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

by

Vinay Kumar Pilly Yadaiah BDS, MPH

© Vinay Kumar Pilly Yadaiah All Rights Reserved (2014)

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Master of Science

2014 Vinay Kumar Pilly Yadaiah

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

ABSTRACT

Composite resins continue to evolve and are increasingly favoured by the people. However,

drawbacks such as decreased longevity, secondary caries and costs make choosing composites a

dilemma. This study evaluated drug release, inhibitory growth against Streptococcus mutans and

drug stability of epigallocatechin-gallate (EGCg) incorporated into 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: placebo, CHX and EGCg.

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

incorporated into paste resins and tested at time points: 24 hours, 7 days, 30 days, 60 days and 90

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

different drugs and drug ratios, which showed stability after 90 days. The results indicate that

drug-based composites may reduce bacterial growth, which may improve its longevity.

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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" (English: Truth Alone Triumphs) derived from the ancient Indian scripture

of Mundaka Upanishad 3.1.6. 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

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

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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 .................................................................................................................................. x

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 .......................................................... 12

2.3.3 Antibacterial dental composites containing silver compounds .................................... 13

2.3.4 Antibacterial dental composites containing physically immobilized quarternary ammonium salts compounds (QAS) ...................................................................................... 14

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

2.3.6 Other quarternary ammonium monomethacrylates/dimethacrylates ............................ 15

2.4 Chlorhexidine (CHX) .......................................................................................................... 16

2.5 Epigallocatechin-3-gallate (EGCg) ..................................................................................... 18

Chapter III: Objectives and Null Hypothesis ................................................................................ 20

3.1 Objectives of the study ........................................................................................................ 20

3.2 Null Hypothesis ................................................................................................................... 21

Chapter IV: Materials and Methods .............................................................................................. 22

4.1 Experimental Design ........................................................................................................... 22

4.2 Determination of minimum inhibitory concentration (MIC) .............................................. 23

4.3 Preparation of experimental resin ....................................................................................... 23

4.4 Preparation of resin samples ............................................................................................... 24

4.5 Determination of drug release rates .................................................................................... 24

4.5.1 Visible spectroscopy using glass quartz ....................................................................... 24

4.5.2 Determination of standard curve: CHX ........................................................................ 25

4.5.3 Determination of standard curve: EGCg ...................................................................... 26

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4.6 Bacterial viability assay ...................................................................................................... 27

4.6.1 Overnight culture of the bacteria .................................................................................. 27

4.6.2 Preparation of phosphate buffered saline (PBS) ........................................................... 28

4.6.3 Preparation of Brain Heart Infusion (BHI) broth ......................................................... 28

4.6.4 Preparation of BHI agar plates ..................................................................................... 28

4.6.5 Release of resin unit into BHI broth and overnight bacterial culture under constant agitation ................................................................................................................................. 29

4.6.6 Bacterial Culture Sonication ......................................................................................... 30

4.6.7 Micro broth dilution...................................................................................................... 31

4.6.8 Colony Forming Units (CFU) determination ............................................................... 31

4.7 Determination of drug stability ........................................................................................... 32

4.7.1 Lyophilization ............................................................................................................... 32

4.7.2 1H NMR spectroscopy .................................................................................................. 33

4.8 Data analysis ....................................................................................................................... 35

Chapter V: Results ........................................................................................................................ 36

5.1 Drug release......................................................................................................................... 36

5.2 Bacterial viability ................................................................................................................ 39

5.3 Drug stability ....................................................................................................................... 43

Chapter VI: Discussion ................................................................................................................. 54

6.1 Policy Implications .............................................................................................................. 63

Chapter VII: Recommendations and future directions ................................................................. 66

Chapter VIII: Conclusion .............................................................................................................. 67

References ..................................................................................................................................... 68

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List of Figures

Figure 1: UV-Vis spectrophotometer ............................................................................................ 25

Figure 2: Standard Curve for CHX ............................................................................................... 26

Figure 3: Standard curve for EGCg .............................................................................................. 27

Figure 4: Rotating laboratory mixer ............................................................................................. 29

Figure 5: Sonicator ........................................................................................................................ 30

Figure 6: Enumeration of bacteria by serial dilution .................................................................... 32

Figure 7: Lyophilizer .................................................................................................................... 33

Figure 8: 1H NMR spectrometer ................................................................................................... 34

Figure 9: Mean drug release rates plotted against time for CHX ................................................. 37

Figure 10: Mean drug release rates plotted against time for EGCg .............................................. 38

Figure 11: Cumulative drug release plotted against time for CHX .............................................. 38

Figure 12: Cumulative drug release plotted against time for EGCg ............................................. 39

Figure 13: Bacterial viability at 24 hours ..................................................................................... 40

Figure 14: Bacterial viability at 7 days ......................................................................................... 41

Figure 15: Bacterial viability at 30 days ....................................................................................... 41

Figure 16: Bacterial viability at 60 days ....................................................................................... 42

Figure 17: Bacterial viability at 90 days ....................................................................................... 42

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

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 ........... 49

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....................................................................................................................................................... 53

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List of Tables

Table 1: Components of phosphate buffered saline solution (200 mL) ........................................ 28

Table 2: Average drug release rates (µg/mL) ............................................................................... 36

Table 3: Bacterial viability at different drug ratios and time points ............................................. 40

Table 4: Cleavage sites for chlorhexidine in 1H NMR ................................................................. 61

Table 5: Cleavage sites for epigallocatechin-3-gallate in 1H NMR .............................................. 62

x

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

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well as for the possibility of performing minimally invasive restorative techniques (Health

Canada, 2012; Lynch, Frazier, McConnell, Blum, & Wilson, 2011). 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

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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 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-

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modified 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

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viewed as a substitute for amalgam 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

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characteristics, wear resistance, polishability and aesthetics. The most recent research also

encompassed common drawbacks 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

8

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.

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

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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 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

10

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 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).

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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).

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).

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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 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).

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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 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).

14

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).

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

15

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 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).

16

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).

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,

17

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.

18

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

19

found that EGCg is capable of non-competitive 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.

20

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.

21

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.

22

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.

23

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, 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

24

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

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

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

25

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 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.

26

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.

27

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.

28

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 dispensed onto the petri dishes (100 × 15mm). A thin layer of BHI agar

29

(~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

30

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

31

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.

32

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

33

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

34

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

35

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).

36

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 ± 1.7

a 188.7 ± 21.5

a 289.9 ± 24.4

a

7 days 0.0 6.1 ± 2.2a 8.5 ± 1.4

b 76.5 ± 11.5

b 159.4 ± 18.2

b

30 days 0.0 7.2 ± 3.1a 10.8 ± 2.3

b 94.9 ± 10.8

b,c 219.1 ± 29.2

a,b

60 days 0.0 6.4 ± 2.0a 10.0 ± 1.3

b 69.7 ± 9.6

b 177.7 ± 42.1

b

90 days 0.0 5.8 ± 1.1 a 10.3 ± 1.3

b 46.7 ± 5.9

b,d 117.3 ± 29.1

b

*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.

37

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

38

Figure 10: Mean drug release rates plotted against time for EGCg Figure 11: Cumulative drug release plotted against time for CHX

39

Figure 12: Cumulative drug release 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

40

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 ± 2.98

a 116.99 ± 23.81

a 156.86 ± 32.82

a

7 days 100.00 243.4 ± 229.68a 0.11 ± 0.06

b 207.97 ± 204.71

a 97.19 ± 56.45

a,c

30 days 100.00 137.24 ± 28.08a 0.18 ± 0.03

b 141.42 ± 84.21

a 77.03 ± 47.99

a,c

60 days 100.00 130.80 ± 22.56a 0.41± 0.47

b 93.16 ± 8.85

a 52.55 ± 11.09

b,c

90 days 100.00 96.23 ± 17.76a 0.37 ± 0.08

b 123.85 ± 17.99

a 28.08 ± 4.48

b,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

41

Figure 14: Bacterial viability at 7 days

Figure 15: Bacterial viability at 30 days

42

Figure 16: Bacterial viability at 60 days Figure 17: Bacterial viability at 90 days

43

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.

44

45

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.

46

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.

47

48

49

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

50

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.

51

52

53

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

54

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±1.7µg/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,

55

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 (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

56

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 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,

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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

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, &

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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).

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

59

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) 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

60

“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.

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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).

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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

adifferent 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

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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

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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 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

65

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 al., 2007). Due to