Upload
others
View
3
Download
0
Embed Size (px)
Citation preview
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
conventional zinc oxide eugenol.
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
conventional zinc oxide eugenol.
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
Review of l iterature
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.
Review of l iterature
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
Review of l iterature
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
Review of l iterature
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
Review of l iterature
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
Review of l iterature
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.
Review of l iterature
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
Review of l iterature
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
Review of l iterature
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.
Review of l iterature
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
Review of l iterature
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.
Review of l iterature
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
Review of l iterature
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
Review of l iterature
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 -
Review of l iterature
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.
Review of l iterature
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
Review of l iterature
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.
Review of l iterature
24
Huth KC et al (2007)5 5
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.
Review of l iterature
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.
Huth KC et al (2009)6 0
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
Review of l iterature
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.
Review of l iterature
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 ,
Review of l iterature
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.
Review of l iterature
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
Review of l iterature
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
Materials and Methods
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
Materials and Methods
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
Materials and Methods
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.
Materials and Methods
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
Materials and Methods
36
Hardness of the mixed sealers was measured using Shore hardness
Durometer type A (ADIS Gauges and Tools Manufactur ing Company,
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
Materials and Methods
37
Solubility 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.
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)
Materials and Methods
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.
Materials and Methods
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
Materials and Methods
40
Figure 4: Eugeno l ozonated by bubbling gases through it .
Materials and Methods
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).
Materials and Methods
42
Figure 7: Samples shaped using stainless steel mou lds
Figure 8: Mett ler AE 260 Delt a range Analyt ical Balance
Materials and Methods
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
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. 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:
Group I : zinc oxide eugeno l Group II : zinc oxide – ozonated eugeno l
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
from the dist illed water compared with the or iginal mass o f the
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:
Group I : zinc oxide eugeno l Group II : zinc oxide – ozonated eugeno l
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:
Group I : zinc oxide eugeno l Group II : zinc oxide – ozonated eugeno l
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
Summary and Conclusion
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
Summary and Conclusion
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