Effectiveness of different matrix metalloproteinase inhibitors on the microtensile bond strength of self etch adhesive: An In Vitro study

METALLOPROTEINASE INHIBITORS ON THE

MICROTENSILE BOND STRENGTH OF SELF ETCH

ADHESIVE – AN IN VITRO STUDY

Dissertation submitted to

THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY In partial fulfilment for the Degree of

MASTER OF DENTAL SURGERY

BRANCH IV

This is to certify that this dissertation titled “Effectiveness of different matrix metalloproteinase inhibitors on the microtensile bond strength of self etch

adhesive – An In Vitro Study” is a bonafide record of work done by Dr.SHALINI. V under my guidance and to my satisfaction during her postgraduate

study period, 2015 – 2018. This dissertation is submitted to THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY, in partial fulfilment for the award of the degree of Master of Dental Surgery in Conservative Dentistry and Endodontics, Branch IV. It has not been submitted (partially or fully) for the award of any other degree or diploma.

________________________ ________________________ Dr. Minu Koshy, MDS Dr. Subha Anirudhan, MDS

Guide, Professor, Co-Guide, Professor

Department of Conservative Dentistry and Department of Conservative Dentistry and

Endodontics, Endodontics,

Sri Ramakrishna Dental College Sri Ramakrishna Dental College and Hospital, Coimbatore. and Hospital, Coimbatore.

_______________________ Dr. V. Prabhakar, MDS,

Principal, Professor and HOD,

Department of Conservative Dentistry and Endodontics, Sri Ramakrishna Dental College and Hospital,

Coimbatore

Date :

NAME OF THE CANDIDATE Dr. Shalini V

TITLE OF DISSERTATION Effectiveness of Different Matrix

Metalloproteinase Inhibitors on the Bond Strength of Self Etch Adhesive – an in Vitro Study

PLACE OF STUDY Sri Ramakrishna Dental College and Hospital

DURATION OF THE COURSE 2015-2018

NAME OF GUIDE Dr. Minu Koshy

HEAD OF THE DEPARTMENT Dr.V.Prabhakar

I hereby declare that no part of the dissertation will be utilized for gaining financial assistance for research or other promotions without obtaining prior permission from the Principal, Sri Ramakrishna Dental College and Hospital. In addition, I declare that no part of this work will be published either in print or in electronics without permission from the guide who has been actively involved in the dissertation. The author solely has the rights for publishing the work with prior permission from the Principal, Sri Ramakrishna Dental College and Hospital, Coimbatore.

Urkund Analysis Result

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CERTIFICATE II

This is to certify that this dissertation work titled “EFFECTIVENESS OF DIFFERENT MATRIX METALLOPROTEINASE INHIBITORS ON THE BOND STRENGTH OF SELF ETCH ADHESIVE – AN IN VITRO STUDY” of the candidate Dr.Shalini V with registration number 241517302 for the award of Masters of Dental Surgery in the branch of Conservative Dentistry and Endodontics. I personally verified the urkund.com website for the purpose of plagiarism check. I found that the uploaded thesis file contains from introduction to conclusion pages and result shows 1 percentage of plagiarism in the dissertation.

This thesis is the result of work done with immense support from many people and it is with immense pleasure that I express my heartfelt gratitude to all of them.

I devote my heartfelt thanks to Dr. V. Prabhakar,MDS, Principal & Head of Department, whose discipline and skills that run deep under his authoritative yet natural care during my post graduate period which enabled me to successfully conclude my thesis.

I would like to thank and acknowledge Dr.Minu Koshy, MDS,Professor, my Guide who has always been a source of support and encouragement at any moment, in and out of the department. I am grateful to Dr. Subha Anirudhan, MDS, Professor, for her innovative ideas, constructive suggestions, valuable criticism and constant encouragement.

I take this opportunity to express my sincere gratitude to Dr. Ambalavanan, MDS, Reader, Dr. Sriman Narayanan, MDS, Reader, Dr.Gayathri V, MDS, Senior Lecturer and Dr.Remya Verghese, MDS, Senior Lecturer for their valuable guidance that enabled me to comprehend this dissertation and supported me at every juncture throughout my postgraduate curriculum.

lending a helping hand with the sectioning of tooth samples .

I am thankful to Dr. Sadhana.K, for her guidance in the statistical works of this study.

I am thankful to my teachers Dr.Sudhakar M.D.S and Dr.Prabhu M.D.S, my seniors, my colleagues and my juniors, who have been together as friends and of great support throughout my period of study here. I am thankful to all other department staff members, my fellow colleagues in other departments, all UG staff members and non-clinical staffs of my department for their great support and encouragement.

I express my dearest gratitude to my mother, my husband and my son, and the special people in my life who contributed in various ways towards my study and this dissertation.

Last but not the least, I am greatly indebted to God the Almighty, for blessing me with all the good things in my life and guiding me throughout.

DR. SHALINI .V

TITLE

PAGE NO.

1

Introduction

1

2

Aim and Objective

4

3

Review of Literature

5

4

Materials and Methods

18

5

Results

32

6

Discussion

43

7

Summary and Conclusion

51

INTRODUCTION

The success of restorations hangs on the adhesion stability between composite

resin and tooth structure. To maintain adhesion stability, the presence of a hybrid

layer is essential so that it forms a resistant structure against bacterial invasion.

Currently, simplified systems such as self-etching primer/adhesive systems have

demonstrated good clinical performance to dentin. This approach is less

technique-sensitive and reduces the time required for the bonding procedure clinically.

Although advances in adhesive dentistry have brought about improvements in

bonding systems and techniques, bond failures at the tooth-restoration interface still

remain a challenge in the dental field 1.

Proper adhesion depends on the proper infiltration of resin monomer or

partial/ complete degradation of dental substrate to allow formation of hybrid layer.

Hybrid layer has a mixture of organic matrix and hydroxyapatite crystal residue of

dentin with resin monomers and solvent adhesives 2. This layer is subject to

hydrolytic or enzymatic degradation. It was observed that hydrolytic degradation of

exposed collagen fibres occurs without the presence of bacterial colonization.

Research on dentin non collagenous proteins has demonstrated that dentin collagen

fibrils contain inactive proforms of proteolytic enzymes called the Matrix

metalloproteinases. Areas not infiltrated by adhesives, expose collagen fibrils that are

slowly degraded by MMP’s. The recent literature revealed that the matrix

metalloproteinase (MMPs) are abundant in the dentin layer after the etching process

Matrix metalloproteinases get activated in an acidic environment , either due

to acid etching or acidic monomers and resin monomers , that leads to increased

digestion of collagen within the hybrid layer , thus decreasing the bond strength 4.

Different strategies have aimed to improve the bond durability by applying

enzyme inhibitors as a pretreatment before resin infiltration or by admixing enzyme

inhibitors to primers. The common MMP inhibitors used are chlorhexidine,

tetracycline, ammonium compounds, green tea polyphenol epigallo-catechin 3 gallate

and chelating agents like ethylene diamine acetic acid etc.

Grape seed extract , a naturally occurring cross linking agent ,is composed of

proanthocyanidin. It has been used to induce exogenous cross links of collagen and

has the property to inhibit MMP’s 5.

Green tea extract has been described as a natural inhibitor of MMPs. It is

made from Camellia sinensis and is composed of polyphenols named catechins, such

as epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and

epigallocatechin gallate (EGCG).It also has antioxidant property 6.

Fluoride (F) has produced a favourable balance in tooth demineralisation and

remineralization and its role in preventing dental caries has been well documented.

More recently, it was found that 1.23% Sodium fluoride (NaF) gel significantly

reduced the demineralised organic matrix degradation. In addition to its established

effect on the demineralisation and remineralization cycle, Fluoride could also inhibit

However, there are only few studies that have evaluated the efficacy of these

MMP inhibitors to prevent dentin bond strength loss over time 6,8,9. Thus, the aim of

the present study was to evaluate the microtensile bond strength (microTBS) of a

self-etching adhesive (Clearfil SE Bond), with pretreatment of different MMP inhibitors

(Grape seed extract , green tea extract and Clearfil SE Protect ) over 7 days , 30 days

AIM AND OBJECTIVE

To evaluate the effectiveness of different Matrix metalloproteinase inhibitors

(Green tea extract , Grape seed extract and Sodium Fluoride containing adhesive ) on

the microtensile bond strength over 90 days using self etch adhesives – an in vitro

REVIEW OF LITERATURE

Ebrahimi-Chaharom et al (2017)10, evaluated the effect of different

concentrations of specific inhibitor of MMPs (galardin) on the shear bond strength of

self-adhesive resin cements to dentin. The samples were divided into 3 groups (with

no galardin, galardin at a high concentration and galardin at a low concentration),

resin blocks were bonded to dentin surface ,stored in water for 24 hrs and shear bond

strength values were determined.Pretreatment with galardin increased the shear bond

strength of self-adhesive resin cements to dentin and this increase had a direct

relationship with the concentration of galardin in the solution.

Gajjela et al (2017)11 , studied the influence of 2 % chlorhexidine (CHX),

6.5% grape seed extract (GSE) and 1 % riboflavin/ chitosan modification on

microtensile bond strength (µTBS) of composite resin to dentin after polymerase

chain reaction (PCR) thermocycling. Composite build ups were constructed over the

specimens and PCR thermocycling (5000 cycles) was performed and tested under

universal testing machine. It was concluded that pretreatment with CHX and GSE

leads to a significant increase in µTBS of composite resin to dentin.

Gerhardt et al (2016)9 , evaluated the effect of dentin pretreatment with

matrix metalloproteinase inhibitors ( 2% green tea extract,2 % chlorhexidine ,2%

epigallocatechin 3-gallate(EGCG ) on long term microtensile bond strength of self

etch adhesive to dentin. Microtensile bond strength testing were carried out

immediately and after 6 months of storage in water.It was concluded that bond

strength increased in dentin pretreatment with EGCG , green tea groups and in non

Correa B et al (2016)12, evaluated the effect of matrix metalloproteinase

(MMP) inhibitors - 2% (CHX) and sodium fluoride (NaF) (5000 ppm) - on

microtensile bond strength (μTBS) of composite resin to Er:YAG laser-irradiated

dentin after chemical degradation of the bond interface using mechanical testing

machine. Fracture pattern was analyzed using SEM.It was found that 2% CHX and

NaF 5000 ppm presented similar μTBS of composite resin to laser-irradiated dentin

before and after chemical degradation.

Carlvaho et al (2016) 6 , evaluated the effect of pretreatment of two matrix

metalloproteinase inhibitors (2 % green tea extract and 2 % chlorhexidine) on the

bonding durability of etch and rinse adhesive to caries affected dentin over 24 hours

and 6 months storage in water.Dentin beams were prepared and subjected to

microtensile bond strength testing.It was concluded that the application of 2% green

tea extract was able to increase bond durability of the etch-and-rinse system to dentin.

Moreira da Silva et al (2015)13, evaluated the degree of conversion ,water

sorption, solubility and bond strength of experimental etch and rinse adhesive

containing Galardin, Batimastat , GM-1489 and cholrhexidine diacetate. Degree of

conversion was measured using FT-IR spectroscopy, water sorption and solubility

calculated based on ISO4049 and tensile bond strength tested using universal testing

machine over 24 hrs,6 months and 12 months storage. They concluded that

experimental adhesive with GM- 1489 inhibitor and chlorhexidine diacetate had better

physiochemical properties and good bond stability over time.

Kim park et al (2015) 14, tested the ability of green tea catechins ( methanol

extract of green tea) and commercially available epigallocatechin 3-gallate (EGCG) to

gelatine zymography. It was concluded that GTX and EGCG completely inhibited

the MMP-9 acitivity and EGCG significantly inhibited the neutrophil release.

Zheng et al (2014)8 , analyzed the effect of different matrix metalloproteinase

(MMP) inhibitors (2% chlorhexidine digluconate [CHX], 0.05% green tea extract, 1

mM ferrous sulfate, or 0.2 mM galardin) on the microtensile bond strength

(microTBS) of an etch-and-rinse (Optibond FL) and a selfetching adhesive (Clearfill

SE bond) immediately and after 9 months of aging. It was concluded that MMP

inhibitors prevented the decrease in microTBS upon aging of the etch-and-rinse but

not of the self-etching adhesive.

Rupam kaur et al (2014)15, determined the effect of pretreatment with grape

seed extract on the bond strength of a self-etch adhesive (All Bond SE ) with dentin.

Occulsal cavities were prepared and restored with hybrid composite.Dentin beams

were prepared and microtensile bond strength were tested using universal testing

machine. It was concluded that pretreatment with grape seed extract leads to

significant increase in the bond strength of composite with dentin.

Srinivasalu et al(2013)5,evaluated the shear bond strength of composite resin

to deep dentin, bonded using a self-etch adhesive, after treatment with two collagen

cross-linkers (10% sodium ascorbate and 6.5% proanthocyanidin) at varying time

intervals. Human incisors were sectioned longitudinally , pretreatment were done for

5 and 10 minutes and shear bond strength were tested using universal testing machine.

It was concluded that dentin surface pre-treatment with both 10% sodium ascorbate

and 6.5% proanthocyanidin resulted in significant improvement in bond strength of

Yi Liu et al (2013)16, evaluated the effects of Proanthocyanidin PAand the

photoinitiator type on the degree of conversion and polymerisation rate of a model

dental adhesive.Three types of photoinitiators were introduced into the Bis-

GMA/HEMA comonomer mixture and for each resin mixture , adhesive containing

0%, 2.5%, 5% and 10% PA with respect to weight of resin were produced. When

light cured , the rate of polymerisation and degree of conversion were analysed using

ATR- FTIR Spectroscopy. It was observed that PA hampered the polymerisation of

all adhesives regardless of the photoinitiators used and also found that photoinitiation

formulations could maintain a satisfactory degree of conversion while a significant

amount of PA is incorporated.

Xijin Du et al (2012)17, studied the antibacterial potential and physiochemical

properties of a dental adhesive incorporated with epigallocatechin-3-gallate(EGCG) in

different concentrations over time. Growth of streptococcus mutans were determined

by direct contact test. Microtensile bond strength and degree of conversion were

evaluated immediately and after 6 months. It was concluded that 200μg/ml EGCG

incorporated adhesive showed better therapeutic value while sustaining the bond

durability of resin- dentin

Epasinghae et al (2012) 18 , studied the effect of varying concentration of

proanthocyanidin incorporation into the dental adhesive resin on resin dentin bond

strength. Experimental hydrophilic adhesives were prepared by combining 50%

weight comonomer with 50% weight ethanol along with 1%, 2 % ,3%

proanthocyanidin concentration respectively with control group free of it. Bonding

procedures were carried out and subjected to tensile forces. They concluded that

effect on bond strength, whereas 3% proanthocyanidin significantly lowered bond

strength.

Fang et al (2012)19, investigated whether transient pretreatment by PA based

preconditioner can improve the resin dentin bonds of various etch and rinse adhesives.

PA based preconditoners were prepared by adding powdered grape seed extract , to

various solvents at different concentrations. Bond strength , failure mode and degree

of conversion were determined via microtensile testing, field emission scanning

electron microscopyand Fourier transform infrared spectroscopy.PA preconditioning

improved the bond strength of tested water / ethanol based adhesive better than the

water/ acetone based system.So it was observed that PA preconditioning improved

resin dentin bonds without compromise on the curing behaviour on the tested

adhesives.

Kato et al (2012)20 , evaluated the impact of protease inhibitors on dentin

matrix degradation by colleganase. Specimens were treated for 1 min with gels

containing epigallocatechin 3-gallate (EGCG), chlorhexidine (CHX, 0.012%), FeSO4

(1 mM), NaF (1.23%), or no active compound (P, placebo) and matrix loss was

analysed using profilometry.It was concluded that treatment with gels containing

EGCG, CHX, or FeSO4 led to significantly lower dentin matrix loss when compared

with the other treatments.

Dourado Loguercia et al (2011)21 , evaluated the effect of the method of

application on the immediate and 3 year resin dentin bond strength for 3 one step self

etch adhesives ( Aper Prompt L- Pop, Clearfil S3 bond and Xeno III. Adhesives were

applied under Inactive application (adhesive layer after application on tooth were left

Dentin sticks were tested for bond strength immediately and after 3 years of water

storage. Nanoleakage was detected using silver nitrate deposition. They observed that

active application improved the immediate bonding strength and showed decreased

degradation rate in active application.

Liu et al(2011)22, analysed the effect of transient proanthocyanidins

preconditioning on the cross-linking and mechanical properties of demineralized

dentin by adding powdered proanthocyanidins-rich grape seed extract to various

solvents at different concentrations. Demineralized dentin specimens were

preconditioned for 20,30, 60 or120 s,followed by the evaluation of their crosslinking

degree, mechanical properties and micromorphology using Ninhydrin assay,

Universal testing machine and field emission scanning electron microscope

(FESEM). It was found that the cross-linking degree of the demineralized dentin

collagen exhibited concentration- and time- dependent increase after preconditioning

treatment, irrespective of the preconditioner and the solvent. 15% proanthocyanidins

preconditioning resulted in thehighestmean ultimate tensile strength and FESEM

confirmed that the demineralized dentin collagen was in a homogeneous and regular

arrangement after preconditioning and maintained expanding.

Castellan et al (2011)23 ,studied the long term stability of dentin matrix

following preptreatment using natural collagen cross linking agent rich in

Proanthocyanidin(PA) from grape seed , cocoa seeds , cranberry, cinnamon and acai

berry. Apparent elastic modulus were analysed over 3 , 6 and 12 months using three

point bend flexural testing. Grape seed extract and cocoa seed extract were able to

De Munck et al(2011)24, evaluated the bonding effectiveness using five

different adhesives to different dentin locations after 1 week, 3, 6 and 12 months of

water storage. The beams were divided into ‘center’ and ‘periphery’ dentin

specimens and were subjected to microtensile bond strength testing. It was determined

that only for the mild self-etch adhesives, µTBS to ‘periphery’ dentin was higher than

for the ‘center’ specimens , whereas for etch and rinse adhesives it was insensitive to

regional variability.

Mutluay et al (2011)25, compared the MMP-inhibitory activity of a series of

alcohols (i.e. methanol, ethanol, propanols, butanols, pentanols, hexanols, the ethanol

ester of methacrylic acid, heptanols and octanol). It was measured against soluble

rhMMP-9 and insoluble matrix-bound endogenous MMPs of dentin in completely

demineralized dentin. The results indicated that alcohols with 4 methylene groups

inhibit MMPs more effectively than methanol or ethanol.

Green et al (2010)26, compared the morphological differences of hybrid layers

created by Bis GMA/HEMA model adhesives with and without the addition of grape

seed extract PA under the conditions of enzymatic collagen degradation. Following

collagenase treatment with 0.1% collagenase solution for 0,1,and 6 days , the

specimens were analysed with SEM/TEM. It was concluded that the presence of

grape seed extract PA in dental adhesives may inhibit the biodegradation of

unprotected collagen fibrils within the hybrid layer.

De Munck et al (2010)27 , evaluated the endogenous enzymatic degradation

for several simplified self-etch adhesives in which primers were modified by adding

two MMP inhibitors: chlorhexidine, a non specific MMP inhibitor and SB-3CT, a

etch-and-rinse adhesives, the involvement of endogenous MMP-2 and MMP-9 in the

bond-degradation process is minimal for mild self-etch adhesives.

Castellan et al (2010)28 , studied the properties of dentin matrix treated with 2

proanthocyanidin rich cross-linking agents (Grape seed extract and Cocoa seed

extract) and their effect on dentin bonded interface. Results showed that both cross

linkers increased elastic modulus of dimeneralised dentin as exposure time increased

and resin dentin bonds also significantly increased following treatment with grape

seed extract regardless of the application time or adhesive used.

Breschi et al (2010)29 , determined the use of a specific MMP inhibitor

(Galardin) for preservation of hybrid layer and its mechanical properties after aging.

Microtensile bond strength and interfacial nanoleakage were analysed. MMP

inhibition were analysed zymographically. Galardin showed inhibition of MMP-2 and

MMP-9. It was also observed that the use of galardin had no effect on immediate

bond strength , while it significantly decreased bond strength after 1 year.

Tezvergil-Mutluay et al (2010)60 , evaluated the effect of different storage

media on changes in matrix stiffness, loss of dry weight or solubilization of collagen

from demineralized dentin beams incubated in vitro for up to 60 days.Dentin beams

were prepared and stored in either media containing both zinc and calcium designated

as complete medium (CM), calcium-free medium, zinc-free medium, a doubled-zinc

medium or water. Quantitation of hydroxyproline (HOP) was taken as an index of

solubilization of collagen by MMPs and measured. It was found that both the storage

medium and the storage time showed significant effects on E, mass loss and HOP

release. The incubation in CM resulted in relatively rapid and significant decrease in

Al Ammar et al (2009)30, investigated the effect of three different

cross-linking agents (5% Glutaraldehyde [GD], 6.5% Grape seed extract [GSE], and 0.5%

Genipin [GE]) on the tensile bond strength (TBS) of resin-dentin bonds. Dentin

surfaces were etched using phosphoric acid and then teeth were pretreated with

collagen cross linkers before adhesive application and restored. After 24 hours,

specimens were tested using universal testing machine. This study concluded that

Chemical modification to the dentin matrix promoted by GD and GSE, but not GE,

resulted in increased bond strength.

Qian xie et al (2008)31, evaluated the effect of 6.5% grape seed extract and

1000 ppm sodium fluoride on the remineralisation of artificially induced root caries

lesions. Fragments were pH cycled through treatment solutions , acidic buffer and

neautral buffer for 8 days at 6 cycles per day. Microhardness and mineral

precipitation were tested using microhardness tester, polarized light microscopy and

confocal laser scanning microscopy. It was concluded that grape seed extract

positively affects the remineralisation/ demineralisation processes of artificial root

caries.

Walter et al (2008) 32, evaluated the effects of dentin collagen modifications

induced by various cross-linkers (5% glutaraldehyde (GA), 0.5% proanthocyanidin

(PA), 0.625% genipin (GE))on the stability of collagen matrix and the inhibition of

root caries. Cross-linker-treated demineralized human root dentin were digested with

bacterial collagenase, centrifuged, and the supernatants were subjected to amino acid

analysis to determine collagen content. The residues were analyzed by SDS-PAGE

and hydroxyproline analysis. Bovine root surfaces were conditioned with phosphoric

acid, treated with the cross-linkers, incubated with Streptococcus mutans and

with confocal microscopy. It was observed that naturally occurring cross-linkers,

especially PA, could be used to modify root dentin collagen to efficiently stabilize

collagen and to increase its resistance against caries.

Carilho et al (2007) 33, analysed the dentin bond preservation using 2%

chlorhexidine pretreatment . Microtensile bond strengths and failure mode distribution

under SEM were analyzed immediately after specimen preparation and 6 months

later.It was observed that significantly better preservation of bond strength was

observed after 6 months and suggested that chlorhexidine might be useful for the

preservation of dentin bond strength.

Bedran Russo et al (2007)34, evaluated the effect of two collagen crosslinking

agents (glutaraldehyde, GD and grape seed extract, GSE) on the modulus of elasticity

of demineralized dentin. Demineralised dentin specimens were immersed in 2.5%

GD; 5% GD, 25% GD; 0.65% GSE; 6.5% GSE and tested at baseline, 10 min, 30

min, 1 h, 2 h, 4 h for modulus of elasticity by three point bend method. It was

concluded that the use of these collagen crosslinkers to increase the stiffness of

demineralized dentin, was both concentration and time dependent.

Shinohara et al (2006) 1, evaluated the influence of a fluoride-containing

adhesive (Clearfill SE Protect bond) on microtensile bond strength to dentin, as well

as analyzed the dentin-adhesive interface after acid-base challenge. Microtensile bond

strength was tested using Universal testing machine.For interfacial analysis, adhesive

system was applied on dentin surface and a low-viscosity resin was placed between

two dentin disks. Then, the specimens were subjected to acid-base challenge,

sectioned, and polished to be observed by SEM. It was observed that a thick acid-base

fluoride-releasing adhesive was used and the presence of fluoride in an adhesive contributed

significantly to prevent secondary caries and did not interfere with dentin-adhesive

bond strength.

Bedran Russo et al (2006)35, evaluated the effect of three collagen

crosslinking agents[5% glutaraldehyde (GD), 0.5% proanthocyanidin PBS solution

(PA), and 0.625% genipin PBS solution (GE)] on the ultimate tensile strength (UTS)

of undemineralized and demineralized dentin. Specimens were prepared and kept in

their respective solutions for either 4 or 40 hour. It was concluded that the application

of two naturally occurring crosslinkers (Proanthocyanidin and Geipin), to dentin

collagen significantly improved the ultimate tensile bond strength.

Nishitani et al (2006)36, compared the ability of all in one adhesives (Clearfil

Tri-S Bond, G-Bond, Adper Prompt L-Pop) or a self etching primer (Clearfil SE Bond

primer ) to activate gelatinolytic and collagenolytic activities in powdered mineralized

dentin made from human tooth. Powered dentin was mixed with adhesive or primer

for varying times and then the reaction was stopped by extracting the adhesives using

acetone. Gelatinolytic activity were recorded using relative fluorescent units (RFU)

per mg dry weight. It was concluded that self-etch adhesives may activate latent

MMP and increase the activity to near-maximum levels and contribute to the

degradation of resin–dentin bonds over time.

Rochaa et al (2006)37, evaluated the effects of dentin surface treatments on

the tensile bond strength (TBS) of the self-etching primer Clearfil SE Bond (CSE) and

the one-step self-etching One-Up Bond F (OUB). The exposed flat dentin surfaces

were prepared with diamond bur at high-speed, carbide bur at low-speed or wet

water for 24 hours, specimens were sectioned and tensile bond strength were tested. It

was observed that the groups treated with CSE exhibited the highest bond strength

for all surface treatments. Dentin surfaces prepared with carbide bur at low speed

reduced tensile bond strength in the CSE group; however, OUB was not affected by

surface treatments.

Mazzoni et al (2006)38, studied the proteolytic activities of mineralised dentin,

acid etched dentin and etch and rinse adhesive treated acid etched dentine. Powdered

dentin prepared from human teeth were treated with 17% EDTA, 10% phosphoric

acid or with five simplified etch and rinse adhesives that were applied to 10%

phosphoric acid etched dentine. Gelatinolytic activity were assayed using fluorescein

labelled gelatine. It was concluded that simplified etch and rinse adhesives can

reactivate endogenous enzymatic activites in dentin that are previously inactivated by

phosphoric acid etching.

Imazato et al(2003)39, investigated the antibacterial activity of an adhesive

resin incorporating the antibacterial monomer 12-methacryloyloxydodecylpyridinium

bromide (MDPB) as well as its bonding characteristics in terms of bond strength into

dentin and curing ability. 2.5% MDPB was incorporated into the bonding agent and

its inhibitory effect against the growth of Streptococcus mutans on its surface was

determined. Bond strength to human dentin and degree of conversion of the

experimental adhesive were evaluated by conventional tensile bond strength test and

Fourier transformation infrared spectroscopy.The results indicated that an adhesive

resin with antibacterial activity after curing could be produced by incorporation of

Demule M et al (2000)49, investigated the effects of different biologically

active components from natural products, including green tea polyphenols (GTP),

resveratrol, genistein and organosulfur compounds from garlic, on matrix

metalloproteinase (MMP)-2, MMP-9 and MMP-12 activities. The inhibition of the

enzymes were measured by Fluorescence assays using gelatin or elastin substrates.

The inhibition were further confirmed using gelatin zymography. The results

indicated that catechins from green tea inhibit MMP activities and proMMP-2

MATERIALS

1. CLEARFIL SE BOND (KURARAY AMERICA, INC)

2. GRAPE SEED EXTRACT ( ZENITH PHARMACEUTICALS)

3. GREEN TEA EXTRACT ( ZENITH PHARMACEUTICALS)

4. CLEARFIL SE PROTECT BOND (KURARAY AMERICA, INC)

5. MICROHYBRID COMPOSITE (VOCO POLOFILL)

6. EXTRACTED HUMAN MOALR TEETH

7. DISTILLED WATER

ARMAMENTARIUM

1. DIAMOND DISC

2. DIAMOND ABRASIVE BUR

3. TWEEZER

4. APPLICATOR TIPS

5. ANODIZED ALUMINIUM COMPOSITE INSTRUMENT

6. POLYWAVE LED CURING LIGHT (IVOCLAR VIVADENT, USA)

7. HARD TISSUE MICROTOME ( LEICA SP 1600)

8. UNIVERSAL TESTING MACHINE (INSTRON)

MATERIALS

Fig 1 - CLEARFIL SE BOND Fig 2- GREEN TEA EXTRACT

ARMANENTARIUM

Fig 5 - INSTRUMENTS USED Fig 6 - LIGHT CURE UNIT

Fig 7 - HARD TISSUE MICROTOME

Fig 8 - UNIVERSAL TESTING MACHINE

METHODOLOGY

1. Preparation of natural human tooth samples

- Selection and preservation of tooth samples

- Grouping of samples

2. Preparation of dentin surface

3. Application of MMP inhibitor / Bonding agent

4. Composite build up

5. Preparation for microtensile bond strength evaluation

6. Statistical evaluation

PREPARATION OF NATURAL HUMAN TOOTH SAMPLES:

SELECTION AND PRESERVATION OF TOOTH SAMPLES

Sound molar teeth (figure 10) were obtained and used for current study. Teeth

with caries , attrition and fracture were excluded. The selected teeth were then

disinfected and stored in physiologic saline for no longer than 4 weeks after

extraction.

GROUPING

The selected teeth were randomly divided into 4 groups. (10 in each group)

according to the material used.

GROUP 1 – Clearfil SE bond

GROUP 2 – Green tea extract

GROUP 3 – Grape seed extract

PREPARATION OF DENTIN SURFACE:

The occlusal enamel of the teeth was removed. A flat mid coronal dentin

surface (figure 12) were prepared by means of a water cooled slow speed diamond

saw(Figure 11). It was then made as an even surface using diamond abrasive bur.

Dentin surface was then checked for the absence of pulp chamber exposure.

APPLICATION

GROUP 1 :

Clearfil SE bond, a 2 step self etch adhesive was used (figure1). Equal drops

of primer and adhesive were added equally and mixed together. It was applied over

the dentin surface using microbrush and light cured for 20 seconds as per

manufacturer’s instructions.(figure- 14,15)

GROUP 2 :

0.5% Green tea extract were prepared by mixing 50 g of green tea extract

powder (ZENITH PHARMACEUTICALS) in 100 ml of distilled water (Figure -2).

The solution was rubbed over the dentin surface for 60 seconds using microbrush and

allowed to dry for one minute .Over the pretreated surface ,a layer of clearfil SE

bond were applied and cured for 20 seconds.(figure – 13,14,15)

GROUP 3 :

6.5% grape seed extract was prepared by mixing 650 mg of grape seed extract

powder (ZENITH PHARMACEUTICALS) in 100 ml of distilled water(Figure 3).

allowed to dry for one minute. Over the pretreated surface, a layer of clearfill SE bond

was applied and cured for 20 seconds.(figure -13,14,15)

GROUP 4 :

Clearfil SE Protect bond , a 2 step self ecth adhesive was used (Figure 4).

Primer and adhesive of equal proportion were mixed and was applied over the dentin

surface using a microbrush. It was then cured for 20 seconds.(Figure – 14,15)

COMPOSITE BUILD UP :

Over the bonded dentin surface, 3 increments (2 mm each) of composite

(Voco Polofil) build up were done using a aluminium coated instrument (Figure 16).

Each increment was cured for 20 seconds. Final cure of the build up was done for 40

seconds according to manufacturer’s instructions.

PREPARATION FOR MICROTENSILE BOND STRENGTH EVALUATION:

Specimen preparation :

The roots of the teeth were sectioned off and the coronal portions were

mounted on acrylic resin blocks (Figure 17). These acrylic blocks were mounted on a

hard tissue microtome (Leica SP 1600 Saw Microtome) and sectioned using water jet

as coolant (Figure 18). The teeth were sectioned longitudinally across the bonded

interface to obtain dentin sticks with a cross sectional area of approximately 1.0 ±

0.1mm2. Three or four resin dentin sticks were acquired from each tooth sample

(Figure 19). 10 sticks per group were stored in separate containers with water as the

Each bonded stick was then attached to a custom made jig with cyanoacrylate

resin (Figure 20 ) for microtensile bond strength testing and subjected to a tensile

force in a Universal Testing machine (Instron,Norwood,USA) at a cross head speed of

0.5mm/min (Figure 21). The load at which the failure occurred was recorded by

specialised software attached to the universal testing machine. The test was done on

the 7th day ,30th day and 90th day.

Thus values were obtained and tabulated. Then were subjected to statistical

evaluation using SPSS software version 21.

STATISTICAL EVALUATION:

The SPSS software version 21 was used for statistical analysis of the

microtensile bond strength values obtained for each sample in the study groups.

The values tabulated were tested for significance using one-way ANOVA and

FIGURE 10 – Sound molar teeth

FIGURE 12- Flat mid coronal dentin surface prepared

Figure 13- Pretreatnent for Group Figure 14- Application of Bonding

2& Group 3 For 60 seconds agents in all four groups

Figure 15 – Curing done for 40 seconds

Figure 17- coronal portion embedded in acrylic resin block

Figure 19 – Dentin Resin Beams

Figure 20 – Dentin beams on custom made jigs

RESULTS

The microtensile bond strength values of 10 samples for each group were

obtained and calculated in MegaPascals(MPa). Values were then subjected to

statistical analysis. The mean and standard deviation values obtained were subjected

to One way ANOVA test and Post Hoc test.

7 th Day

Table – 1 Bond strength values in Mega Pascals

Sample Group 1 Clearfil SE Bond Group 2 Green tea extract Group 3 Grape seed extract Group 4 Clearfil SE Protect

1 6.1 6.2 7 6.7

2 5.3 5.9 6.9 5.9

3 5.7 7.5 9.1 7.1

4 6.2 6.7 7.8 7.4

5 5.5 8.4 7.5 6.3

6 7.1 8.1 6.2 5.2

7 6.5 7.6 8 6.4

8 6 5.3 6.5 6.5

9 5.5 5.7 5.9 7.1

Table – 2 Mean and standard deviation values

N Minimum Maximum Mean

Std.

Deviation

Group 1 10 5.300 7.100 6.050 .577

Group 2 10 5.300 9.100 7.050 1.271

Group 3 10 5.900 9.200 7.410 1.133

Group 4 10 5.200 7.400 6.540 .644

Table – 3 Oneway ANOVA test

Sum of Squares

Df Mean

Square

F Sig.

Between Groups

10.591 3 3.530 3.860 .017

Within Groups 32.923 36 .915

Total 43.514 39

Graph - 1 Bardiagram representing mean values of 4 groups on 7th day

M

P

Post Hoc Tests

Multiple Comparisons

Table – 4 Tukey HSD

(I) GROUP (J) GROUP Mean Difference (I-J) Std. Error Sig.

95% Confidence Interval

Lower

Bound

Upper

Bound

1

2 -1.000 .427 .108 -2.151 .151

3 -1.360* .427 .015 -2.511 -.208 4 -.490 .427 .664 -1.641 .661

2

1 1.000 .427 .108 -.151 2.151

3 -.360 .427 .834 -1.511 .791

4 .510 .427 .635 -.641 1.661

3

1 1.360* .427 .015 .2081 2.511 2 .360 .427 .834 -.791 1.511

4 .870 .427 .195 -.281 2.021

4

1 .490 .427 .664 -.661 1.641

2 -.510 .427 .635 -1.661 .641

3 -.870 .427 .195 -2.021 .281

The mean difference is significant at the 0.05 level.

When comparing the Group 4 (Clearfil SE Protect) with other groups, there

was no statistical difference between the mean microtensile bond strength values.

When comparing the Group 3 (Grape seed extract) with Group 1 (Clearfil SE ), there

30th day

Table – 5 - Bond strength values in Mega Pascals

Sample Group 1 Group 2 Group 3 Group 4

1 8.2 12.1 15.1 11.2

2 7.9 10.1 13.4 9.3

3 10.5 15.3 18.5 14.4

4 9.1 12.1 11.1 16.3

5 7.9 16.2 16.2 12.1

6 11.3 13.4 13.5 11.2

7 10 9.2 17.9 13.1

8 8.5 8.9 13.2 17.1

9 7.9 12 14.1 15.2

10 9.3 14.3 18.0 11.9

Table – 6 Mean and standard deviation values

N Minimum Maximum Mean

Std.

Deviation

Group 1 10 7.900 11.300 9.060 1.207

Group 2 10 8.900 16.200 12.360 2.484

Group 3 10 11.100 18.900 15.190 2.604

Table – 7 One way ANOVA test

Sum of Squares

Df Mean

Square

F Sig.

Between

Groups

195.407 3 65.136 12.601 .000

Within Groups 186.093 36 5.169

Total 381.500 39

Graph – 2 Bar diagram representing mean values of 4 groups on 30th day

M

P

Multiple Comparisons

Table – 8 Tukey HSD

(I) Group (J) Group Mean Difference (I-J) Std. Error Sig.

95% Confidence Interval

Lower Bound

Upper Bound

1

2 -3.300* 1.016 .013 -6.038 -.561 3 -6.130* 1.016 .000 -8.868 -3.391 4 -4.1200* 1.016 .001 -6.858 -1.381

2

1 3.300* 1.016 .013 .561 6.038 3 -2.830 1.016 .041 -5.568 -.091 4 -.820 1.016 .851 -3.558 1.918

3

1 6.130* 1.016 .000 3.391 8.868 2 2.830* 1.016 .041 .091 5.568 4 2.0100 1.016 .216 -.728 4.748

4

1 4.120* 1.016 .001 1.381 6.858 2 .820 1.016 .851 -1.918 3.558

3 -2.010 1.016 .216 -4.748 .728

* The mean difference is significant at the 0.05 level.

On 30th day, Mean microtensile bond strength values were higher in Grape seed

extract (15.19) when comparing with Clearfil SE (9.06), Green Tea extract (12.36)

and Clearfill SE Protect (13.18)

When comparing Clearfil SE group with other three groups , there was a significant

difference.

When comparing Clearfil SE Protect group with Green tea extract and Grape seed

90 th day

Table – 9 Bond strength values in Mega Pascals

Sample Group 1 Group 2 Group 3 Group 4

1 7.1 11.2 14.2 11.3

2 6.9 10.2 12.8 9

3 9.1 14.5 16.3 13.4

4 8.5 11.8 11 13.3

5 7.5 15.1 15.8 11.8

6 10.5 13.1 13.2 10.6

7 9 8.9 16.4 12.5

8 7.9 8.7 10.1 16.4

9 7.6 11.6 11.1 15.3

10 8.6 12.5 16.4 11.2

Table – 10 Bond strength values in Mega Pascals

N Minimum

Maximu

m

Mean

Std.

Deviation

Group 1 10 6.900 10.500 8.270 1.094

Group 2 10 8.700 15.100 11.760 2.142

Group 3 10 10.100 16.400 13.730 2.450

Table – 11 One way ANOVA Test

Sum of Squares

Df Mean

Square

F Sig.

Between Groups 164.194 3 54.731 13.123 .000

Within Groups 150.142 36 4.171

Total 314.336 39

Graph – 3 Bar diagram representing mean values of 4 groups on 90th day

M

P

Multiple comparison

Post Hoc analysis

Table- 12 Tukey HSD

(I) group (J) group Mean Difference (I-J) Std.

Error Sig.

95% Confidence Interval

Lower

Bound

Upper

Bound

1

2 -3.490* .913 .003 -5.949 -1.030

3 -5.460* .913 .000 -7.919 -3.000

4 -4.210* .913 .000 -6.669 -1.750

2

1 3.490* .913 .003 1.030 5.949

3 -1.970 .913 .155 -4.429 .489

4 -.720 .913 .859 -3.179 1.739

3

1 5.460* .913 .000 3.000 7.919

2 1.970 .913 .155 -.489 4.429

4 1.250 .913 .527 -1.209 3.709

4

1 4.210* .913 .000 1.750 6.669

2 .720 .913 .859 -1.739 3.179

3 -1.250 .913 .527 -3.709 1.209

On 90th day , the mean microtensile bond strength values were higher in Grape

seed extract group(13.73) comparing to Clearfil SE group(8.27), Green tea extract

group(11.76) and Clearfil SE Protect group(12.48)

When comparing the Clearfil SE group with Green tea extract, Grape seed

extract and Clearfil SE Protect group , there was a significant difference. When

comparing the Clearfil SE Protect group with Green tea extract and Grape seed

Graph – 4 Bond strength values in 3 different time periods.

0 2 4 6 8 10 12 14 16

group 1 group 2 group 3 group 4

7 th day

30 th day

90 th day

From the above line diagram comparing the 7th day, 30th day and 90th day

mean microtensile bond strength values of all four groups, there was a significant

increase in bond strength after 30 and 90 days comparing to 7 days. Among the

groups, Grape seed extract had shown increase in bond strength followed by Clearfil

DISCUSSION

Adhesive restorations are routinely used to replace the lost dental tissue.

Current adhesive systems bond to dentin through a micromechanical mechanism

based on the formation of a hybrid layer. The hybrid layer, a collagen-resin interface,

is the most vulnerable portion of the bonded interfaces where stress tends to

concentrate and most failures take place40. While bonding to enamel substrate has

been shown to be reliable over-time, bonding to dentin substrate is a great challenge41.

Dentin represents the bulk of the tooth and a reliable long-term bond is essential for

the success of adhesive restorations. It has been speculated that a decreasing

concentration gradient of resin monomer diffusion within the acid-etched dentin, and

a subsequent resin elution from hydrolytically unstable polymeric hydrogels within

the hybrid layers leave the collagen fibrils unprotected and vulnerable to degradation

by endogenous metalloproteinases (MMPs) 20.

MMPs are a group of 23 mammalian enzymes capable of degrading all

extracellular matrix components. Human dentin contains collagenase (MMP-8),

gelatinases MMP-2 and -9, and enamelysin MMP-2042. MMPs were shown to be

expressed during tooth development and to be necessary for normal dentin formation .

After dentin mineralization, they remain trapped in the calcified matrix either under

active or proenzyme forms, which may explain their persistent presence within the

dentin of adult teeth 43. It was found that their exposure and activation during the

caries process would allow these enzymes to promote matrix degradation and caries

progression. Dentin collagenolytic and gelatinolytic activities can be suppressed by

protease inhibitors indicating that MMP inhibition could be beneficial in the

Two main methods to increase the dentin/resin interface properties have to be

considered: the continuing improvement/development of new adhesive systems and

the establishment of tissue engineering/biomimetics approaches to improve the

intrinsic properties of the substrate. Intrinsic collagen cross-links provide the tensile

properties of collagen molecules. The use of extrinsic collagen cross-linking agents

can induce additional formation of inter and intramolecular cross-links44. Selective

cross-linking agents have been demonstrated to increase the ultimate tensile strength

and elastic modulus of demineralized dentin45. This can be achieved by the use of

various collagen cross-linkers, both synthetic and natural, on the dentin substrate prior

to the bonding procedure. Naturally occurring collagen cross-linkers such as sodium

ascorbate, epigallectonin and proanthocyanidin have been reported to increase the

collagen cross-linking in sound and caries-affected dentin46. Several synthetic

collagen cross-linkers (Glutaraldehyde, Carbodiimide, formaldehyde and epoxy

resins) have been investigated to improve on the mechanical properties of dentin and

the resin-dentin interface. The major drawback with these are they are toxic to the

tissues.

The two main strategies that are currently in use for adhesive bonding to

enamel and dentin are the total-etch technique and the self-etch technique. Although

etch-and-rinse technique is still considered as the most effective approach to achieve

efficient and stable bonding to enamel and dentin, the multiple application steps, the

critical rinsing step and the frustratingly high incidence of post-operative sensitivity

led to the evolution of more simplified, less technique-sensitive and user-friendly

self-etch adhesives47 .

The importance of fluoride (F) in preventing dental caries by favorably

its ability to inhibit matrix metalloproteinases (MMPs), has been investigated

recently and found to be effective 7. Thus a fluoride containing self etch

adhesive was studied on the bond strength against different naturally occuring MMP

inhibitors for a period of 7 days, 30 days and 90 days, if effective this could minimize

the clinical step of pretreating with mmp inhibitors as they are incorporated in the

adhesive.

Proanthocyanidins (PA) are naturally occurring bioflavonoids found in high

concentrations in grape seed, pine bark, cranberries, lemon tree bark and hazel nut

tree leaves. PA from grape seed extract has been shown to effectively cross-link

collagen in few vitro studies28,46. Their effect on the bond strength of resin composite

bonded using a self-etch adhesive to deep dentin has proved to be effective. It

interacts with proteins to induce cross-links by four different mechanisms: covalent

interaction, ionic interaction, hydrogen bonding interaction, or hydrophobic

interactions 48.

Green tea, has been described as a natural inhibitor of MMPs. It is made from

Camellia sinensis and is composed of polyphenols named catechins, such as

epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and

epigallocatechin gallate (EGCG)49. EGCG ,a major polyphenol of green tea has

known to contain potential health ingredients including antioxidant , antimicrobial,

antidiabetic, anti inflammatory and cancer preventing properties. Significant

antimicrobial activity in vitro has been demonstrated against a variety of gram

positive , gram negative and fungal pathogens. It was also found to have distinct

A two-step self-etching primer/adhesive system, Clearfil Protect Bond－

composed of an antibacterial primer containing MDPB

12-methacryloyloxydodecylpyridinium bromide） and a fluoride-releasing adhesive

－ has shown the potential in artificial secondary caries inhibition around

restorations51. NaF, the most common F compound in oral hygiene products,has

the ability to completely inhibit the activity of MMPs in clinically relevant

concentrations The possible mechanism by which NaF inhibits the MMPs is not

known clearly 7.

Bond strength is referred to the force per unit area that is required to debond

the adhesive or adherent interface. To measure the ultimate tensile strength and

modulus of elasticity of dentin . Schreiner et al (1994) 52 introduced microtensile

bond strength testing to dentistry. According to Pashley et al (1999) 53, the

microtensile bond strength test presented several advantages in comparison to the

macro and shear testing as it permitted a greater number of adhesive failures and

the measurement of regional bond strengths. Of all the in- vitro tests, microtensile

bond strength testing is deemed better despite its technical limitations, as the

bonded interface of smaller cross sectional area of specimens has a better stress

distribution during loading 54. Considering these factors microtensile bond

strength testing methodology was selected for the current study. The dimension of

the specimen also plays an important role in the determination of the bond

strength. In this study , stick shaped dentin beam specimens were produced and it

has a more favourable stress distribution as investigated by Phrukkanom et al

(1998)58 regarding specimen geometry. The specimen’s dimensions were

In this study , microtensile bond strength values on 7 th day showed a

significant difference between grape seed extract- 0.015 Mpa (group 3 ) and

Clearfil SE bond (group 1 ) . There was no statistical difference between the

group 4 (Clearfil SE Protect) and other groups. On 30th day the mean bond

strength were greater in grape seed extract followed by Clearfill SE Protect and

Green Tea extract. Group 4 (clearfill SE Protect) showed a statistical similar

difference in comparison with Grape seed extract and Green tea extract group. On

the 90th day testing, Group 3 (Grape seed extract) mean bond strength values were

still higher. But the test Group 4 had no statistical difference with group 2 and

group 3 . On 90th day aging the control group seemed to have lower bond strength

values.

A study done by Kaur et al (2014)15 showed that microtensile bond strength

of grape seed extract group were higher and gave a significant difference

comparing to control group without grape seed extract pretreatment using self etch

adhesives. Bond strength of 1 hr treated Grape seed extract 6.5 % showed bond

strength values that were statistically higher than glutaraldehyde treated and

control group27. In a study by Castellan et al(2010) 28, comparing the effect of

Grape seed extract and Cocca seed extract for 10 minute pretreatment , only

Grape seed extract pretreatment showed higher bond strength values than control

group. On the contrary in a study done by Epasinghae et al (2012) 18, there was

significant decrease in bond strength when 3% Grape seed extract was

incorporated into an experimental adhesive . But there was no significant change

in groups with no treatment or 1 % or 2% proanthocyanidin incorporation.

Pretreatment for 1 hour or 10 minutes is not clinically possible so in this study

strength was evaluated. Shortening the pretreatment time did influence the bond

strength. It had a higher bond strength in comparison with other groups.

A study by Carvalho et al (2016)6 found that 2% Green tea extract increased

microtensile bond strength values using etch and rinse system after 6 months of

storage in water.The bond remained stable, without showing any determental or

beneficial effects. 2% green tea extract showed an increase in bond strength after

24 hour and 6 month water storage using self etch adhesive system 9. This increase

may be due to the hydrophobic interaction and antioxidant property of EGCG that

is present as 42.3 % in Green tea extract17 .

Zheng et al (2014)8 in a study determined the effect of different inhibitor

solution effects using etch and rinse and self etch technique. In their study the

bond strength of Clearfil SE immediately and after 9 months was found to be

statistically similar. Clearfil SE contains 10-MDP which is completely

hydrophobic and relatively resistant to hydrolysis monomer that promotes strong

ionic bond with calcium. So in addition in micromechanical retention ,clearfil se

also provides chemical adhesion with calcium, thus explaining why there was no

significant decrease in bond strength in all the groups after 9 months. Andre et al

(2015)56 compared a etch and rinse adhesive with 4 self etch adhesive and found

that adhesive interface formed by self etching primer 10- MDP functional

monomer, showed the highest bond stability among the adhesive systems after 12

months of storage.

Theoretically higher levels of MMP-2 and MMP-9 activity were demonstrated

for etch-and-rinse compared with self-etching adhesives.This might therefore

is affected to a higher extent than that of the Clearfil SE Bond(Self etch).

Moreover , it is likely that with longer aging times, the effect of enzymatic

degradation might become more apparent for Clearfil SE Bond as well57.

In this study, Group 4 (Clearfil SE Protect) seemed to maintain a stable bond

strength over 3 months of aging .The possible mechanism by which NaF inhibits

the MMPs is not known clearly. Considering that MMPs are Zn2+- and Ca2+

dependent enzymes, and F is highly electronegative, these excess F could make

these cations unavailable to participate in the catalytic process and inhibiting the

MMP activity to an extent7 . However group 4 (Clearfil SE Protect ) bond

strength seemed to be less compared to Grape seed extract but similar to that of

Green tea extract. So Group 4 can a provide stable bond strength over long time

in comparison with Clearfil SE and Green tea extract pretreatment. In a study by

Shinora et al (2006)1, Clearfil SE Protect gave increased bond strength values

compared to Clearfill SE .

According to De Munck, et al (2012)58, the simple water storage of specimens

has a clear bond degrading effect. However, the literature makes no definitive

conclusion in regard to the minimal period of water storage that promotes

degradation in the hybrid layer and a consequent decrease in bond strength. By

applying an adhesive system under simulated intrapulpal pressure and storing

specimens for 2 years in artificial saliva Mobark et al (2011)59 observed that the

bond strength to caries-affected dentin was similar between 2%

chlorhexidine-treated and non-chlorhexidine-treated dentin. But, at a concentration of 5%, chlorhexidine was

able to prevent loss in bond strength after 2 years. MMPs require zinc and calcium

Tezvergil-Mutluay, et al(2010)60 reported that using water as a storage medium

underestimates the hydrolytic activity of endogenous dentin MMPs, because it

promoted the loss of calcium and zinc ions from dentin matrices, rather than

restoring them. The use of better solutions that simulate oral fluids and longer

storage periods should be researched in future studies. So the use of water as a

SUMMARY AND CONCLUSION

The current study evaluated the microtensile bond strength of two naturally

occurring Matrix metalloproteinase inhibitors (Grape seed extract and Green Tea

extract ) and a two step self etch adhesive containing a synthetic MMP inhibitor

Sodium Fluoride (Clearfil SE Protect) against a Self etch adhesive (Clearfil SE ).

Four groups of tooth samples were evaluated Group 1- Clearfil SE, Group 2-0.5%

Green Tea Extract, Group 3 – 6.5% Grape seed extract , Group 4 – Clearfil SE

Protect . Methods were strictly based on manufacturer’s instructions and

standardisation. A flat ground section was prepared and pretreated according to

groups and bonding procedure was carried out and composite restorations were

done. The samples were then sectioned to obtain resin dentin sticks and were

mounted in a custom made jig and tested for microtensile bond strength with

samples n =10 per group on 7th day, 30th day and 90th day using Universal Testing

Machine.

The results of the study showed that on 7th day analysis Grape seed extract had

the highest bond strength followed by Green tea extract , Clearfil SE Protect and

Clearfil SE Bond. On 30th day Grape seed extract group had a significant

difference with Green tea extract group and Clearfil SE group but had no

significant difference with Clearfil SE Protect group. On 90th day Grape seed

extract showed higher bond strength followed by Clearfil SE Protect , Green Tea

Within limitations, it is concluded from this study that Grape seed extract on

pretreatment for 60 seconds can provide a better bond strength but it needs a

additional step before bonding procedure and also requires more research to either

make it readily available or to incorporate into an adhesive . Many previous

studies done on grape seed extract had a prolonged pretreatment time which was

not feasible clinically. Clearfil SE Protect ,which had a good bond strength and

durability than Clearfil SE and Green tea extract can be used clinically without

any additional procedure and is readily available with antibacterial properties.

However more research are required to incorporate natural MMP inhibitors to the