AN IN VITRO STUDY OF ANTICARIOGENIC COMPOUNDS INCORPORATED ... · i AN IN VITRO STUDY OF...

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AN IN VITRO STUDY OF ANTICARIOGENIC COMPOUNDS INCORPORATED INTO Bis-GMA/TEGDMA COPOLYMER

by

Vinay Kumar Pilly Yadaiah BDS, MPH

A major project submitted in conformity with the requirements for the degree of Master of Science

Discipline of Dental Public Health, Faculty of Dentistry University of Toronto, Toronto, Canada

Vinay Kumar Pilly Yadaiah BDS, MPH

Faculty of Dentistry University of Toronto

2014

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© Vinay Kumar Pilly Yadaiah All Rights Reserved (2014)

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AN IN VITRO STUDY OF ANTICARIOGENIC COMPOUNDS INCORPORATED INTO Bis-GMA/TEGDMA COPOLYMER

ABSTRACT

Composite resins continue to evolve and are increasingly favoured by the public and dental professionals

for a number of interrelated reasons. However, there are several drawbacks such as decreased longevity,

risk of secondary caries and increased costs that make choosing composite resin a clinical and public

health dilemma. In combination with an aging population who are retaining their natural teeth (many

without dental insurance), this warrants a need for advancements in dental composite sciences. In this

regard, this in vitro study evaluated the drug release rates, inhibitory growth activity against

Streptococcus mutans and drug stability of epigallocatechin-gallate (EGCg) incorporated into a dental

copolymer compared to resins containing chlorhexidine (CHX). Resin discs (5mm × 3mm) were prepared

from 70 mol% Bis-GMA and 30 mol% TEGDMA comonomers containing: a) placebo, b) CHX, c)

EGCg. Two corresponding concentrations in weight% of each drug for 0.5 × MIC and 1.0 × MIC were

incorporated in the paste resins and tested in the following time points: 24 hours, 7 days, 30 days, 60 days

and 90 days. For drug delivery and bacteria viability tests, data within the different time points were

evaluated by one-way ANOVA and Tukey’s tests (p <0.05). The 1H NMR spectra were evaluated

qualitatively. There is a significant difference in the 90 days drug delivery and bacterial growth inhibition

rates among different drugs and drug ratios. Both CHX and EGCg showed sustained release rates over

90-day period. 1.0 × MIC CHX retained ability to inhibit bacterial growth over 90-day period. All drugs

showed chemical stability after being released by resin matrix after 90 days. The results indicate that

CHX and EGCg-based experimental restorative materials may reduce bacterial growth around composite

restorations, which may improve the longevity of resin-based restorations.

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ACKNOWLEDGEMENTS

I would like to express my deepest appreciation to my committee members, Dr. Anuradha

Prakki; Dr. Céline M. Lévesque and Dr. Carlos Quiñonez. No amount of praise and gratitude

will be sufficient in thanking Dr. Prakki, who not only agreed to be my supervisor, but also stood

by me during very stressful, undeserving and painful phase of my speciality training. I will fail in

my duty if I do not equally thank Dr. Lévesque, who has ensured that my dream of achieving this

very special degree, from this very special university was still a reality. My special thanks to Dr.

Morris Manolson and Dr. Herenia Lawrence.

I appreciate the constant encouragement of my peers: Dr. Elaine Cardoso; Dr. Sonica Singhal;

Dr. Rafael Figueiredo; Dr. Abeer Khalid; Dr. Carlos Brito Jr.; Dr. Jodi Shaw; Dr. Faahim

Rashid; Dr. Sojung Lee; and Ms. Julie Farmer for helping me throughout the program. My

special appreciation for Dr. Alexandra Nicolae for her unconditional support and guidance

during my program. I am grateful to Dr. Tim Burrows, from the department of Chemistry.

Without all your help, it would not have been possible to accomplish that amount of research that

we did. I am indebted to Keying Li and Halyna Hrynash from Dr. Prakki’s lab; Stephanie

Koyanagi; Vincent Leung and Alexandra Mankovskaia from Dr. Lévesque’s lab, who provided

invaluable assistance, help and guidance throughout. A very special mention about Dr. Delphine

Dufour, who has transformed someone like me who has no business to be in a microbiology lab,

especially coming from a public health background and without prior laboratory skills, learn

some work and appreciate quality research, which is not only an addition to my skill set, but also

helped me fulfill a long awaited desire to work in a microbiology lab.

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The Pilly brothers, Kiran Pilly and Praveen Pilly for their support and encouragement. Lastly,

and most importantly, I thank my mom (Jumbi), who continue to be a source of inspiration and

love for me. I really miss my little niece Siri who is going to turn 3 soon. Behind every

successful man, there stands a woman and behind my victory is my loving wife and best friend

Shanthi, for her encouragement, support and inspiration in achieving what I truly wanted in life.

The mantra for success, which is also reflective of my efforts during the M.Sc. program is

"Satyameva Jayate" (स यमेव जयते; English: Truth Alone Triumphs) derived from the ancient

Indian scripture of Mundaka Upanishad 3.1.6. The mantra is as follows:

“स यमेव जयते नानृ तं स येन प था वततो देवयानः | येना म यृषयो ा कामा य तत ्स य य परम ं नधानम ्||६||”

The English translation is as follows:

“Truth alone triumphs; not falsehood. Through truth the divine path is spread out by which the

sages whose desires have been completely fulfilled, reach where that supreme treasure of Truth

resides”

Finally, I would like to dedicate this major project to Dr.Gerry Uswak, my guru, well wisher and

a very kind person. This dream and an opportunity to study at this prestigious university could

not have been envisioned without his initiation and unconditional support. I am honoured to be

his student and it was a privilege knowing and working with him. My elevation and steep rise in

dentistry in Canada would have been impossible without him. Thank you Dr.Uswak for being

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such an important person in my life, instrumental in guiding and shaping my career. Lastly, my

special gratitude to Mrs. Leslie Topola for my being my constant source of encouragement.

As I begin a new phase of my life, I would like to recollect one of my favorite quotes by, Robert

Goddard, “It is difficult to say what is impossible, for the dream of yesterday is the hope of today

and the reality of tomorrow”. As Swami Vivekananda rightly said “Knowledge is Power”, I shall

arise, awake and stop not till the goal is reached.

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Table of Contents

Abbreviations ............................................................................................................................ xi

Chapter I: Introduction ................................................................................................................1

Chapter II: Literature Review ......................................................................................................4

2.1 Direct aesthetic restorative materials ..................................................................................4

2.2 Failure of composite resin restorations ...............................................................................6

2.3 Antimicrobial polymers ................................................................................................... 11

2.3.1 Classification ............................................................................................................. 11

2.3.2 Antibacterial fluoride releasing dental materials ........................................................ 11

2.3.3 Antibacterial dental composites containing silver compounds .................................... 12

2.3.4 Antibacterial dental composites containing physically immobilized quarternary ammonium salts compounds (QAS) ................................................................................... 13

2.3.5 Antibacterial dental composites containing chemically immobilized QAS compounds .......................................................................................................................................... 14

2.3.6 Other quarternary ammonium monomethacrylates/dimethacrylates ........................... 14

2.4 Chlorhexidine (CHX)....................................................................................................... 15

2.5 Epigallocatechin-3-gallate (EGCg) .................................................................................. 17

Chapter III: Objectives and Null Hypothesis ............................................................................ 19

3.1 Objectives of the study ..................................................................................................... 19

3.2 Null Hypothesis ............................................................................................................... 20

Chapter IV: Materials and Methods ........................................................................................... 21

4.1 Experimental Design ........................................................................................................ 21

4.2 Determination of minimum inhibitory concentration (MIC) ............................................. 21

4.3 Preparation of experimental resin ..................................................................................... 22

4.4 Preparation of resin samples ............................................................................................ 23

4.5 Determination of drug release rates .................................................................................. 23

4.5.1 Visible spectroscopy using glass quartz ..................................................................... 23

4.5.2 Determination of standard curve: CHX ...................................................................... 24

4.5.3 Determination of standard curve: EGCg .................................................................... 25

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4.6 Bacterial viability assay ................................................................................................... 26

4.6.1 Overnight culture of the bacteria................................................................................ 26

4.6.2 Preparation of phosphate buffered saline (PBS) ......................................................... 27

4.6.3 Preparation of Brain Heart Infusion (BHI) broth ........................................................ 27

4.6.4 Preparation of BHI agar plates ................................................................................... 27

4.6.5 Release of resin unit into BHI broth and overnight bacterial culture under constant agitation ............................................................................................................................. 28

4.6.6 Bacterial Culture Sonication ...................................................................................... 29

4.6.7 Micro broth dilution .................................................................................................. 30

4.6.8 Colony Forming Units (CFU) determination .............................................................. 30

4.7 Determination of drug stability ........................................................................................ 31

4.7.1 Lyophilization ........................................................................................................... 31

4.7.2 1H NMR spectroscopy ............................................................................................... 32

4.8 Data analysis.................................................................................................................... 34

Chapter V: Results .................................................................................................................... 35

5.1 Drug release ..................................................................................................................... 35

5.2 Bacterial viability ............................................................................................................ 38

5.3 Drug stability ................................................................................................................... 42

Chapter VI: Discussion ............................................................................................................. 53

6.1 Policy Implications .......................................................................................................... 61

Chapter VII: Recommendations and future directions ................................................................ 64

Chapter VIII: Conclusion .......................................................................................................... 65

References ................................................................................................................................ 66

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List of Figures

Figure 1: UV-Vis spectrophotometer ......................................................................................... 24

Figure 2: Standard Curve for CHX ............................................................................................ 25

Figure 3: Standard curve for EGCg ........................................................................................... 26

Figure 4: Rotating laboratory mixer........................................................................................... 28

Figure 5: Sonicator .................................................................................................................... 29

Figure 6: Enumeration of bacteria by serial dilution .................................................................. 31

Figure 7: Lyophilizer................................................................................................................. 32

Figure 8: 1H NMR spectrometer ................................................................................................ 33

Figure 9: Mean drug release rates plotted against time for CHX ................................................ 36

Figure 10: Mean drug release rates plotted against time for EGCg ............................................. 37

Figure 11: Cumulative drug release rates plotted against time for CHX ..................................... 37

Figure 12: Cumulative drug release rates plotted against time for EGCg ................................... 38

Figure 13: Bacterial viability at 24 hours ................................................................................... 39

Figure 14: Bacterial viability at 7 days ...................................................................................... 40

Figure 15: Bacterial viability at 30 days .................................................................................... 40



Figure 18: 1H NMR spectra of (a) placebo at baseline; (b) placebo at 7 days; (c) placebo at 30 days; (d) placebo 60 days; (e) placebo 90 days time points. ....................................................... 44

Figure 19: 1H NMR spectra of (a) pure CHX with associated molecular structure; (b) CHX released from copolymer at baseline; (c) CHX released from copolymer at 7-day time point; (d) CHX released from copolymer at 30-day time point; (e) CHX released from copolymer at 60-day time point; and (f) CHX released from copolymer at 90-day time point. .................................... 48

Figure 20: 1H NMR spectra of (a) EGCg 99% purity with associated molecular structure; (b) EGCg released from copolymer at baseline; (c) EGCg released from copolymer at 7-day time point; (d) EGCg released from copolymer at 30-day time point; EGCg released from copolymer at 60 time point ; and EGCg released from copolymer at 90-day time point. .............................. 52

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List of Tables

Table 1: Components of phosphate buffered saline solution (200 mL) ....................................... 27

Table 2: Average drug release rates (µg/mL) ............................................................................. 35

Table 3: Bacterial viability at different drug ratios and time points ............................................ 39

Table 4: Cleavage sites for chlorhexidine in 1H NMR ............................................................... 59

Table 5: Cleavage sites for epigallocatechin-3-gallate in 1H NMR ............................................. 60

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Abbreviations ANOVA: Analysis of Variance BHI: Brain Heart Infusion Bis-GMA: Bisphenol A-Glycidyl Methacrylate CDFF: Constant Depth Film Fermentor CHX: Chlorhexidine CQ: Camphorquinone DMAEMA: 2- (Dimethylamino) Ethyl Methacrylate DMAE-CB: Methacryloyloxyethyl Cetyl Ammonium Chloride EC: Epicatechin ECG: Epicatechin-3-gallate EGC: Epigallocatechin EGCg: Epigallocatechin-3-gallate EVA: Ethylene Vinyl Acetate GIC: Glass Ionomer Cement HEMA: Hydroxyl Ethyl Methacrylate HSD: Honestly Significant Difference MDPB: Methacryloyloxy Dodecyl Pyridinium Bromide MIC: Minimum Inhibitory Concentration MMPs: Metalloproteinases NIHB: Non-Insured Health Benefits NMR: Nuclear Magnetic Resonance MPS: Methacryloxy Propyltrimethoxy Silane PBS: Phosphate Buffered Saline PEI: Polyethyleneimine PEMA: Poly Ethyl Meth Acrylate PVA: Polyvinyl Alcohol RMGIC: Resin-modified Glass Ionomer Cements RPM: Rotations Per Minute QADM : Quaternary Ammonium Dimethacrylates QAS: Quaternary Ammonium Salt SD: Standard Deviation TEGDMA: Triethylene Glycol Dimethacrylate THFMA: Tetra Hydrofurfuryl Methacrylate UA: Ursolic Acid UDMA: Urethane Dimethacrylate WT: Wild Type

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Chapter I: Introduction

Composite resins have evolved greatly over the last several decades, including their mechanical

properties, esthetics, abrasive resistance, and bonding to tooth structure (Delaviz, Finer, &

Santerre, 2014). Importantly, they are increasingly favoured over dental amalgam due to

political, public, socio-cultural and professional reasons. This explains why dental composites

are currently one of the most preferred restorative materials of choice for various applications.

Amalgam has traditionally been used for the past 150 years (Bernardo et al., 2007). Despite its

proven clinical success in terms of longevity, durability, strength and cost-effectiveness,

politically, there is a growing international criticism against amalgam usage, leading to decline in

public confidence in its safety (Health Canada, 2012). The worldwide ban on dental amalgam

may not necessarily impact worldwide environmental mercury pollution (Jones, 2008b), but it

may lead to an increase in dental expenditures, as amalgam alternatives such as dental

composites are more expensive (Jones, 2008b).

Dental composites are increasingly sought by the public for their aesthetic or tooth coloured

nature (Quiñonez, 2012). This is under-girdled by a socio-cultural milieu, which privileges

clean-straight-white teeth as a social prerogative (Quiñonez, 2013). Dental practitioners prefer

composites due to competing incentives and business tradeoffs, as well as for the possibility of

performing minimally invasive restorative techniques (Health Canada, 2012; Lynch, Frazier,

McConnell, Blum, & Wilson, 2011).

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The above is also coupled to edentulous rates among those aged 60-70 years that have dropped

from 43% in 1990 to 21% in 2009 (Health Canada, 2010). The life expectancy at birth for

Canadians over a span of three decades increased from 74.9 years in 1979 to 81.1 years in 2009

(Statistics Canada, 2014). Moreover, only 38.6 % of seniors aged 60-79 have private dental

insurance, compared to 62.6% of the general Canadian population. This means that there will be

an increasingly larger senior population who are living longer, are dentate and many without

private dental insurance. The dental materials of the future will need to last longer, require less

repair or replacement and retain function and aesthetics. (Forss & Widstrom, 2004; Manhart,

Garcia-Godoy, & Hickel, 2002), resulting in a direct benefit to the patient as well as to the dental

health care system (Pereira-Cenci, Cenci, Fedorowicz, & Azevedo, 2013).

Despite efforts aimed at improving the longevity of dental composites, the impact of the

biological agent is a significant factor in the failure of the restoration (Deligeorgi, Mjör, &

Wilson, 2001). In order to improve the longevity of composites, it is important that the integrity

of the tooth-restoration interface is maintained. Inhibiting bacterial growth is one of the

alternatives to sustain the seal of the tooth-restoration interface. In this regard, chlorhexidine has

been investigated and has shown to be capable of arresting caries when applied to dentin, due to

its well established wide-spectrum antibacterial activity as well as antiproteolytic activity

(Gendron, Grenier, Sorsa, & Mayrand, 1999). However, certain drawbacks such as tooth

staining, its synthetic nature, bacterial resistance and mild toxicity to odontoblastic cells had led

researchers to look for alternatives to CHX (De Souza et al., 2007). One such natural plant

derived alternative is EGCg, which is a catechin derived from the green tea leaves. In a study

conducted by Mankovskaia and colleagues (2013), EGCg demonstrated its ability to inhibit

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cariogenic bacterial growth, biofilm formation and acid production in dental plaque. Apart from

the anticariogenic property, catechins are antioxidant and promote tumour suppression, cancer

prevention, protection against heart disease, prevention of oxidation of lipoproteins in

macrophages and have anti-inflammatory properties (McKay & Blumberg, 2002; Rasheed &

Haider, 1998; Rietveld & Wiseman, 2003).

The development of antimicrobial polymers, which are resin systems consisting of either

biocidal or biostatic groups or inherent repeats units in their chemical structure, would be crucial

in the prevention of colonization caused by microorganisms in the surroundings of a restoration.

Those polymers avoid the inconvenience of delivering the antimicrobials manually, thereby

reducing the toxicity of the agent to the surrounding tissues (Muñoz-Bonilla & Fernández-

García, 2012). Also, they have the advantage of being non-volatile, chemically stable and

minimize any loss to photolytic decomposition (Muñoz-Bonilla & Fernández-García, 2012).

However, in order to predict the long-term efficacy, it is important to test the amount of drug

release, bacterial viability and chemical stability after incorporation of these biological agents

into dental materials.

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Chapter II: Literature Review

2.1 Direct aesthetic restorative materials

The standard treatment for dental caries is to remove the decayed tooth structure and restore it

with an inert material. The dental restorations are a clinic based strategy to prevent dental caries

and a form of disease stabilization which is achieved by gaining control of the disease by

restoring cavities.

The available direct restorative materials are the glass-ionomer cements, amalgam and resin-

based composites for small to moderate sized cavities. Of these, the conventional glass ionomers

cements (GICs) consist of fluoroalumino-silicate glass fillers and an aqueous solution of

polyalkenoic acid. Once set the GICs exhibit moisture sensitivity, delaying their final strength.

Due to low mechanical properties such as moderate compressive strength and poor wear

resistance their use as a posterior load-bearing restoration is limited (Burke, Ray, & McConnell,

2006).

The resin-modified glass ionomer cements (RMGICs) were introduced to improve some of the

mechanical properties of GICs. They are chemically similar to conventional GICs, but with

additional photopolymerizable monomers, such as 2-hydroxyethylmethacrylate (HEMA)

(Culbertson, 2001) . Due to the resin modification, RMGICs are subject to some of the same

limitations that affect resin-based materials, such as polymerization shrinkage and heat

generation (Musanje, Shu, & Darvell, 2001). Similarly, compomers or polyacid-modified

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composite resins were introduced to overcome some of the limitations of GICs. However, they

too exhibited lower mechanical properties compared to dental composites and their usage is

mostly restricted for restoration of primary teeth or non-stress bearing areas (Piwowarczyk, Ottl,

Lauer, & Buchler, 2002).

The amalgam on the other hand when compared to composites and glass ionomers is cheaper and

more durable on teeth subjected to extensive wear due to chewing. In addition, the safety of

amalgam and their longevity in posterior restorations is extensively studied more than any other

restorative materials (Health Canada, 2006). However, due to some significant disadvantages,

such as, aesthetics, non-adhesive nature and strict requirements for cavity preparation,

composites have replaced amalgam restorations and are currently considered as the “material of

choice” for most types of cavities, especially for use in direct minimal intervention approaches to

the restoration of posterior teeth (Fedorowicz, Nasser, & Wilson, 2009; Lynch, Blum, Wilson, &

Brunton, 2008).

The resin-based composites offer the practitioner to practice minimally invasive dentistry,

avoiding the need to remove healthy tooth structure to achieve resistance and retention form

(Lynch et al., 2008). The modern dental curriculum in operative dentistry has now moved

towards minimal invasive techniques and procedures. Due to this, amalgam is considered to be

outdated for its mechanical concepts of treating dental caries. The state-of-the-art of dental

composites is rapidly evolving and with the increasing appropriateness, popularity and

effectiveness of composites in load-bearing areas it is now viewed as a substitute for amalgam

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and not as an alternative material anymore for its usage in all areas of the mouth (Lynch et al.,

2011).

2.2 Failure of composite resin restorations

The dental composites, by definition, consist of two or more distinct phases which are combined

to produce a product with properties greater than the individual constituents (Dogon, 1990). The

dental composite consists of reinforcing glass particles as fillers (commonly glass, quartz, or

ceramic oxides) and silane coupling agent such as methacryloxy propyltrimethoxy silane (MPS),

embedded in a resin matrix which can be polymerizable (Ferracane, 1995). The resin matrix

comprises of monomers, the most common being Bis-GMA (Bisphenol A-Glycidyl

Methacrylate), UDMA (Urethane Dimethacrylate) and TEGDMA (Triethylene Glycol

Dimethacrylate). The polymerization process can be self, light or a combination of both

(Drummond, 2008).

In the past several years, there has been an increase in the use of resin composites as posterior

restorations (Christensen, 2005). This is attributed to its predictable performance, improved

physical properties, aesthetics and demand. There is evidence in the literature that supports its

use as a direct restorative material in load bearing areas of posterior teeth (Burke & Shortall,

2001; Roeters, Shortall, & Opdam, 2005). The dental composites are now a feature of

contemporary dental practice. The current state of art of dental composites includes a variety of

materials with improved mechanical properties, handling characteristics, wear resistance,

polishability and aesthetics. The most recent research also encompassed common drawbacks

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associated with dental composites such as polymerization shrinkage. The dental restorations in

spite of meticulous care taken by the patient and use of best technique by the dental professional,

do not last forever and dental composites are no exception (Christensen, 2007). Although there

are recent advancement and success using dental composites, repair and replacement is still a

significant limitation, contributing to a phenomenon known as ‘repetitive restoration cycle’

(Elderton, 1988), because the composite resins are unable to avoid dental caries from occuring

due to the modern day food which is rich in fermentable carbohydrates and frequent food

ingestion (Ten Cate, 2006). The composites also tend to accumulate more amount of plaque than

the enamel or other restorative materials (Eick, Glockmann, Brandl, & Pfister, 2004).

From a patient, dentist and funding agency perspective, it is of interest and importance to know

the longevity of dental restorations. The longevity of a dental restoration is dependent on many

factors, namely, the patient and the problem, which includes: type and position of tooth, site and

size of restoration, age, gender, reason for replacement and type of caries; the material used and

technique; the clinician skill; and, the outcome measures - rate of failure, level of wear or patient

usage (Chadwick, 2001). A systematic review conducted by (Delaviz et al., 2014), assessed the

relationship between composite resin degradation and clinical failure, catalyzed by biological

factors. They concluded that all commercially available composite resin products are subject to

biological degradation and volumetric shrinkage in the oral cavity despite varied vinyl acrylate

compositions. The authors based their finding on the ability of salivary enzymes, especially

esterases, to degrade the polymeric matrix of resin composites and adhesives. Upon further

degradation, acid-producing bacteria such as S. mutans can infiltrate, accumulate and increase in

numbers causing post-operative sensitivity, secondary caries, pulp necrosis and inflammation.

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Among these, secondary caries is the most significant and a common cause for restoration

replacement (Bourbia, Ma, Cvitkovitch, Santerre, & Finer, 2013; Kermanshahi, Santerre,

Cvitkovitch, & Finer, 2010). The bacteria S. mutans have shown high affinity for resin

composites compared to other materials such as metals and ceramics, which is also proved in in

vitro studies (Beyth, Bahir, Matalon, Domb, & Weiss, 2008; He, Soderling, Osterblad, Vallittu,

& Lassila, 2011).

Additionally, current reports have shown that the mechanism of resin restoration failure due to

dentinal caries and bond degradation is initiated from beneath the bonded interface, with the

breakdown of demineralized and denatured collagen matrices by host derived matrix

metalloproteinases (MMPs) (Hiraishi, Yiu, King, Tay, & Pashley, 2008) present within dentin

and from saliva (Chaussain-Miller, Fioretti, Goldberg, & Menashi, 2006). It has been reported

that MMPs digest components of the extracellular matrix (Brinckerhoff & Matrisian, 2002)

contributing to many biological and pathological processes. When activated by low pH, they

have been suggested to play an important role in the degradation of dentin (Chaussain-Miller et

al., 2006) organic matrix, and therefore, in the control and progression of carious decay. Overall,

there is a strong evidence to conclude that MMPs along with bacteria and salivary enzymes can

breakdown the marginal interface and limit the longevity of resin composites leading to

secondary caries formation.

There are four dominant strategies for enhancing the bio-stability and longevity of resin

composites, namely, enhancing the bio-stability by reducing volumetric shrinkage; enhancing the

bio-stability by shielding susceptible bonds; enhancing bio-stability by reducing the rate of

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collagen degradation by MMPs and enhancing the bio-stability by reducing adherence of bacteria

(Delaviz et al., 2014). Despite recent recommendations aimed at improving the longevity of

composites, there are several biological variables that are beyond the control of the clinician. Of

the many different factors mentioned above, secondary caries due to recurrent marginal decay is

the most significant factor, accounting for nearly 88% of all composite restoration failures

(Bernardo et al., 2007). It is clear that the success of resin composites depends on the integrity of

tooth-restoration interface, which can be accomplished by inhibiting bacterial growth. Hence,

there is a need to supplement the dental composites. It is therefore proposed that that future

research in restorative dental composites should focus on development of materials with bio-

active functions, such as antibacterial functions, enabling the material to be self-repairable and

the tooth to re-mineralize (Ferracane, 2011).

The factors that determine failure of a restoration differs depending on the clinical assessment

used and diagnostic criteria applied. Unfortunately, there is no standardized diagnostic criterion

for replacing a dental restoration (Chadwick et al., 2001), which makes it difficult for a dentist to

determine if a new lesion is in fact a new lesion or residual caries that was not excavated or

removed completely from a previous carious lesion. This contributes to the interpreter variability

(inter or intra) of different clinicians. In general, a distinction must be made between early and

late failures of a dental restoration (Hickel & Manhart, 2001). The tooth-coloured nature of

composite restorations makes it difficult to locate signs of tooth decay such as, the

discolouration, wear and marginal breakdown. By the time symptoms of decay are evident the

tooth would have passed through all the progressive stages of pulp disease and developed apical

periodontitis. A systematic review conducted by University of York (Chadwick, 2001) stated that

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subjective decision making is common in daily practice or in other words a group of examiners

following up on a restoration would determine failure differently and at different points in time.

It is possible that the longevity of a restoration is influenced more so due to lack of clarity

between objective and subjective factors in clinical decision making than due to physical and

chemical properties of the material itself (Chadwick, 2001). A review conducted by Jokstad and

colleagues (2001) proposed that dental restorations must be evaluated in terms of preservation of

tooth structure, esthetics and function, instead of durability and serviceability. The author’s state

that the purpose of any restoration is to restore tooth morphology and as long as this pre-set aim

is achieved and lasts, contribute to the success of the restoration. Apart from that patient

satisfaction, is also considered to be an important determinant of quality (Jokstad et al., 2001).

Clearly there is an urgent need for development of reliable, accurate, validated and a more

objective criterion for replacing dental restorations. This will certainly have an impact on the

longevity of a restoration and implications for patient, practitioner and dental care system.

In assessing the longevity of restorations there are two possible outcome measures, namely, time

until restoration replacement and time until failure. The time until making a decision to replace a

restoration is very subjective in nature. Although subjective opinions are valid in clinical

practice, they do not allow for comparisons and therefore not ideal for setting or improving

standards in diagnosis and treatment planning. On the other hand, time until restoration failure is

an end point if no intervention is undertaken. But, it would be unethical if not intervened during

the disease process. It is therefore necessary to devise parameters that can indicate the stages in

the progression of disease (Chadwick, 2001).

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It is anticipated that the strategy to enhance the longevity of composite resin restorations by

preventing secondary caries and by improving the durability of resin-dentin bonds may benefit

from the development of multi-functional restorative materials such as, drug-loaded dental

resins, capable of releasing compounds with antibacterial and/or antiproteolytic properties.

2.3 Antimicrobial polymers

2.3.1 Classification

The antimicrobial polymers are group with covalent linkages with antimicrobial properties. They

are classified into four groups: a) polymers with antimicrobial activity; b) polymers that undergo

chemical modifications to achieve antimicrobial activity; c) polymers containing antimicrobial

organic compounds and; d) polymers incorporating antimicrobial inorganic compound. The

various antimicrobial polymers are used based on the need and application (Muñoz-Bonilla &

Fernández-García, 2012).

2.3.2 Antibacterial fluoride releasing dental materials

The use of antibacterial delivery system in the oral cavity is quite known. In the past, efforts to

improve the antibacterial action of dental materials focussed on slow releasing low molecular

weight agents such as zinc ions, silver ions, iodine, dodecylamine, bipyridine, tannic acid

derivatives, polyhexanide, amphilic lipids, quaternary ammonium salt (QAS), phosphonium salt

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groups and fluoride (Craig & Power, 2002; Kazuno et al., 2005; Kudou et al., 2000; Osinaga et

al., 2003; Takahashi et al., 2006; Wiegand, Buchalla, & Attin, 2007; Yamamoto et al., 1996).

The glass ionomer cement, polycarboxylate cement (Riggs, Braden, & Patel, 2000), compomers

(Braden, 1997), orthodontic wires (Lee & Kim, 1995) and few methacrylate based systems

(Patel, Pearson, Braden, & Mirza, 1998) are reported to release fluoride ions to prevent dental

caries. But most of these systems release fluoride by diffusion, requiring water as a medium to

facilitate release of fluoride ions which carries the risk of resin plasticization. Due to this,

fluoride ions get depleted quickly, reducing their long-term effectiveness (Delaviz et al., 2014).

Moreover, the fluoride released from such composites is much lower (< than 45ppm) than the

levels required for bacterial growth inhibition (Xu, Wang, Liao, Wen, & Fan, 2012).

2.3.3 Antibacterial dental composites containing silver compounds

The silver ions (Ag+) are known for their antibacterial properties and are used in medical and

dental sciences for their ability to react with proteins, anions and receptors present on bacterial

cell surface (Lansdown, 2002). These ions appear to act by disrupting enzyme systems

irreversibly and block bacterial DNA replication (Morones et al., 2005). They can be introduced

into dental materials by several means such as, direct mixing of silver salts (Kawahara, Tsuruda,

Morishita, & Uchida, 2000) or by the addition of silver nanoparticles (Jia, Hou, Wei, Xu, & Liu,

2008). When used in the form of nanoparticles prepared by mixing AgNO3 in isopropanol with

an acrylic liquid (Kassaee, Akhavan, Sheikh, & Sodagar, 2008), silver nanoparticles have a high

surface area to volume ratio (Fan et al., 2011). The silver nanoparticles have also shown a

13

controlled release of silver ions under aqueous conditions similar to oral cavity, without loss of

mechanical properties of the composite resin (Kawashita et al., 2000). A major issue with curing

(hardening of a polymer material by cross-linking of polymer chains) silver nanoparticles is that

it takes at least 2 minutes to achieve a suitable degree of conversion. If the curing is inadequate,

polymerization is compromised resulting in elution of monomers from the resin (Burgers et al.,

2009). Another limitation using silver nanoparticles is aesthetics, as both chemical and light

cured resins show amber or dark brown colour staining instead of tooth colour (Fan et al., 2011).

2.3.4 Antibacterial dental composites containing physically immobilized quarternary ammonium salts compounds (QAS)

The QAS are positively charged due to the presence of ammonium groups in its molecule, which

interacts with the negatively charged cell membrane of the bacteria, disrupting its electrical

balance and cell membrane, causing cell lysis. In contrast with other antibacterials, QAS are

generally immobilized in the dental resin matrix and are expected to last longer (Xie et al.,

2011). Due to immobility, such compounds offers only surface antibacterial effect. In order to

overcome this problem, polyethyleneimine (PEI) quaternary ammonium nanoparticles were

introduced as an alternative method for immobilizing QAS in a dental composite resins.

However, the antibacterial component failed to be mobilized physically from the resin and

therefore proved ineffective against bacteria (Beyth, Yudovin-Farber, Bahir, Domb, & Weiss,

2006; Beyth et al., 2008; Kesler Shvero et al., 2013).

14

2.3.5 Antibacterial dental composites containing chemically immobilized QAS compounds

Despite some recent advances in QAS compounds, the major focus has long been on the

development and testing of quaternary ammonium monomers. One such monomer is 12-

methacryloyloxydodecyl pyridinium bromide (MDPB) which possessed antibacterial properties

and is commonly used in dental adhesives, primers and composites (Imazato, 2003; Thome,

Mayer, Imazato, Geraldo-Martins, & Marques, 2009). It was later discovered that MDPB acted

as an ‘immobile antibacterial’ with bacteriostatic properties as it inhibited the growth of bacteria

only when in contact with its surface (Imazato, 2009). The MDPB has only bacteriostatic effect

as the amount required for bacteriocidal effect (>40 mg/mL) is cytotoxic in nature. Therefore,

MDPB incorporated into composite monomer compositions is limited to 0.4 wt% (Ebi, Imazato,

Noiri, & Ebisu, 2001).

2.3.6 Other quarternary ammonium monomethacrylates/dimethacrylates

In an attempt to improve the antibacterial, physical and mechanical properties of QAS

containing dental materials, several new monomers were developed. One such monomer which

has shown promising antibacterial effect without compromising on its mechanical properties is

methacryloyloxyethyl cetyl ammonium chloride (DMAE-CB) (Xiao et al., 2008). However, the

loading of the monomers must be kept at low levels, as increase in its content can decrease the

degree of crosslinking, which will have negative effects due to elution of unreacted monomer

(Ebi et al., 2001; Huang et al., 2011). In order to overcome this, quaternary ammonium

dimethacrylates (QADM) were introduced to allow greater monomer loading without significant

uncured monomer elution or detriment effects to its mechanical properties. However, a high

15

concentration of QADM loading is required (10 wt%) to achieve antibacterial effects. This may

be due to the absence of long aliphatic chains, leading to low lipophilicity and subsequent low

bactericidal effect (Zhang et al., 2012). Despite recent advancements in this field, chlorhexidine

is still considered to be a “gold standard” among the various polymeric dental materials that

release antibacterial agents (Leung, Spratt, Pratten, Gulabivala, Mordan, & Young, 2005).

2.4 Chlorhexidine (CHX)

Chlorhexidine (CHX) is a bisguanide cationic broad spectrum antimicrobial compound, active

against both gram-negative and gram-positive bacteria. It is commonly used in dentistry, in

preventing oral diseases. Because of its positive charge, CHX binds electrostatically to the

negatively charged bacterial surface, forming pores and disrupting the bacterial cell membrane.

CHX can exert both bacteriostatic and bactericidal effects depending on the concentration of the

drug. At low concentration, it can cause low molecular weight substances to leak out of cell

membrane without damaging it irreversibly. At high concentrations, CHX causes cytoplasm to

precipitate causing permanent damage to the bacterial cell (McDonnell & Russell, 1999; Puig

Silla, Montiel Company, & Almerich Silla, 2008). A study (Chaussain-Miller et al., 2006) has

demonstrated that CHX applied to dentin is capable of preventing caries. More recently, several

researchers have demonstrated the ability of CHX to inihibit MMPs (Gendron, Grenier, Sorsa, &

Mayrand, 1999; Hannas, Pereira, Granjeiro, & Tjaderhane, 2007). Both in vivo and in vitro

studies have shown that CHX when applied before resin restorative procedure, arrests the self-

destruction of tooth organic matrix, possibly due to MMP inhibition (Carrilho et al., 2007;

Hebling, Pashley, Tjaderhane, & Tay, 2005).

16

Based on the positive effects mentioned above, CHX has been added to restorative cements, both

provisional and permanent conventional cements, and polymethyl methacrylate-based resin

cements (Hiraishi, Yiu, King, & Tay, 2010; Lewinstein, Chweidan, Matalon, & Pilo, 2007; Orug

et al., 2005; Takahashi et al., 2006). It has been suggested that positively charged antibacterial

agents such as CHX, released underneath a restoration, can remain trapped and could potentially

provide long-term benefits (Leung, Spratt, Pratten, Gulabivala, Mordan, & Young, 2005).

Despite all the uses mentioned above, there are several disadvantages associated with CHX, such

as, bacterial resistance (Papas et al., 2012; Slot, Vaandrager, Van Loveren, Van Palenstein

Helderman, & Van der Weijden, 2011), cytotoxicity and being synthetic in origin (Bagis,

Baltacioglu, Ozcan, & Ustaomer, 2011; Lessa, Nogueira, Huck, Hebling, & Costa, 2010;

Paraskevas, 2005; Thomas, Maillard, Lambert, & Russell, 2000).

Although CHX is favoured widely in dental sciences, there are other antibacterial compounds

which are releasable such as, triclosan and octenidine. Despite the fact that triclosan has shown

good bactericidal properties and is releasable from dental materials, it failed to show complete

bacterial inhibition in in vivo studies (Wicht, Haak, Kneist, & Noack, 2005). Octenidine was

previously used as an antibacterial in mouth washes, until it was discovered to be a releasable

agent from dental composites (Rupf et al., 2012). The current research is looking for alternatives,

which are natural in origin such as plant and fruit extracts, capable of inhibiting S.mutans

bacteria. One such functional compound that is attracting wide attention is green tea extract.

17

2.5 Epigallocatechin-3-gallate (EGCg)

The beverage tea is derived from the cured leaves of Camellia sinensis plant. After water, tea is

the most commonly consumed beverage around the world. There are three major types of tea,

namely, green, black and oolong. Among these, green tea has shown promising results in the

prevention of caries (Sharma, Bhattacharya, Kumar, & Sharma, 2007). Green tea is rich in

flavonoids which include catechins such as epichatechin (EC), epicatechin-3-gallate (ECG),

epigallocatechin (EGC), and epigallocatechin-3-gallate (EGCg) (Sakanaka, Kim, Taniguchi, &

Yamamoto, 1989). The black and oolong tea have also been studied in relation to dental health.

Although similar in many aspects, the black and oolong tea differ in certain parameters such as

structure, molecular weight and method of oxidation. The oolong tea is considered to be an

intermediate between green and black tea (Arab et al., 2011; Sumpio, Cordova, Berke-Schlessel,

Qin, & Chen, 2006).

The green tea catechin EGCg has shown to exhibit anticariogenic properties, and to be

antibacterial against S.mutans (Hirasawa, Takada, & Otake, 2006; Taylor, Hamilton-Miller, &

Stapleton, 2005; Xu, Zhou, & Wu, 2011). The antibacterial (against S.mutans) mode of action of

catechins is still under investigation. In a recent study (Xu et al., 2011), it has been demonstrated

that EGCg has the potential to disrupt the formation and disturb the integrity of S.mutans

biofilms, due to possible interactions between EGCg and S.mutans glucosyltransferase enzymes.

More recently, researchers have demonstrated that EGCg can suppress gtfB, gtfC, gtfD genes,

responsible for the formation of extracellular polysaccharides in S.mutans (Xu, Zhou, & Wu,

2012). A recent study (Hara et al., 2012) has found that EGCg is capable of non-competitive

18

inhibition (molecule binds to an enzyme somewhere other than the active site) of alpha-amylase,

indicating EGCg action against fermentable carbohydrates. Moreover, in situ experiment has

shown that the ability of EGCg to inhibit MMP activity results in the reduction of dental erosion

and abrasion (Magalhaes et al., 2009). With EGCg showing capability to inhibit the MMPs

(MMP-2, 8 and 9), which are known to be associated with caries progression in dentin (Hannas

et al., 2007), a new pathway in the prevention of dental caries may open.

19

Chapter III: Objectives and Null Hypothesis

3.1 Objectives of the study

The aim of the present study was to evaluate long term (90 days) properties of the bio-active

drugs chlorhexidine (CHX) and epigallocatechin-3-gallate (EGCg) incorporated into an

experimental resin. The specific objectives are:

I. To determine drug release rates at different times of a dental resin incorporated at different

concentrations with CHX or EGCg.

II. To examine the growth inhibitory effect at different times of a dental resin incorporated at

different concentrations with CHX or EGCg on S.mutans.

III. To determine whether the drugs (CHX and EGCg) after incorporation into dental resins

remain stable at different times and at different concentrations.

20

3.2 Null Hypothesis

I. There would be no difference in the drug delivery rates at different times among different

drugs (placebo, CHX and EGCg) and drug ratios incorporated into a dental resin.

II. There would be no difference in the bacterial growth inhibition at different times among

different drugs (placebo, CHX and EGCg) and drug ratios incorporated into a dental resin.

III. The different drugs (placebo, CHX and EGCg) released from a dental resin at different drug

ratios would not present chemical stability at different times.

21

Chapter IV: Materials and Methods

4.1 Experimental Design

This study examined the drug release rates, bacterial growth inhibition, and drug chemical

stability of experimental resin-based materials, as a response to three factors:

I. Drugs incorporated into a dental resin at three levels (placebo, CHX, and EGCg).

II. Drug ratios at two levels (0.5 × MIC and 1.0 × MIC).

III. Resin storage periods at five levels (24 hours, 7 days, 30 days, 60 days, 90 days).

A total of 115 (15 for first assay; 75 for second assay and 25 for third assay) resin samples were

prepared for this study. For the first assay, each specimen (n=3) underwent repeated

measurements at five different time points for the evaluation of drug release rates. For the second

assay, each specimen (n=3) underwent a single measurement for the evaluation of bacterial cell

viability. For the third assay, each specimen (n=1) underwent a single measurement for the

evaluation of drug stability.

4.2 Determination of minimum inhibitory concentration (MIC)

The minimum inhibitory concentrations (MICs) are considered to be the ‘gold standard’ in

determining the susceptibility of microorganisms to antimicrobials (Andrews, 2001). Using the

broth microdilution technique (Suntharalingam, Senadheera, Mair, Levesque, & Cvitkovitch,

22

2009) that determines the lowest concentration of test agent that prevents the appearance of

turbidity, Mankovskaia and colleagues (2013), calculated the MICs for CHX and EGCg to be

2µg/mL and 700µg/mL respectively using S.mutans UA159. They estimated the MIC to be the

lowest concentration needed to inhibit more than 90% of bacterial culture relative to the original

bacterial density. Based on that, 0.53wt% of 0.5 × MIC CHX and 1.05wt% of 1.0 × MIC CHX

was calculated and used for the preparation of the resin samples. Similarly, 7.02wt% of 0.5 ×

MIC EGCg and 14.04wt% of 1.0 × MIC EGCg was calculated and used.

4.3 Preparation of experimental resin

An experimental resin formulation was prepared combining Bis-GMA (bisphenol glycidyl

dimethacrylate, Sigma-Aldrich, St. Louis, MO, USA) and TEGDMA (triethyleneglycol

dimethacrylate, Sigma-Aldrich, St. Louis, MO, USA) at 70/30 mol% ratio. Comonomers were

activated by the addition of DMAEMA (2-(dimethylamino) ethyl methacrylate, Sigma-Aldrich,

St. Louis, MO, USA) at 0.2 wt% and the photosensitizer CQ (camphorquinone, Sigma-Aldrich,

St. Louis, MO, USA) at 0.2 wt% each. Except for the placebo groups, with no drugs added,

comonomers were mixed with two ratios (0.5 × MIC or 1.0 × MIC) of either CHX

(chlorhexidine diacetate salt hydrate, Sigma-Aldrich, St. Louis, MO, USA) or 99.0% pure EGCg

(epigallocatechin-3-gallate, Selleckchem, Houston, Texas).

23

4.4 Preparation of resin samples

A small drop from the prepared experimental resin mixtures were taken using a spatula, loaded

inside a cylindrical acrylic matrix (5mm diameter × 3mm height). The unpolymerized material

was sandwiched between two polyester mylar strips over a glass-mixing tablet. The

polymerization was done on both sides by a visible light curing unit (Demi LED, Kerr Co., WI,

USA) that delivered uninterrupted 540mW/cm2, verified by a radiometer (Model 100, Demetron

Research Co., CT, USA) for 40 seconds. The cured excess material was slightly trimmed using a

fine sand paper and stored in glass containers.

4.5 Determination of drug release rates

The amount of drug which is leached in 1mL of distilled water from the resin was measured by

determining its optical density and converted into drug release expressed in µg/mL based on a

standard linear equation determined as explained below.

4.5.1 Visible spectroscopy using glass quartz

A UV-Vis Spectrophotometer (Figure 1; Synergy HT BioTEK, Winooski, VT, USA) was used to

confirm the maximum absorption peak of CHX diacetate at 257nm and EGCg at 297nm. The

specimens were individually stored in 1mL of deionized water at 370C at the following time

intervals: 24 hours, 7 days, 30 days, 60 days, and 90 days. The absorbance peak heights of the

24

storage solutions were obtained and converted to drug release rates based on the established

linear calibration.

Figure 1: UV-Vis spectrophotometer

4.5.2 Determination of standard curve: CHX

A series of solutions containing 50, 25, 10, 7.5, 5.0, 2.5, 1.0 µg/mL CHX diacetate was prepared

using distilled water. A linear relationship between absorption peak height and CHX

concentration in the reference solutions was established for each solution. The absorbance peak

heights of the replaced solutions at 257 nm were converted to the quantities of CHX released,

based on the straight line equation Y= 0.0565X-0.0204 (Figure 2) obtained from the linear

relationship.

25

Figure 2: Standard Curve for CHX

4.5.3 Determination of standard curve: EGCg

A series of solutions containing 100, 75, 50, 25, 10 µg/mL of EGCg was prepared using distilled

water. A linear relationship between absorption peak height and EGCg concentration in the

reference solutions was established for each solution. The absorbance peak heights of the

replaced solutions at 297 nm were converted to the quantities of EGCg released, based on the

straight line equation Y= 0.0094X+0.0111 (Figure 3) obtained from the linear relationship.

26

Figure 3: Standard curve for EGCg

4.6 Bacterial viability assay

4.6.1 Overnight culture of the bacteria

S.mutans UA159 (Ursolic Acid), wild type (WT) strain used in this study was isolated from a

child with active caries (Ajdić et al., 2002). The frozen bacterial stock was cultivated overnight

in 5 mL of BHI (Brain Heart Infusion) broth at 37 °C and in air with 5% CO2.

27

4.6.2 Preparation of phosphate buffered saline (PBS)

The different measured components of phosphate buffered saline (PBS) solution (Table 1) were

dissolved in 200 mL of distilled water and the pH adjusted to 7.4 with the addition of HCl or

NaOH.

Table 1: Components of phosphate buffered saline solution (200 mL)

Components Measurement (grams)

NaCl (BioShop® Canada Inc., Burlington ,ON) 1.6 KCl (EMD chemicals, Gibbstown, NJ) 0.04 KH2PO4 (BioShop® Canada Inc., Burlington ,ON) 0.048 Na2HPO4 (BioShop® Canada Inc., Burlington ,ON) 0.288

4.6.3 Preparation of Brain Heart Infusion (BHI) broth

To a laboratory glass bottle of 1 litre volume, 19 grams of BHI powder (BioShop® Canada Inc.,

Burlington, ON) was added. To this 500mL of water was added, mixed properly and autoclaved.

4.6.4 Preparation of BHI agar plates

To a wide mouthed glass flask of 1 liter volume, 19 grams of BHI powder and 7.5 grams of agar

powder (Becton, Dickinson and Company, Sparks, MD) were combined. To this 500mL of water

was added so that the resultant BHI broth agar plates were 1.5% (w/v) agar. A magnetic stirrer

was dropped in the flask before autoclaving the solution, which remained until the solution was

28

dispensed onto the petri dishes (100 × 15mm). A thin layer of BHI agar (~20 mL) was poured

into each plate under sterile conditions. The solidified plates were inverted and kept at 4ºC until

used.

4.6.5 Release of resin unit into BHI broth and overnight bacterial culture under constant agitation

Under aseptic conditions, resin samples were individually transferred into sterile eppendorf tube

containing 1mL of BHI broth and 50µl of overnight culture (1 in 20). Later these tubes were

mounted on a rotating laboratory mixer (Figure 4) to undergo constant agitation @20 RPM

(Rotations Per Minute) at 370C overnight.

Figure 4: Rotating laboratory mixer

29

4.6.6 Bacterial Culture Sonication

S. mutans grows in pairs and/or small chains. The cultures were gently sonicated for 5 seconds to

disrupt the streptococcal chains. The sonicator (Figure 5; XL-2000, QSonica LLC, Newtown,

CT, USA) was set in remote mode before tuning on. Once turned on, the output wattage was

adjusted and set at level ‘5’. Before using the sonicator, the probe was cleaned first with 70%

ethyl alcohol and then with distilled water. The probe was inserted into the eppendorf tube,

without touching its walls and culture was sonicated for 5 seconds, before placing it in the ice

box.

Figure 5: Sonicator

30

4.6.7 Micro broth dilution

Under aseptic conditions, the wells of a 96-well microtitre plate were filled with 270µL of sterile

PBS. To the first well, 30µL of undiluted cell suspension was added and mixed by pipetting up

and down. 30µL from the first well was then transferred to the second well (dilution factor 10^2)

and this was repeated until the sixth well (dilution factor 10^6).

4.6.8 Colony Forming Units (CFU) determination

Under aseptic conditions, the solution present in a 96 plate well was thoroughly mixed before

placing three drops of 20µL solution on the quadrant marked for that particular well on the petri

dish (Figure 6). The same procedure was repeated for each of the next five wells, i.e., from

dilution factor 10^2 to 10^6. The loaded petri dish was allowed to dry before incubation. They

are turned upside down during incubation in order to prevent moisture condensation. Colonies

(between 50-200) are counted after 48 hours of incubation at 37ºC in air with 5%CO2. The

equation used to determine the CFU/mL is: CFU/mL = number of bacteria ×50 ×dilution factor.

The percentage of cell survival corresponded to the number of live cells after each counting

divided by the total number of live cells in the untreated sample.

31

Figure 6: Enumeration of bacteria by serial dilution

4.7 Determination of drug stability

The drug which was leached into the distilled water from the resin was measured qualitatively

for assessing the structural stability, first by concentrating the residual distilled water using a

lyophilizer and then by subjecting the reminder to 1H NMR (Nuclear Magnetic Resonance)

spectroscopy as explained below.

4.7.1 Lyophilization

A lyophilizer (Figure 7; Savant SC100 Speed Vac®, GMI Inc., MN, USA) was used to

dehydrate the solution remaining after the removal of resin unit until a small visible amount of

32

liquid (~0.1 mL) was present at the bottom of the eppendorf tube. The remaining liquid was

taken to the NMR facility to be loaded onto the 1HNMR machine to be run overnight.

Figure 7: Lyophilizer

4.7.2 1H NMR spectroscopy

A single resonance, 1H NMR spectra of unloaded Bis-GMA/TEGDMA comonomer extract;

CHX and EGCg alone; CHX and EGCg extracted from the drug loaded comonomer was

analyzed at 24 hours, 7 days, 30 days, 60 days, and 90 days. The spectroscopy (Figure 8; nyago-

vnmrs500, Agilent Technologies, Burlington, ON, Canada) was operated at 500 MHz, proton, at

a temperature of 250C. The lyophilized resin extracts was dissolved in deuterium oxide (D2O) at

5% (w/v) in 5mm tubes. In these spectra, signal intensity (vertical axis) is plotted versus the

chemical shift (symbolized by δ), measured in ppm (horizontal axis). The chemically different

protons (1H nuclei) in the samples resonate at different frequencies because they are shielded

33

more or less by the electrons that surround them. In order to confirm if the NMR spectra belong

to the drug (CHX or EGCg) released from the resin, a pure NMR spectra for CHX and EGCg

was plotted prior to that for comparison.

Figure 8: 1H NMR spectrometer

34

4.8 Data analysis

Distribution of data was evaluated visually using a histogram, and also using Shapiro-Wilk test,

and the data were found to be normal. Levene’s test indicated that the data presented equal

variances. One-way ANOVA (Analysis of Variance), followed by Tukey’s post-hoc test were

used for drug delivery and bacteria viability assays, to compare results among different evaluated

time points. The level of significance was set at 0.05. Collected data were compiled and

examined for relevance with the SPSS version 20.0 (SPSS Inc., Chicago, IL) statistical program.

The 1H NMR spectra were analyzed qualitatively using Mnova version 9.0.0 (Mestrelab

Research, Escondido, CA).

35

Chapter V: Results

5.1 Drug release

The average drug release rates for CHX and EGCg at two different ratios and at five different

time points are shown in Table 2 and Figures 9, 10. The placebo resins showed no detectable

amount of the test drugs. The drug release rates for all the experimental resins at the end of 24

hours were the highest compared to the remaining time points (7 days, 30 days, 60 days, and 90

days), except for 0.5 × MIC CHX.

Table 2: Average drug release rates (µg/mL)

Average drug release rates (µg/mL)

Placebo 0.5×MIC CHX 1.0×MIC CHX 0.5×MIC EGCg 1.0×MIC EGCg

24 hours 0.0 7.4 ± 1.7a 15.6 ± 1.7a 188.7 ± 21.5a 289.9 ± 24.4a

7 days 0.0 6.1 ± 2.2a 8.5 ± 1.4b 76.5 ± 11.5b 159.4 ± 18.2b

30 days 0.0 7.2 ± 3.1a 10.8 ± 2.3b 94.9 ± 10.8b,c 219.1 ± 29.2a,b

60 days 0.0 6.4 ± 2.0a 10.0 ± 1.3b 69.7 ± 9.6b 177.7 ± 42.1b

90 days 0.0 5.8 ± 1.1 a 10.3 ± 1.3b 46.7 ± 5.9b,d 117.3 ± 29.1b

*ANOVA and Tukey’s test; α=0.05; s.d.: standard deviation; same letters indicate no statistical difference within each column.

The average 90 days cumulative drug release rates for CHX and EGCg are shown in Figures 11

and 12.

36

The Tukey’s HSD (Honestly Significant Difference) has shown that the 24 hour drug release rate

for 1.0 × MIC CHX and 0.5 × MIC EGCg was significantly higher compared to the drug release

rates at 7 days, 30 days, 60 days, and 90 days. Similarly, the drug release rates for 0.5 × MIC

EGCg at day 30 was significantly higher compared to day 90. For 1.0 × MIC EGCg, the 24 hour

drug release rate was significantly higher compared to rates at 7, 60 and 90 days.

Figure 9: Mean drug release rates plotted against time for CHX

37

Figure 10: Mean drug release rates plotted against time for EGCg

Figure 11: Cumulative drug release rates plotted against time for CHX

38

Figure 12: Cumulative drug release rates plotted against time for EGCg 5.2 Bacterial viability

The bacterial viability for the experimental resins: placebo and drugs at two different ratios and

at five different time points is presented inTable 3 and Figures 13-17. None of the drugs

incorporated at 0.5 × MIC level exhibited antibacterial activity against S.mutans. The resins

containing 1.0 × MIC CHX was able to significantly (p<0.05) inhibit bacterial growth during the

90 days period. For this group, 24 hour inhibition percentages were significantly lower than for

the other time points evaluated. The resin containing 1.0 × MIC EGCg showed a significant

bacterial growth inhibition only at 60 and 90 days time points evaluated (p= 0.012).

39

Table 3: Bacterial viability at different drug ratios and time points Bacterial Viability % ± s.d.

Placebo 0.5×MIC CHX 1.0×MIC CHX 0.5×MIC EGCg 1.0×MIC EGCg

24 Hours 100.00 138.19 ± 1.09a 7.62 ± 2.98a 116.99 ± 23.81a 156.86 ± 32.82a

7 days 100.00 243.4 ± 229.68a 0.11 ± 0.06b 207.97 ± 204.71a 97.19 ± 56.45a,c

30 days 100.00 137.24 ± 28.08a 0.18 ± 0.03b 141.42 ± 84.21a 77.03 ± 47.99a,c

60 days 100.00 130.80 ± 22.56a 0.41± 0.47b 93.16 ± 8.85a 52.55 ± 11.09b,c

90 days 100.00 96.23 ± 17.76a 0.37 ± 0.08b 123.85 ± 17.99a 28.08 ± 4.48b,c

*ANOVA and Tukey’s test; α=0.05; s.d.: standard deviation; same letters indicate no statistical difference within each column.

Figure 13: Bacterial viability at 24 hours

40

Figure 14: Bacterial viability at 7 days


41



42

5.3 Drug stability

A single resonance 1H NMR spectra was collected for the compounds in their pure form and for

those extracted from the resins in deionized water after 24 hours, 7 days, 30 days, 60 days, and

90 days. The following are representative images (Figure 18a-e) of 1H NMR spectra of placebo

at baseline (Fig. 18a), at 7 days (Fig. 18b), at 30-days (Fig. 18c), at 60-days (Fig. 18d) and 90-

days (Fig. 18e) time points.

43

44

Figure 18: 1H NMR spectra of (a) placebo at baseline; (b) placebo at 7 days; (c) placebo at 30 days; (d) placebo 60 days; (e) placebo 90 days time points.

45

The following are representative images (Figure 19a-f) of 1H NMR spectra of pure CHX (Fig.

19a) and 1.0 × MIC CHX released from the experimental copolymers at baseline (Fig. 19b), 7-

day (Fig. 19c), 30-day (Fig. 19d), 60-day (Fig. 19e) and 90-day (Fig. 19f) time points. After the

assessment of 1H NMR of the leached media for both 0.5 × MIC (not represented in this section)

and 1.0 × MIC CHX incorporated copolymers, all expected CHX signals, as compared to pure

compound signals, were detected.

46

47

48

Figure 19: 1H NMR spectra of (a) pure CHX with associated molecular structure; (b) CHX released from copolymer at baseline; (c) CHX released from copolymer at 7-day time point; (d) CHX released from copolymer at 30-day time point; (e) CHX released from copolymer at 60-day time point; and (f) CHX released from copolymer at 90-day time point.

49

The following are representative images (Figure 20a-f) of 1H NMR spectra of pure EGCg (Fig.

20a) and 1.0 × MIC EGCg released from the experimental copolymers at baseline (Fig. 20b), 7-

day (Fig. 20c), 30-day (Fig. 20d), 60-day (Fig. 20e) and 90-day (Fig. 20f) time points. Likewise,

after the assessment of 1H NMR of the leached media for both 0.5 × MIC (not represented in this

section) and 1.0 × MIC EGCg incorporated copolymers, all expected EGCg signals, as compared

to pure compound signals, were detected.

50

51

52

Figure 20: 1H NMR spectra of (a) EGCg 99% purity with associated molecular structure; (b) EGCg released from copolymer at baseline; (c) EGCg released from copolymer at 7-day time point; (d) EGCg released from copolymer at 30-day time point; EGCg released from copolymer at 60 time point ; and EGCg released from copolymer at 90-day time point.

53

Chapter VI: Discussion

The Bis-GMA monomer, with its high viscosity is still being used widely in dentistry. To obtain

adequate filler loading and ease in handling (Davy, Kalachandra, Pandain, & Braden, 1998), it is

often mixed with TEGDMA which has a relatively lower viscosity. Several polymers such as

Hydron (polymer of HEMA), ethylene vinyl acetate (EVA) and polyvinyl alcohol (PVA) have

shown the ability to release bio-active molecules for a period extending 100 days. The

phenomenon of ‘Fickian diffusion’, which is the controlled release of solutes from a region of

higher to a lower concentration caused by the concentration gradient, has been subject to

research for many years and applied also to the release of drugs or bioactive agents from dental

resins (Ritger & Peppas, 1987). A past study (Anusavice, Zhang, & Shen, 2006) has shown that

CHX release from UDMA-TEGDMA resin can be effectively controlled by providing an acidic

environment (lower pH) and by lowering the filler concentration of the resin.

The present results have shown that there is a statistically significantly difference in the amount

of drug released from different experimental resins, except for 0.5 × MIC CHX, thus rejecting

the first null hypothesis. The 24 hour mean drug release rates in deionized water were 7.4±1.7

µg/mL, 15.6±1.7 µg/mL , 188.7±21.5 µg/mL and 289.9±24.4 µg/mL for 0.5 × MIC CHX, 1.0 ×

MIC CHX, 0.5 × MIC EGCg and 1.0 × MIC EGCg respectively. All the resins presented a

significantly higher release rates in the first 24 hours, except for 0.5 × MIC CHX, due to ‘burst

release’, which is an inherent property of diffusion-controlled systems (Allison, 2008), followed

by a steady release up to day 90. The steady release after the initial ‘burst release’ is due to the

slow nature of diffusion into the resin matrix, requiring greater time for complete saturation

54

(Pearson, 1979). This is in agreement with the study conducted by Pallan and colleagues (2012)

who found similar results using three different resins. The fact that the highest amount of drug is

released within the first 24 hours for both CHX and EGCg and at two different ratios (0.5 × MIC

and 1.0 × MIC) compared to any other time points (24 hours, 7 days, 30 days, 60 days, and 90

days) is due to the hydrophilic nature of the Bis-GMA/TEGDMA resin. It is also due to the

release of surface bound drug from the micro voids present in the matrix (Wilson & Wilson,

1993) or due to drug not homogenously distributed in the sample (Kalachandra, Dongming, &

Offenbacher, 2002). This is in agreement with other studies that have shown similar results using

different drugs such as tetracycline, nystatin and minocycline (Hiraishi et al., 2008; Kalachandra,

Lin, Stejskal, Prakki, & Offenbacher, 2005; Riggs et al., 2000). The average amount of drug

released decreased from 24 hours to 90 days for all the drugs and their ratios.

The process of drug release is affected by many factors, such as type of monomer, degree of

conversion, cross linking density, type and concentration of drug, and extracting media (Hiraishi

et al., 2008; Riggs et al., 2000). The CHX released from polymeric dental materials depends on

the affinity of polymer matrix to water (Leung, Spratt, Pratten, Gulabivala, Mordan, & Young,

2005). In this study, the fact that CHX and EGCg incorporated into resins at 1.0 × MIC released

more drug than CHX at 0.5 × MIC is due to the higher amount of drug which is diffusing

through the polymer chains into the BHI medium. The TEGDMA with its linear ether linkages is

a very hydrophilic monomer that allows water to penetrate more efficiently into the resin matrix

(Prakki, Cilli, Vieira, Dudumas, & Pereira, 2012). This allows larger expansion of voids

between the polymer chains, allowing incorporated drugs to be easily released due to the osmotic

55

gradient between the resin matrix and external solution until an equilibrium is achieved between

the two (Riggs et al., 2000).

The CHX used in this study was incorporated into the polymer matrix via direct mixing with the

copolymer. As the drug is used as it is received from commercial sources, the effect of particle

size on the release characteristics is unknown. By grinding CHX particles frozen with liquid

nitrogen, 0.62 µm sized particles can be obtained from 40 µm sized received particles. The

smaller size of the particles is known to have an increasing effect on the release rates of cured

composites as well as on their antibacterial properties (Cheng et al., 2012).

The drug loading is another factor that is known to affect drug release and antibacterial activity.

The drug release rate is known to be directly proportional to drug loading. However, the nature

of polymer matrix is also known to affect drug release as a function of drug loading. By

changing the drug loading slightly (within the range of 4.5 wt% to 12 wt%), a significant

increase in drug release was noticed in a self-curing system based on poly ethyl methacrylate

(PEMA) and tetrahydrofurfuryl methacrylate (THFMA) (Patel et al., 2001). This is in contrast

with an ethyl methacrylate/hexyl methacrylate copolymer system, where increase in drug release

rates is noticed only by doubling the drug loading from 2.5 wt% to 5.0 wt% (Tallury, Airrabeelli,

Li, Paquette, & Kalachandra, 2008).

The results have shown that drug released from experimental resins containing either 1.0 × MIC

CHX and 1.0 × MIC EGCg at 60 and 90 days had a significant effect on bacterial growth

inhibition, thus rejecting the second null hypothesis. The bacterial viability assay results have

56

shown that, with the exception of 1.0 × MIC CHX and 1.0 × MIC EGCg , sub-MIC of CHX and

EGCg did not inhibit bacterial growth. This is very likely due to higher amounts of drug added to

the experimental resins. This compares well with a study conducted by Maezono and colleagues

(2011) in which erythromycin was found to be ineffective in inhibitng Porphyromonas gingivalis

at sub-MIC levels. Sometimes the effect of sub-inhibitory concentration of antimicrobial drugs is

controversial as Marani and Jamil (2011) have found that sub-MICs of vancomycin promoted

bacterial growth. Similar results were found using sub-MIC concentrations of several drugs such

as aminoglycosides, beta-lactams and macrolides on Pseudomonas aeruginosa (Hoffman et al.,

2005). In this study, almost all the experimental resins at 0.5 × MIC concentrations seem to

promote bacterial growth compared to placebo resins samples.

In the past CHX containing resins have been tested for bacterial growth inhibition using disc

diffusion method. In this sensitive method, test samples are applied directly onto the agar plates.

After which, bacterial growth inhibition was measured based on the appearance and diameter of

zones of growth inhibition around the test samples (Esteves, Ota-Tsuzuki, Reis, & Rodrigues,

2010; Hiraishi et al., 2008; Jedrychowski, Caputo, & Kerper, 1983). The interpretation of results

using disc diffusion method requires high precision (EUCAST, 2013). For the purpose of this

study, the experimental resins (placebo, CHX, EGCg) were immersed into a suspension of

S.mutans that best replicates the conditions in vivo. It was reported that by allowing to do so, the

polymerized resin samples absorb water, swell and release drugs by diffusion from all surfaces,

yet maintaining their antibacterial activity (Mankovskaia et al., 2013).

57

The antibacterial susceptibility testing is performed with either phenotypic or genotypic methods.

The genotypic method, which is to investigate the genetic constitution of an individual organism,

is useful for rapid and accurate determination of antibacterial resistance. However, due to lack of

standardization, cost and labor-intensiveness, genotyping assays have limited ability to detect

bacterial susceptibility accurately. In view of this, phenotypic approach is used and one of the

most commonly used phenotypic method for this purpose is the determination of minimum

inhibitory concentration (Louie & Cockerill, 2001). The MIC values for CHX and EGCg against

S.mutans were shown to be within the reference ranges of 0.25 - 4.0 µg/mL for CHX (Gronroos

et al., 1995; Kang, Oh, Kang, Hong, & Choi, 2008) and 31.25 – 625 µg/mL for EGCg (Xu et al.,

2011), depending on S.mutans and culture medium (solid agar).

The qualitative analysis of drug stability indicated that placebo, CHX and EGCg, remained

stable over time, thus rejecting the third null hypothesis. In this study, the stability of released

compounds into aqueous media was chosen to be analyzed by 1H NMR, which is treated like a

fingerprint of the chemical structure of the molecule (Kalachandra et al., 2005). The NMR

spectroscopy is commonly used to determine the structural stability of a molecule in a non-

destructive and non-invasive manner based on the chemical shift values. This is done by

exposing the sample to a strong magnetic field which acts on the spin active atomic nuclei,

creating an energy differential which can be measured. This change in energy is specific for each

nucleus in a molecule. It is also used in quality control in assessing the purity of mixtures of

molecules based on relative signal intensities. The presence or absence of a molecule can be

determined by comparing the NMR of a mixture with the NMR of pure compound (Nowicki &

Sem, 2011; Thomas & Sem, 2010). For complicated molecules, either a two-dimensional (2D)

58

NMR spectroscopy, continuous-wave spectroscopy, fourier transform spectroscopy, multi-

dimensional spectroscopy or a solid-state NMR spectroscopy may be used that employ a variety

of isotopes such as, 1H, 2H, 3He, 11B, 13C, 14N, 15N, 17O, 19F, 31P, 35Cl, 43Ca, 113Cd and 195Pt. For

the purposes of this study one-dimensional (1D) 1H NMR spectroscopy was conducted. The

proton NMR (1H NMR) is the most commonly used form of NMR spectroscopy because

hydrogen is abundantly present in the biological systems and has a nucleus which is highly

sensitive to NMR signals (Silverstein, Bassler, & Morrill, 1991).

Hrynash and colleagues (2014) evaluated the 1H NMR plot for CHX (Table 4) and it is used as a

reference in this study. The CHX molecule (C22H30CI2N10 . 2C2H4O2) with its characteristic

“doublet” pattern has shown peaks at 7.31 ppm and 7.14 ppm corresponding to positions 1 and 2

on the structure of CHX. The methylene proton adjacent to guanidine nitrogen is plotted at 3.08

ppm. The remaining peaks around 1.43 ppm and 1.24 ppm corresponded to the 8 methylene

protons in the hexane diamine linkage.

59

Table 4: Cleavage sites for chlorhexidine in 1H NMR

Chlorhexidine

Proton δ(1H) Molecular Structure

1 7.31

2 7.14

3 3.08

4 1.43

5 1.24

An article by Peres and colleagues (2010) is used as the reference for EGCg (C22H18O11) 1H

NMR plot (Table 5).

60

Table 5: Cleavage sites for epigallocatechin-3-gallate in 1H NMR

Epigallocatechin-3-gallate

Proton δ(1H) Molecular Structure

1,2 7.018

3,4 6.604

5,6 6.17

7 5.567

8 2.984

In this study deuterium oxide was used because the solvent of interest is water (dehydrated

residue after lyophilization) and the nuclide of interest is hydrogen. This is because the signals

from the water solvent may interfere with the signals from the molecule of interest. By adding

D2O, some of the labile hydrogen atoms present on a compound are exchanged or substituted by

deuterium (2H) atoms from D2O which do not show up on a 1H NMR spectrum, due to a

different magnetic moment, thereby effectively suppressing water signals (Price, 1999).

However, a strong line at ∼4.65 ppm is observed in all the 1H NMR plots. This is because

deuterated solvents are magnetically active and hygroscopic in nature, which meant that a small

amount of residual water is present in NMR solvents. Also, the samples contained

exchangeable protons such as OH (EGCg) and NH2 (CHX) groups (RSC, n.d.). For both CHX

and EGCg all the expected signals were observed in the leached media as compared to the

61

NMR of the pure compounds, which suggests the stability of the released compounds (Hrynash

et al., 2014; Peres et al., 2010). Other signals observed might correspond to non - reacted

monomers and impurities released by the comonomer.

6.1 Policy Implications

There are still several drawbacks such as longevity and risk of secondary caries that make

choosing composite resin a dilemma. Most amalgam restorations can be expected to serve

clinically for 10 to 12 years, while resin-based composite restorations are expected to last on

average 7.8 years (Van Nieuwenhuysen, D'Hoore, Carvalho, & Qvist, 2003). There is a problem

to solve when studies in the United Kingdom suggest that 70% of all procedures in general

dental practice is restoration replacement and 60% of those are due to secondary caries (Chen,

Shen, & Suh, 2012). In 2001, it was estimated that in United States the cost of restoration

replacement was $5 billion/year and in Canada $500 million/year (Jokstad, Bayne, Blunck, Tyas,

& Wilson, 2001). Resins also tend to fail in high caries risk or poor periodontal conditions,

requiring retreatment, which only exacerbates the already high costs associated with it.

Dental caries is a condition that is widespread, since more than 50% of Canadian children in the

age group 6-19 years have had a cavity and 96% of adults have had a history of tooth decay

(Health Canada, 2010). It can be deduced that some of these cases may be due to restoration

failure caused by secondary caries, and since composites resins carry a greater risk of secondary

caries compared to amalgam, and since their usage is increasingly common, restoration failure

due to secondary caries is therefore a condition that is likely very prevalent. Dental caries is also

62

a potential cause of morbidity, since an estimated total of 40.36 million hours were lost from

normal activities, school or work during a period of 12 months due to oral diseases and

conditions (Health Canada, 2010). Some of that estimated lost time may be due to re-treatment

of secondary caries due to composite resin placement. Lastly, due to the high costs and less

returns on longevity, durability and strength associated with composites compared to amalgam,

there is a perception on part of the public, government, or dental public health authorities, that

there exists a public health problem from a financing perspective in the absence of public and

practitioner acceptance of a cost-effective alternative, i.e. amalgam. The current dilemma with

the usage of dental composites satisfies the definition of a public health problem as defined by

Burt and Eklund (2005), signifying the importance of the need to improve the properties of

dental composites such that they deliver ‘the best bang for their buck.’

From this research several policy implications concerning dental composite resins are evident.

There are various political, public, socio-cultural and professional factors that have contributed

to making composite resins one of the most preferred restorative materials of choice. For

example, there is a move towards mercury-free health centers, which includes dental clinics, in

much of Europe and in North America. However, this will have implications for the financing of

composite resins by private insurers and public programs, and may have implications for access

to dental care for publicly insured patients needing care who may only be eligible for amalgam

restorations under public schemes (Rustagi & Singh, 2010).

The last decade has witnessed an increase in the usage of composites despite its clinical

contraindications, such as its use in patients with high caries risk or poor hygiene (Bernardo et

63

al., 2007). Due to the costs associated with increased usage, composite restorations are driving

dental expenditures in Canada. In 2008/09, for example, one of Canada’s largest public dental

care programs, the Non-Insured Health Benefits (NIHB), had dental expenditures ($176.4

million) which ranked as the third highest, preceded by pharmacy costs ($419 million) and

medical transportation costs ($ 275 million). Among the dental related expenses, restorative

services (crowns, fillings, etc) were the highest, which saw an increase of 12.6% over the

previous fiscal year. Interestingly, posterior and anterior composite restorations saw an increase

of 15.5% compared to the previous fiscal year and a seven-fold increase compared to those for

amalgam restorations in the same year (Health Canada, 2012). This clearly reflects the patient

and practitioner preference in choosing dental composites over amalgam. Although composites

offer a choice for the patient and clinician, from a public financing perspective and also to

certain extent private insurer perspective, the rising costs due to composite resins will have

budgetary implications given current trends in usage. Yet if the incorporation of antibacterials

into composite resins represents an additional cost that translates into increased longevity and

clinical stability, they may eventually prove a cost-effective option for funders.

64

Chapter VII: Recommendations and future directions

1. The evaluation of drug release and its effectiveness in different media (i.e., saliva or artificial

saliva) should be performed.

2. It is known that CHX, as a 2% gel, has a known mild toxicity towards odontoblast-like cells

(Lessa et al., 2010). Such an effect using EGCg must also be evaluated in future studies before it

can be considered as a viable alternative to CHX.

3. The 90-day CHX and EGCg drug release rates, anti-bacterial activity and drug stability is

presented in this study. It would be interesting to examine the effectiveness, either synergistic or

antagonistic, by combining CHX and EGCg.

4. In order to better replicate the conditions within the oral cavity, a constant depth film

fermentor (CDFF) should be used as it is particularly suited for studying multi-species biofilms

similar to those in the oral cavity.

5. The composite restorations consist of two major parts: composite resin and bonding agent.

Since the bonding agent is responsible for disinfecting the cavity preparation, it is logical to

incorporate the same antibacterials used in this study and test for its drug release, bacterial

viability and drug stability.

65

Chapter VIII: Conclusion

1. The drug release rates varied significantly within the 24 hours, 7 days, 30 days, 60 days and 90

days time periods, except for 0.5 MIC × CHX, and higher drug concentration was associated

with higher release rates.

2. The placebo groups and groups with lower drug concentrations did not inhibit bacterial

growth. The bacterial growth inhibition was higher for CHX than for EGCg.

3. The chemical structure of the placebo and the groups with both drugs (CHX and EGCg) at two

different concentrations (0.5 × MIC and 1.0 × MIC) was found to be stable at 24 hours, 7 days,

30 days, 60 days and 90 days time periods.

66

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