comparative evaluation of the physical properties...

COMPARATIVE EVALUATION OF THE PHYSICAL

PROPERTIES AND BIOCOMPATIBILITY OF ZINC

OXIDE-OZONATED EUGENOL AND CONVENTIONAL

ZINC OXIDE EUGENOL AS AN ENDODONTIC SEALER

– AN INVITRO STUDY

A Dissertation submitted

in partial fulfilment of the requirements

for the degree of

MASTER OF DENTAL SURGERY

BRANCH – IV

CONSERVATIVE DENTISTRY AND ENDODONTICS

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

CHENNAI- 600032

2014 – 2017

ADHIPARASAKTHI DENTAL COLLEGE AND HOSPITAL

MELMARUVATHUR – 603319 .

DEPARTMENT OF CONSERVATIVE DENTISTRY AND

ENDODONTICS

CERTIFICATE

This is to certify that DR.C.RAVI VARMAN, Post Graduate

student (2014-2017) in the Department of Conservative Dentistry and

Endodontics, Adhiparasakthi Dental College and Hospital ,

Melmaruvathur – 603319, has done this dissertation titled

“Comparative evaluation of the physical properties and

biocompatibility of zinc oxide-ozonated eugenol and conventional

zinc oxide eugenol as an endodontic sealer” – An invitro study

under our direct guidance and supervision in partial fulfilment of the

regulations laid down by the Tamilnadu Dr.M.G.R Medical University,

Chennai – 600032 for MDS., (Branch-IV) CONSERVATIVE

DENTISTRY AND ENDODONTICS degree examination.

Dr.S. Thillainayagam MDS.,

Principal

Guide

Dr.S.Thillainayagam MDS.,

Professor & Head

Co-Guide

Dr.A.Jayasenthil MDS.,

Reader

ACKNOWLEDGEMENT

I am extremely grateful to Dr.S.Thillainayagam MDS . , Guide,

Professor and Head, Department of Conservative Dentistry and

Endodontics, Adhiparasakthi Dental College and Hospital ,

Melmaruvathur. Words cannot express my gratitude for his quiet

confidence in my ability to do the study, his willingness to help clear

the stumbling blocks along the way and his tremendous patience t ill the

end of the study.

It is my duty to express my thanks to my Co -Guide

Dr.A.Jayasenthil MDS . , Reader for his expert guidance and moral

support during the completion of this study. I consider myself

privileged, to have studied, worked and completed my dissertatio n

under them in the department.

My sincere thanks to our Correspondent Dr.T.Ramesh MD., for

his vital encouragement and support.

I am extremely thankful to my teachers Dr.D.S.Dinesh MDS.,

Professor , Dr.N.Bharath MDS., Reader, Dr.S.Karthikeyan MDS.,

Senior lecturer, for their valuable suggestions, constant encouragement

and timely help rendered throughout this study.

I am extremely grateful to Dr.Mohankumar , Lab testing, Cipet,

Guindy and Dr.K.Thirugnanasambandam , Research director, PCBS,

Puducherry for granting me permission to conduct the study in their

department and helping me to bring out my study.

I thank Dr.S.Pandian, Madras Veterinary College and hospital,

Vepery, Chennai, for helping me with the statistics in the study.

I also wish to thank my post graduate colleagues and my juniors

for their help and support.

I owe my grati tude to my father (late) Dr.T.Chandramohan

MD., and my mother Dr.R.Manoranjitham MS., who stood beside me

during my hard time and sacrificed so much to make me what I am

today. A special thanks to my wife Dr.R.J.Shalini and my son master

R.Dishaanchand for their support and encouragement throughout my

career.

My acknowledgement wouldn’t be complete without mentioning

my teaching and non-teaching staffs, Office staffs, and l ibrary staffs

for being my strength, by giving me continual support all the time.

Before and after everything I thank the Almighty for guiding me

through the ups and downs in my life. I humbly bow before Him for his

blessings.

Dr.C.Ravivarman

Post graduate student

DECLARATION

TITLE OF THE DISSERTATION “Comparative evaluation of the

physical properties and

biocompatibility of zinc oxide-

ozonated eugenol and

conventional zinc oxide eugenol

as an endodontic sealer” – An

invitro study

PLACE OF THE STUDY Adhiparasakthi Dental College and

Hospital, Melmaruvathur – 603319

DURATION OF THE COURSE 3 years

NAME OF THE GUIDE Dr.S.Thillainayagam, MDS.,

NAME OF CO-GUIDE Dr.A.Jayasenthil , MDS.,

I hereby declare that no part of the dissertation will be uti lized

for gaining financial assistance or any promotion without obtaining

prior permission of the Principal, Adhiparasakthi Dental College and

Hospital, Melmaruvathur – 603319. In addition, I declare that no part

of this work will be published either in print or in electronic media

without the guides who has been actively involved in dissertation. The

author has the right to reserve for publish work solel y with the

permission of the Principal, Adhiparasakthi Dental College and

Hospital, Melmaruvathur – 603319

Co-Guide Guide & Head of department Signature of candidate

ABSTRACT

BACKGROUND:The challenge to the prognosis of endodontic therapy

lies on the quality of root canal fi lling. The zinc oxide eugenol (ZOE)

based sealers were introduced in Endodontics by Grossman in 1936

and they have been used in dentistry over a long period of time. It has

been reported that eugenol released from ZOE is an irritant to the

periapical tissues and has cytotoxic properties . To overcome these

disadvantages, there is a continued research for chemical compounds

with better physical properties and more biocompatibility. Ozone

therapy has a long history of research and clinical application with

humans because of its interesting biological properties.

AIM:The aim of the study is to evaluate and compare the physical

properties and biocompatibili ty of zinc oxide - ozonated eugenol and

conventional zinc oxide eugenol.

MATERIALS AND METHODS:Following physical properties of

conventional zinc oxide eugenol and zinc oxide-ozonated eugenol were

evaluated and compared. [Group I : Zinc oxide – eugenol; Group II :

Zinc oxide - ozonated eugenol]. Hardness of the mixed samples was

measured using Shore hardness Durometer type A.Solubili ty was tested

as a percentage of the mass of specimen removed from the distil led

water compared with the original mass of the specimens using Mettler

analytical balance. pH of the fresh samples and the set samples were

measured using calibrated pH meter after predetermined time intervals.

Biocompatibility of the tested samples was evaluated using the MTT

tetrazolium reduction assay.

RESULTS: The results of the present study have shown that the

hardness and solubil ity of the tested samples were almost similar in

both the groups. pH and biocompatibility of the samples in Group II

where eugenol is ozonated was higher when compared to the samples in

Group I. The proportional values obtained were statistically analysed

by unpaired t -test.

CONCLUSION: Within the limitations of the study, it could be

concluded that ozone with interesting biological properties seems to be

a better treatment modality in improving the properties of eugenol.

However further clinical trials and randomized control studies are

required in this field to use ozonated eugenol in combination with zinc

oxide powder to use it as an endodontic sealer invivo.

CONTENTS

S.NO TITLE PAGE

No.

1. INTRODUCTION 1

2. AIM AND OBJECTIVES 5

3. REVIEW OF LITERATURE 6

4. MATERIALS AND METHODS 31

5. RESULTS 44

6. DISCUSSION 52

7. SUMMARY AND CONCLUSION 60

8. REFERENCES 63

9. ANNEXURE 73

LIST OF PICTURES

Figure

No.

TITLE Page No.

1.

Zinc oxide powder, Eugenol, Glass slab

and stainless steel spatula.

39

2.

Stainless steel ring of dimension

1.5mmx10mm.

39

3. Ozonator machine. 39

4.

Eugenol ozonated by bubbling gases

through it.

40

5. Shore hardness Durometer type A. 41

6. Calibrated digital pH meter. 41

7.

Samples shaped using stainless steel

molds.

42

8.

Mettler AE 260 Delta range Analytical

Balance.

42

9. MTT Reagent 43

10. Micro-plate Reader 43

11. Biocompatibility Testing Group I 43

12. Biocompatibility Testing Group II 43

LIST OF TABLES

Table

No.

TITLE Page No.

1.

Mean hardness values of the tested

samples

45

2. Mean pH values of the tested samples 46

3.

Mean solubility values of the tested

samples

47

4.

Mean biocompatibili ty values of the tested

samples

48

LIST OF CHARTS

Chart

No. TITLE Page No.

1. Hardness values of tested samples after

12hrs 49

2. Hardness values of tested samples after

24hrs 49

3. Mean hardness values of the tested samples 50

4. Mean pH values of the tested samples 50

5. Mean solubility values of the tested

samples 51

6. Mean biocompatibili ty values of the tested

samples 51

LIST OF ABBREVIATIONS

ADA : American dental association

Al : aluminium

ANSI : American national standards institute

ATP : adenosine triphosphate

COX : cyclooxygenase

DNA : deoxyribose nucleic acid

DUWLs : dental unit water lines

IC : inhibitory concentration

IL-β : Interleukin-1β

IRM : intermediate restorative material

LDH : lactic dehydrogenase

LPS : Lipopolysaccharide

MTT : (3-(4, 5-dimethylthiazol-2-yl)-2, 5-

diphenyltetrazolium bromide)

NaOCl : sodium hypochlorite

NF : nuclear factor

O3 : ozone

OSHA : occupational safety and health administration

ZOE : zinc oxide eugenol

PDGF : platelet derived growth factor

PDT : photodynamic therapy

PRCL : primary root carious lesions

pH : potential of hydrogen

ppm : parts per million

RNA : ribose nucleic acid

TNF : tumour necrosis factor

TGF-β : transforming growth factor

VEGF : vascular endothelial growth factor

Introduction

1

INTRODUCTION

Success in Endodontic therapy is based on diagnosis, treatment

planning, knowledge of tooth anatomy and the traditional concepts of

debridement and obturation. Schilder describes the final objective of

endodontic procedure as “the complete obturation of the root canal

space”. It is the sealing of complex root canal system from the

periodontal bone that ensures the health of attachment apparatus

against the breakdown of endodontic origin ¹ .

Endodontic sealers are used in the obturation of root canal

system to achieve a fluid tight seal throughout the canal including the

canal irregularities. An ideal root canal sealer should provide excellent

seal when set, should have dimensional stability, biocompatibility and

help to resolve periapical lesions ² .

The Zinc Oxide Eugenol (ZOE) based sealers were introduced in

Endodontics by Grossman in 1936. ZOE sealers have a history of

successful use over extended period of t ime and has both anaesthetic

and antimicrobial properties ³ . However the eugenol released from ZOE

is reported to be an irritant to periapical tissues and said to have

cytotoxic properties4. Every attempt made to overcome these

disadvantages and to replace ZOE completely has failed, so there is

continued research for chemical compounds with better physical

properties and biocompatibility.

Introduction

2

In search of other therapeutic modalities, ozone therapy has a

long history of research and clinical applications with hum ans because

of its interesting biological properties. The word ozone was first

introduced by Schonbein in 1840 when he subjected oxygen to electric

discharge and noted the odour of electric matter5. Ozone is said to have

antimicrobial, immune-modulatory, immune-stimulating, anti -

inflammatory, analgesic and haemostatic property. Ozonation is an

energetic process that results in an oxygen molecule being split into

singlet oxygen, the singlet oxygen then combines with diatomic oxygen

to form ozone. Three systems of generating ozone are: 1.Ultraviolet

system 2.Cold plasma system 3.Corona discharge system. The modes of

applications of ozone can be in gaseous form, aqueous solution or in

the form of ozonated oils.

Antimicrobial action : Being a superoxide, ozone i s 5 times more

effective than chlorine. It affects only the unhealthy cells such as

cancer cells, organisms like bacteria, viruses, fungi, parasites which

have lost their protective mechanism and those devoid of anti -oxidants

and free radical scavengers.

Anti-inflammatory and Analgesic action : Ozone helps in the

synthesis of biologically active substance such as interleukins,

leukotriene and prostaglandins thereby reducing the inflammation and

pain.

Anti-cariogenic action : The strongest naturally occurrin g acid

produced by acidogenic bacteria is pyruvic acid. Ozone has the ability

Introduction

3

to decarboxylate pyruvic acid to acetic acid and said to enhance the

remineralisation of teeth.

Antihypoxic action : ozone brings about the rise of partial pressure of

oxygen and acts as super oxygenator by bringing oxygen to tissues,

assisting in its natural healing process6.

In dentistry, ozonated oil applied on herpes labialis infection and

osteomyelitis has demonstrated faster healing rate. It has been also

used in the treatment of avascular osteonecrosis of the jaw. Ozone has

strong bactericidal activity against plaque biofilm and can be used to

disinfect denture bases from Candida albicans. Zinc oxide -ozonated

sesame oil when used as a root filling material has demonstrated good

success rate(93.3%) when compared to ZOE(63.3%)7. In restorative

dentistry, gaseous ozone has been used in the treatment of incipient

lesions since it causes reduction in the number of microorganisms8. In

Endodontics, ozonated water had nearly the same antimicrobial activity

as 2.5% NaOCl with fewer side effects when used as an irrigant9.

Ozonator D-400 uses a highly efficient corona discharge system

rated at 400mg/hr. of constant ozone output. It can be used to ozonate

water and oil. Due to the highly unstable nature of ozone in aqueous

form, we opted use ozone in the form of oils. Eugenol is a member of

phenyl propanoids, a yellow oily liquid extracted especially from clove

oil (80-85%), which can be ozonated to overcome its disadvantages

like irritating action on periapical tissues and cytotoxic properties.

Introduction

4

Hence, the purpose of the study is to evaluate and compare the

physical properties and biocompatibility of ozonated ZOE and

conventional ZOE for being used as an endodontic sealer.

Aim and Objectives

5

AIM AND OBJECTIVES

Aim of the study:

To evaluate and compare the physical properties and

biocompatibility of zinc oxide -ozonated eugenol and that of


Objectives of the study:

1. To evaluate the physical properties such as hardness, solubil ity

and pH of zinc oxide- ozonated eugenol and conventional zinc

oxide eugenol.

2. To evaluate the biocompatibili ty of zinc oxide -ozonated eugenol

and conventional zinc oxide eugenol.

3. To compare and correlate the physical properties and

biocompatibility of zinc oxide- ozonated eugenol and


Review of l iterature

6

REVIEW OF LITERATURE

Root canal therapy depends on the integrally related t reatment

phases: microbial control, cleaning and shaping, and effect ive sealing

of the root canal system. The success of t reatment depends on the

execut ion o f the final phase i.e. sealing of the root canal. Endodont ic

filling mater ials may be considered like t rue implants as they touch and

are based in vital t issues o f the body. The main components of a root

filling are: a so lid core mater ial and a sea ler. ZOE sealers have a

history o f successful use over extended per iod o f t ime. It has both

anaesthet ic and ant imicrobia l propert ies1 0

.

Torabinejad M (1993)1 1

stated that sealers help to prevent leakage,

reduce the possibilit y of residual bacter ia from the canal to invade the

per iapical t issues and to resolve per iapical lesions.

McMichen FR et al (2003)1 2

stated that the handling propert ies o f root

canal sealers and it s clinical behaviour may be character ised by lab

tests on their physical propert ies. These include flow, so lubilit y,

working t ime and set t ing t ime. He also stated that these mater ials

should possess adequate working t ime for clinical handling and

t reatment procedures.

ISO standards: ISO 6876: 20011 3

Flow o f endodont ic sealer should have a diameter not less than

20mm.

Working t ime o f the sealer to be less than 30 min


7

Sett ing t ime o f the sealer should be greater than 30 min and

upto72hrs.

Film thickness should not be more than 50µm

Dimens ional change should not to exceed 1.0 % in shr inkage or

0.1% in expansion.

Solubilit y o f the sealer shall not exceed 3% of mass fract ion.

Radio-opacity should be equivalent to or not less than 3mm of

aluminium.

Grossman stated that Zinc oxide eugeno l sealer have a history o f

successful use over an extended per iod of t ime. An ideal root canal

sealer should possess fo llowing propert ies 1 4

.

1. It should be tacky when mixed, to be able to provide

adhesion/sealing between the ca nal walls and the core obturat ing

mater ial.

2. Flowabilit y in it s non-set state to promote lut ing.

3. It should be non- irr itat ing to the per iapical t issues.

4. It should possess a hermet ic sealing abilit y.

5. It should be radiopaque.

6. It should possess minimal set t ing shr inkage.

7. Should be non-staining on the dent in / tooth st ructure.

8. It should be ideally bacter icidal or at least control the bacter ial

growth.

9. Should have long working t ime.

10.Inso luble in t issue flu ids.

11.It should be easy to remove if necessary.


8

12.It should be neither mutagenic nor carcinogenic.

13.It should not provoke an immune react ion.

Grossman‟s or iginal formula for zinc oxide eugeno l based sealers

contained ZnO, stabylit e resin, bismuth sub carbonate, bar ium sulphate

and sodium borate with eugeno l as the l iquid component .

Roth’s sol sealer (Roth‟s pharmacy, Chicago, inc.) is essent ially t he

same as Grossman‟s or ig inal formulat ion with the subst itut ion o f

bismuth sub-nit rate for bismuth sub-carbonate.

Ricket’s formula marketed as Kerr‟s pulp canal sealer ( Sybron endo/

Kerr, Orange, CA) has long been accepted mater ial. But its use has

been rest r icted for staining o f tooth st ructure from the silver used for

radio-opacity.

Tubliseal(Sybronendo/ Kerr) is a two paste system contained in two

separate tubes developed as non-staining alt ernat ive with bar ium

sulphate for radiopac ity. It has the disadvantage of rapid set t ing t ime.

Wach’s cement (Roth internat ional Inc. Chicago) consists o f powder of

ZnO, bismuth sub- iodide, bismuth sub-nit rate, magnesium oxide and

calcium phosphate. It has the advantage of smooth consistency and the

Canada balsam make the sealer tachy1 4

.

Hauman CH et al (2003)1 5

reported that there are many formulat ions

and brands of sealer that have zinc oxide as pr imary ingredient ,

differ ing only by other components added to the sealer. Zinc oxide


9

sealers allow for t he addit ion o f other chemicals, such as

paraformaldehyde, rosin, Canada balsam all o f which may enhance

their propert ies.Endodont ic mater ials can be broadly categor ized as

those used to maintain pulp vita lit y and those used in pulp canal

therapy for disinfect ion o f the pulp space ( irr igants and intra-canal

medicament s) and root cana l filling (solid mater ials and sealers).

Biocompat ibilit y o f these endodont ic mater ials is character ized by

many parameters such as genotoxicity, mutagenic ity, carcinogenicit y,

cytotoxicity, histocompat ibilit y or microbial effects. Hence it is

impossible to bio log ically character ize the mater ials by a single test

method alone and the ir propert ies need to be invest igated by a ser ies of

var ious in vit ro and in vivo tests ina st ructured approach.

Candeiro et al (2012)1 6

reported that nowadays var ious kinds o f

endodont ic sealers are available such as sealers based on glass

ionomer, calc ium hydroxide, silicone, resin based and bioceramic

mater ials.

Lacey S (2005)1 7

stated that as yet no root canal sealers has all t he

ideal propert ies and most leak with t ime, either through poor init ial

adaptat ion to the canal walls or due to solubilit y and dis integrat ion of

the sealer. They also stated that successful obturat ion is achieved by

the adhesion and stabilit y o f t he seale r to the canal walls which,

amongst other factors, is affected by its rheo logical propert ies. They

reported that narrower tubes produced increased velocit y, which was

significant for all sealers, and reduced vo lumetr ic flow, which was


10

significant for all sealers except Grossman‟s sealer. Reduct ion in

powder: liquid rat io o f Grossman‟s significant ly increased flow in

narrow tubes and at higher st rain rate.

Eugenol (4- acyl-2-methoxy pheno l) is an extract of clove o il widely

used in dent ist ry as a therapeut ic agent , most commonly as a

component o f zinc oxide eugeno l cement applied as a ba se or

temporary dressing to dent ine, or as a root canal sealer1 0

.

Lindquist et al (1981)1 8

reported that eugeno l that leaches out of zinc

oxide eugeno l based root canal sealers, may part icipate in the

development o f per iapical inflammat ion or the cont inua t ion of a pre-

exist ing per iapical lesion.They conducted a study to check the amount

of eugeno l released from four different dental mater ials immersed in

phosphate buffer measured by gas liquid chromatography. Maximal

release o f eugeno l from zinc oxide -eugenol cement (ZOE) and IRM was

attained within 5hrs and corresponded to 5% and 4%, respect ively, o f

the total amount of eugeno l in each mater ial. Eugeno l applied to

growing cultures o f human diplo id fibroblasts reduced the number o f

cells recovered to a value which was 4% of t hat found for control

cultures which grew to form mono layers. Confluent mono lay ers o f H-

Uridine labeled cellcultures which were incubated for 1hr in t he

presence o f 4 mm eugeno l lo st approximately 100% of t his cytoplasmic

label, indicat ing total cell death.

Branstetter J (1982)1 9

stated that endodont ic sealers are used in the

obturat ion o f root canal system to achieve a fluid t ight or hermet ic sea l


11

throughout the canal including the apical foramen and canal

irregular it ies.

Bedoukian (1986)2 0

reported that eugenol was first iso lated in 1929

and commercia l product ion commenced in the United States in the

1940s. Eugeno l can be produced synthet ically, t he most pract ica l

method being the allylat ion o f guaiaco l with allyl chlor ide. However,

eugeno l is predominant ly prepared from natural o il sources by mixing

the essent ial o il with an exces s o f aqueous sodium (3%) or potassium

hydroxide so lut ion and shaking, leading to the format ion of a pheno lic

alkali salt . The inso luble non-pheno lic port ion is then extracted with a

so lvent or via steam dist illat ion. The un- disso lved port ion is removed,

the alkali so lut ion is acidified at low temperatures and the liberated

eugeno l pur ified by fract ional dist illat ion.

Thompson et al (1990)2 1

stated that the eugeno l induced cytotoxicity

is influenced by the format ion o f glutathione conjugates dur ing

oxidat ion o f eugeno l by microsomal fract ion of rat liver and lung.

Jeng et al (1994)2 2

invest igated the effect of eugeno l on mouse

fibroblast at with a concentrat ion higher than 3 mmol/L, and reported

that eugeno l was cytotoxic to oral mucosal fibroblasts in a

concentrat ion and t ime-dependent manner. They reported that cell

death was associated with intracellu lar deplet ion o f glutathione. Most

of the glutathione was depleted pr ior to the onset of cell death. At

concentrat ions o f 3 mmol/L and 4 mmol/L, eugeno l dep leted about 45%

and 77% of the cells aft er one-hour incubat ion. In addit ion, eugeno l


12

decreased cellular ATP (Adenosine tr i-phosphate) level in a

concentrat ion and t ime dependent manner and they concluded that

eugeno l is cytotoxic to oral mucosal fibroblast s.

Tai et al (2002)2 3

stated that zinc oxide eugeno l based sealers irr itated

per iapical t issues invivo and exhibit ed cytotoxic effects exvivo. In this

study, tet razolium bromide reduct ion assay, DNA precipit at ion assay,

and DNA fragmentat ion analys is were performed to invest igate the

cytotoxic and genotoxic effects o f four different root canal sealers on

cultured V79 cells. These findings suggest that root canal sealers

containing formaldehyde and bispheno l-A dig lyether proved as not

only cytotoxic but also genotoxic. They also stated that mammalian test

system might be used rout inely for evaluat ion o f the genotoxicity o f

dental mater ials in future invest igat ions.

Ito et al (2006)2 4

invest igated the ant i-oxidant act ivity o f eugeno l and

it s isomer isoeugeno l using iron-mediated lipid peroxidat ion and auto -

oxidat ion o f Fe 2+. The inhibitory effect of eugeno l on lipid

peroxidat ion was less potent with an IC 50 value o f ≈80 μm, which was

eight -fo ld the value o f isoeugeno l. The mechanism o f act ion o f the two

compounds was invest igated and it was suggested that the ant i-oxidant

capacity o f eugeno l could be expla ined by the format ion of complexes

with reduced metals. The potent inhibitory effect of isoeugeno l on lipid

peroxidat ion may be related to the decr eased format ion o f the per -

ferryl ion or iron-oxygen chelate complex as the init iat ing factor of

lipid peroxidat ion.


13

van Zyl et al (2006)2 5

conducted a study in which twenty essent ial o il

const ituents were evaluated for their ant imalar ial act ivity against the

chloroquine-resistant st rain P.falciparum (FCR-3). Eugeno l exhibited

some act ivity with an IC 50 value o f 753 μm which was lower than for

other const ituents such as nero lido l, lina lylacetae, α -pinene and

pulegone (IC 50 values ranging from 0.9 to 1.4 μ m)

Singh et al(2007)2 6

invest igated the ant ibacter ial effect of eugeno l on

the growth of Gram-posit ive (B.cereus; B.subtilis; S.aureus ) and Gram-

negat ive (E.coli; S.typhi; P.aeruginosa) bacter ia using the agar well

diffusion method. At 1,000 ppm, eugenol inhibit ed the growth o f the

bacter ia and complete inhibit ion was obtained against P.aeruginosa at

a high concentrat ion o f 2,000 ppm. This inhibit ion was high in

compar ison to ampicillin (1 mg/mL) when used as a posit ive control.

Tragoolpua et al (2007)2 7

tested the in vit ro ant iviral act ivity o f

eugeno l against the herpes simplex-1: HSV-1 and HSV-2 viruses. The

replicat ion o f these viruses was inhibit ed with IC 50 values o f 25.6

μg/mL and 16.2 μg/mL against HSV-1 and HSV-2, respect ively.

Addit ional invest igat ions revealed synergist ic interact ions with a

combinat ion o f eugeno l and acyc lovir, a known ant iviral drug. Studies

have shown that applicat ion o f eugeno l delayed the development of

herpes virus- induced kerat it is in a mouse model. Eugeno l was

evaluated for its ant i-HSV propert ies on standard HSV-1(F), standard

HSV-2(G) and ten HSV iso lates using the plaque reduct ion assay. Only

HSV-1 iso lates 1 and 30 were inhibited by eugeno l and the inhibit ion


14

against these iso lates was greater than for the extract obtained from the

flower buds o f E. caryophyllata.

Barceloux et al (2008)2 8

reported that Clove has been used in ancient

China as a spice and fragrance for more than 2,000 years. Medic inally,

the well-known t radit ional remedy o f applying clove o il to t reat a

toothache was documented for the first t ime in 1640 in „Pract ice o f

Phys ic‟ in France. In Chinese t radit ional medicine clove o il has been

used as carminat ive, ant ispasmodic, ant ibacter ial and ant parasit ic

agent , while t he buds were used to t reat dyspepsia, acute/chronic

gastr it is and diarrhoea.

Daniel et al (2009)2 9

invest igated about Eugenia caryophyllata, which

contains eugeno l and acetyleugeno l as major const ituents for potent ial

ant i- inflammatory act ion on COX-2 (cyclooxygenase) and LOX

enzymes. The o il exhibited st rong inhibitory act ivity against COX -2

(58.15%) and 15-LOX (86.15%) enzymes at 10μg/mL and 25μg/mL,

respect ively. The ant i- inflammatory act ivity o f eugeno l was evaluated

by inflammatory exudate vo lume and leukocyte migrat ion in

carrageenan- induced pleur isy and carrageenan- induced paw oedema

tests in rats. Eugeno l (200 and 400 mg/kg) was found to reduce the

vo lume o f pleural exudates without changing the total blood leukocyte

count indicat ing the ant i- inflammatory potent ial o f eugeno l.

Okamoto et al (2009)3 0

evaluated the anaesthet ic property o f eugeno l

in juvenile Lepor inusmacrocephalus, a fish species also known as

“piavuçu”. Eugeno l had st rong anaesthet ic effect on L. macrocephalus


15

and a dose o f 37.5 mg/L o f eugeno l was recommended for the fast and

safe anaesthesia o f piavuçu juveniles (n = 72). In a similar

invest igat ion, t he efficacy o f benzocaine and eugeno l as anaesthet ics in

Trachinotusmarginatus juvenile fish was studied. Benzocaine and

eugeno l, at 50 ppm, induced fast anaesthesia and recovery (3 min and 5

min, respect ively). In European sea bass (Dicentrarchuslabrax) (n =

240), eugeno l induced deep anaesthesia without substant ially affect ing

their blood profile as measured by serum cort iso l, g lucose and

haematocr it va lues. Induct ion was rapid but recovery was very slow

even at low concentrat ions.

Hussain et al (2011)3 1

tested eugeno l alone and in combinat ion with a

chemotherapeut ic drug (gemcitabine) to evaluate it s inhibit ory effect

against cancer cells. Eugeno l showed dose-dependent select ive

cytotoxicity towards HeLa cells in compar ison to normal cells.

Furthermore, eugeno l and gemcit abine in combinat ion induced growth

inhibit ion and apoptosis (programmed cell death) at lower

concentrat ions in compar ison to the individual compounds indicat ing

synergist ic int eract ions. Eugeno l (150 μm) and gemcitabine (15 μm)

resulted in a decrease in cell viabilit y from 84% (eugeno l a lone) and

51% (gemcit abine alone) to 47% in combinat ion. Treatment o f co lon

cancer cells with eugeno l resu lted in the reduct ion o f int racellular non -

protein thio ls and an increase in the ear lier lipid layer break. In

addit ion, dissipat ion o f mitochondr ial membrane potent ial and

generat ion of react ive oxygen species accompanied the eugeno l -

induced apoptosis.


16

Marcos-Arias et al (2011)3 2

evaluated the effect o f eugeno l against 38

Candida species iso lated from denture -wearers and 10 co llect ion

Candida st rains using the CLSI M27-A3 broth micro dilut ion method.

It was found that eugeno l exhibit ed ant ifungal act ivity with the MIC

value ranging from 0.06 to 0.25% (v/v), while t he minimum

concentrat ion o f drug that inhibit ed 50% of the iso lates tested (defined

as MIC 50 ) ranged from 0.06 to 0.12% (v/v) . MIC and skin lesion

scor ing tests were performed in vivo to evaluate the ant ifungal effect

of eugeno l in a guinea pig model infected by Microsporum gypseum.

Eugeno l exhibit ed promising act ivit y with the MIC value ranging from

0.01 to 0.03%. The sk in lesion scor ing test showed that eugeno l was

clinically effect ive at improving the lesion dur ing the first week o f

applicat ion. However, eugeno l did not improve the skin lesions

infected by M. gypseum as determined in the hair culture test .

The word ozone (O3) is der ived from the Greek word ozein

(odorant). It is one o f t he most powerful ant imicrobia l agents available

for use in medicine and dent ist ry. In 1920, Dr.Edwin Parr, a Swiss

dent ist started the use of O 3 as a part of his disinfect ion system3 3

.

Mudd JB et al (1969 )3 4

conducted a study on the react ion of ozone

with amino acids and proteins and stated that ozone acts on bacter ial

cell membranes by oxidat ion o f their lipid and lipoprotein component .

Roy D et al (1981)3 5

conducted a study on the mechanism o f

enteroviral inact ivat ion with ozone by compar ing the sedimentat ion


17

profiles o f nucleic acids o f ozone-exposed viruses with those o f control

viruses, they observed that viral nucle ic acid was damaged as a result

of ozonat ion. It would appear that the major cause o f viral inact ivat ion,

as determined with po liovirus 1, by ozone using a residual

concentrat ion less than 0.3 mg/liter and a contact t ime up to 2 min is

damage o f the RNA. They concluded that all viruses are suscept ible to

ozone; yet differ widely in their suscept ibilit y and also stated that the

analys is o f viral components showed damage to the po lypept ide and

envelope proteins impair ing viral at tachment capabilit y.

Alvarez et al (1982)3 6

studied the germic idal propert ies o f the

ozonized sunflower o il (OLEOZON), they decided to make an

invest igat ion in 80 children between 1 and 14 year o ld, with t he main

object ive o f eva luat ing the effect iveness of the OLEOZON in pr imary

pyoderma, compar ing this with Gentamycin cr eam. The invest igat ion

was carr ied out in areas o f health o f the general teaching hospital

"Comandante Pinares ", San Cr istoba l, Pinar del Rio, last ing 6 months.

Pat ients were randomly assigned to both group o f t reatment ;

exper imental (using OLEOZON) and control (using Gentamycin) .To

all pat ients bacter io logical studies were done, at the beginning and

after 15 days o f t reatment . The result s demonstrated that the most

frequent iso lated germ was the Streptococcus haemolyt ic o f group A.

Healing effect iveness aft er 15 days o f treatment , was 68 % for the

OLEOZON and 28 % in those pat ients where Gentamycin was used. No

side effects were observed.


18

Morgolis HL et al (1985)3 7

reported that ozone had a severely

disrupt ive effect on car iogenic bacter ia, result ing in eliminat ion o f

acidogenic bacter ia. The st rongest naturally occurr ing ac id, produced

by acidogenic bacter ia, pyruvic acid is decarboxylated to acet ic acid,

which in turn encourages remineralisat ion.

Arsalane K et al (1995)3 8

conducted a study on ozone st imulat ion o f

inflammatory med iators and reported that ozone behaves as a weak

cytokine such as tumour necrosis factor (TNF-α), inter leukin (IL-2, IL-

6) and t ransforming growth factor -beta (TGF-β). In t he present study,

the in vit ro effect of low concentrat ions of O3 (0.1 to 1 ppm) was

evaluated on cell viabilit y and cytokine secret ion by alveo lar

macrophages (AM) from guinea pigs and healthy subjects. Cell injury

was est imated immediately after O 3 exposure by evaluat ion o f ATP cell

content as measured by bio luminescence and lact ic dehydrogenase

(LDH) release in the culture medium.

Mudd JB et al (1997)3 9

invest igated the effect of ozone on blood cells

and concluded that ozone reduces or eliminates clumping o f red blood

cells and it s flexibilit y is restored along with oxygen carrying abilit y.

Ozone was applied to sealed red cell ghost membranes at the rate of 95

mmol/min for per iods up to 20 min. Acetylcho line esterase, on the

outer face o f the membrane, was inhibited up to 20%. Glyceraldehyde -

3-phosphate dehydrogenase, on the inner surface o f the membrane, was

inhibited up to 87%. These differences reflect ed the inherent

suscept ibilit ies o f t he two enzymes and the presence or absence o f the


19

membrane barr ier. They also stated that there was no decline in the

amount of cho lestero l in the lip ids der ived from ozone -t reated red cell

membranes.

Liu Z et al (1998)4 0

stated that the applicat ion o f ozonized o ils o f

vegetal source const itutes an appropr iate method for the local t herapy

of several diseases. The interact ion o f ozone with these o ils produces a

mixture o f chemical compounds namely ozonoides and peroxides with

bet ter germicidal power. The result s o f topical applicat ion of ozonized

oil (OLEOZON) in dermatological diseases o f viral, fungi and bacter ial

origin, dur ing 9 years o f study, were presented. Among the diseases o f

bacter ial or igin (pyoderma), 600 pat ients were t reated, obtaining

healing cr iter ion in 87%. In case o fmycot ic diseases

(epidermophythosis and onychomycosis), 1000 pat ients received the

t reatment , with 91% of healing. In viral diseases (herpes simplex), 300

pat ients were t reated and in 74% the relapses disappeared. No side

effects were observed in any o f the cases after t reatment .

Oizumi M et al (1998)4 1

reported that oral microorganisms and no

viable C.a lbicans were detected after exposure to flowing ozonated

water (2 or 4 mg/L) for 1 min, suggest ing the applicat ion o f ozonated

water might be useful in reducing the number o f C.albicans on denture

bases.

Velano HE et al (2001)4 2

conducted invit ro studies and reported that

ozone was effect ive over most of the ba cter ia found in cases of pulp


20

necrosis. Ozone is effect ive when it is used in suffic ient concentrat ion,

for an adequate t ime and delivered appropr iately.

Ebensberger U et al (2002)4 3

exper imented the use o f ozonated water

in decontaminat ion o f avulsed toot h befo re replantat ion and concluded

that 2 minute irr igat ion o f avulsed teeth with non- isotonic ozonated

water not only provides mechanical cleansing but also decontaminated

the root surface with no negat ive effect on per iodontal cells.

Reth et al (2002)4 4

reported that ozone reacts with unsaturated fat ty

acids of the lipid layer in cellu lar membranes, forming hydrogen

peroxide, one o f the most significant cytokine inducers. It also plays an

important role as a secondary messenger in the init iat ion and

amplificat ion of signaling at the ant igen receptor. The ant igen

receptors themselves are H 2O2-generat ing enzymes and that the

oxidat ive burst in macrophages seems to play a ro le not only in

pathogen killing but also in the act ivat ion of these cells as well as

neighbor ing cells.

Holmes J (2003)4 5

studied the c linical reversal o f root car ies by ozone

using double blinded randomized control study and reported that ozone

is capable o f clinically reversing the leathery root carious lesion. He

assessed the effect o f an ozone delivery system, combined with the

daily use of a re-mineralizing pat ient kit , on the clinical sever it y o f

non-cavit ated leathery pr imary root carious lesions (PRCL’s) , in an

older populat ion group. He also reported that leathery non -cavit ated

pr imary root car ies can be arrested non-operat ively with ozone and re -


21

mineralizing products and this t reatment regime is an effect ive

alternat ive to convent iona l “dr illing and filling”.

Walker JT et al (2003)4 6

reported that in model dental unit water

lines, ozone achieved a 57% reduct ion in bio film and a 65% reduct ion

in viable bacter ia in spite o f being used in a very low dose and with a

short t ime o f applicat ion. They also concluded that dental

manufacturers should be invited to design dental units that incorporate

automated devices to disinfect DUWLs.

Nagayoshi M et al (2004)47

reported that ozonated water had strong

bactericidal act ivity against bacteria in plaque biofilm. It was found that

ozonated water (0.5–4mg/L) was highly effect ive in killing of both gram-

posit ive and gram-negat ive microorganisms. Gram-negat ive bacteria, such

as P.gingivalis P.endodontalis were more sensit ive to ozonated water

than gram-posit ive oral st reptococci and C .albicans in pure culture.

Baysan A et al (2004)4 8

conducted a study on the efficacy o f ozone on

pr imary root car ies lesion (PRCL) and found that ozone applicat ion for

eit her for 10 or 20 s dramat ically reduced most o f t he microorganisms

in PRCLs without any side effects recorded at recall int ervals between

3 and 5.5 months.

Young SB et al (2004)4 9

examined the mechanism o f killing B.subti lus

spores by ozone and reported that it seems to render the spores

defect ive in germinat ion perhaps because o f damage to the s pore‟s

inner membrane.


22

Huth KC et al (2005)5 0

conducted in vit ro studies with short term

fo llow-up and assessed the effect of ozone on pit and fissure car ies and

pr imary root caries with result s showing significant reduct ions in t he

number of microorganisms in t he car ious lesions. In this study gaseous

ozone (HealOzone) was applied once for 40 seconds to the randomly

assigned test molar o f each pair without the use o f re-mineralizing

so lut ions. Lesion progression or reversal was monitored by the laser

fluorescence system DIAGNOdent for up to 3 months and the

deter iorat ion or improvement compared between the ozone -t reated

lesions and the untreated control lesions

Celiberti P et al (2006)5 1

stated that ozone had no influence on the

phys ical propert ies o f the enamel to enhance or hinder the sealing

abilit y. Thus, ozone can be applied over intact and prepared enamel

dur ing the restorat ion process. They also stated that the applicat ion o f

gasiform ozone does not affect the modulus o f elast icit y and the

Vicker‟s hardness o f dent in.

Polydorou OPK et al (2006)5 2

exper imented the ant imicrobial efficacy

of ozone and reported that long exposure to ozone gas has a st rong

ant i-bacter icidal effect on microorganisms within the dent inal tubules

of deep cavit ies, which could result in increasing the clinical success

of the restorat ion. In this study thirty- five human molars were divided

into 5 groups. Cavit ies were then cut into the teeth (n=28 cavit ies pe r

group). After ster ilizat ion, the teeth were left in broth cultures o f 106

co lony-forming unit s (CFU) ml) o f S. mutans at 36ºC for 48hrs. The


23

appropr iate t reatment fo llowed (group A, control; group B, Clearfil SE

Bond; group C, Clearfil Protect Bond; grou p D, 40 s of t reatment with

ozone; and group E, 80 s o f t reatment with ozone), and the cavit ies

were then filled with composite resin. After 72hrs, the restorat ions

were removed, dent in chips were co llected with an excavator, and the

total number o f microo rganisms was determined. All t reatments

significant ly reduced the number o f S.mutans present compared with

the control group. The ant imicrobia l effect of both bonding systems

and t reatment with 80 s o f ozone was significant ly higher t han the 40 s

ozone t reatment . They concluded that Heal Ozone and the bonding

systems showed st r iking ant imicrobial effects against S.mutans.

Baysan A et al (2007)5 3

assessed the effect of the ozone-mediated

killing o f bacter ia in infected dent ine associated with non -cavitated

occlusal car ious lesions and stated that the longer the contact t ime, the

bet ter the microbio logical kill rate. It was reported that by increasing

the contact t ime from 10 to 20 s, the bacter ial kill rate changed from

ozone being a disinfectant to acquir ing ster ilizing effect .

BaysanA et al (2007)5 4

suggested that the reversal o f car ious lesions

after t reatment with ozone depend on the size and localizat ion o f the

car ious lesion. Non-cavit ated lesions were more likely to reverse than

cavitated lesions. The hardness values improved significant ly in the

ozone-t reated test lesions aft er 4, 6, and 8 months (P <0.05) compared

with baseline while t he control lesions had no significant change in

hardness at any recall int erval.


24


stated that Ozonated water has an excellent

ant i- inflammatory capacity on NF (nuclear factor) -kappa system, a

paradigm for inflammat ion-associated signalling/t ranscr ipt ion. Their

result s showed that NF-kappaB act ivity in oral cells in per iodontal

ligament t issue from root surfaces o f per iodontaly damaged teeth was

inhibited fo llowing incubat ion with ozonized medium.

Muller et al(2007)5 6

compared the influence o f ozone gas with

photodynamic therapy (PDT) and known ant isept ic agents (2%

Chlorhexid ine, 0,5 and 5% hypochlor ite solut ions) on a mult ispecies

oral bio film in vit ro. The fo llowing bacter ia were studied –

A.naeslundii, F.nucleatum, S.sobrinus, S.oralis and C.albicans .

Gasiform ozone was produced by vacuum ozone deliver y system Kavo

Heal ozone. They concluded that the matr ix-embedded microbial

populat ions in bio film are well protected towards ant imicrobia l agents.

Only 5 % hypochlor ite so lut ion was able to eliminate all bacter ia

effect ively. Usage o f gasiform ozone or PDT was notable to reduce

significant ly or completely eliminate bacter ia in the bio film.

Petrucci MT et al (2007)5 7

conducted a study in which they compared

the use o f ozonated oil in an exper imental group to a control group in

which ant ibiot ic t herapy was used in the t reatment o f alveo lit is , it was

found that pat ients t reated with ozonated o il healed more quickly.

There was complete healing o f t he lesions with the disappearance o f

symptoms.


25

Nogales CG et al (2008)5 8

conducted an exper imental study on

Ozonated o il and concluded that when applied on herpes labia lis and

mandibular osteomyelit is demonstrated faster healing t imes than

convent ional protocols. Ozone in these cases neutralises herpes vir ions

by direct act ion, thus inhibit ing bacter icidal supra - infect ions and

st imulat ing the healing o f t issues through circulatory prompt ing.

Noguchi et al (2009)5 9

reported that irr igat ion o f the root surface o f

avulsed teeth with ozonated water did not reveal a negat ive effect on

per iodontal ligament cell pro liferat ion. In this study aft er the pre -

t reatment of LPS ( Lipopolysacchar ide) with aqueous ozone, effects o f

LPS and aqueous ozone -t reated LPS on cell viabilit y; calc ificat ion

abilit y; expression o f cyc looxygenase 2 (COX-2), inter leukin 6 (IL-6),

and tumour necrosis factor α (TNF -α) ; and act ivat ion of p38 o f KN -3

cells were examined. Their results suggest that odontoblast ic cells

exhibit inflammatory responses against LPS and that ozonated wa ter

has the abilit y to improve LPS- induced inflammatory responses and

suppression o f odontoblast ic propert ies of KN -3 cells through direct

inhibit ion o f LPS.


demonstratedant imicrobia l act ion against

bacter ial st rains, such as Mycobacteria, E.faecalis, and C.albicans.

Notably, when the specimen was irr igated with sonicat ion, ozonated

water had near ly the same ant imicrobia l act ivity as 2.5% NaOCl. This

study invest igated whether gaseous ozone (4 · 106 l gm.) and aqueous

ozone (1.25–20 l gm.) exert any cytotoxic effects on human oral


26

epithelia l cells and gingival fibroblast (HGF-1) cells compared with

established ant isept ics [chlorhexidine digluconate (CHX) 2%, 0.2%;

sodium hypochlor ite (NaOCl) 5.25%, 2.25%; hydrogen peroxide (H2O2)

3%), over a t ime of 1 min, and compared with the ant ibiot ic,

metronidazo le, over 24hrs. Cell counts, metabo lic act ivity, Sp -1

bind ing, act in levels, and apoptosis were evaluated. Ozo ne gas was

found to have toxic effects on both cell types. Essent ially no cytotoxic

signs were observed for aqueous ozone suggest ing it s biocompat ibilit y

when compared to other chemica ls.

Kim HS et al (2009)6 1

has undertaken a study to evaluate the

therapeut ic effects o f topical ozonated olive o il on acute cutaneous

wound hea ling in a guinea pig model and also to elucidate its

therapeut ic mechanism. Aft er creat ing full -thickness skin wounds on

the backs o f guinea pigs by using a 6 mm punch biopsy, we examined

the wound healing effect of topically applied ozonated olive o il (ozone

group), as compared to the pure o live o il (oil group) and non -treatment

(control group). Immunohistochemical staining demonstrated up

regulat ion o f platelet der ived growth factor (PDGF), t ransforming

growth factor-beta (TGF beta) and vascular endothelial growth factor

(VEGF) expressions, but not fibroblast growth factor expression in t he

ozone group on day 7, as compared with the o il gro up. He concluded

that these results demonstrate that topical applicat ion o f ozonated o live

oil can acce lerate acute cutaneous wound repa ir in a guinea pig in

associat ion with the increased expression of PDGF, TGF -beta, and

VEGF.


27

Atabek et al (2011)6 2

conducted a study to evaluate the efficiency o f

ozone alone and with a re-mineralizing solut ion fo llowing applicat ion

on init ia l pit and fissure car ies lesions in permanent mandibu lar

molars. In their study forty children (9 -12 years) having non-cavit ated

fissure car ies lesions on bilateral 40 first permanent mandibular molar

teeth were part ic ipated in the study. Pat ients were randomly allocated

to 2 exper imental groups consist ing of 20 subjects. In the first group,

ozone was applied once for 40 seconds to the assigned test teeth o f

each pair. In the second group, ozone was applied once for 40 seconds

to the assigned test teeth o f each pair with the use o f re -mineraliz ing

so lut ion. Progression or improvement of the car ies was assessed at

base line, immediately a ft er t reatment and at 1-,2-,3-, and 6 month

fo llow-up by compar ing the DIAGNOdent va lues, Clinical Sever it y

Indexes, Oral Hygiene Scores. They concluded that ozone t reatment

eit her alone or combined with a re -mineraliz ing so lut ion was found to

be effect ive for remineralisat ion o f init ial fissure car ies lesions.

Gerspach et al (2012)6 3

invest igated the ant imicrobia l efficacy o f

gaseous ozone on bacter ia adhered to var ious t itanium and zirconia

surfaces and to evaluate adhesion o f osteoblast like MG-63 cells

to ozone-t reated surfaces. Saliva-coated t itanium (SLA and po lished)

and zirconia (acid etched and po lished) disks served as substrates for

the adherence of S.sanguinis and P.gingivalis . The test specimens were

t reated with gaseous ozone (140 ppm; 33 mL/s) for 6 and 24 s. Bacter ia

were re-suspended using ult rasonicat ion, ser ially diluted and cultured.

MG-63 cell adhesion was analysed with reference to cell at tachment ,


28

morpho logy, spreading, and pro liferat ion. Surface topography as well

as cell morpho logy o f the test specimens was inspected by SEM. They

concluded that gaseous ozone showed select ive efficacy to reduce

adherent bacter ia on t itanium and zirconia without affect ing adhesion

and pro liferat ion of osteoblast ic cells. This in vit ro s tudy may provide

a so lid basis for clinical studies on gaseous ozone t reatment of per i-

implant it is and revealed an essent ial base for suffic ient t issue

regenerat ion.

Chandra SP et al (2014)6 4

conducted a study to evaluate the success

rate of mixture o f ozonated o il and zinc oxide as a pr imary teeth root

filling mater ial. The study inc luded 60 infected pr imar y mandibu lar

molars which were equally divided into study group (ozonated o il -zinc

oxide) and control group (zinc oxide -eugeno l). Pulpectomy procedure

was performed and the children were fo llowed at regular intervals. All

the children were available for eva luat ion at the end o f 12 months. The

teeth were evaluated for success or failure based on clinical and

radiographic cr iter ia by a blinded invest igator. Clinical and

radiographic evaluat ion suggested that teeth obturated with ozonated

oil-zinc oxide demonstrated good success rate (93.3 %) as compared to

zinc oxide eugeno l (63.3 %). However , no stat ist ically significant

var iat ion (p = 0.408) was observed between the groups. They

concluded that Ozonated o il-ZnO demonstrated a good clinical and

radiographic success at 12 months fo llow -up and it can be considered

as an alt ernat ive obturat ing mater ial in infected pr imary teeth.


29

Noites et al (2014)6 5

conducted a study to determine whether irr igat ion

with sodium hypochlor ite, chlorhexidine, and ozone gas, alone or in

combinat ion, were effect ive against E.faecalis and C.albicans and

concluded that only the combined act ion of 2% CHX and ozone gas for

short period promotes the complete eliminat ion o f both microorganisms

tested in the tooth model. The result s of this study could lead to

redirect efforts towards new protocols for reducing microbial load in

infected root canals looking for synergisms between new moda lit ies

(ozone) or already known ant imicrobial products.

Anand et al (2015)6 6

evaluated the ant ibacter ial property o f ozonated

water, whit e wine (14%) and de-alcoho lised white wine against

S.mutans and E.faecalis and concluded that ozonated water has the best

ant ibacter ial property among the study groups and the ant ibacter ia l

act ion o f Calcium hydroxide is enhanced when it is mixed with de -

alcoho lised white wine.

Ahmedi et al (2016)6 7

assessed the efficacy o f ozone gas (O 3) on the

reduct ion o f dry socket (DS) occurrence fo llowing surgical extract ion

of lower jaw third molars and concluded that O 3 gas has a posit ive

effect on reducing the development of DS and pain fo llowing third

molar surgery. The microbio logical and metabo lic capabilit ies o f

O3 for promot ing haemostasis, increasing the supply o f oxygen, and

inhibit ing bacter ial pro liferat ion increase the opportunit ies for its use

in all fields o f surgery. These changes on the cellular level exp lain

elevat ion o f funct ional act ivity and regene rat ion potent ial of t issues


30

and organs which may account for the increased percentage of viabilit y

in mouse fibroblasts when t reated with ozonated eugeno l. It also

act ivates aerobic processes like glyco lys is and Krebs cycle at cellular

level to st imulate blood circulat ion; hence ozone is also used for

t reatment of circulatory disorders.

Materials and Methods

31

MATERIALS AND METHODS

Materials used

1. Zinc oxide powder (Dental Products o f India, Mumbai). Batch

no: LC 01

2. Eugeno l (Dental Products of India, Mumbai). Batch no: LG 01

3. Stainless steel r ings (dimensions : height - 1.5 mm; inner

diameter- 10mm)

4. Glass slab.

5. Stainless steel spatula.

6. Ozonator machine (Eltech Ozonators, Mumbai). Model no:D-400

7. Humidity chamber (calibrated Labtherm Humidity chamber).

8. Mett ler AE 260 Delta range Analyt ical Balance (Mett le r- Toledo

Internet INC, Columbus OH).

9. Shore A Hardness Durometer.

10.pH meter ( Eutech Instrument , pH tester).

11.Petri dish.

12.Fibroblast culture medium.

13.MTT reagent

14.Spectrophotometer


32

Methods

This study was conducted to evaluate the phys ical propert ies and

biocompat ibilit y o f z inc oxide – ozonated eugeno l and convent ional

zinc oxide eugeno l as an endodont ic sealer. Invit ro lab test ing was

conducted to evaluate and compare the hardness, so lubilit y, pH and

biocompat ibilit y o f zinc oxide-ozonated eugeno l and convent ional zinc

oxide-eugeno l.

Eugeno l was ozonated using ozonator machine with an output of

400mg/hr. 100ml o f eugeno l is taken in a glass beaker and subjected to

ozonat ion for a t ime per iod of 30 min.

Zinc oxide powder and eugeno l were weighed be fore

manipulat ion us ing Mett ler analyt ical balance with an accuracy o f

0.0001g. Pre-weighed quant it ies o f zinc oxide powder was mixed with

ozonated and convent ional eugeno l in t he rat io 6:1 by weight ( P:l

ratio). Manipulat ion was done as per manufacturer’s inst ruct ions and

fo llowing physical propert ies were evaluated. Samples were d ivided

into two groups Zinc oxide-eugeno l (Group-I) and Zinc oxide-ozonated

eugeno l (Group-II).

Hardness of the mixed samples was measured using Shore hardness

Durometer type A (ADIS Gauges and Tools Manufactur ing Company,

Mumbai) with an intender t ip Hardened steel rod 1.1 mm – 1.4 mm

diameter, with a t runcated 35° cone, 0.79 mm diameter. The samples

were mixed and 10 stainless steel r ing molds o f inner diameter 10mm

and height 1.5mm were used to shape the samples. After predetermined


33

per iods o f 12hrs and 24hrs the hardness of the sealer samples were

measured using Shore hardness Durometer t ype A. The load applied

was 0.822 Kg with a result ing force of 8.064 N.

pH change of the samples was measured using a previously calibrated

digit al pH meter (Eutech Instrument , pH tester). The samples mixed

immediately after manipulat ion were denoted as fresh samples, and the

samples mixed and stored in the incubator ( 37°C, >95% relat ive

humid ity) for a per iod o f 12hrs and 24hrs were denoted as set samples.

Stainless steel r ings of inner diameter 10mm and height 1.5mm were

used to shape the molds. Each set sample was put in a flask containing

10ml o f dist illed water and stored at 37°C througho ut the study per iod.

For fresh samples, the same vo lume as the set samples o f mixed sealer

was placed in a flat bottom container containing 10ml o f dist illed water

at 37°C. After predetermined per iods o f 12, 24 and 48hrs the pH o f t he

so lut ion was measured using a calibrated digit al pH meter.

Solubi lity was tested as a percentage o f the mass o f specimen removed

from the dist illed water compared with the or iginal mass o f the

spec imens. Three 1.5 mm thick stainless steel r ing o f inner diameter

10mm were used for each group. The r ings were filled with sealers and

supported by a glass plate. The filled molds were placed in an

incubator (37°C, >95% relat ive humidity) for a per iod o f 24 hrs. The

samples were removed from the mold and weighed three t imes each

with an accuracy o f 0.0001g (Mett ler analyt ical balance). Each sample

was put in a petr i dish, which was weighed before use with 50ml


34

dist illed water. Then the samples were discarded, and the p etr i dish

were dr ied in an oven at 110 c, cooled in the desiccator to room

temperature, and reweighed. The amount of sealer removed from each

spec imen was calculated as t he difference between the init ia l mass and

final mass of the petr i dish.

Biocompatibi lity o f the tested samples was evaluated using the MTT

(3-(4,5-dimethylthiazo l-2-yl)-2, 5-diphenyltet razo lium bromide)

tet razolium reduct ion assay. It was the first homogeneous cell viabilit y

assay developed for a 96-well format that was suitable for high

throughput screening (HTS).The MTT substrate was prepared in a

phys io logica lly balanced so lut ion, added to cells in culture at a final

concentrat ion o f 0.2 - 0.5mg/ml, and incubated for 1 to 4 hours. After

24hrs biocompat ibilit y was evaluated as the percentage o f viabilit y o f

mouse fibroblast cell line L929 when test samples were added to it .

The quant it y o f formazan (presumably direct ly proport ional to the

number o f viable cells) is measured by recording changes in

absorbance at 570 nm using a plate read in g spectrophotometer.


35

Flow chart:

Ozonation

Eugeno l is taken in a 100ml glass container

Ozone gas is bubbled through eugeno l using ozonator machine

400mg/hr/30mins

Concentrat ion o f ozone is calculated using ozone analyzing st r ips

(under 0.01ppm)

Manipulation

Zinc oxide (powder)

6:1

Convent ional eugeno l ozonated eugeno l

Group I Group II


36

Hardness of the mixed sealers was measured using Shore hardness


Mumbai)

pH change o f the samples was measured using a previously calibrated

digit al pH meter ( Eutech Instrument , pH tester).

3 samples for each group were subjected to pH test ing

Subjected to pH test ing

Stainless steel r ings of inner diameter 10mm and height 1.5mm -

shape the molds.

10 samples for each group were subjected to hardness test ing

After 12hrs and 24hrs the hardness o f the sealer samples were

measured.

Samples mixed immediately

Samples mixed and stored in

the incubator (set samples)

After 12hrs After 24hrs Fresh samples


37

Solubility was tested as a percentage o f the mass o f specimen removed


spec imens.

Stainless steel r ings of dimens ions 1 .5x10mm were used to

shape the samples.

3 samples for each group were subjected to solubilit y test ing

Samples were removed from the mold

Weighed three t imes each with an accuracy 0.001g

Each sample was put in a petr i dish - weighed before use

with 50ml d ist illed water.

Samples discarded from petr i dish,

Dr ied in an oven at 110° C, cooled in the desiccator

and re-weighed.

Amount of sea ler removed from each specimen

(w1 – w2 of the petr i dish)


38

Biocompatibi lity o f the tested samples were evaluated using the MTT

(3-(4,5-dimethylthiazo l-2-yl)-2, 5-diphenyltet razo lium bromide)

tet razolium reduct ion assay.

Mouse fibroblast cells (L929) and test compounds prepared

in 96-well plates

Incubated for desired per iod of exposure.

10µl MTT so lut ion added per well - final concentrat ion

of 0.45 mg/ml.

Incubated for 1 to 4hrs at 37°C.

100 µl so lubilisat ion so lut ions added to each well to

disso lve formazan crystals.

Absorbance recorded at 570 nm to check

the % of viabilit y.


39

Armamentarium

Figure 1: Zinc oxide powder, Eugeno l, Glass slab and stainless steel

spatula.

Figure 2 :Stainless steel r ing. Figure 3: Ozonator D-400

Dimension : 1.5 x 10 mm


40

Figure 4: Eugeno l ozonated by bubbling gases through it .


41

Figure 5: Shore hardness Durometer t ype A (ADIS Gauges and Tools

Manufactur ing Company, Mumbai)

Figure 6: Calibrated digital pH meter (Eutech Instrument , pH tester).


42

Figure 7: Samples shaped using stainless steel mou lds

Figure 8: Mett ler AE 260 Delt a range Analyt ical Balance


43

Figure 9 : MTT Reagent Figure 10: Micro plate reader

Figure 11 :Biocompat ibilit y

Group I

Figure 12: Biocompat ibilit y

Group II

Results

44

RESULTS

This study was conducted to evaluate the phys ical propert ies and

biocompat ibilit y o f zinc oxide – ozonated eugeno l and to compare it

with convent ional zinc oxide eugeno l as an endodont ic sealer. Eugeno l

was ozonated using ozonator machine by bubbling ozone through it .

The powder and liquid were mixed in the rat io o f 6:1 by weight and the

mixed samples were divided into two groups: Group I (zinc oxide

eugeno l) and Group II (zinc oxide – ozonated eugeno l). Phys ical

propert ies such as hardness, pH, so lubilit y and biocompat ibilit y o f t he

samples were evaluated and compared. The obtained results were

tabulated and the data co llected were stat ist ically analysed us ing

unpaired T test with P value significant at 5% level (p<0.05).

Results

45

Hardness of the mixed samples was measured using Shore A hardness


Mumbai) with an intender t ip Hardened steel rod 1.1 mm – 1.4 mm

diameter. A total o f 10samples for each group were mixed and shaped

using stainless steel r ing molds o f inner diameter 10mm and height

1.5mm. After 12hrs the mean hardness values o f the samples in Group I

and Group II was 55.9100 and 57.3600 respect ively. After 24hrs the

mean hardness values o f the samples in Group I and Group II was

73.1700and 73.5000 respect ive ly. On compar ison there was no

stat ist ically significant difference between the groups with p values

0.064 and 0.545 after 12hrs and 24hrs respect ive ly.

Table 1:

Group I : zinc oxide eugeno l Group II : zinc oxide – ozonated eugeno l

Shore A

Hardness

No.of

samples

Mean

Std.

Deviation

P-Value

After 12hrs

Group I 10 55.9 1.95587 P=.064

NS Group II 10 57.3 1.24829

After 24hrs

Group I 10 73.1 .90068 P=.545

NS Group II 10 73.5 1.43295

Signif icant at the 5% level. (p<0.05) .Statistical ly no signif icant

difference using unpaired t’test

Results

46

pH change of the samples was measured using a previously calibrated

digit al pH meter (Eutech Instrument , pH tester). A total o f three

samples from each group were subjected to pH test ing after

predetermined t ime per iods: freshly mixed, 12hrs and 24hrs. The mean

pH values o f the freshly mixed samples in Group I and Group II was

6.5667and 7.5667 respect ively with stat ist ically s ignificant difference

between them (P=.013). Aft er 12hrs the mean pH values o f the samples

in Group I and Group II was 6.1000 and 6.7667 respect ively with

stat ist ically significant difference between them (P=.007). A fter 24hrs

the mean pH values o f the samples in Group I and Group II was

5.7667and 6.2667 respect ively with no stat ist ically significant

difference between them (P=.123).

Table 2:


pH of the

test samples

No of

samples Mean Std. Deviation P-Value

Freshly

mixed

Group I 3 6.5667 .20817 P=.013

Sig Group II 3 7.5667 .35119

After 12hrs Group I 3 6.1000 .17321 P=.007

Sig Group II 3 6.7667 .15275

After 24hrs Group I 3 5.7667 .30551 P=.123

NS Group II 3 6.2667 .32146

Signif icant at the 5% level.(p<0.05).Statistically signif icant dif ference

using unpaired t’test in freshly mixed samples and af ter 12

hrs.Statistically no signif icant dif ference af ter 24 hrs.

Results

47

Solubility was tested as a percentage o f the mass o f specimen removed


spec imens. 1.5 mm thick stainless steel r ing of inner diameter 10mm

and were used to shape the samples. A total o f three samples from each

group was subjected to pH test ing after 12hrs and 24hrs. After 12hrs

the mean so lubilit y values o f the samples in Group I and Group II was

0.6233and 0.6100 respect ively. After 24hrs the mean hardness values

of t he samples in Group I and Group II was 0 .5500 and 0.5400

respect ively. On compar ison there was no stat ist ically significant

difference between the groups with p values 0.612 and 0.513 after

12hrs and 24hrs respect ively.

Table 3:


Solubility of

the test

samples (wt%)

No of

samples

Mean

Std.

Deviation

P-Value

After 12hrs

Group I 3 .6233 .05033 P=.612

NS Group II 3 .6100 .02000

After 24hrs

Group I 3 .5500 .02000 P=.513

NS Group II 3 .5400 .02000

Signif icant at the 5% level.(p<0.05). Statistically no signif icant

difference using unpaired t’test

Results

48

Biocompatibi lity o f the tested samples were evaluated using the MTT

(3-(4, 5-dimethylthiazo l-2-yl)-2, 5-diphenyltet razo lium bromide)

tet razolium reduct ion assay. A total o f three samples from each group

were subjected to biocompat ibilit y test ing aft er 24hrs. Aft er

24hrsbiocompat ibilit y was evaluated as the percenta ge o f viabilit y o f

mouse fibroblast cell line L929 when test samples were added to it .

After 24hrs the mean biocompat ibilit y values o f the samples in Group I

and Group II was 52.6000 and 68.4333 respect ively. On compar ison

there was stat ist ically significant difference between the groups with p

value set at leve l 5% after 24hrs (p=0.002).

Table 4:


Biocompatibi lity

(% of viabi lity)

No of

samples

Mean

Std.

Deviation

P-Value

After 12hrs

Group I 3 52.6000 1.00167 P=.002

Sig Group II 3 68.4333 1.15326

Signif icant at the 5% level (p<0.05). Statistically signif icant dif ference

using unpaired t’test

Results

49

Group I: Zinc oxide eugeno l Group II: Zinc oxide – ozonated eugeno l

Chart 1: Hardness values of tested samples after 12hrs

Chart 2: Hardness values of tested samples after 24hrs

50

51

52

53

54

55

56

57

58

59

60

1 2 3 4 5 6 7 8 9 10

12hrs Group I

12hrs GroupII

Results

50

Chart 3: Mean hardness va lues of tested samples

Chart 4: Mean pH values of tested samples

0

10

20

30

40

50

60

70

80

12hrs 24hrs

Group I

Group II

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

Freshly mixed 12hrs 24hrs

Group I

Group II

Results

51

Chart 5: Mean so lubilit y values o f tested samples

Chart 6: Mean biocompat ibilit y values of tested samples

0.50

0.52

0.54

0.56

0.58

0.60

0.62

0.64

12hrs 24hrs

Group I

Group II

0

10

20

30

40

50

60

70

Group I (24hrs) Group II (24hrs)

Group I (24hrs)

Group II (24hrs)

Discussion

52

DISCUSSION

Root canal sealers along with so lid core mater ial plays a vital

role in achieving the three dimensional sealing o f the root canal

system. These sealers are bind ing agents which are used to bind the

r igid gut ta-percha to the canal walls and to fill up the vo ids, accessory

canals and irregular it ies in the canal1. Endodont ic sealers, alt hough

used only as adjunct ive mater ials in the obturat ion o f root canals, they

also have shown to influence the outcome of endodont ic t reatment . A

perfect combinat ion o f sea ling abilit y, ant imicrobial act ivity and

biocompat ibilit y is what an ideal root canal sealer should possess.

An ideal root canal sealer should possess fo llowing propert ies1 4

.

1. It should be tacky when mixed, to be able to provide

adhesion/sealing between the canal walls and the core obturat ing

mater ial.

2. Flowabilit y in it s non-set state to promote lut ing.

3. It should be non- irr itat ing to the per iapical t issues.

4. Should possess a hermet ic sealing abilit y.

5. It should be radiopaque.

6. Minimal set t ing shr inkage.

7. Should be non-staining on the dent in / tooth st ructure.

8. It should be ideally Bacter icidal or at -least control Bacter ia l

Growth.

9. Should have Long Working Time.

10.Inso luble in Tissue Flu ids .

11.It should be easy to remove if necessary.

Discussion

53

12.It should be neither Mutagenic nor Carcinogenic.

13.It should not provoke an Immune react ion.

ZOE sealers have a history o f successful use over long per iod o f

t ime. It has both anaesthet ic and ant imicrobial propert ies. The qualit y

of the seal achieved with gut ta -percha (GP) and convent ional zinc

oxide eugeno l (ZOE) sealers is not quite perfect6 8

. Despite o f its

advantages, GP and convent ional sealer combinat ion st ill has it s own

drawbacks, like its inabilit y to st rengthen the root , as it does not

adhere to dent in, inabilit y to control micro leakage, and the so lubilit y

of sealer makes the prognosis quest ionable6 9

. Also, the eugeno l

released from ZOE is reported to be an irr itant to per iapical t issues and

has cytotoxic propert ies with limited ant imicrobial act ivity.

In search o f modern endodont ic therapeut ic modalit ies with

efficient and broad ant imicrobial act ivity, ozone has proved to elicit

super ior act ion in var ious fields o f dent ist ry. Ozone (O 3) is an

energised form o f oxygen and dissociates readily back to oxygen,

liberat ing a sing let oxygen which is responsible for its so called

oxidising potent ial. Ozone is 1.6- fo ld denser and 10-fo lds more so luble

in water (49.0 mL in 100 mL water at 0° C) when comp ared to that of

oxygen. Though ozone is not a radical molecule, it is t he third most

potent oxidant (E_ 5 12.076 V) after fluor ine and per sulphate. Ozone

is an unstable gas that cannot be stored and should be used

immediately because it has a ha lf - life of 40 min at 20 °C3 3

. In the

clinical set t ing, an oxygen/ozone generator simulates lightning through

Discussion

54

an electr ical discharge fie ld to generate ozone. Ozone gas has a high

oxidising potent ial and it is 1.5 t imes greater than chlor ide when used

as an ant imicrobia l agent against bacter ia, viruses, fungi, and protozoa.

It also has the abilit y to st imulate blood circulat ion and the immune

response. These features just ify t he current interest in it s applicat ion in

medicine and dent ist ry and have been suggested for t he t reatment of

260 different pathologies.

There is an evidence o f use o f ozone as a disinfectant from 1881,

which was ment ioned in a book on Diphther ia. In 1893, the wor ld’s

first water t reatment plant ut ilizing ozone was installed in Ousbaden,

Holland. Dur ing World War 1, ozone was used medically for t reat ing

wounds and other infect ions. Medical grade Ozone is a combinat ion o f

pure O2 and pure O3 in the rat io of 0.1% to 5% of O 3 and 95%-99.5%

of O27 0

.

Interest ingly enough, in 1930, a German dent ist , Dr. E.A. Fisch ,

t reated Austr ian surgeon Ernst Payr for a gangrenous pulpit is using

ozone and thereafter inspired him to begin a new line o f researches

dedicated to ozone use in health care. However, ozone seems to have

disappeared from pract ice in dental ca re unt il 2001 when the first

scient ific study was published examining the biomolecules present in

dental car ies, before and after t reat ing with ozone7 1

. As dental

demographics changed and in the era o f minimally invasive therapy,

the new challenges o f managing and t reat ing an aged populat ion those

who have retained rather than lost their teeth needed to be addressed.

Discussion

55

This was of part icular concern in reg ard to elder ly pat ients with the

increased suscept ibilit y o f root surfaces car ies. In dent ist ry, the

applicat ions of ozone are as fo llows5 7

:

1. Reversal o f incipient car ies.

2. Prevent ion o f pulpa l infect ion in deep car ies affected dent in.

3. Disinfect ion dur ing root canal t reatment .

4. Disinfect ion o f cavit y preparat ions pr ior to restorat ions.

5. Treatment of cervical sensit ivity.

6. Prior to placement of all fissure sealant .

7. For bleaching teeth.

8. Combined with other approaches to manage car ies such as ART.

9. For treat ing apthus ulcer and herpes simplex lesions.

Ozone (O3) can be used in gaseous, aqueous form or as ozonated

oils. In order to control t he decomposit ion o f O 3 into oxygen it can be

combined with a vehicle with aqueous propert ies to improve the

conversion more quick ly or with a vehicle with relat ively more viscous

propert ies to retard the conversion. In a study conducted to evaluate

the biocompat ibilit y o f ozone, the aqueous form o f Ozone was found to

be less cytotoxic than gaseous Ozone. Due to the unstable nature of

ozone in gaseous and aqueous form, we opted to use ozone in the form

of o ils.

Estrela C et al used ozonated o ils to ster ilize the root canal

systems and to clear t he canals o f necrot ic debr is by virtue o f ozone’s

bacter icidal and effervescent propert ies. In t heir study they used

Discussion

56

ozonated oils for dis infect ion and concluded that irr igat ion with

ozonated oil is more quick and efficient in canal ster ilizat ion than that

of convent ional irr igat ion by the sodium hypochlor it e so lut ion7 2

.

Chandra SP et al conducted a study to evaluate the success rate

of mixture of ozonated sesame o il and zinc oxide as a pr imary teeth

root filling mater ial. They conc luded that Ozonated sesame o il -ZnO

demonstrated a good clinical and radiographic success rate at 12

months fo llow-up and it can be considered as an alt ernat ive obturat ing

mater ial in infected pr imary teeth7.

Eugeno l that has been used in dent ist ry fo r a long t ime due to its

analgesic and ant isept ic property is a colour less to pale yellow o ily

liquid extracted from certain essent ial o ils especia lly from clove o il,

nutmeg, cinnamon, basil and bay leaf. In Endodont ics studies have

focused on the use o f ozonated water and o ils as an intra -cana l

medicament and as an irr igant . But none of t he studies have conducted

by ozonat ing eugeno l for being used as an endodont ic sea ler.

In the present study, ozone was used to overcome the drawbacks

of ZOE sealer by bubbling ozone gas through eugeno l using an

ozonator machine which has an output of 400mg/hr. The concentrat ion

of ozone in eugeno l was checked using ozone ana lysing st r ips and was

kept under 0.05ppm (safety dose o f ozone as per OSHA: less t han

0.1ppm)7 3

. Invit ro studies were conducted to evaluate and compare the

hardness, so lubilit y, pH and biocompat ibilit y o f conven t ional zinc

oxide-eugeno l (Group I) and zinc oxide-ozonated eugeno l (Group II).

Discussion

57

Hardness o f t he tested samples was evaluated using Shore A

hardness Durometer t ype A (ADIS Gauges and Tools Manufactur ing

Company, Mumbai). The samples were mixed as per manufacturer’s

inst ruct ions and subjected to hardness test ing at the end of 24hrs and

48hrs. The hardness values obtained were almost similar for both the

groups with no stat ist ically significant difference between them.

(p>0.05)

pH of the mixed samples was evaluated using calibrated pH

meter after predetermined t ime intervals ( freshly mixed, 24hrs and

48hrs). In Group II where eugeno l is ozonated the pH o f freshly mixed

samples was relat ively higher than that o f the samples in Group I. This

may be due to format ion o f hydroxyl ions readily due to the unstable

nature of ozone. This t rans ient radical anion rapidly becomes

protonated, generat ing hydrogen t rioxide (HO 3), which, in turn,

decomposes to an even more powerful oxidant , the hydroxyl rad ical

(OH)5 7

. There was stat ist ically significant difference between the two

groups (p < 0.05) in t he freshly mixed samples and set samples after

12hrs. However there was no stat ist ically significant difference

between the two groups in the set samples after 24hrs whi ch may be

due to the deplet ion o f singlet oxygen with t ime.

Solubilit y o f the tested samples was evaluated by the difference

in the percentage o f the mass o f the specimen before and after

subject ing to solubilit y test ing. So lubilit y o f the tested samples after

12hrs and 24hrs were almost similar in both the groups with no

Discussion

58

stat ist ically significant difference between them (p>0.05). In this

study, the samples in both the groups demonstrated good results, none

of the sealers exceeded 3% mass fract ion after immersion in water for

24 hours, in accordance with ISO 6876 and ANSI/ADA Specificat ions

No. 57 and No. 30 13

.

Biocompatibi lity o f the tested samples was evaluated using the

MTT (3-(4, 5-dimethylthiazo l-2-yl)-2, 5-diphenylt et razolium bromide)

tet razolium reduct ion assay. In the present study biocompat ibilit y was

evaluated as the percentage o f viabilit y of mouse fibroblast cell line

L929 when test samples were added to it . Aft er 24hrs, the percentage

of viabilit y was measured by recording the changes in absorbance at

570 nm using a plate reading spectrophotometer.

In Group II where eugeno l is ozonated, there was higher

percentage o f viabilit y when compared to convent ional eugeno l with

stat ist ically significant difference between them (p<0.05). These

findings were similar to the studies of Ebensberger U and Huth KC in

which a high level o f biocompat ibilit y of aqueous ozone has been

found on human oral epithelial cells , ging ival fibroblast cells, and

per iodontal cells4 3

. The metabo lic act ivity o f L-929 mouse fibroblasts

was high when the cells were t reated with ozonated water, whereas that

of significant ly decreased when the cells were t reated with 2.5%

NaOCl5 9

.

As a response to inflammatory process, Ozone st imulates the

release o f inter leukins, leukotriene and prostaglandins, to promote

Discussion

59

wound healing. It act ivates mechanisms of protein synthesis, increases

amount of r ibosomes and mitochondr ia in cells7 4

. These changes on the

cellular level expla in elevat ion o f funct ional act ivity and regenerat ion

potent ial o f t issues and organs which may account for the increased

percentage o f viabilit y in mouse fibroblasts when t reated with ozonated

eugeno l. It also act ivates aerobic processes like glyco lys is and Krebs

cycle at cellular level to st imulate blood circulat ion; hence ozone is

also used for t reatment of circulatory diso rders.

Ishizaki K et al explor ing the effect of ozone on E. coli , they

stated that ozone penetrated t hrough cell membranes, react ing with

cytoplasmic contents, cleaving the circular plasmid DNA, thus

impair ing bacter ial procreat ion. They also stated that higher organisms

have developed mechanisms for protect ing DNA and repair ing them

when disrupted while subject ing to ozone therapy7 5

. This could provide

a part ial exp lanat ion for why ozone at doses prescr ibed, is toxic to

pathogens and not to the healthy cells and these features just ify t he

current interest in its app licat ion in dent ist ry.

Summary and Conclusion

60

SUMMARY AND CONCLUSION

Dent ist ry is changing, as we are now using modern science to

pract ice dent ist ry. In Endodont ics, the sealers are responsible for the

pr incipal funct ions o f the final root filling: sealing o ff o f the root cana l

system, entombment of remaining bacter ia and the filling o f

irregular it ies in t he prepared canal. The Zinc Oxide Eugeno l (ZOE)

based sealers were introduced in Endodont ics by Grossman in 1936.

ZOE sealers have a history o f successful use over extended per iod of

t ime. However it s use has been rest r icted in recent years due to its few

drawbacks including the cytotoxic and ir r itat ing property o f eug eno l3.

Eugeno l (4- acyl-2-methoxy pheno l) is an extract of clove (Eugenia

caryophyllata) commonly used as an anaesthet ic and ant imicrobial

agent in the field o f medic ine and dent ist ry.

Several at tempts that have been made to overcome these

disadvantages have failed and there is cont inued research for chemical

compounds with bet ter physical propert ies and biocompat ibilit y. The

evo lut ion o f sealers is from the convent ional ZOE to the contemporary

ones like epoxy-based resin and methacrylate - resin based sealers, and

to the most recent MTA sealer and bioceramic sealer. However t il l

date, none o f the sealers has proved to be the ideal except a few which

can come c loser to being one.

Cons ider ing the beneficial effects of eugeno l with few

drawbacks, we decided to improve it s propert ies by other modalit ies. In


61

search o f other t reatment modalit ies, ozone therapy has a long history

of research and clinical applicat ions with humans because o f its

int erest ing bio logical propert ies. In Endodont ics studies have focused

on the use o f ozonated water and o ils as an intra -canal medicament and

as an irr igant . But none o f the studies have conducted by ozonat ing

eugeno l for being used as an endodont ic sealer. In the present study,

ozone was used to overcome the drawbacks o f ZOE sealer by bubbling

ozone gas through eugeno l using an ozonator machine.

In vit ro lab test ing was done to evaluate and compare the

phys ical propert ies and biocompat ibilit y of z inc oxide eugeno l (Group

I) and z inc oxide- ozonated eugeno l (Group II). The proport iona l

values obtained were tabulated and stat ist ically analysed by unpaired t

test . Solubilit y and hardness o f the tested samples were almost similar

in both the groups wit h no stat ist ical difference between them. pH o f

freshly mixed samples in Group II was relat ively higher than the

samples in Group I which may due to the increased format ion o f

hydroxyl ions.

Biocompat ibilit y o f t he tested samples in Group II was

comparat ively more than that of the samples in Group I. The increased

percentage o f viabilit y may be due to the act ivat ion o f protein

synthesis, increased amount of r ibosomes and mitochondr ia synthesis

in ce lls. These changes on the ce llular level expla in the ele vat ion o f

funct ional act ivity and regenerat ion potent ial o f t issues and organs.

Ozone br ings about the r ise o f pO 2 in t issues and improves


62

t ransportat ion o f oxygen in blood, which result s in change o f cellular

metabo lism – act ivat ion o f aerobic processes (glyco lys is, β-oxidat ion

of fat ty ac ids and Krebs cycle) and use o f energet ic resources.

Within the limitat ions o f the study, it can be concluded that

consider ing the beneficia l propert ies o f eugeno l with few drawbacks:

ozone with interest ing bio logical propert ies seems to be a bet ter

t reatment modalit y in improving the propert ies o f eugeno l. However

further clinical t r ials and randomized control studies are required in

this field to use ozonated eugeno l in combinat ion with zinc oxide

powder to use it as an endodont ic sealer invivo.

References

63

REFERENCES

1. Ingles Endodont ics. Obturat ion of the Radicular space, 6t h

edit io n

CBS publishers and dist r ibutors.1053

2. Hui-min Zhou et al. phys ical propert ies of 5 root canal sealers. J

Endod 2013; 39:1281-6.

3. Marín-Bauza GA, Silva-Sousa YT, Cunha SA, Rached-Junior FJ,

Bonet t i-Filho I, Sousa-NetoMD, Miranda CE. Phys icochemica l

propert ies o f endodont ic sealers o f d ifferent bases. Journal o f

Applied Oral Science. 2012 ;20(4):455-61.

4. Ranade MP, Kamra Al. A comparat ive t issue toxicit y evaluat ion

of four endodont ic mater ials. Endodontology; 2003; 15:7 -12.

5. Anil kumar, SharnammaBhagawat i, Prashantyagi, Pr ince Kumar.

Current interpretat ions and scient ific rat ionale o f the ozone usage

in dent ist ry: A systemat ic review o f l iterature. European Journa l

of General Dent ist ry; 2014; 3: 175-80.

6. SushmaDas. Applicat ion o f ozone therapy in dent ist ry. IJDA

2011; 3(2):538-42.

7. Chandra SP, et al Success o f root fillings with zinc oxide -

ozonated oil in pr imary molars:preliminar yresults. E ur Arch

Pediatr Dent 2014; 15:191-5

8. BikashPat tanaik et al; Ozone therapy in Dent ist ry: A literature

review. Journal o f inter disciplinary Dent ist ry; Jul -Dec 2011 ;

2:87-91.

References

64

9. Nagayoshi M, Fukuizumi T, Kitamura C,Yano J, Terashit a M,

Nishihara T. Efficacy o f ozone on survival and permeabilit y o f

oral microorganisms.OralMicrobio lImmunol 2004;19:240 -6.

10. Grossman LI. Endodont ic pract ice. 8th. ed. Philadelphia: Lea

&Febiger ; 1974.

11. Walton RE, Torabine jad M. Pr inciples and Pract ice o f

Endodont ics, 3rd ed. Philadelphia:Saunders;2002

12. McMichen FR, Pearson G, Rahbaran S, Gulabivala K. A

comparat ive study o f selected phys ical propert ies of five root -

canal sealers. Int Endod J 2003; 36:629 –35.

13. ANSI/ADA. Specificat ion No. 57 Endodont ic sealing mater ials.

Chicago, IL; 2000.

14. Grossman LI. Phys ical propert ies o f root canal cement s. J Endod

1976; 2:166-75

15. Hauman CH, Love RM. Biocompat ibilit y of dental mater ials used

in contemporary endodont ic therapy: a review. Part 2.Root -canal-

filling mater ials. Int Endod J. 2003 Mar; 36( 3):147-60.

16. Candeiro GT, Correia FC, Duarte MA, et al. Evaluat ion o f

radiopacity, pH, release o f calcium ions and flow of a

bioceramic root cana l sealer. J Endod 2012; 38: 842 –5.

17. Lacey S, Pit t Ford TR, Watson TF, Sherr iff M. A study o f the

rheo logical propert ies o f endodont ic sealers. Int Endod J 2005;

38: 499–504.

References

65

18. Lindquist , I . , Otteskog, P. Eugeno l: liberat ion from denta l

mater ials and effect on human diplo id fibroblast cells. Scan J

Dent Res. 1980; 88: 552–556.

19. Branstet ter J, von Fraunhofer JA. The phys ical propert ies and

sealing act ion o f endodont ic sealer cements: a review o f the

lit erature. J Endod.1982; 8: 312 –6.

20. Bedoukian, P.Z. Perfumery and Flavour ing Synthet ics, 3r d ed.;

Allured Publishing Corporat ion: Caro l Stream, IL, USA, 1986.

21. Thompson D1, Constant in-Teodosiu D, Egestad B, Mickos H,

MoldéusP.Format ion o f g lutathione conjugates dur ing oxidat ion

of eugeno l by microso mal fract ions of rat liver and lung.

BiochemPharmaco l. 1990 May 15; 39(10):1587 -95

22. Jeng JH, Hahn LJ, Lu FJ, Wang YJ, Ku o MY. Eugeno l t r iggers

different pathobio logical effects on human oral mucosa l

fibroblasts. J Dent Res. 1994; 73(5):1050 -5.

23. Tai KW, Huang FM, Huang MS, Chang YC. Assessment of the

genotoxicity o f resin and zinc‐oxide eugeno l‐based root cana l

sealers using an in vit ro mammalian test system. Journal o f

biomedical mater ia ls research. 2002 Jan 1; 59(1):73 -7.

24. Ito , M.; Murakami, K. ; Yoshino, M. Ant i-oxidant act ion o f

eugeno l compounds: ro le o f metal ion in the inhibit ion o f lipid

peroxidat ion. Food Chem. Toxico l. 2005; 43: 461–6.

25. vanZyl, R.L. ; Seat lho lo, S.T.; van Vuuren, S.F. ; Viljoen, A.M.

The bio logical act ivit ies o f 20 nature ident ical essent ial o il

const ituents. J. Essent . Oil Res. 2006; 18: 129 –33.

References

66

26. Singh, G. ; Maurya, S.; deLampasona, M.P.; Catalan, C.A.N. A

compar ison o f chemical, ant i-oxidant and ant imicrobia l studies

of cinnamon leaf and bark vo lat ile o ils, o leoresins and their

const ituents. Food Chem. Toxico l. 2007; 45: 1650 –61.

27. Tragoolpua,Y. ; Jat isat ienr, A. Ant i-herpes simplex virus

act ivit ies o f Eugenia caryophyllus (Spreng.) Bullock & S. G.

Harr ison and essent ial o il, eugeno l. Phytother Res.2007; 21:

1153–58.

28. Barceloux, D.G. Medical Toxico logy of Natural Substances.

Foods, Fungi, Medic inal Herbs, Plants and Ve nomous

Animals; Wiley: Hoboken, NJ, USA, 2008 .

29. Daniel, A.N. ; Sartoretto , S.M.; Schmidt , G.; Caparroz -Assef,

S.M.; Bersani-Amado, C.A.; Cuman, R.K.N. Ant i -

inflammatory andant inocicept ive act ivit ies o f eugeno l

essent ial o il in exper imental animal models. Rev. Bras.

Farmacogn. 2009; 19: 212–17.

30. Okamoto, M.H.; Tesser, M.B. ; Louzada, L.R. ; dos Santos, R.A. ;

Sampaio, L.A.Benzocaine and eugeno l as anaesthet ics for

pompano juvenile Trachinotusmarginatus. Cienc. Rural 2009; 39:

866–70.

31. Hussain, A. ; Brahmbhat t , K.; Pr iyani, A. ; Ahmed, M. ; Rizvi,

T.A.; Sharma, C. Eugeno l enhances the chemotherapeut ic

potent ial o f gemcit abine and induces ant icarcinogenic and ant i -

inflammatory act ivity in human cervical cancer cells. Cancer

Biother. Rad iopharm.2011 ; 26: 519–27.

References

67

32. Marcos-Arias, C.; Eraso, E.; Madar iaga, L.; Qu indos, G. In vit ro

act ivit ies o f natural products against oral Candida iso lates fro m

denture wearers. BMC Complement . Altern. Med. 2011; 11: 119.

33. Bocci VA. Sc ient ific and medical aspects of ozon e therapy. State

of the art . Arch Med Res.2006; 37: 425-35.

34. Mudd JB, Leavit t R, Ongun A, McManus TT. React ion o f ozone

with amino acids and proteins. Atmos Environ . 1969; 3: 669-82.

35. Roy D, Wong PK, Engelbrecht RS, Chian ES. Mechanism o f

enteroviral inact ivat ion by ozone. Appl Environ Microbio l . 1981;

41: 718-23.

36. Alvarez ME, O'Br ien RT. Mechanisms o f inact ivat ion o f

poliovirus by chlor ine d ioxide and iodine. Applied and

Environmental Microbio logy. 1982 Nov; 44(5): 1064 -71.

37. Margo lis HC, Moreno EC, Murphy BJ. Importance o f high pKa

acid in car iogenic potent ial o f plaque. J Dent Res 1985; 64: 786 –

92.

38. Arsalane K, Gosset P, Vanhee D, Voisin C, Hamid Q,

TonnelAB,et al . Ozone st imulates synthesis o f inflammator y

cytokines by alveo lar macrophag es in vit ro . Am J Respir Cel l

MolBio l. 1995;13: 60-8.

39. Mudd JB, Dawson PJ, Santrock J. Ozone does not react with

human erythrocyte membrane lipids. Arch BiochemBiophys .

1997; 341: 251-8.

40. Liu Z, Carpenter SB, Bourgeo is WJ, Yu Y, Constant in RJ,

Falcon MJ, Adams JC. Var iat ions in the secondary metabo lit e

References

68

camptothecin in relat ion to t issue age and season in

Camptothecaacuminata. Tree Physio logy. 1998 Apr ; 18(4):265 -

70.

41. Oizumi M, Suzuki T, Uchida M, Furuya J, Okamoto Y. In vit ro

test ing of a denture cleaning method using ozone. J Med Dent

Sci. 1998; 45: 135-9.

42. Velano HE, Nascimento LC, Barros LM, Panzer i H. In vit ro

assessment of ant ibacter ial act ivity o f ozonized water against

Staphylococcus aureusPesqu iOdonto l Bras . 2001; 15: 18-22.

43. Ebensberger U, Pohl Y, Filippi A. PCNA expres sion o f

cementoblasts and fibroblasts on the root surface after extraora l

r insing for decontaminat ion. Dent Traumatol.2002; 18: 262 -6.

44. Reth M. Hydrogen peroxide as second messenger in lymphocyte

act ivat ion. Nat Immunol. 2002; 3: 1129-34.

45. Holmes J. Clinical reversal o f root car ies using ozone,

double‐blind, randomised, controlled 18 ‐month t r ial.

Gerodontology. 2003 Dec; 20(2):106-14.

46. Walker JT, Bradshaw DJ, Fulford MR, Marsh PD.

Microbio logical evaluat ion o f a range o f disinfectant products to

control mixed-species bio film contaminat ion in a laboratory

model o f a dental unit water system. Applied and environmenta l

microbio logy. 2003 Jun1; 69(6):3327-32.

47. Nagayoshi M, Kit amura C, Fukuizumi T, Nishihara T,

TerashitaM. Ant imicrobial effect of ozonated water on bacter ia

invading dent inal tubules. J Endod 2004; 30:778 -81

References

69

48. Baysan A, Lynch E. Effect of ozone on the oral microbiota and

clinical sever it y o f pr imary root car ies. Am J Dent 2004;

17: 56-60.

49. Young SB, Set low P. Mechanisms o f kill ing o f Bacillus subt il is

spores by Decon and Ozone, two general decontaminants fo r

bio logical agents. J ApplMicrobio l. 2004; 96: 289 -301.

50. Huth KC, Paschos E, Brand K, Hickel R. Effect o f ozone on

noncavit ated fissure car ious lesions in permanent molars —a

controlled prospect ive clinical study. Am J Dent 2005; 18: 223 -8.

51. Celibert i P, Pazera P, Lussi A. The impact of ozone t reatment on

enamel phys ical propert ies. Am J Dent 2006; 19: 67 -72.

52. Polydorou O, Pelz K, Hahn P. Ant ibacter ial effect of an ozone

device and it s compar ison with two dent in‐bonding systems.

European journal o f oral sciences. 2006 Aug 1; 114(4):349 -53.

53. Baysan A, Lynch E. Clinical reversal o f root caries using ozone:

6-month result s. Am J Dent 2007; 20: 203 -8.

54. Baysan A, Beighton D. Assessment o f the ozone -mediated k illing

of bacter ia in infected dent ine associated with non- cavitated

occlusal car ious lesions. Car ies Res 2007; 41: 337 - 41.

55. Huth KC, Saugel B, Jakob FM, Cappello C, Quir ling M, Paschos

E, Ern K, Hickel R, Brand K. Effect of aqueous ozone on the NF-

κB system. Journal o f dental research. 2007 May 1; 86 (5):451 -6.

References

70

56. Müller P, Guggenheim B, Schmidlin PR. Efficacy o f gasifor m

ozone and photodynamic therapy on a mult ispecies oral bio film in

vit ro. European journal o f oral sciences. 2007 Feb 1;115(1):77 -

80.

57. Petrucci MT, Gallucci C, Agr illo A, Mustazza MC, Foà R. Ro le

of ozone therapy in the t reatment o f osteonecrosis o f the jaws in

mult ip le myeloma pat ients. Haematologica . 2007; 92: 1289-90.

58. Nogales CG, Ferrar i PH, Kantorovich EO, Lage -Marques JL.

Ozone therapy in medic ine and dent ist ry. J Contemp Dent Pract .

2008; 9: 75-84.

59. Noguchi F, Kit amura C, Nagayoshi M, Chen KK, Terashit a M,

Nishihara T. Ozonated water improves lipopo lysacchar ide -

induced responses o f an odontoblast - like ce ll line. Journal o f

endodont ics. 2009 May 31;35(5):668-72.

60. Huth KC, Jakob FM, Saugel B, Cappello C, Paschos E, Hollweck

R, et al . Effect of ozone on oral cells compared with established

ant imicrobia ls. Eur J Oral Sci 2006; 114: 435 -40.

61. Kim HS, Noh SU, Han YW, Kim KM, Kang H, Kim HO, Park

YM. Therapeut ic Effects o f topical applicat ion o f Ozone on

Acute Cutaneous Wound Healing. J Korean Med Sci.

2009;24:368-74.

62. Atabek D, Oztas N. Effect iveness o f ozone with or without the

addit ional use o f re-mineraliz ing so lut ion on non-cavitated

fissure car ious lesions in permanent molars. European journal o f

dent ist ry. 2011 Oct ;5(4):393.

References

71

63. Hauser-Gerspach I, Vadaszan J, Deronjic I, Gass C, Meyer J,

Dard M, Walt imo T, Stübinger S, Mauth C. Influence o f gaseous

ozone in per i- implant it is: bacter icidal efficacy and c ellular

response. An in vit ro study using t itaniu m and zirconia. Clinica l

oral invest igat ions. 2012 Aug ;16(4):1049 -59.

64. Campanat i A, De Blasio S, Giuliano A, Ganzet t i G, Giuliodor i K,

Pecora T, Consales V, Minnet t i I , Offidani A. Topical ozonated

oil versus hyaluronic gel for the t reatment of part ial -to full-

thickness second-degree burns: A prospect ive, comparat ive,

single-blind, non-randomised, controlled clinical t r ial. Burns.

2013 Sep 30; 39 (6):1178-83.

65. Noites R, Pina-Vaz C, Rocha R, Carvalho MF, Gonçalves A,

Pina-vaz I. Synergist ic ant imicrobia l act ion of chlorhexidine and

ozone in endodont ic t reatment . BioMed research internat ional.

2014 May; 28: 592-8.

66. Anand SK, Ebenezar AR, Anand N, Mary AV, Mony B. A

Comparat ive Analys is o f Ant imicrobial Prope rty o f Wine and

Ozone with Calc ium Hydroxide and Chlorhexidine. Journal o f

clinical and diagnost ic research: JCDR. 2015 Jun; 9(6): ZC04.

67. Ahmedi J, Ahmedi E, Sejfija O, Agani Z, Hamit i V. Effic iency o f

gaseous ozone in reducing the development of dry socket

fo llowing surgical third molar extract ion. European Journal o f

Dent ist ry. 2016 Jul; 10(3):381.

68. Schäfer E, Zandbig lar i T. So lubilit y o f root canal sea lers in water

and art ific ial saliva. Int Endod J 2003;36: 660 ‑ 9.

References

72

69. Sunnen GV (1988) Ozone, Nit r ic oxide, and Avian influenza:

Preliminary theoret ical considerat ions and poss ible therapeut ic

direct ions. Ozonics internat ional.

70. Chandrasekhar T, A Ratnaditya, Chait anya Ram Kandregula,

Gunt ipalli M Naidu. Ozone therapy: Applicat ions in Prevent ive

Dent ist ry. J Res Adv Dent 2015; 4(1):103-106.

71. Komali G. Ozone Therapy-A Revo lut ionary Non- invas ive

Therapy in Dent ist ry. Open Access Scient ific Reports.

2012;1(10):473.

72. Estrela C, Estrela CR, Decurcio DA, Hollanda AC, Silva JA.

Ant imicrobia l efficacy o f ozonated water, gaseou s ozone, sodium

hypochlor it e and chlorhexidine in infected human root canals.

Internat ional Endodont ic Journal. 2007 Feb 1; 40 (2):85 -93.

73. Occupat iona l health hazard for ozone. U.S department of Healt h

and Human services.

74. Cech TR. A model for t he RNA-catalyzed replicat ion o f RNA.

Proceedings o f the Nat ional Academy o f Sciences. 1986 Jun 1; 83

(12):4360-3.

75. Ishizaki K, Sawadaishi K, Miura K, Shinr iki N. Effect of ozone

on plasmid DNA of Escher ichia co li in sit u. Water research. 1987

Jul 31;21 (7):823-7.

Annexure

73

INSTITUTIONAL ETHICS COMMITTEE AND REVIEW BOARD

ADHIPARASAKTHI DENTAL COLLEGE AND HOSPITAL

Melmaruvathur, Tamilnadu-603019

MEMBERS

Prof.Dr.A.Momon Singh,MD

Prof.Dr.H.Murali, MDS

Dr.Muthuraj, MSc, MPhil, PhD

Prof.Dr.T.Ramakrishnan, MDS

Prof.Dr.T.Vetriselvan, MPharm,

PhD

Prof.Dr.A.Vasanthakumari, MDS

Prof.Dr.N.Venkatesan, MDS

Prof.Dr.K.Vijayalakshmi, MDS

Shri.Balaji, BA, BL

Shri.E.P.Elumalai

CHAIR PERSON

Prof.Dr.K.Rajkumar, BSc,MDS, PhD

MEMBER SECRETARY

Dr.S.Meenakshi, PhD

This ethical committee has undergone the research protocol

submitted by DR.C.RAVI VARMAN, Post Graduate Student,

Department of Conservative Dentistry and Endodontics under the

title "COMPARATIVE EVALUATION OF THE PHYSICAL

PROPERTIES AND BIOCOMPATIBILITY OF ZINC OXIDE-

OZONATED EUGENOL AND CONVENTIONAL ZINC

OXIDE EUGENOL AS AN ENDODONTIC SEALER – AN

INVITRO STUDY" Reference No: 2014-MD-BrIV-SAT-09 under

the guidance of DR.S.THILLAINAYAGAM MDS., for

consideration of approval to proceed with the study.

This committee has discussed about the material being

involved with the study, the qualification of the investigator, the

present norms and recommendation from the Clinical Research

scientific body and comes to a conclusion that this research protocol

fulfils the specific requirements and the committee authorizes the

proposal.

Date: Member secretary

comparative evaluation of the physical properties...

Documents