ rpfpr;irf;fhfxl;lg;gLk;

cnyhfbghj;jhd;fspd; kPJgluToaEz;zpaph; fpUkpfspd;

tsh;r;rpiajLf;Fk; K:d;W tifahdgpnuhgahof;fspd; (Probiotic)

bray;jpwid xg;gPL bra;jy@bjhlh;ghfMa;t[ bra;acs;nsd;/

Ma;tpd; nehf;fk;:

K:d;W tifahdgpnuhgaof;fspd; (Probiotic)bray;jpwidxg;gPLbra;jy;/

bra;Kiw:

Muha;r;rpf;fhfnjh;e;bjLf;fgl;lth;fSf;FtHf;fk; nghynt

gy;rPuikg;g[ rpfpr;irnkw;bfhs;sg;gLk; gpd;dh; mth;fSf;F

njh;e;bjLf;fg;gl;l gy;ypd; cnyhfbghj;jhd;fspy; ,Ue;J nrhjid

khjphpvLf;fg;gLk;/ gpd;dh; mth;fisehd;Fgphpt[fshfgphpj;J. K:d;W

gphptpdUf;F. K:d;W tifahd Probiotic-fis Kiwna gad;gLj;j

mwpt[Wj;jg;gLk;/ xUkhjk; fHpj;JKd;dh; nghd;Wmnjgy;ypy; ,Ue;J

nrhjid khjphpvLf;fg;gLk;/ ,uz;L nrhjid khjphpfSk; cs;s

Ez;Qqaph; fpUkpfspd; vz;zpf;if mstplg;gLk;/

ed;ikfs;:

Prfobioticfs; tha; neha; gut[k; jPaEz;Qqaph;fs; gut[tijjLf;Fk;/

,ufrpajd;ik:

nehahspfs; gw;wpaFwpg;g[fs; Muha;r;rp Koa[k; tiu ufrpakhf

ghJfhf;fg;gLk;/ ,e;jMuha;r;rpiabtspapLk; nghJnehahspfspd;

jdpg;gl;l tptu’;fs; vJt[k; ghjpf;fg;glkhl;lhJ/

Annexure

Page ii

g’;FbgWnthhpd; chpik:

,e;jMuha;r;rpapy; g’;FbgWtJnehahspfspd; jdpg;gl;ltpUg;gk;/

nkYk;. nehahspfs; ,e;j Muha;r;rpapypUe;J vg;nghJ ntz;Lbkd;why;

tpyfpf; bfhs;syhk;/ nehahspfspd; ,e;jKotpdhy; mtUf;nfh my;yJ

Muha;r;rpahsUf;nfh vt;tpjghjpg;g[k; fpilahJ/

,e;j Muha;r;rpapd; Kot[fs; nehahspfSf;F Muha;r;rp Koa[k;

jUthapnyh my;yJ ,ilapnyh bjhptpf;fg;gLk;/ Muha;r;rpapd;

bghGJ VJk; gpd; tpist[fs; Vw;gl;lhy; mij rhpbra;ajFe;j cjtpfs;

my;yJ njitahd rpfpr;irfs; cldoahf nkw;bfhs;sg;gLk;/

,Hg;gPL: vJt[k; tH’;fg;glkhl;lhJ/

Ma;t[ gw;wpajftiybgw

kU/ff;fl; fpUghypepjpd; gha;

,uz;lhk; Mz;LMDS,

KJepiykhztp.

jkpH;ehLgy; kUj;Jtfy;Y}hp kw;Wk; kUj;Jtkid.

brd;id?600 003/

bry;ngrp: _________________

nehahspapd; bgah; ifbahg;gk; - ifnuif

njjp

Muha;rprahshpd; bgah; ifbahg;gk; - ifnuif

njjp

Annexure

Page iii

ANNEXURE - II

PARTICIPANT INFORMATION SHEET

TITLE OF THE STUDY: “Comparing effectiveness of 3 different probiotics in

inhibition of streptococcus mutansin plaque around orthodontic brackets: A

Randomized controlled trial”

Name of the research institution: Tamilnadu Government Dental College & Hospital

Purpose and procedure of the study:

To evaluate and compare the efficacy of 3 different Probiotics in inhibition

of streptococcus mutans in dental plaque around orthodontic brackets.

Clinically, 1. Plaque specimens will be collected from the labial surfaces

immediately surrounding orthodontic bracket of maxillary lateral incisor

using sterilised scalar before probiotic therapy using four pass technique,

2. Phase of probiotic therapy, 3. Sample collection with the same method

after probiotic therapy of 1 month. And check for the difference in the

bacterial count before and after probiotic therapy.

Risk of participation:

• Patients are selected only according to inclusion and exclusion criteria

hence there will be negligible risk.

Benefits of participation:

• Reduces the risk of other oral problems associated with fixed orthodontic

treatment.

Annexure

Page iv

1. Confidentiality:

The privacy of the patients in the research will be maintained throughout

the study. In the event of any publication or presentation resulting from the

research, no personally identifiable information will be shared.

2. 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.

3. Compensation: NIL

Contacts:

For queries related to the study:

PRMARY INVESTIGATOR: DR. KAKKAD KRUPALI NITINBHAI

CONTACT DETAILS: PG SECTION, DEPT OF ORTHODONTICS AND

DENTOFACIAL ORTHOPEDICS,

TAMILNADU GOVT DENTAL COLLEGE &

HOSPITAL,

FRAZER BRIDGE ROAD, Chennai-600003.

PHONE NUMBER: 9884273509

For queries related to the rights as a study participant, please write to:

The Chairperson,

TAMILNADU GOVT DENTAL COLLEGE & HOSPITAL,

FRAZER BRIDGE ROAD, Chennai-600003

Annexure

Page v

ANNEXURE - III

Ra xg;g[jy; gotk;

bgah; : Muha;r;rpnrh;f;ifvz; :

taJ: ghy; :

Muha;r;rpbra;ag;gLk; jiyg;g[

@gy;rPuikg;g[ rpfpr;irf;fhfxl;lg;gLk; cnyhfbghj;jhd;fspd;

kPJgluToaEz;zpaph; fpUkpfspd; tsh;r;rpiajLf;Fk; K:d;W

tifahdgpnuhgahof;fspd; (Probiotic) bray;jpwidxg;gPLbra;jy;@/

Muha;r;rpepiyak; : muRgy; kUj;Jtf; fy;Y}hp. brd;id?600 003/

g’;FbgWgthpd; gpwe;jnjjp: njjp________khjk; ________ - tUlk; ________

,e;jMa;t[ rk;ge;jkhfehd; nknyTwg;gl;ljfty; gotj;ij KGikahf

goj;Jg; ghh;j;njd; vd;WcWjpTWfpnwd;/

ehd; ,J bjhlh;ghf midj;J nfs;tpfSf;Fk; epiwthd gjpy;fs;

bgwg;gl;nld;/

,e;j Ma;tpy; vdJg’;Fjd;dpr;irahdJvd;Wk; ve;jneuj;jpYk; ,e;j Ma;tpy;

,Ue;J rl;lchpikfs; ghjpf;fg;glhky; tpyfpf; bfhs;srk;kjpf;fpnwd;/

kUj;Jt Ma;t[ mjpfhhpfs;. vdJ rpfpr;ir bjhlh;ghd gjpntLfis

ghh;itaplt[k;. ve;j neuj;jpYk;. Ma;tpy; ,Ue;Jehd; tpyfpdhYk; ghh;itapl

rk;kjpf;fpnwd;/ vdJ milahsFwpg;g[fs; K:d;whtJ egUf;F

bjhptpf;fg;glkhl;lhJ vd;W g[hpe;Jbfhz;nld;/

,e;jMa;t[ mwpf;iffis gad;gLj;jt[k;. btspaplt[k; ehd; rk;kjpf;fpnwd;/

Ma;thsh; vdJ kUj;JtFwpg;g[fis btspapl jilahf ,Uf;fkhl;nld; vd

cz;ikahfrk;kjpf;fpnwd;/

g’;nfw;gthpd; ifbahg;gk; : ____________________ ,lk; _______________ njjp

fl;iltpuy; nuif

g’;nfw;gth; bgah; kw;Wk; tpyhrk;

Ma;thshpd; bgah; :

Ma;thshpd; ifbahg;gk; :

Annexure

Page vi

ANNEXURE - IV

Annexure: AF 06/004/01.0

Informed Consent Form

“Comparing effectiveness of 3 different probiotics in inhibition of

streptococcus mutans in plaque around orthodontic brackets: A Randomized

Controlled Trial”.

Participant ID No:

“I have read the foregoing information sheet given to me about the methods and

procedures to be followed for the study, 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 participan t, 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 interviewer Signature of the interviewer

Annexure

Page vii

ANNEXURE - V

Figure 2: Armamentarium (Sterile scalar)

Figure 3: Test tube and Glass rods

Figure 4: Thioglycolate Broth/Agar

Figure 5: Blood Agar

Figure 6: Incubator

Figure 7: Candle Jar

Figure 8: Digital colony counter

Figure 9: Probiotic lozenges (BIFILAC lozenges)

Figure 10: Probiotic sachets (DAROLAC- Z sachets)

Figure 11: Probiotic drink (Yakult)

Figure 12: Collection of plaque samples from the labial surfaces immediately

surrounding the orthodontic brackets of the maxillary lateral incisors with a sterilized

scalar using a four pass technique

Figure 13: Mixed growth of sample

Figure 14: Growth of sample in controls

Figure 15: Pure isolate of Streptococcus Mutans

Figure 16: Colony counting method

Figure 17: Colony counting

Chart 1: Groups and no of participants in each group

Chart 2: Distribution of study participants according to Gender

Chart 3: Comparison of S. mutans CFU/ml before and after 30 days in all four groups

0

2

4

6

8

10

12

14

16

Group 1

Probiotic

Lozenges

Group 2

Probiotic

Sachet

Group3

Probiotic

Drink

Group 4

Control

no. of

Part

icip

an

ts

Male

Female

0

50

100

150

200

250

300

Group 1

Probiotic

Lozenges

Group 2

Probiotic Sachet

Group 3

Probiotic Drink

Group 4 Contorl

no o

f s

mu

tan

s C

FU

1000

S mutans CFU at baseline S mutans CFU after 30 days

comparing effectiveness of three different …repository-tnmgrmu.ac.in/5257/1/240502017kakkad... ·...

Documents