a study to investigate antibacterial activity and...
TRANSCRIPT
A STUDY TO INVESTIGATE ANTIBACTERIAL ACTIVITY
AND PHYSICAL PROPERTIES OF NEWLY DEVELOPED
CHITOSAN BASED DENTAL COMPOSITES
A Thesis
Submitted to Medical Research Center
Liaquat University of Medical & Health Sciences
Jamshoro Sindh, Pakistan
For the Degree
Of
Doctor of Philosophy
(Science of Dental Materials)
By
Dr Shahid Ali
BDS
Under the Supervision of
Dr Naresh Kumar
PhD
2016
i
ii
ABSTRACT
Across the globe dental RBCs are being widely used by clinicians for restoration of posterior teeth.
This increased use of RBCs is due to concerns about safety of amalgam among clinicians as well
as researchers. However, RBCs clinical failure due to secondary caries has been widely reported.
This failure may be attributed to the tendency of RBCs to accumulate more dental plaque compared
to other restorative materials.
The potential solution for this problem is to modify and improve antibacterial properties of existing
RBCs. Therefore various soluble and other insoluble or immobilized antimicrobial agents have
been added to RBCs in order to achieve optimum antibacterial activity without compromising
physical properties. However several studies have reported negative effect of antimicrobial agents
on physical and mechanical properties of RBCs.
Chitosan (CS) a natural polysaccharide present in crustaceans’ shells has been used in various
commercial products including tooth pastes, mouth washes and desirable antibacterial activity has
been reported. Therefore, in this study 0.25, 0.5 and 1 % CS was incorporated into commercial
RBCs to equip the material with antimicrobial activity and subsequently reduce the rate of
secondary caries and increase the durability of restoration.
This study determined antibacterial activity of CS based and control RBCs via agar disc diffusion
test (ADT) and direct contact test (DCT) method against three test bacteria including Streptococcus
mutans, Lactobacilli casei and Actinomyces viscous. However, it was found that CS based RBCs
had no antibacterial effect against these test bacteria.
iii
The mean water sorption and solubility values of control and experimental flowable as well as
micro hybrid RBCs were found to be within ISO accepted range. In addition, flowable RBCs
containing 1 % CS had significantly higher hardness values compared to control and other
experimental flowable RBC groups. The micro hybrid RBCs consisting of 0.50 % CS exhibited
significantly higher hardness values compared to experimental micro hybrid RBC group
containing 1 % CS.
Key words: chitosan, resin based composites, antibacterial activity, physical properties
iv
ACKNOWLEDGEMENTS
I praise Almighty Allah, who is all knowing and source is of wisdom. Many Durood Sharif for
Holy Prophet Hazrat Muhammad (PBUH), who is a great guide for the mankind forever.
I am glad to say my sincere thanks to my supervisor and mentor Dr. Naresh Kumar for his
valuable supervision and guidance during research work. Indeed, his scholarly comments and
corrections during thesis write up enabled me to successfully complete this task.
I would like to thank staff of Dental Materials department LUMHS Jamshoro for their moral
support especially Assistant Professor Dr. Mohammad Muslim, Lecturer Dr Waqas and dental
technician Mr. Shahid Ali. I would to like to thank Mr. Falak, Mr. Robin as well.
I am indebted to Dr. Ali Mohammad Waryah and his research team for continuous moral support
during my experimental work in research lab, medical research center. In addition, I would like to
thank Mr. Qamar Bhatti lab technician for his assistance in antimicrobial activity testing. I am
obliged to Dr. Kefi Iqbal for his assistance in preparation of composite specimen mould.
I am thankful to all PhD faculty members of LUMHS Jamshoro for moral support and guidance
especially Professor Aneela Atta u Rehman, Dr Samreen, Dr Binafsha, Dr Ameer, Dr
Suleman and Dr Baby Kanwal. Moreover, I am thankful to staff and administration of medical
research center for their assistance.
Furthermore, I would appreciate company of my all PhD colleagues especially Dr Abdul Bari
Memon, Dr Vikram Pal, Dr Sikander Memon, Dr Shakeel Shaikh, Dr Yaqoob Shahani and Dr
Asif. In addition, I would like to thank Dr Syed Yousuf Shah and Dr Ameen for motivation and
moral support.
v
I would like to acknowledge care and affection which I always received from my loving parents Mr.
and Mrs. Amanullah. They have always been great source of inspiration for me. I am thankful to my
siblings for their continuous moral support and love. I would like to thank my lovely brother Dr. Dildar
Ali who inspired me to pursue PhD; indeed he has been very kind and generous to me during all testing
times of this PhD journey.
I would like to thank and appreciate Rahila (my wife) and her family for motivation and best wishes
during this academic journey, without their moral support I would never have achieved this milestone.
I would always remain very grateful to them for their unconditional support and encouragement during
my stay at Hyderabad.
Last but not the least; I would like to thank Shaheed Mohtarma Benazir Bhutto Medical University
(SMBBMU) Larkana for allowing me to pursue PhD, special thanks to Prof Abdul Hakim Arain Ex
Principal Bibi Aseefa Dental College Larkana for his motivation and moral support.
vi
DEDICATED TO MY PARENTS AND TEACHERS
vii
Section
Page
number
Chapter 1 Development of resin based dental composites (RBCs)
1.1 Historical development of Resin based composites (RBCs) 1
1.2 Composition of Resin based composites (RBCs) 5
1.2.1 Resin matrix 5
1.2.2 Fillers and coupling agent 7
1.2. 3 Photo initiators 8
1.2.4 Inhibitors 10
1.2.5 Pigments and Ultra violet (UV stabilizers) 10
1.3 Classification of Resin based composites (RBCs) 10
1.3.1 Traditional or Conventional Resin based composites (RBCs) 12
1.3.2 Micro filled Resin based composites (RBCs) 12
1.3.3 Hybrid Resin based composites (RBCs) 13
1.3.4 Nanofilled Resin based composites (RBCs) 14
1.3.5 Flowable and Packable Resin based composites (RBCs) 15
1.3.6 Chapter Summary 17
Chapter 2
Literature Review
2.1 Introduction 19
2.2 Secondary Caries 21
2.2.1 Micro leakage and Secondary Caries 23
TABLE OF CONTENTS
viii
2.2.2 Prevention of Secondary Caries 24
2.3 Treatment of Caries 24
2.4 Antibacterial agents in Resin based composites (RBCs)
25
2.5 Introduction of Chitosan 36
2.5.1 Antimicrobial properties of chitosan 38
2.5.2 Biocompatibility and safety of chitosan 40
2.6 Antibacterial activity testing methods for Resin based composites
(RBCs)
42
2.7 Physical Properties of Resin based composites (RBCs) 43
2.8 Unanswered Questions in Literature 45
2.9 Aim of Study 46
2.10 Objectives 46
2.11 Hypothesis 46
Chapter 3
Material and Methods
3 Introduction 48
3.1 Materials used during this research work 48
3.2 Methodology 50
3.2.1 Experimental Resin based composites (RBCs) preparation 50
3.2.2 The Resin based composites (RBCs) specimen preparation 53
3.3 Test Microorganism Growth 57
3.3.1 Streptococcus mutans Growth 57
3.3.1.1 Mitis Salivarius Agar (MSA) Preparation 57
ix
3.3.1.2 Brain Heart Infusion (BHI) Broth Preparation 57
3.3.1.3 Phosphate buffer saline (PBS) Preparation 58
3.3.1.4 Inoculation and incubation of Streptococcus mutans 58
3.3.1.5 Transfer of bacterial culture 61
3.3.1.6 Centrifuge and vortex 61
3.4 Preparation of 0.5 Mc Farland turbidity standards 62
3.5 Growth of Lactobacilli casei 65
3.6 Growth of Actinomyces viscous 67
3.7 Antibacterial activity test 69
3.7.1 Agar disc diffusion test (ADT) 69
3.7.2 Direct contact test (DCT) 71
3.7.2.1 Miles-Misra technique 73
3.8 Water sorption and solubility 75
3.9 Vickers Hardness test 79
3.10 Statistical Analysis 79
Chapter 4 Results
4.1 Antibacterial activity test 82
4.2 Water sorption and solubility 92
4.3 Vickers hardness 95
Chapter 5 Discussion
5 Discussion 99
5.1 Antibacterial activity 99
5.2 Water sorption and solubility 102
x
5.3 Vickers hardness 105
5.4
5.5
Conclusion
Future work
108
109
REFERENCE 110
xi
DECLARATION
I hereby declare that this thesis comprised of my original research work. However, different
sources of information have been used and are acknowledged. This thesis consists of total five
Chapters. I have not submitted this work before for any degree Program.
xii
LIST OF MAIN ABBREVIATIONS
Abbreviation Full form
ADT Agar disc diffusion test
ANOVA Analysis of variance
ATCC American type culture collection
BHI Brain heart infusion
BisGMA Bisphenol A glycidyl dimethacrylate
Bis-EMA Ethoxylated bisphenol A dimethacrylate
CFU Colony forming unit
CHX Chlorhexidine
CQ Camphorquinone
CS Chitosan
DCT Direct contact test
DD Degree of deacetylation
DMAEMA Dimethyl amino ethyl methacrylate
DMAEMA N,N- dim ethyl amino ethyl methacrylate
EGDMA Ethylene glycol dimethacrylate
HEMA 2 hydroxy ethyl methacrylate
LED Light emitting diodes
MBC Minimum bactericidal concentration
MDPB Methacry-loyloxydodecylpyridinium bromide
MIC Minimum inhibitory concentration
MPTMS 3-methacryloxypropyltrimethoxysilane
xiii
MRS deMan, Rogosa and Sharpe
MSA Mitis Salivarius Agar
MW Molecular weight
PBS Phosphate buffer saline
PEGDMA Poly ethylene glycol di methacrylate
PEI Poly ethylene imines
PMMA Poly methyl methacrylate
QTH Quartz tungsten halogen
RBCs Resin based composites
SD Standard deviation
TEGDMA Triethylene glycol dimethacrylate
UDMA Urethane dimethacrylate
UV Ultra-violet
v/v Volume by volume
w/v Weight by volume
γ-MPTS γ-methacryloxypropyltrimethoxysilane
xiv
LIST OF FIGURES
Figure Page number
3.1 Analytic weight balance 51
3.2 RBCs specimen aluminum mould 54
3.3 Elipar LED curing light 54
3.4 RBCs specimen discs for antibacterial activity test (ADT) 55
3.5 RBCs specimen teflon mould 55
3.6 RBCs specimen discs for antibacterial activity test (DCT) 56
3.7 Streptococcus mutans kwik stik 59
3.8 CO2 Incubator 59
3.9 Streptococcus mutans growth on MSA agar plate 60
3.10 Gram stain slides 60
3.11 Wire loop and Flame 63
3.12 Centrifuge machine 63
3.13 Vortex equipment 64
3.14 Mcfarland 0.5 turbidity tube and bacterial suspension 64
3.15 Growth of Lactobacilli casei on MRS agar plate 66
3.16 Growth of Lactobacilli casei in MRS broth 66
3.17 Growth of Actinomyces viscous on BHI agar plate 68
3.18 Growth of Actinomyces viscous in BHI broth 68
3.19 The RBCs specimen placed on surface of inoculated agar for ADT 70
3.20 RBCs specimen fit into bottom of eppendorf tube for DCT 72
xv
3.21 The CFU of Lactobacilli casei via miles and misra technique 74
3.22 RBCs specimen in distilled water 77
3.23 RBCs specimen in desiccator 77
3.24 Micro meter screw gauge 78
4.1A No inhibition zone is evident around experimental flowable RBCs
groups against Streptococcus mutans
83
4.1B No inhibition zone is evident around experimental micro hybrid
RBCs groups against Streptococcus mutans
83
4.2A No inhibition zone is evident around experimental flowable RBCs
groups against Lactobacilli casei
84
4.2 B No inhibition zone is evident around experimental micro hybrid
RBCs groups against Lactobacilli casei
84
4.3A No inhibition zone is evident around experimental flowable RBCs
groups against Actinomyces viscous
85
4.3B No inhibition zone is evident around experimental micro hybrid
RBCs groups against Actinomyces viscous
85
xvi
LIST OF TABLES
Table Page
1.1 Summary table of different Resin based composites (RBCs) used in
clinical practice with their advantages and disadvantages
4
1.2 Classification of Resin based composites (RBCs) 11
2.1 Antibacterial agents incorporated into RBCs, type of study and
antimicrobial effects
26-28
3.1 Composition of Resin based composites (RBCs) used in present study 52
4. 1 CFU count of Streptococcus mutans bacteria per 20 µl after 48 hours of
contact with experimental flowable RBCs
86
4. 2 CFU count of Streptococcus mutans bacteria per 20 µl after 48 hours of
contact with experimental micro hybrid RBCs
87
4.3 CFU count of Lactobacilli casei bacteria per 20 µl after 48 hours of
contact with experimental flowable RBCs
88
4.4 CFU count of Lactobacilli casei bacteria per 20 µl after 48 hours of
contact with experimental micro hybrid RBCs
89
4.5 CFU count of Actinomyces viscous bacteria per 20 µl after 48 hours of
contact with experimental micro hybrid RBCs
90
4.6 CFU count of Actinomyces viscous bacteria per 20 µl after 48 hours of
contact with experimental flowable RBCs
91
xvii
4.7 Mean water sorption & solubility values of experimental flowable RBCs 93
4.8 Mean water sorption & solubility values of experimental micro hybrid
RBCs
94
4.9 Mean vicker hardness values of experimental flowable RBCs 96
4.10 Mean vicker hardness values of experimental micro hybrid RBCs 97
xviii
CHAPTER 1
Development of Resin based dental composites (RBCs)
1
1.1 Historical Development of Resin based composites (RBCs)
In the late 1930s, poly methyl methacrylate (PMMA) was introduced for fabrication of denture
base. Initially heat cured PMMA denture base material namely Vernoite (Rohm and Hass,
Philadelphia, Pennsylvania) was made commercially available. The PMMA was also used for
fabrication of indirect restorations such as inlays, crowns and bridges (Peyton, 1943). In Germany
during early 1940s, the discovery of the benzoyl peroxide-tertiary amine redox initiator-accelerator
system (Henriks Eckerman et al. 2004) facilitated the marketing of chemical or self-polymerizing
PMMA at room temperature, which paved the way for its use as direct filling material (Philips,
1982).
The PMMA exhibited drawbacks such as high coefficient of thermal expansion, serious pulp
damage, color instability, high polymerization shrinkage leading to marginal leakage and high
incidence of recurrent caries. In addition, PMMA was not strong enough to support masticatory
forces (Bayne and Thompson 2013). These demerits of PMMA motivated R.L. Bowen to develop
other synthetic resins for use as a direct restoration. Subsequently, a few indirectly placed heat-
cured epoxy resin restorations were developed which exhibited good esthetics. However, slow
setting of epoxy resins prevented their use as a direct filling material (Peutzfeldt, 1997). Later on
R.L Bowen synthesized a new high molecular weight difunctional monomer known as bisphenol
A glycidyl dimethacrylate (BisGMA or Bowens resin). This new monomer was strong with high
elastic modulus, had lower polymerization shrinkage and quicker setting compared to PMMA and
2
epoxy resin. This newly developed resin resembled epoxy resin except that the epoxy groups were
replaced by methacrylate groups. The synthesis of BisGMA resulted in the production of
commercial resin-based composites (RBCs) containing inorganic fillers (Bowen, 1958). In 1970
another difunctional monomer urethane dimethacrylate (UDMA) was adopted for use in RBCs
(Burgess et al. 2002). The BisGMA and UDMA continue to be most frequently used resins in
existing commercial RBCs. These resins exhibited low polymerization shrinkage of 6.1 and 6.7
volume %, and formed polymer networks with suitable mechanical charactistics. Because of
highly viscous nature of BisGMA and UDMA addition of adequate content of filler in resin
matrices was not possible. Therefore, low viscosity resins such as ethylene glycol dimethacrylate
(EGDMA) and triethylene glycol dimethacrylate (TEGDMA) have been used to dilute these
viscous resins and ensure addition of ample quantity of fillers (Moszner N and Hirt T 2012).
The earliest commercial RBCs included Adaptic (Johnson and Johnson, New Brunswick, N.J,
USA) and Addent RBCs (3M ESPE Dental USA). These RBCs were chemically cured two paste
system and were made available after pioneering work of Robert Chang and Henry lee in late
1960s (Chang R, 1969; Lee H, 1970). However, chemically activated RBCs had inferior properties
such as no control on working time, color instability and poor strength. Therefore, to solve such
problems light cured RBCs were introduced in 1970, these RBCs exhibited superior strength and
color stability compared to chemical activated RBCs (Powers et al. 1978). The superior strength
and color stability of light activated RBCs were attributed to decreased oxygen incorporation
compared to self-curing RBCs. Initial photo activated RBCs consisted a photo initiator namely
benzon methyl ether and required ultra-violet (UV) light to initiate polymerization. However the
harmful biologic effects of UV light were reported, consequently low energy radiation visible light
3
cured RBCs containing camphorquinone (CQ) initiator and co-initiator namely dimethyl amino
ethyl methacrylate (DMAEMA) were developed (Dart et al. 1978).
The properties of RBCs depend not only on proportion of its various components but also on nature
of the interfaces between these components. Without strong internal interfaces the clinical
performance of RBCs is poor. Therefore, to develop durable RBCs, silica filler particles were
chemically coated with a coupling agent; so that their surfaces could be closely bonded to resin
matrix during curing. The preliminary coupling agent used by R.L Bowen was tris 2-
methoxyethoxy vinyl silane (Bowen, 1962). The coupling agent chemically bridges the interfaces
of fillers and resin matrix. The functional groups at the ends of the coupling agent must match
chemically with the resins and fillers on its either side (Bowen, 1963). The coupling agent 3-
methacryloxypropyltrimethoxysilane (MPTMS) has been employed to coat the filler particle
surfaces in commercial RBCs (Antonucci JM et al. 2005).
Initially, almost all commercial RBCs had filler particle sizes of approximately 10 to 50 μm
diameter range. Since the early filler particles were relatively large, RBCs based on those large
fillers became known as macro filled RBCs. These earliest RBCs had superior strength but were
poor to polish. Therefore, desired surface smoothness was not possible to achieve, this led to
aesthetically inferior restoration (Khaled AN, 2011). To solve this problem, micro filled RBCs
were introduced around 1977 (Ferracane, 2010). The micro filled RBCs were good to polish which
resulted in smooth surface. This smooth surface of micro filled RBCs resisted deposition of stains
hence exhibited improved color stability. In addition, micro filled RBCs had improved wear
resistance (Schulein, 2005). By 1980 to meet the strength and aesthetic requirements of RBCs,
combination of fillers with variable size was attempted (Ferracane, 2010). This led to development
of hybrid universal RBCs suitable for restoration of both anterior and posterior teeth. The universal
4
RBCs comprise a mixture of minifillers and nanofillers. These combinations of fillers improve
wear resistance, strength, appearance and polymerization shrinkage of RBCs (Moszner N and Hirt
T 2012). The different RBCs used in clinical practice have been summarized in table 1.1 with their
advantages and disadvantages.
Table 1.1: Summary table of different Resin based composites used in clinical practice with
advantages and disadvantages
Type of Resin based
composites (RBCs)
Advantages Disadvantages
Chemically activated
Resin based
Composites
No need of Light for curing
Could be used in areas not
accessible to light
Increased polymerization
shrinkage
No control on working time
Poor aesthetics
Poor strength
Increased plaque accumulation
leading to secondary caries
Light activated Resin
based Composites
Superior strength
Superior aesthetics
Control on working time
Concerns about biologic harmful
effects of curing light
Polymerization shrinkage
Flowable Resin based
Composites
Desired handling Increased polymerization
shrinkage
Packable Resin based
Composites
Decreased polymerization
shrinkage
Superior strength
Superior aesthetics
Polymerization shrinkage
Concern for materials adaptation in
line and point angles of tooth
cavity
5
1.2 Composition of Resin based Composites (RBCs)
Modern RBCs comprise of hard inorganic filler particles bound together by soft resin matrix and
a coupling agent. The resin matrix consists of a monomer system, an initiator system and
stabilizers. The inorganic filler consists of particulates such as glass, quartz, and/or fused silica
and the coupling agent such as MPTMS that chemically bonds the reinforcing filler to the resin
matrix. The clinical performance of RBCs depends upon nature of its constituents. Some of the
RBCs properties are mainly related to the filler and the coupling agent, whereas others to the resin
matrix. The strength, high elastic modulus, abrasion resistance and coefficient of thermal
expansion mainly depend on fillers and coupling agent. While color stability may be attributed to
resin matrix. Polymerization shrinkage and water sorption are influenced by both fillers and resin
matrix (Bayne and Thompson 2013: Khaled AN 2011; Ferracane, 2010).
1.2.1 Resin Matrix
The resin matrix of most of existing commercial RBCs comprises a mixture of conventional
dimethacrylate. The BisGMA and UDMA are most widely used dimethacrylate which exhibit low
polymerization shrinkage during curing. The BisGMA and UDMA based RBCs have suitable
mechanical properties. Since the BisGMA and UDMA resins are highly viscous. Therefore
manufacturers add TEGDMA and EGDMA to reduce viscosity and improve handling (Moszner
N and Hirt T 2012).
Another alternative resin is the ethoxylated version of BisGMA known as ethoxylated bisphenol
A dimethacrylate (BisEMA). The hydroxyl groups of BisEMA reduce viscosity and allow higher
degree of conversion and better mechanical properties (Gajewski VES et al. 2012). Since the
6
polymerization of RBCs involve a degree of shrinkage depending on the resin matrix. Hence,
several attempts have been made to address polymerization shrinkage problem of existing RBCs.
Apart from traditional dimethacrylate resin matrix new siloxane-oxirane based resins have been
patented by 3M-ESPE company (Tilbrook DA, 2000). These resins manage to achieve 90-100%
conversion by reducing the carbon to carbon double bonds. These siloxane-oxirane resins utilize
an innovative approach of ring-opening chemistry (Ernst et al. 2004; Weinmann et al. 2005). These
resin monomers connect by opening, flattening and extending towards each other which result in
a significantly lower polymerization shrinkage. In contrast traditional methacrylate which are
linear, connect by actually shifting closer together which results in a greater shrinkage (Eick JD et
al, 1993). Despite introduction of several new resins, the manufacturers of commercial RBCs still
utilize traditional resin matrix systems such as BisGMA/TEGDMA monomer or
BisGMA/UDMA/TEGDMA combination (Hervás García A et al. 2006).
The RBCs can be polymerized in a variety of ways. The earliest RBCs had self-curing chemistry
similar to dental denture base. These RBCs have been known as self-cured or chemically cured or
two component systems (Kramer et al, 2008). The amine accelerators that were used to increase
polymerization rates of self-curing RBCs contributed to discoloration after 3 to 5 years of service
in patient’s mouth. An alternative system was introduced that used UV (Ultra violet) rays to initiate
polymerization. However, UV curing units had limited reliability because of inconsistent light
energy densities and led to some negative effects on vision and skin of dental personnel (Bayne
and Thompson 2013). Therefore, visible light curing systems were introduced for polymerization
which is yet most popular method for polymerization of resin matrix of RBCs among clinicians
and researchers.
7
1.2.2 Fillers and coupling agent
The filler particles are incorporated into resin matrix of RBCs to improve the physical and
mechanical properties. The primary purpose is addition of as high content of filler to resins as
possible. The filler decreases RBCs polymerization shrinkage and coefficient of thermal
expansion. It also makes the material radio-opaque, improves handling and cosmetic
characteristics (Hervás García A et al. 2006). There is a wide variation in RBCs filler particles
with respect to their chemical composition, shape and size. The silicon dioxide, lithium aluminum
silicate and boron silicate are frequently used fillers in commercial RBCs. In many RBCs, the
quartz is partially replaced by heavy metal particles such as barium, strontium, zinc, aluminum or
zirconium, which are radio-opaque (Hervás García A et al. 2006). The filler particles are subjected
to a special pretreatment prior to blending with the resin. This involves a surface coating of a
coupling agent on the particles to enhance bonding between filler and resin matrix.
The coupling agent most commonly used is γ-methacryloxypropyltrimethoxysilane (γ-MPTS).
This coupling agent is a difunctional molecule comprising of methacrylate monomer at one end while at
the other end it has a silane group which bonds with quartz or glass filler surfaces (Antonucci JM et al.
2005). Hence, it is able to establish a chemical bond between the resin and filler components of RBCs
(McCabe and walls, 2008). The effective bonding between resin matrix and filler via coupling agent has
been reported to reinforce the filler surface against fracture (Mohsen and Craig, 1995). The superior
coupling between resin and filler enables stress distribution and transmission from the flexible resin matrix
to filler component (Calais and Soderholm, 1988).
The filler particles are considered macro fillers in the range of 10 to 100 μm, midi fillers from 1 to
10 μm, mini fillers from 0.1 to 1 μm, micro fillers from 0.01 to 0.1 μm. The expanding field of
nanotechnology has made the development of new RBCs possible containing zirconium/silica
8
filler nanoparticles of around 25 nm size and 75 nm diameter nanoaggregates. The approximately
80% of these nano filler (aggregates and nanoparticles) may be loaded into RBCs. Since size of
fillers is so small it results in fine finishing and polishing. Subsequently, this equips RBCs with
superior properties for instance improved strength, decreased curing shrinkage, reduced cusp wall
deflection, acceptable appearance and less bacterial biofilm formation (Labella et al. 1999;
Ferracane, 2010; Moszner N and Hirt T, 2012).
While finishing RBCs, the harder filler particles have tendency to be pulled out of resin matrix
resulting in rough and soft surface which is rich in resin. This resin rich surface could quickly
undergo wear. In contrast, soft filler particles are more likely to wear rather than to be pulled out
of the matrix when contacts hard objects. Hence, soft fillers have an overall greater abrasion
resistance and wears at a slower rate (Ferracane, 2010).
1.2.3 Photo initiators
The photo initiators for existing commercial RBCs must meet certain requirements. The photo
initiators must exhibit absorption of visible light mainly in the blue region of visible light spectrum
(400–500nm) (Neumann et al. 2005; Obici et al. 2005). Since this spectrum of visible light matches
the light emission of currently available light curing units for instance quartz tungsten halogen
(QTH) lights and blue light emitting diodes (LEDs) which are quite efficient (Kramer N et al,
2008). The excellent photo reactivity, solubility and compatibility of photo initiators with the resin
matrix is required (Rueggeberg et al. 1993; Mills, 1999). In addition, the photo initiator should
exhibit storage stability, safety and do not form colored by products. These requirements for photo
initiators are met by mixtures of diketone type camphorquinone (CQ) with tertiary amines as co
initiators. Therefore, CQ and tertiary amines are most frequently used photo initiator systems in
9
commercial RBCs. This photo initiator system starts polymerization when activated by visible
light (Kramer N et al, 2008). When CQ is sufficiently excited upon exposure to visible light it
reacts with the amine to begin a chain reaction for the formation of free radicals. Subsequently
these free radicals initiate polymerization of the resin monomers converting them into a cross
linked polymer. As a result, RBCs paste is transformed into strong restoration (Neumann et al.
2005; Obici et al. 2005).
One of important disadvantages of CQ is that it leads to yellowing of RBCs, especially in
restorations of very light shades (Neumann et al. 2005). This yellowing is mainly due to photo-
oxidation process that can occur after curing of RBCs. This drawback of CQ has motivated
researchers for discovery of new photo initiator with faster curing, significantly improved depth
of the cure and no negative effect on appearance of RBCs. Other photo initiators reported in
literature includes cylphosphine oxides, alpha diketones and lucrin TPO (Neumann et al. 2005).
The CQ absorbs light in 400-500nm range with peak absorption at 468nm (Uhl et al. 2004;
Neumann et al. 2005). This range falls in the blue emissions of the visible light spectrum. The
content of CQ ranging from 0.2 to 1.0 % is added into commercial RBCs as reported by
manufacturers (Powers, 2002).
10
1.2.4 Inhibitors present in Resin based composites
Inhibitors present in RBCs prevent polymerization of monomers thereby increase the shelf life.
The inhibitors react with the free radicals at the onset of initiation step of polymerization (Hadis,
2011). This compensates shrinkage stress before the cross-linking attains level at which molecular
displacement is not possible. Hydroquinone or butyl hydroxytoluene (BHT) are commonly used
inhibitors in commercial RBCs (Braga and Ferracane, 2002).
1.2.5 Pigments and Ultra violet (UV) stabilizers of Resin based Composites
These components of RBCs provide suitable appearance to aid its color matching to natural
dentition. The metal oxides for instance titanium and aluminum oxides are used as pigments. Since
RBCs may undergo discoloration due to UV absorption (below 400 nm). Therefore, benzophenone
is incorporated into commercially available RBCs, which absorb UV light to prevent discoloration
(Awan, 2010).
1.3 Classification of Resin based composites (RBCs)
During the course of RBCs evolution, classification systems have not been consistent (Lutz and
Phillips1983; Lang et al. 1992). The RBCs have been classified on the basis of their constituents
or handling characteristics. The filler particle size based classification method is most commonly
used. Based on polymerization method RBCs are classified as self-curing, ultra violet (UV) light
curing, visible light curing and dual curing RBCs (Khaled AN 2011). Several scientists have
contributed to classification of RBCs (Lutz and Phillips 1983; Lang et al.1992; Willems et
11
al.1992). Based on filler particles size RBCs include mega fill, macro fill, midi fill, mini fill, micro
fill, and nano fill. The RBCs with combination of two types of filler particle sizes are called
hybrids. The hybrid type is defined by the largest particle size for instance mini fill hybrid RBCs
comprise of micro fillers and mini fillers, since mini fill particle size is larger therefore it is known
as mini fill hybrid RBCs. The RBCs consisting of filler particles and unpolymerized resin matrix
is known as homogeneous. The RBCs containing pre polymerized resin or some distinct filler, it
is called heterogeneous (Bayne and Thompson 2013). Depending on site of application in oral
cavity, RBCs have been classified as anterior, posterior or universal RBCs. The universal RBCs
are used for restoration of anterior as well as posterior teeth (Prasad and Sarkar, 2004). The various
classes of RBCs based on filler particle size, method of curing and handling characteristics have
been listed in table 1.2.
Table 1.2: Classification of Resin based Composites (RBCs)
Classification based on
Fillers
Classification based on
Curing
Classification based on
consistency/ handling
characteristics
Mega fill RBCs Chemically activated RBCs Flowable RBCs
Macro fill RBCs Light activated RBCs Packable/ Condensable RBCs
Midfill RBCs Dual-cured RBCs
Minifill RBCs
Microfill RBCs
Nanofill RBCs
12
1.3.1 Traditional or Conventional Resin based Composites
The traditional RBCs comprised of macro size quartz or glass fillers ranging from 1–100 μm
diameter (Lutz & Philips 1983). Traditional RBCs contained approximately 60–80% by weight
macro fillers. Although the particle size distribution may vary within this range from one product
to another. Some traditional RBCs contain relatively greater amounts of larger particles
approaching 100 μm whereas others contain small size filler particles in greater quantity (McCabe
and walls, 2008). These macro size filler containing traditional RBCs had poor surface finish. This
resulted in a restoration with rough surface. The rough surface of these RBCs increased plaque
accumulation and staining compared to other types of RBCs. Traditional RBCs have little clinical
importance nowadays. However some orthodontists still use these RBCs for bonding brackets or
appliances. Since the rough surface makes easy detection during the removal (Gladwin et al. 2009).
1.3.2 Micro filled Resin based Composites
In the late 1970s, micro filled RBCs were marketed. These RBCs exhibited smooth and glossy
surface appearance very similar to tooth enamel. Despite their good aesthetic appearance and
polish retention these materials were generally weak due to reduced filler content (Ferracane,
2010). Therefore, further improvements were made to fillers in order to achieve adequate strength
and aesthetics which resulted in developments of micro hybrid RBCs (Ferracane, 2010). Micro
filled RBCs comprised of fillers with a mean particle size of 0.04-0.1μm (Ferracane, 2010). The
surface area of these filler particles requires more resin to wet them. Consequently, it is not possible
to incorporate very high filler amount and products which are available contain only 30–60% filler
by weight. Even this low level of fillers in micro filled RBCs did not disperse well into matrix and
13
many filler particles are present as agglomerates and not as individual particles surrounded by resin
(McCabe and walls, 2008). Therefore, increased resin content lead to high coefficient of thermal
expansion and lower strength. Micro filled RBCs were used for the restorations Class I and II
cavities, but the performance of most of the products was comparable to traditional RBCs. Micro
filled RBCs have been indicated when cosmetics has been the main demand. The grossly carious
Class IV cavities are restored with RBCs of different shades and translucencies. The initial layer
of hybrid RBCs is used for strength whereas the final layer of micro filled RBCs is placed for
acceptable surface (Gladwin et al. 2009). The micro filled RBCs exhibit lower modulus of
elasticity therefore, have been used for restoration of caries at gingival third of clinical crown.
Clinical research has shown Class V restorations with micro filled RBCs are more likely to be
retained compared to other types of RBCs (Gladwin et al. 2009).
1.3.3 Hybrid Resin based Composites
Hybrid RBCs were developed in the late 1980s and contained a blend of both traditional glass or
quartz particles together with some submicron particulate silica. The hybrids RBCs utilize filler
quantity of about 75% traditional size and 8% submicron size. Total filler content of 83% by
weight or greater can be incorporated into hybrid RBCs. Some hybrids RBCs contain a blend of
at least three different filler particle sizes. These RBCs allow efficient packing of filler and enable
filler loadings of up to 90% by weight (McCabe and walls, 2008). The hybrid RBCs demonstrates
desired strength, abrasion resistance and suitable appearance. Hence, these RBCs have been widely
used by clinicians for small to medium size restorations in posterior teeth. The hybrid RBCs
finishing is as good as that of micro fills. Thus, they are also used for Class III and IV restorations.
14
The hybrid RBCs have been further classified into micro hybrid and nanohybrid. The Micro hybrid
RBCs comprise a combination of mini fillers, whereas nanohybrid is mainly based on nanoscale
filler particles (Moszner N and Hirt T, 2012).
1.3.4 The Nanofilled Resin based Composites
One of the notable event witnessed during RBCs development is the introduction of nanofilled
RBCs. The nanofilled RBCs filler size is less than 10 nm (0.01 μm). The main purpose of using
fillers with nano size dimension is to improve strength, wear resistance, polymerization shrinkage
and surface characteristics of RBCs (Khaled AN 2011; Karimzadeh A 2014). The nanofilled RBCs
are marketed as nanohybrid which comprise of glass fillers and distinct nanoparticles called
nanomers. These nanomers particles have been arranged in groups and are known as
“nanoclusters” (Mitra, et al. 2003). It has been reported that nanoclusters equip nanofilled RBCs
with improved strength via unique reinforcing mechanism (Khaled AN 2011; Curtis, et al. 2009).
The resin and filler coupling is of greater importance in nanofilled RBCs in comparison to
conventional RBCs (Khaled AN 2011). Since the nano filler dimension provides greater surface
area and require a high degree of silanization than macro size filler of conventional RBCs (Wilson
et al. 2005; Wilson et al. 2006). The quality of coupling ultimately influences physical and
mechanical properties of nanofilled RBCs (Khaled AN 2011).
Nanofilled RBCs based restoration is appealing to clinicians as well as patients. Since, these RBCs
exhibit improved strength of the hybrid (Mitra SB et al. 2003: Moszner N and Klapdohr S 2004)
and the high polishibility of the micro fill (Turssi CP et al. 2000). The high wear resistance (Turssi
CP et al. 2005; Terry DA. 2004) improved optical properties (Mitra SB et al. 2003) and reduced
polymerization shrinkage (Mitra SB et al. 2003; Chen MH et al. 2006) of nanofilled RBCs have
15
also been reported. These newly developed RBCs provide clinicians with the choice for restoration
of anterior as well as posterior teeth (Mitra et al. 2003; Sideridou ID et al. 2011).
1.3.5 Flowable and Packable Resin based Composites
The RBCs with specific handling characteristics such as flowable and packable RBCs were
introduced to dental practice to meet the demand of dentists. The flowable RBCs due to reduced
viscosity exhibit suitable flow into narrow pits and fissures on tooth surface. The flowable RBCs
are dispensed by a syringe into tooth cavity. The viscosity of flowable RBCs has been controlled
either by increasing size of fillers or decreasing overall content of filler particles. Most of flowable
RBCs contain mini fill particle sizes; some contain micro fill fillers (Wakefield and Kofford 2001).
The rate and extent of flow varies significantly from one product to another (Burgess et al. 2002).
The first generation of flowable RBCs were introduced in 1996 prior to condensable RBCs. The
reduced content of fillers in flowable RBCs resulted in poor properties such as increased
polymerization shrinkage, lower wear resistance and low radio opacity compared to the traditional
hybrid RBCs (Bayne et al. 1998; Wilkerson et al. 1998). Therefore, to improve these inferior
properties second-generation flowable RBCs have been developed. The properties of second
generation flowable RBCs are nearly identical to those of conventional RBCs. The flowable RBCs
modulus of elasticity is low this makes them more suitable for restoration of teeth at cervical sites
and avoids fracture due to abfraction. The first-generation flowable RBCs are used as pit and
fissure sealant or restorations of small cavities in anterior teeth. Although second-generation
flowable RBCs have been recommended for restoration of anterior as well as posterior dentition,
yet these RBCs are preferred for conservative restorative procedures in dental practice. The most
popular applications for flowable RBCs continue to be as the first increment during RBCs
16
restoration procedure and for repair of disintegrated margins of old restorations (Bayne and
Thompson 2013; Pongprueksa et al. 2007).
Since, it was not possible to establish adequate interproximal contact with conventional RBCs this
led to problem of food stagnation and secondary caries (Burgess et al. 2002). Therefore, packable
RBCs also known as condensable RBCs were developed in order to achieve two goals including
restoration of a proximal surface of teeth with suitable interproximal contact and handling
characteristics identical to amalgam (Leinfelder and Prasad, 1998). The size of filler particle in
packable RBCs is greater compared to conventional hybrid RBCs, filler sizes vary in range from
0.04 to 20 microns (Combe and Burke, 2000; Craig and Powers, 2002).
In literature the earliest packable RBCs composition and characteristics have been reported to be
similar or slightly improved compared to traditional hybrid RBCs (Ruddell et al. 1999; Leinfelder
et al. 1999). The packable RBCs provide easier placement and packing in posterior dentition
(Leinfelder et al. 1999; Papadogiannis et al. 2007). The packable RBCs increased cure depth and
decreased polymerization shrinkage make them suitable for bulk-fill technique (Aw and Nicholls,
2001). However, there have been concerns among clinicians with respect to ability of packable
RBCs to properly adapt to internal line and point angles and cavosurface margins of tooth cavity
(Leevailoj et al. 2001). To address this problem, use of flowable RBCs have been recommended
as liners. As flowable RBCs exhibit higher fluidity hence may adapt to the cavity walls better than
packable RBCs (Bayne et al. 1998).
The packable RBCs characteristics include increased degree of cure, deceased polymerization
shrinkage, coefficient of thermal expansion close to that of the tooth structure, modulus of
elasticity similar to that of amalgam, radiopacity and low wear rate (Tung et al. 2000; Blalock et
al. 2006).
17
1.3.6 Chapter Summary
The earliest commercial RBCs included chemically activated RBCs, which had many
drawbacks. Later in 1970, light activated RBCs were introduced with improved properties.
The modern RBCs consist of resin matrix, fillers, coupling gent, photo initiators, inhibitors,
pigments and UV stabilizers.
RBCs have been classified on the basis of filler particle size, method of activation and
handling characteristics.
The RBCs superior properties and concern over mercury toxicity of amalgam has led to its
increased use in clinical practice.
The polymerization shrinkage and secondary caries are two major reasons for RBCs
clinical failure.
18
CHAPTER 2
Literature Review
19
2.1 Introduction
Across the globe dental RBCs are being widely used by clinicians for restoration of posterior teeth.
This increased use of RBCs is because of the concern over safety of dental amalgam,
environmental issues related to mercury disposal and increased demand of patients for tooth-
matching restorations (Burke et al. 2000). However, in vitro (Beyth et al. 2007) and in vivo studies
(Auschill et al. 2002) have reported that more plaque accumulation occurs on RBCs compared to
other restorative materials for instance amalgam and glass ionomers (Sevinc BA and Hanley,
2010) or dental structures such as enamel (Hahn et al. 1993). This growth of plaque adjacent to
the restoration margins in vivo lead to secondary caries and is one of the leading limitations to the
longevity of RBCs (Burke et al. 2001; Sousa et al. 2009).
The increased deposition of dental plaque on modern RBCs have been attributed to four main
factors; namely greater surface roughness (Beyth N et al. 2008; Ono M et al. 2007), biodegradation
(Khalichi P et al. 2004), composition altering wet ability or surface free energy and lack of
antibacterial activity (Gyo M et al. 2008; Carlen A et al. 2001).
So far, several studies have investigated the antibacterial activity of commercial RBCs as well as
its various constituents against microorganisms associated with dental caries (Schmalz 1977;
Orstavik and Hensten Pettersen 1978; Hansel et al. 1998; Yap et al. 1999). In most studies, it was
found that commercial RBCs did not demonstrate antibacterial activity. In addition, constituents
of commercial RBCs exhibited negligible antibacterial activity against microorganisms of oral
cavity. The silica-based filler and monomers BisGMA, TEGDMA or UDMA frequently used in
RBCs exhibited no growth inhibition of Streptococcus mutans. However, among the initiator and
20
activator component, amine compounds showed antibacterial effects but at much higher
concentrations (Bundy et al.1980; Kawai 1988). The lack of antibacterial activity increases
commercial RBCs tendency to accumulate more dental plaque leading to secondary caries and
restoration failure. Although, it has been reported by some researchers that polishing of RBCs
restrict Streptococcus mutans accumulation (Ono et al. 2007; Ionescu et al. 2012). But no
consensus has been found in research reports in this regard. Since it is not always possible to keep
the restoration surface ideally smooth, therefore, to equip RBCs with antibacterial activity its
composition has been modified by addition of antimicrobial agents. However, the desired results
have yet to be achieved.
Although the mechanical properties of commercial RBCs have been improved substantially since
their development (Neuman, 1999). However their antibacterial properties still remain a cause of
concern among clinicians as well as researchers (Matalon et al. 2004). Hence, to equip RBCs with
antibacterial activity research work is being conducted on the biological properties. The research
work about modification of RBCs with either organic compounds for instance chlorhexidine,
antibiotics or inorganic ions such as silver and zinc has been published. Initial research aimed to
equip RBCs with antibacterial function was carried out during 1960s to 1970s (Imazato S et al.
1994 & 2012). The preliminary research focused on incorporation of soluble antibacterial agents
to existing RBCs. Such soluble agents exhibited antibacterial activity by releasing into surrounding
agar media. However, this strategy could not win much support of clinicians and researchers.
Because the release of antibacterial agents resulted in porosity and consequently limited the life of
restoration (Jedrychowski JR et al. 1983).
21
Therefore, new concept of the immobilized antimicrobial agent was introduced to address
problems of soluble antibacterial agents (Imazato S et al. 1994). This idea of immobilized
antimicrobial agent attracted great attention in dentistry and other fields. In this approach
antimicrobial agent reacts chemically and forms strong chemical bonds such as covalent bond with
components of carrier material. This immobilized antibacterial agent has capacity to inhibit
bacterial growth upon contact and it does not leach out from carrier material (Imazato S, 2003).
Following this novel concept of immobilized antimicrobial agent, Imazato S and his co
investigators made commendable efforts and successfully invented commercial adhesive with
antibacterial activity (Imazato S et al. 1994 & 2012). The two research groups led by Imazato and
Beyth respectively have carried out intensive research work on the two promising antimicrobial
molecules methacry-loyloxydodecylpyridinium bromide (MDPB) and poly ethylene imines (PEI).
They incorporated these agents into RBCs and explored its antibacterial activity. Although the
MDPB and PEI modified RBCs exhibited antibacterial activity but it was not as robust compared
to RBCs containing soluble antibacterial agents (Imazato S et al. 2012; Beyth N et al. 2014).
Hence, to equip RBCs with antimicrobial activity search for new antibacterial agents is ongoing.
One potential candidate for incorporation in RBCs is chitosan (CS), which has been used as
antimicrobial agent in various commercial medical and dental products (Morgana et al. 2011).
2.2 Secondary Caries
The phenomenon of secondary caries has been known since the earliest days of restorative
dentistry. The G.V. Blacks well-established idea of extension for prevention has been attributed to
secondary caries (Black, 1908). The Fédération Dentaire Internationale defined secondary caries
as a ‘positively diagnosed carious lesion, which occurs at the margins of an existing restoration’’
22
(Fédération Dentaire Internationale, 1962). Despite the decreasing trends in prevalence of primary
caries, due to use of fluoridated water and oral hygiene products. The prevalence of secondary
caries is significant. Even in modern day dentistry, burden of restoration replacement due to
secondary caries is huge. It has been reported that either in permanent or primary teeth 50 to 60 %
of restorations replacement irrespective of its type is due to secondary caries (Mjör and Toffenetti,
2000). In the United States yearly budget of approximately $5 billion is spent on restoration
replacement in dental practice (Jokstad A et al. 2001).
Secondary caries is associated with all type of restorative materials (Burke et al. 1999; Forss and
Widström E, 2004). However, compared to amalgam more RBCs restoration replacement is
associated with diagnosed recurrent caries. The lack of clinical durability for 87.6 % and 66.7 %
RBCs and amalgam respectively have been found due to secondary caries in randomized control
trials (Bernardo et al. 2007).
The Hals et al. made a great contribution by doing extensive investigation of the secondary caries
associated with restorative materials both in vitro and in vivo experiments. They studied the
secondary caries in permanent teeth restored with silicate and amalgam. They concluded that
histologic presentation of secondary caries was similar irrespective of type of restorative material.
In addition, the frequency and severity of secondary caries was low with silicate compared to
amalgam. This decreased incidence of secondary caries was attributed to fluorides elution from
the silicate (Hals 1975; Hals et al. 1974; Hals and Norderval, 1973).
The secondary caries mainly affected the gingival and proximal margins of Class II and Class III
restorations. Whereas Class I restorations and the occlusal part of Class II restorations was spared
mostly (Mjör, 2005). With the development of evidence based dentistry and adoption of minimal
23
invasive dentistry. It is highly recommended that clinicians should differentiate the affected and
infected tooth tissue so that only infected tooth structure may be removed. Since this approach will
conserve tooth tissue which will provide ample resistance and retention form thereby increase the
lifespan of restoration and teeth (Fusayama, 1988; Kidd, 2010).
It is indeed a difficult task for clinicians to accurately diagnose secondary caries since another kind
of lesion that is residual caries may occur adjacent to the restorations. The diagnosis of secondary
caries is yet a challenge for clinicians as there is no standard protocol available. However, with
advancement in diagnosis of dental caries. It has been advised that use of sharp explorer/ probe for
detection of secondary caries should be avoided. Several other suitable methods available for
diagnosis of early secondary caries include microradiograph (Arends et al. 1987), confocal laser
scanning microscopy (Fontana et al. 1996) and light-induced fluorescence (Ando et al. 2004).
At present etiology of secondary caries is unclear and there is no consensus whether it is the same
as that of primary caries. The Kidd et al. reported that no significant differences exist between the
microorganisms associated with primary and secondary caries adjacent to amalgam (Kidd et al.
1993). Yet the area of microbiology of secondary needs to be explored further for better
understanding and management of secondary caries.
2.2.1 Micro leakage and secondary caries
The clinically undetectable leakage between the tooth cavity wall and the restorative material is
referred as micro leakage (Kidd, 1976). This allows a favorable environment for accumulation of
cariogenic bacteria including Streptococcus mutans and Lactobacilli which demineralize the tooth
structure (González-Cabezas et al. 2002; Splieth et al. 2003). At the interface of RBCs and tooth
structure a gap of 6-10 μm developed even after applications of acid etch and a bond (Irie et al.
2002). It has been reported that micro leakage following restorative treatment is inevitable
24
(Mousavinenasab and Jafary, 2004). There is evidence that chances of secondary caries increase
with relation to the size of the micro space (Totiam et al. 2007; Piwowarczyk et al. 2005). There
is a consensus among researchers and clinicians that micro leakage is associated with recurrent
caries (Thomas et al. 2007).
2.2.2 Prevention of Secondary Caries
The clinicians and researchers have given ample importance to prevention of secondary caries so
as to increase the durability of restoration. The secondary caries may be prevented by maintenance
of good oral hygiene and use of restorative materials with antibacterial activity. The restorative
materials with antibacterial activity may inhibit the growth of cariogenic bacteria in the plaque or
/and the carious dentin beneath the restorative material (Mjör, 2005).
2.3 Treatment of Caries
Dental caries treatment traditionally comprises of the diagnosis followed by immediate restoration
treatment. The restoration treatment options include use of number of direct restorative materials
for instance amalgam, RBCs and indirect restorations such as inlays and crowns. The restoration
of decayed tooth with restorative materials does not always guarantee a sound tooth in future.
However, it may lead to a restorative cycle in which the restoration may be replaced number of
times (Van Amerongen et al. 2001).
25
The silicate was the first fluoride releasing tooth matching restorative material. Other fluoride-
releasing restorative materials include conventional glass ionomers, compomers and resin
modified glass ionomers (Burgess, J.O. 2001). There is increasing evidence from clinical trials
that glass ionomers decrease the development of secondary caries. The association between dose
and duration of fluoride release from these materials with decreasing prevalence of caries has been
established. Although the fluoride releasing materials are not ideal materials but they can arrest
caries through enhancement of remineralization (Niessen LC and Gibson G, 1997).
The replacement of restoration has been mostly advocated as only treatment option for secondary
caries. However, there is growing evidence that repair instead of total replacement should be the
goal of secondary caries treatment (Blum et al. 2003).
2.4 Antibacterial agents in Resin based composites (RBCs)
The addition of an antibacterial agent into RBCs is a simple way to equip the material with
antibacterial activity (Shay D.E et al. 1956; Leung D et al. 2005). It has been reported that
antibacterial agents incorporated into RBCs eliminate bacteria via either by disruption of cell wall
and/or cell membrane, interacting with surface-adsorbed components, blocking of protein or
nucleic acid synthesis and inhibition of enzyme activity through oxidation (Cowan 1999).
Several types of antibacterial agents have been incorporated into RBCs so far and their impact on
antimicrobial activity has been evaluated. The published literature about antimicrobial properties
of RBCs was reviewed and the findings are summarized in table 2.1. The table lists the type of
study, antibacterial agent added, outcome in terms of antimicrobial activity and bacteria tested.
26
Table 2.1: Antibacterial agents incorporated into RBCs, type of study and antimicrobial effect
S.No Antibacterial agent Reference Type of
study
Bacteria/
Biofilm tested
Result
1.
Chlorhexidine
Apel C et
al. 2013
In Vitro
Streptococcus
mutans
Positive
Cheng L
et al. 2012
In Vitro
Streptococcus
mutans
Positive
Mehdawi I
et al. 2009
In Vitro
Streptococcus
mutans,
Lactobacillus
casei,
Actinomyces
naeslundii
Positive
2.
Quaternary ammonium
polyethylenimine
(QPEI)
Beyth N et
al. 2006
In Vitro
Streptococcus
mutans
Positive
Beyth N et
al. 2010a
In Vitro
Streptococcus
mutans
Positive
Beyth N et
al. 2010b
In Vivo Saliva
Biofilm
Positive
3.
Methacryloyloxydodecylpyridiniu
m bromide (MDPB)
Imazato
S. et al.
1995 and
2003
In Vitro Streptococcus
mutans
Positive
Ebi N et
al. 2001
In Vitro Streptococcus
mutans
Positive
4. Furanone Weng Y et
al. 2012
In Vitro Streptococcus
mutans
Positive
5.
Quaternary
ammoniumdimethacrylate
(QADM), nanoparticles of silver,
nanoparticles of amorphous
calcium phosphate (NACP).
Lei Cheng
et al. 2012
In Vitro Streptococcus
mutans
Positive
27
Table 2.1(Continued): Antibacterial agents incorporated in RBCs, study and antimicrobial effect
S. No Antibacterial agent Reference Type of
study
Bacteria/
Biofilm tested
Result
6.
Ursolic acid Kim JS et al.
2013
In Vitro Streptococcus
mutans
Positive
7. Benzalkonium chloride Sehgal et al.
2007
In Vitro Streptococcus
mutans
Positive
8. Zinc oxide Sara et al.
2013
In Vitro Streptococcus
mutans
Positive
9. Triclosan or
Irgasan (5-chloro-2-4-
Dichlorophenoxy phenol)
Apel C et al.
2013.
In Vitro Streptococcus
mutans
Positive
Rüttermann S
et al. 2013
In Vitro
Streptococcus
sanguinis,
Streptococcus
oralis,
Streptococcus
mitis,
Actinomyces
viscosus,
Actinomyces
naeslundii
Positive
10. Cetylpyridinium Chloride Namba et al.
2009
In Vitro
Streptococcus
mutans
Positive
11. Fluoride Pandit et al.
2011
In Vitro
Plaque Positive
12. Chitosan Kim JS et al.
2013
In Vitro Streptococcus
mutans
Positive
28
Table 2.1(Continued): Antibacterial agents incorporated in RBCs,study and antimicrobial effect
S.No Antibacterial agent Reference Type of
study
Bacteria/
Biofilm tested
Result
13 Dimethacrylate quaternary
ammonium compounds N,N-
bis[2-(3
(methacryloyloxy)propanamid
o)ethyl]-N-methyldodecyl
ammonium iodide (QADMAI-
12),(QADMAI-16),
(QADMAI-18)
Jingwei et
al. 2014
In Vitro Streptococcus
mutans
Positive
14 Benzyl chloride quaternized
dimethylaminoethyl
methacrylate (DMAEMA-BC).
Benzyl chloride quaternized
diethylaminoethyl
methacrylate (DEAEMA-BC)
Beigi
Burujeny
S, et al.
2015
In Vitro Streptococcus
mutans
Positive
15 Dimethyl amino dodecyl
methacrylate (DMADDM)
Dimethylaminohexane
methacrylate (DMAHM) and
dimethylaminododecyl
Methacrylate (DMADDM)
Melo
MAS et al.
2014
In Vitro
Saliva bacteria
Positive
Zhou C et
al. 2013
In Vitro Streptococcus
mutans
Positive
16. Carolacton Apel C et
al. 2013
In Vitro Streptococcus
mutans
Positive
17.
Octenidine dihydrochloride Stefan
Rupf et al.
2012
In Situ Biofilm/
Plaque
Positive
18.
Dimethylaminohexadecyl
methacrylate (DMAHDM)
Zhang N
et al.2015
Wu J et al.
2015
In Vitro
In Vitro
Plaque
Plaque
Positive
Positive
29
1. Chlorhexidine
The chlorhexidine (CHX) is a soluble antimicrobial agent incorporated into RBCs. The CHX
containing RBCs exhibited inhibition of oral Streptococci growth in laboratory based experiments.
The release of CHX from RBCs have been confirmed and continued for five days on immersion
of experimental specimens in water (Takemura et al. 1983). The CHX release at low concentrations
have been reported for about five weeks. Moreover, incorporation of 1% chlorhexidine gluconate
into RBCs have been attributed to decreased tensile and compressive strength (Jedrychowski et al.
1983). The decrease in strength of these RBCs may be attributed to disturbance of curing of
monomers and porosity in material due to release of chlorhexidine gluconate (Addy, 1981).
Since addition of CHX into RBCs decreased its mechanical properties including tensile and
compressive strength, its use for permanent restorative materials such as RBCs is not suitable.
However, it may be used for temporary restorative materials that are used for short period. In
addition the release of CHX from RBCs is not controlled, large amount of CHX is lost within a
few days leading to significant decrease in antibacterial activity (Addy and Thaw. 1982; Wilson
and Wilson, 1993).
2. Poly ethylene imines (PEI)
The antibacterial activity of RBCs containing quaternary ammonium polyethylenimine (PEI) has
been investigated and positive results have been reported (Beyth et al. 2010). The antibacterial
activity of experimental RBCs containing PEI agent has been attributed to direct close contact
mechanism similar to MDPB containing RBCs. In addition, the mechanical characteristics of these
experimental RBCs were found to be similar to control RBCs. Moreover, the antimicrobial activity
lasted for at least one month. Although, the antibacterial activity of PEI agents has not been
30
elaborated in detail, yet the literature describes the action mechanism of PEI via its interaction
with bacterial cell membrane. This interaction with cell membrane leads to increased permeability
and rupture of bacterial cell membrane leading to death of bacteria (Lin et al. 2002). The use of
polyethylenimine has been questioned as there are concerns about its safety (Bruno Sarmento and
José das Neves, 2012).
3. Methacryloyloxy dodecyl pyridinium bromide (MDPB)
This monomer has been synthesized by reaction of the quaternary ammonium and a methacryloyl
group. The quaternary ammonium component of MDPB exhibits antibacterial activity. This
antibacterial agent has been incorporated into RBCs (Imazato S et al. 1994; Imazato S and
McCabe. 1994; Imazato S et al. 1999). The MDPB has been reported to copolymerize with RBCs
and form the polymer network after curing. The MDPB containing RBCs antibacterial activity
has been reported to last for longer period of time. It was found that incorporation of MDPB at
0.2% into RBCs exhibited growth inhibition of plaque accumulation by Streptococcus mutans.
This effect is reported to last even after 3 months immersion of experimental RBCs specimen in
water (Imazato S et al. 1994).
Another advantage is that no negative effect has been reported on the mechanical properties and
curing behavior of the MDPB containing RBCs (Imazato S and McCabe, 1994; Imazato S et al.
1999). The MDPB based RBCs exhibited mechanical characteristics for instance flexural strength,
compressive strength and flexural modulus identical to the control RBCs even after ageing of
specimens in water (Imazato S and McCabe, 1994). The antibacterial effects of the MDPB-
containing RBCs are mainly bacteriostatic and not as robust as the RBCs which contain releasing
antibacterial agents. In contrast to releasing antimicrobials agents, MDPB is unable to penetrate
31
fully through the cell wall or membrane of cariogenic bacteria. Another drawback of these
experimental RBCs is decreased antibacterial activity due to adsorption of surface proteins. This
drawback needs to be solved to achieve clinically long lasting RBCs (Ebi et al. 2001).
The mechanism of antibacterial action of experimental RBCs containing MDPB against
Streptococcus mutans stated in literature includes by establishment of direct close contact without
releasing into surrounding environment. Therefore, these MDPB containing RBCs are more
advantageous than agent releasing RBCs because they exhibit favorable mechanical properties
even after aging. Furthermore, incorporation of MDPB is preferred to another immobilized
antibacterial agent such as silver agents because of its color stability. Since this is an important
property for esthetic restorative materials (Imazato S et al. 2003).
4. Furanone
The furanone has been added to RBCs to study its effect on antibacterial activity and strength of
material. The RBCs modified with furanone showed a significant antibacterial activity against
Streptococcus mutans. The 16–68 % reduction in viability of Streptococcus mutans has been
reported. Moreover, the ageing of these experimental RBCs in saliva exhibited no negative
influence on its antibacterial effect indicating antibacterial activity for longer period of time (Weng
et al. 2012). The mode of furanone antibacterial activity is yet to be explored (Lattmann et al.
2005). Experimental RBCs containing 5–30 % furanone demonstrated compressive strength
similar to control. However with increase in quantity of furanone resulted decrease in strength
(Weng et al. 2012).
32
5. Silver
Since long time silver has been used in medicine as an antimicrobial agent due to promising results
(Chen X and Schluesener HJ 2008). The silver ion releasing materials have demonstrated
antibacterial activity against oral Streptococci (Hernandez-Sierra JF et al. 2008). Because of
antimicrobial characteristics of silver, this agent has also been incorporated into RBCs. The
antimicrobial results have been encouraging. The antimicrobial activity of RBCs containing silver
nanoparticles at two different concentrations was explored and found that the number of adhered
bacterial cells to experimental RBCs were smaller compared to control specimens (Bürgers et al.
2009). In another study, silver modified RBCs exhibited bactericidal effect against oral bacteria
including Streptococcus oralis, Streptococcus sanguinis and Streptococcus mitis (Yamamato et al.
1996). The bacteriostatic effects against Streptococcus salivarius have also been reported (Ohashi
et al. 1995). The number of studies have confirmed elution of silver ions from RBCs (Tanagawa
et al. 1999; Yoshida et al. 1999). Therefore, due to release behavior of silver in experimental RBCs
the antimicrobial activity lasted for short duration (Kawabata and Nishiguch, 1988).
The antibacterial activity of low viscosity RBCs containing silver- zinc zeolite and silver- zinc
apatite has been evaluated. It has been reported that both experimental RBCs inhibited the
Streptococcus mutans growth up to seven days (Syafiuddin et al. 1995). Furthermore, silver-apatite
modified RBCs effect on recurrent caries was investigated and found that RBCs incorporating
10% silver-apatite efficiently decreased rate of recurrent caries. However, the silver-zeolite and
silver-apatite addition had significant negative effect on RBCs strength (Syafiuddin et al. 1995 and
1997). The silver modified RBCs poor color stability is great hurdle to its acceptance in clinical
practice (Syafiuddin et al. 1995). Moreover, RBCs containing silver nanoparticles (Ag NPs) have
been reported to reduce biofilm viability (Cheng L, et al. 2012). A pilot study by Fan et al. in year
33
2011 indicated that Ag NPs incorporated into RBCs inhibited biofilm growth in an in situ
experiment (Fan C et al, 2011).
6. Ursolic acid
One recent investigation about antibacterial properties of RBCs reported incorporation of ursolic
acid (UA). It was found that experimental RBCs groups resulted in a complete growth inhibition
of Streptococcus mutans (Kim S et al. 2013). The antibacterial activity action of UA against
Streptococcus mutans is not thoroughly elaborated. However, one study evaluated the effect of
UA against Listeria monocytogenes. The results revealed UA inhibited peptidoglycan synthesis
of Listeria monocytogenes (Kurek et al. 2010).
7. Benzalkonium chloride
The antimicrobial properties of this compound have been stated in literature. Hence, it has been
added in small amounts to RBCs to achieve antimicrobial properties. The benzalkonium chloride
modified RBCs exhibited antibacterial activity against Streptococcus mutans (Sehgal et al. 2007).
The mode of action of these antibacterial agents is considered to be like other common quaternary
ammonium compounds. Although the details of action mechanism have not been known (Gilbert
and Moore, 2005), yet it has been proposed that benzalkonium chloride bearing positive charge
may attach to surface of cell membrane of bacteria. Subsequently this binding to cell membrane
may result in the disruption of the cell membrane. Hence, components within the bacterial cell
may leak out of cell and this might lead to the death of bacteria (McDonnell and Russell, 1999).
34
8. Zinc oxide
The antibacterial effect of zinc oxide (Zn O) has been evaluated, it has been reported that zinc
oxide inhibits the growth of Lactobacillus and Streptococcus mutans (Sheng J et al. 2005).
Moreover, the antibacterial activity has also been demonstrated against other gram-positive and
gram-negative bacteria. Therefore, the commercial products for oral hygiene for instance dentifrice
have been reported to contain zinc oxide along with citrate (Cummins D, 1991). The zinc oxide
has been shown to kill several oral microorganisms known to contribute to caries (Fang et al.
2006). The Zinc oxide nanoparticles have been found to be more effective than larger particles
against both gram negative as well as gram positive bacteria. The proposed antibacterial action
mechanism of zinc oxide is that they generate active oxygen species such as H2O2 which inhibit
growth of plank tonic microorganisms (Adams et al. 2006; Jones et al. 2008).
The antibacterial properties of RBCs containing zinc oxide have been investigated using different
tests. Although the ADT test method revealed no significant antibacterial effect by experimental
RBCs (Sevinc BA and Hanley, 2010;Sara et al. 2013), however the direct contact test method
demonstrated that by increasing the zinc oxide nanoparticles content in RBCs, the bacterial growth
significantly diminished. In the aging test the antibacterial properties of zinc oxide containing
RBCs reduced significantly. Moreover, the mechanical characteristics of these experimental RBCs
revealed that flexural strength and compressive modulus remained unchanged compared to
control, while the compressive strength and flexural modulus increased significantly. The zinc
oxide containing RBCs showed significantly lower depth of cure (Sara et al. 2013).
9. Triclosan
Triclosan antibacterial agent has been reported to kill cariogenic bacteria by acting on its cell wall
(Wicht et al. 2005). This antibacterial agent has been widely used in commercial dentifrices and
35
mouth washes. Because of its non-releasing nature, addition of triclosan to RBCs has been reported
to be superior compared to addition of releasing agent for instance fluoride, chlorhexidine and
benzalkonium chloride (Imazato S et al. 1995; Kalyon BD and Olgun U 2001).
10. Cetylpyridinium Chloride
This antimicrobial agent has been frequently added to commercial oral hygiene maintenance
products for instance dentifrices and mouth washes. The cetylpyridinium chloride has been added
to RBCs and its release has been confirmed by ultra violet (UV) spectrophotometer (Namba et al.
2009). It has been reported that 3% cetylpyridinium chloride containing RBCs significantly
inhibited the growth of Streptococcus mutans. However, due to soluble nature of cetylpyridinium
chloride when experimental RBCs were immersed in water the antibacterial activity proved to be
short lived (Al-Musallam et al. 2006).
11. Fluorides
Fluorides have been incorporated into RBCs to attain antimicrobial activity. However, fluoride
like CHX leached from RBCs but at a much lower level (Arends and Ruben, 1988; Cook and
Youngson, 1989). Compared to conventional and resin-modified glass-ionomers, fluoride in RBCs
had no robust antimicrobial effect. The prospective clinical studies about fluoride releasing RBCs
revealed it had no significant effect on the incidence of secondary caries (Wiegand et al. 2007).
Fluoride releasing filler systems for instance strontium fluoride and ytterbium tri fluoride have
been investigated and found to produce antibacterial effect. Antibacterial effect of these fluoride
containing fillers have been attributed to water diffusion into RBCs resin matrix and fluoride
release from the filler particles (Temin and Csuros, 1988; Sonis and Snell, 1989; Xu and Burgess,
2003). The major drawback of fluoride release from RBCs is that it leads to voids in the matrix
36
thereby decreasing strength. In addition, most of the fluoride is released during the setting reaction
followed by a smaller amount of long-term fluoride release (Xu and Burgess 2003; Kawashita et
al. 2000). It has been proposed that the fluoride releasing RBCs may decrease deposition of dental
plaque, provided ample quantity of fluoride is released during the start of plaque formation (Pandit
et al. 2011; Sousa et al. 2009). The fluoride-releasing RBCs were marketed by number of
manufacturers aimed at arresting demineralization and promote remineralization of dental decay
and were expected to exhibit antibacterial effects due to the release of fluoride ions. However, it
was found that the amount of fluoride released from these RBCs was smaller than those of glass
ionomers cements. Clinically fluoride containing RBCs could not demonstrate desired results as
far as significant growth inhibition of cariogenic bacteria is concerned (Yap et al. 1999; Momoi
and McCabe, 1993).
2.5 Introduction of Chitosan
The chitin was first isolated by French scientist Henri Braconnot from mushrooms in 1811, which
proved to be a precursor of chitosan (CS) biopolymer. The isolation of chitin compound led to
discovery of CS in year 1859. Two methods used to obtain CS from chitin include thermo
chemical deacetylation and enzymatic hydrolysis (Singla and Chawla, 2001). The CS differs from
chitin with respect to degree of deacetylation (DD) which is comparatively low. The research about
potential applications of CS gained momentum during 1930s to early 1940s. However, rise in
production of synthetic materials led to decreased interest in natural products including chitin and
CS. Moreover, due to limitations of synthetic materials use of natural products including CS
37
flourished in 1970s and continues to expand until recently (Braconnot H. 1811; Rouget C.1859;
Muzzarelli RAA. 1973).
In nature, chitin is one of the most abundantly present bio polysaccharide. It is primarily found in
the outer skeletons of crustaceans for instance lobster, crab, shrimp and is available in worms,
insects, fungi and mushrooms in different quantity. Moreover, latest innovations in fermentation
methods indicate that the fungi may be grown and could provide an alternative source of CS
(Hafdani FN and Sadeghinia N 2011).
The CS is an important highly basic polysaccharide biopolymer consisting of two monosaccharide
that are GlcNAc and D glucosamine (GlcN) bound together through glycosidic bonds. The
quantity of these two monosaccharides in CS may vary. This produces commercial CS with
variable viscosity, degree of deacetylation, molecular weight (MW) and pKa values. These
characteristics of CS greatly influence its physicochemical properties and applications (Singla and
Chawla, 2001; Dina and Hans 2009; Zileinski BA and Acbischer P 1994).
The evidence suggests that MW is an important determinant of properties of CS (Rabea et al.
2003). The MW of CS is influenced by its deacetylation process. Therefore, characterization of
each batch of CS is of utmost importance (Rhoades and Roller, 2000). Another important feature
of CS is its degree of deacetylation which influences its moisture absorption, solubility and
viscosity (Singla and Chawla, 2001). The CS is usually insoluble in water, organic solvents and
alkaline media (Singla and Chawla, 2001). The viscosity of commercial CS varies from 10 to 1000
mPas (Kumar, 2000).
The chemistry of CS indicates that it consists of three reactive functional groups, an amino group,
primary and secondary hydroxyl groups. These reactive functional groups readily allow for
38
chemical interaction with some drugs and determine physicochemical properties for instance CS
improved solubility at neutral pH (Singla and Chawla, 2001). Commercially CS is available with
degree of deacetylation greater than 85%, and molecular weights (MW) between 100 to 1000 kDa.
There is no a specific standard to classify CS on the basis of MW. However it is accepted that Low
MW CS < 50 kDa, Medium MW CS 50 –150 kDa, and High MW CS > 150 kDa (Rejane C.G,
2009).
2.5.1 Antimicrobial Properties
Antimicrobial activity of CS is well recognized. The antimicrobial action of CS against algae,
fungi and bacteria has been reported. These antimicrobial characteriscs were confirmed via
experiments involving in vivo and in vitro interaction of different forms of CS such as films,
solutions and composites (Choi et al. 2001).
The preliminary research studies determining antimicrobial activity of CS were carried out during
1980-1990s (Young, DH. et al. 1982; Shahidi F. et al. 1999; Hadwiger LA et al. 1981). These
studies reported that CS exhibits both bactericidal (kills the live bacteria) and bacteriostatic activity
(inhibit the growth of bacteria). However, latest literature characterize CS as bacteriostatic (Coma,
V et al. 2002). Its antibacterial effect against Streptococcus mutans, Actinobacillus
actinomycetemcomitans and Porphyromonas gingivalis have been reported (Fujiwara et al. 2004;
Choi et al. 2001; Jervinen et al. 1993). The CS has demonstrated low toxicity and no resistance by
microorganisms (Morgana et al. 2011). The factors that may influence the antimicrobial action of
the CS include the type of microorganism, CS intrinsic characteristics and environmental factors
for instance temperature, time and pH (Kong et al. 2010).
39
The attachment of cariogenic bacteria to the tooth surface is initial step in dental caries process.
This binding of bacteria to tooth is quite complicated and involves electrostatic and hydrophobic
interactions. Therefore, prevention of bacterial attachment to tooth surface could be a promising
approach to control and prevention of dental caries. To achieve this objective either ionic or non-
ionic molecules have been used which modify the hydroxyapatite surface, thus reducing
attachment of cariogenic bacteria (Sano et al. 2003). It has been suggested that CS anti-adherence
activity may change hydroxyapatite ionic properties leading to reduced attachment of cariogenic
bacteria. In addition, CS may inactivate bacterial enzyme and also the block the formation of
teichoic acid (Tarsi et al. 1997; Busscher et al. 2008).
The antimicrobial mechanism of action of the CS is not yet completely clear; however several
action mechanisms have been stated in literature. Some authors report that the amino groups of the
CS when interact with microbial physiological fluids are protonated and on attachment to cell
membrane of the microorganisms lead to agglutination and inhibition of microorganisms growth
(Avadi et al. 2004). Moreover, it has been reported that CS may lead to displacement of calcium
from cell membrane resulting in damage to bacteria (Bhise and Yadav, 2004). Some other studies
suggest that the antimicrobial action is closely related to the characteristics of CS and nature of
cell wall of the microorganism (Costa Silva et al. 2006). The interaction between the positive
charge of the CS and its opposite charge on the microbial cell wall results in cell wall rupture and
loss of internal components of microorganisms.
It has been proposed that CS may enter into nuclei of microorganisms and bind with its DNA. This
inhibits the mRNA and synthesis of protein (Sebti I et al, 2005; Hadwiger L.A et al, 1983;
Sudarshan NR et al, 1992). The CS compound is assumed to be able to pass through the bacterial
cell wall, composed of multilayers of cross-linked murein, and reach the plasma membrane.
40
Observation by confocal laser scanning microscopy (Liu et al. 2001) confirmed the presence of
CS oligomers (a chain with few number of monomer units) inside Escherichia coli exposed to
chitosan under different conditions. Whereas CS of high molecular weight act as a chelating agent.
This binds trace metals selectively, thereby inhibiting toxin production and microbial growth
(Pedro et al. 2009).
The minimum bactericidal concentration (MBC) of CS often depends on its molecular weight and
type of bacterial species. It has been reported that the MBC of water-soluble chitosan (70% degree
of deacetylation) against Streptococcus mutans is 1,250 μg/ml (Bae et al. 2006). The CS has been
reported to decease deposition of dental plaque and promote production of saliva in oral cavity.
This potential of CS suggests is use for control and cure of dental caries (Hayashi et al. 2007; Miao
et al. 2009).
The minimum inhibitory concentration (MIC) of CS has been reported to range from 0.005 to 0.1
μg/ml with respect to bacterial species, MW and pH. Pure CS can dissolve only in acetic acid
solution. Since acetic acid has also antimicrobial activity therefore, this needs to be taken into
consideration in the experiment investigating the antimicrobial activity of CS (No et al. 2002). The
antibacterial activity of various weight percentages of CS having different MW (55 to 155 kDa)
but similar degrees of deacetylation (80%) have been explored against Enterococcus faeclis. It was
concluded that CS exhibited antibacterial activity at concentrations over 200 part per million (ppm)
(Liu et al. 2001; Liu et al. 2006).
2.5.2 Biocompatibility and safety of chitosan (CS)
Biocompatibility and safety of CS is an important characteristic for its various applications. This
indicates that CS does not have any harmful local or systemic effect on biological systems (Dutta
PK. 2005; Keong LC and Halim AS. 2009). The CS component N-acetyl glucosamine is identical
41
to heparin, hyaluronic acid, glycosaminoglycans and chondroitin sulphate structure. These
compounds are biocompatible and exhibit harmless interactions with growth factors, receptors and
adhesion proteins in all mammals (Li Q et al. 1992; Suh FJK and Matthew HWT. 2000). The CS
compatibility with living structures including eye, skin and nasal epithelium has been reported.
Therefore it has been used in drug delivery and tissue engineering to prepare tissue scaffolds that
has promising applications in healthcare (Shigemasa and Minami, 1995; Felt et al. 1999).
The available evidence suggest safety of CS. Therefore, it has been permitted for use in food
products in Japan, Finland and Italy (Illum L, 1998). It has been permitted by the food and drug
administration (FDA) for use in wound dressings as well. The commercially available CS is safe
compared to a toxic polymer such as polyethylenimine (Bruno Sarmento and José das Neves,
2012). The side effects of CS ingestion in human studies (for up to 12 weeks) have been explored
and found to produce no adverse reaction (Ylitalo et al. 2002).
42
2.6 Antibacterial activity testing methods for Resin based composites (RBCs)
The antibacterial behavior of experimental and commercial RBCs have been investigated using
agar disc diffusion test (ADT) and direct contact test (DCT) method (Beyth N et al. 2014; Farrugia
C and Camilleri J, 2015). The other antibacterial activity testing methods for RBCs reported in
literature include methyl thiazolytetrazolium (MTT) assays, spectrophotometry, determination of
colony forming units and scanning electron microscope (SEM) (Farrugia C and Camilleri J, 2015).
Although ADT has often been used to evaluate antibiotics or materials containing soluble
antibacterial agents, it has certain limitations. Despite the limitations, ADT method has been
widely used for investigation of antibacterial properties of restorative materials including RBCs.
One primary disadvantage of ADT method is that it depends on the solubility and diffusion
properties of both the test material and agar media (Farrugia C and Camilleri J, 2015; Imazato S
2009). Hence, this method is not suitable for testing antibacterial activity of restorative materials
containing insoluble agents.
The direct contact test (DCT) was introduced to evaluate antibacterial activity of non soluble
antibacterial agent containing materials (Weiss et al. 1996). This test method is based on measuring
the antibacterial activity following physical contact between bacteria and the experimental
material. The DCT method used to determine antibacterial activity of RBCs involved use of
spectrophotometer or colony forming unit count of test bacteria. The spectrophotometer use is
based on the turbidometric determination of bacterial growth in micro titer plates. The bacterial
growth in each well is recorded continuously at specific wavelength at 37 0C every 30 minutes
using a temperature-controlled spectrophotometer. Auto-mixing prior to each reading ensures a
43
homogeneous bacterial cell suspension. The DCT method has been used to determine the
antibacterial properties of endodontic sealers, pit and fissure sealants, luting cements and RBCs
(Weiss et al. 1996; Shirani et al. 2008; Matalon S et al. 2003; Sara et al. 2013).
Furthermore, the DCT method which relies on direct and close contact between the test micro-
organism and the test material is independent of the diffusion properties. This method is more
reliable for testing antibacterial activity of restorative materials and cements compared to ADT.
The DCT method simulates the clinical situation to some extent in which the cariogenic
microorganisms are in close contact with the experimental material. However, it must be noted
that the DCT experimental design employed is still far from the clinical situation in terms of oral
environment, margin location and surface area of material exposed at restoration margins
(Lewinstein et al. 2005).
2.7 Physical Properties of Resin based composites (RBCs)
The researchers have investigated physical properties of RBCs containing antimicrobial agents.
However the negative effects of antimicrobial agents on physical properties of RBCs have been
identified in different studies. This suggests that antimicrobial agents based RBCs may not perform
better in clinical oral environment. Hence the search for new antibacterial agents which do not
compromise physical characteristics of RBCs is ongoing. Since, it is well known that RBCs are
susceptible to hydrolytic degradation. In order to determine hydrolytic stability of RBCs
gravimetric analysis is commonly used method across the research community. By gravimetric
analysis stability of RBCs in oral environment can be predicted.
44
The various mechanical properties of RBCs such as strength, hardness, elastic modulus and
dimensional stability are affected by water sorption (Ferracane et al. 1998). The reductions of
mechanical properties of experimental RBCs containing antibacterial agents have been attributed
to the solubility and release of antibacterial agent leading to porosity and decreased strength. In
addition, it has been reported that antibacterial agent might have negative influence on curing of
RBCs. This results in deceased hardness of RBCs (Beyth N et al. 2014).
The water sorption of experimental RBCs containing up to 20% synthesized fluoride-releasing
monomer was evaluated and found to be similar to that of control RBCs (Ling L et al. 2009). In
another study, water sorption and solubility of control and experimental RBCs comprising of 2-
Dimethyl-2-dodecyl-1-methacryloxyethyl ammonium iodine (DDMAI) antibacterial agent was
investigated. The significant increase in water sorption and solubility values of experimental RBCs
containing DDMAI has been reported. The DDMAI structure consists of both positive and
negative charges and has potential for more water uptake. Therefore, increased water sorption of
DDMAI containing RBCs has been reported. In addition, these experimental RBCs exhibited
higher water solubility compared to control group (Jingwei et al. 2013). The Rüttermann et al,
investigated the water sorption and solubility of experimental RBCs containing Irgasan/ triclosan
antibacterial agent using ISO 4049 standard. The water sorption and solubility of experimental
RBCs did not differ from control RBCs (Rüttermann S et al, 2013). The hardness of triclosan
modified RBCs was evaluated using knoop hardness tester. It was found that addition of triclosan
negatively influenced the hardness of RBCs (Paula AB et al 2013).
45
2.8 Unanswered Questions in Literature
The antibacterial properties of RBCs remain a cause of concern among clinicians as well as
researchers. Moreover, negative effects of various antimicrobial agents on physical characteristics
of RBCs have been reported in literature. Hence, search for suitable antimicrobial agent which
may equip RBCs with antibacterial activity against cariogenic bacteria without compromising its
physical properties is ongoing. Therefore, the unanswered questions addressed in this study
included does the addition of antimicrobial chitosan into RBCs equip the material with
antibacterial activity against cariogenic bacteria namely Streptococcus mutans, Lactobacilli casei
and Actinomyces viscous. In addition, does the addition of antimicrobial chitosan into RBCs would
significantly affect its physical properties?
46
2.9 Aim of study
The aim of this study was synthesis of RBCs with antimicrobial activity against cariogenic bacteria
with suitable physical properties.
2.10 Objectives
1. To prepare experimental micro hybrid and flowable RBCs containing 0.25, 0.5 and 1 %
chitosan.
2. To test antibacterial activity of control and experimental micro hybrid and flowable RBCs
containing chitosan using agar disc diffusion (ADT) and direct contact test (DCT) method.
3. To determine water sorption and solubility of control and experimental micro hybrid and
flowable RBCs.
4. To evaluate vicker hardness of control and experimental micro hybrid and flowable RBCs.
2.11 Hypothesis
Our hypothesis was that addition of CS into RBCs may render antibacterial effect, without
compromising physical (water sorption, solubility) and mechanical (Vickers hardness) properties.
47
CHAPTER 3
Material and Methods
48
3. Introduction
The experimental RBCs containing different weight percentages of chitosan were prepared. Then
for investigation of antibacterial activity and physical properties control and experimental RBCs
specimen were fabricated. The antibacterial activity of control and experimental RBCs specimens
was determined via agar diffusion test as well as direct contact test. The antibacterial activity test
included cultivation of cariogenic bacteria i.e. namely Streptococcus mutans, Lactobacilli casei
and Actinomyces viscous in current study. The water sorption, solubility and hardness of control
and experimental RBCs specimen was investigated following ISO standard. The materials used
during this study are described in following section 3.1.
3.1 Materials used during this research work were:
i. Two commercial RBCs (Table 3.1)
A. Conventional micro hybrid RBCs (Filtek Z350 XT 3M ESPE USA)
B. Flowable RBCs (Filtek Z350 3M ESPE USA)
ii. High molecular weight Chitosan (CS) coarse ground flakes and powder (Sigma Aldrich,
MO USA) with >75% degree of deacetylation
iii. Growth media for bacteria namely
A. Mitis Salivarius Agar (MSA) (Fluka, Sigma Aldrich, MO USA)
B. Brain heart infusion (BHI) broth (Fluka, Sigma Aldrich, MO USA)
C. Phosphate buffer saline (PBS) (Sigma Aldrich, MO USA)
D. Bacitracin (Sigma Aldrich, MO USA)
E. deMan, Rogosa and Sharpe (MRS) agar (Oxoid Ltd Hampshire, England)
49
F. deMan, Rogosa and Sharpe (MRS) broth (Oxoid Ltd Hampshire, England)
G. Brain heart infusion (BHI) agar (Oxoid Ltd Hampshire, England)
iv. Three strains of Cariogenic bacteria
A. Actinomyces viscous ATCC (American type culture collection) #15987
Microbiologiscs USA
B. Streptococcus mutans ATCC# 25175 Microbiologiscs USA
C. Lactobacilli casei ATCC#343 Microbiologiscs USA
v. Glass and Plastic wares used
A. Petri dishes
B. Test tubes
C. Glass beaker
D. Glass rod
E. Glass slide
F. Glass slab
G. Glass flask
H. Glass bottle
vi. Instruments used
A. Analytic balance (Ay-Z20 Japan)
B. Elipar LED curing unit (3M ESPE, Germany)
C. Autoclave (Thermo Scientific, USA)
D. Dry heat Oven (Tuttingen Germany)
E. CO2 Incubator (Thermo Scientific, USA)
F. Refrigerator (GABA Pakistan)
50
G. Vortex (Gyro mixer China)
H. Incubator (Tuttingen Germany)
I. Micrometer Screw gauge (Aiishil International Thane West, Thane)
J. Centrifuge machine (Hermle Labnet Z 200 A)
K. Desiccator (Humboldt Mfg. Co. USA)
L. Vickers hardness tester (Wolpert 402MVD USA)
M. Light microscope (Leica DM 500 Switzerland)
N. Digital camera(Sony DSCW 830 Japan)
O. Wire loop
P. Juster (Micropipette)
3.2 Methodology
3.2.1 Experimental Resin based composites (RBCs) preparation
Six experimental RBC groups were prepared by adding 0.25, 0.5, and 1.0% (w/w) CS to each
commercial micro hybrid and flowable RBCs. The RBCs and CS were weighed using analytic
balance (Ay-Z20 Japan) (Figure 3.1) accurate to 0.001 gram. In a 50 ml glass beaker CS was
incorporated into the RBCs and homogeneously mixed in a dark room with glass rod. The CS
mixed RBCs in glass beaker was protected from surrounding light by use of silver foil. The
commercial micro hybrid and flowable RBCs without CS served as control groups.
51
Figure 3.1: Analytic weight balance
52
Table 3.1: Composition of RBCs used in present study
Material Classification Resins Filler Filler weight %
(Volume)
Filtek Z 350 XT
micro hybrid
Micro hybrid dentin
A3 shade
BIS-GMA
UDMA
TEGDMA
PEGDMA
Bis-EMA
Zirconia/ Silica
4 nm-10 microns
78.5 % (63.3 %)
Filtek Z 350 XT
flowable
Nano filled A3
shade
BIS-GMA
TEGDMA
Bis-EMA
Zirconia / Silica
5nm- 1.4 microns
65% (55%)
53
3.2.2 The Resin based composites (RBCs) Specimen preparation
For determination of antibacterial activity of experimental RBCs via agar disc diffusion (ADT)
test, total five disc-shaped specimens (15 mm diameter, 2 mm thickness) of each RBC group were
prepared using aluminum mold (Figure 3.2) as specified by ISO 4049, 2000. The cellulose acetate
strip was used to cover top and bottom surfaces of each specimen (0.1 mm thickness) to decrease
the inhibition of specimen polymerization from environmental oxygen (Shawkat et al. 2009). All
specimens were light polymerized from top surface by Elipar LED curing unit (3M ESPE,
Germany) (Figure 3.3). The curing light tip diameter was 10 mm and it was placed in contact with
the acetate strip and specimen was cured in an orbital sequence four times for 40 seconds each in
overlapping shots (Wolf H. et al. 2012). Then each specimen (Figure 3.4) was taken out of mold
and flash cut away using a blade.
The RBCs specimens were sterilized by using ethanol for total one hour. This method was used
following established methodology reported in literature (Kraigsley et al. 2012). Initially, ethanol
was diluted in distilled water to 70 % (by Volume). The control and experimental RBCs specimens
were immersed in 70 % ethanol for half an hour, followed by another 30 minutes immersion of
same specimens in 100 % ethanol. Then specimens were ready for antibacterial activity testing
(Kraigsley et al. 2012).
54
Fig 3.2: Aluminium mould for RBCs specimen
Fig 3.3: Elipar LED curing light
55
Fig 3.4: RBCs specimen discs for ADT
Figure 3.5: Teflon mould
56
Figure 3.6: RBCs specimen discs for DCT
57
3.3 Test Microorganism Growth
Three ATCC (American type culture collection) bacteria namely Streptococcus mutans,
Lactobacilli casei and Actinomyces viscous were grown. Initially suitable agar and broth media
were prepared following manufacturer instructions. Then test ATCC bacteria were inoculated on
appropriate agar plate followed by incubation. To prepare bacterial suspension few colonies of
bacteria were transferred aseptically to suitable broth and incubated overnight. The details for
growth media used for each test bacterium and its incubation temperature and time are given on
the following pages (Section 3.3.1 to 3.6).
3.3.1 Streptococcus mutans Growth
3.3.1.1 Mitis Salivarius Agar (MSA) Preparation
Initially, distilled water (dH2O) was measured with measuring cylinder and poured into 500 ml
flask. Then weighed MSA powder was added to it, flask swirled gently to dissolve powder
completely followed by autoclaving at 121°C for 15 minutes. It was then allowed to equilibrate to
room temperature and poured into petri dishes. As growth media solidified completely into petri
dishes, they were stored upside down at 2-8 0C, until inoculation of streptococcus mutans.
3.3.1.2 Brain Heart Infusion (BHI) Broth Preparation
Distilled water (dH2O) was measured and poured into 500 ml flask. The weighed BHI powder was
added to it, flask swirled to ensure BHI powder dissolved homogenously. Prepared BHI broth was
58
transferred to test tubes and sterilized by autoclaving at 121°C for 15 minutes. It was allowed to
cool down at room temperature. The BHI containing test tubes were stored at 2-8 0C until
inoculation of Streptococcus mutans.
3.3.1.3 Phosphate buffer saline (PBS) Preparation
One sachet of PBS powder (Sigma, St. Louis, MO, USA) was mixed in 1 liter of distilled water.
Note: MSA, BHI broth and PBS were supplemented with 0.0625 g/ml bacitracin (Sigma, St.
Louis, MO, USA) to minimize contamination (Beyth et al. 2006).
3.3.1.4 Inoculation and incubation of Streptococcus mutans
The pure culture of Streptococcus mutans ATCC#25175 kwik-stik were obtained from
Microbiologiscs USA (Figure 3.7). The kwik stik were chosen because they are easy to use since
the hydrating fluid and inoculating swab are all in one device hence risk of contamination is less
and storage is easy. The Streptococcus mutans kwik-stik pouch which was stored in a refrigerator
at 2-8 0C was allowed to cool down to room temperature prior to tearing the pouch at notch and
removing the kwik-stik unit. The label on the kwik stik was removed and attached to the primary
culture plate. The ampoule at the top of the kwik-stik (just below the fluid meniscus of the
ampoule) was pinched once to release the hydrating fluid. Kwik stik was held vertically and tapped
on a hard bench surface to facilitate flow of hydrating fluid through shaft into bottom of unit
containing bacterial pellet. Using a pinching action on the bottom portion of the kwik stik unit, the
pellet was crushed in the fluid until the pellet suspension is homogenous. The swab heavily
saturated with the hydrating fluid was immediately transferred to MSA agar medium for
59
inoculation. Using a sterile loop, streaking was performed to facilitate colony isolation. The
inoculated primary culture plate was immediately incubated at 37 0C in 5 % CO2 Incubator for 24
hours (Figure 3.8). Following 24 hours of incubation, growth of Streptococcus mutans was evident
(Figure 3.9). Then purity of bacterial growth was confirmed by gram-staining prior to preparation
of bacterial suspension in phosphate buffer saline (Figure 3.10).
Figure 3.7: Streptococcus mutans kwik stik
Figure 3.8: CO2 Incubator
60
Figure 3.9: Streptococcus mutans growth on MSA agar
Figure 3.10: Gram stain slides
61
3.3.1.5 Transfer of bacterial culture
Flame sterilization method was used for sterilization of an inoculating wire loop during transfer of
bacterial culture to avoid contamination (Figure 3.11). This method was chosen because it is
quicker and easy way of destroying microorganisms. The wire loop was held inside a flame for a
few seconds to bring it to redness and then cooled. Using sterile wire loop 3-4 identical colonies
of Streptococcus mutans from MSA plates were transferred to 5 ml of BHI broth and incubated
over night at 37 0C in 5 % CO2 Incubator ( Thermo Scientific USA).
3.3.1.6 Centrifuge and vortex
Following overnight incubation of inoculated BHI broth, to prevent formation of bacterial
aggregates the top 4 ml BHI broth with bacteria was passed to another test tube and centrifuged
for 10 minutes at 3000 rpm (Figure 3.12). The bacterial pellet was formed at the bottom of test
tube as a result of centrifuge, then superficial broth was discarded. The bacterial pellet was
suspended in 5ml of phosphate-buffered saline (PBS) (Sigma, St. Louis, MO, USA) and vortexed
gently for ten (10) seconds (Figure 3.13). The bacterial suspension in PBS was stored at 2-8 0C
prior to antibacterial activity test. The turbidity of bacterial suspensions was adjusted to 0.5 Mc
Farland Standard equivalents to 1.5 × 108 colony forming unit per milliliter (CFU/ml). The Mc
Farland Standard is a barium sulphate standard against which the turbidity of the test bacterial
suspension is compared. The standard and bacterial suspension was shaken
62
immediately before use. When the bacterial suspension turbidity matched with the standard (Figure
3.14), it was spread on MSA agar plates for incubation.
3.4 Preparation of 0.5 Mc Farland turbidity standard
Initially 1% v/v solution of sulphuric acid was prepared by adding 1 ml of concentrated sulphuric
acid to 99 ml of water. Then 1% w/v solution of barium chloride was prepared by dissolving 0.5
gram of dihydrate barium chloride (BaCl2.2H2O) in 50 ml of distilled water. The turbidity standard
0.5 Mc Farland was prepared by addition of 0.6 ml of prepared barium chloride solution to 99.4
ml of the sulphuric acid solution. Small volume of the Mc Farland solution was transferred to
screw-cap test tube of the same type as used for preparing the bacterial suspension. The Mc Farland
standard may be preserved for period six (6) months.
63
Figure 3.11: Wire loop and flame
Figure 3.12: Centrifuge machine
64
Figure 3.13: Vortex equipment
Figure 3.14: Mcfarland 0.5 turbidity tube (left) and bacterial suspension (right)
65
3.5 Growth of Lactobacilli casei
The Lactobacilli casei kwik stik (ATCC#343) was inoculated on MRS agar and incubated for 24
hours in 5 % CO2 Incubator (Thermo Scientific USA) at 37 0C. Following 24 hours of incubation
growth of lactobacilli casei was evident (Figure 3.15). Then 3-4 colonies of Lactobacilli casei were
aseptically transferred to MRS broth and incubated in 5 % CO2 Incubator at 37 0C overnight to
prepare bacterial suspension. Overnight incubated MRS broth (Figure 3.16) was centrifuged at
3000 rpm for ten minutes and supernatant was discarded. Then the PBS was added to pellet of
microorganism formed as a result of centrifuge and it was vortex mixed for few seconds. Bacterial
suspension turbidity was matched to 0.5 McFarland standards prior to its use for antibacterial
activity test via ADT and DCT.
66
Figure 3.15: Growth of Lactobacilli casei on MRS agar
Figure 3.16: Lactobacilli casei growth in MRS broth
67
3.6 Growth of Actinomyces viscous
Actinomyces viscous kwik stik (ATCC#15987) was grown on BHI agar plate and incubated for 24
hours at 37 0C in 5 % CO2 Incubator (Thermo Scientific USA). The growth was evident following
24 hours of incubation (Figure 3.17). Then few colonies of Actinomyces viscous were transferred
to BHI broth and incubated overnight at 37 0C in 5 % CO2 Incubator. Then BHI broth containing
Actinomyces viscous (Figure 3.18) was centrifuged at 3000 rpm for 10 minutes then supernatant
was discarded. The bacterial pellet formed as a result of centrifuge was suspended in 5 ml of PBS
and vortex mixed gently for 10 seconds and its turbidity was adjusted to 0.5 McFarland prior to
antibacterial activity experiments via ADT and DCT.
68
Figure 3.17: Growth of Actinomyces viscous on BHI agar
Figure 3.18: Actinomyces viscous growth in BHI broth (left) plain BHI broth (right)
69
3.7 Antibacterial activity test
3.7.1 Agar disc diffusion test (ADT)
The antibacterial test via ADT method was conducted following the procedures stated in literature
(Beyth et al. 2006). The bacterial suspension of Streptococcus mutans, Lactobacilli casei and
Actinomyces viscous (200 μl volume) turbidity equivalent to 0.5 Mc Farland standards was spread
on a Petri dish (20x100 mm) containing suitable agar for each bacterium as has been described in
section 3.3.1, 3.5 and 3.6. Once bacterial suspension was uniformly distributed, petri dishes were
allowed to dry for 1 hour before placing experimental and control RBCs discs. The RBCs test and
control specimens (n=5) were placed on the inoculated agar surface (Figure 3.19) with the help of
sterilized tweezers. Then these petri dishes were incubated for 48 hours at 37°C in 5% CO2
Incubator (Thermo Scientific, USA). After 48 hours of incubation the diameter of inhibition zone
around each specimen disc was measured in mm using a metric ruler, by placing the ruler on the
bottom of the plate (Beyth N et al. 2006).
70
Figure 3.19: The RBCs specimen placed on surface of inoculated agar for ADT
71
3.7.2 Direct Contact Test
Each specimen (n=5) of experimental and control RBCs group was fit into the bottom of 1.5 ml
sterile eppendorf tube (Figure 3.20). Then 10 µl of bacterial suspension of Streptococcus mutans,
Lactobacilli casei and Actinomyces viscous was placed over the specimen followed by incubation
for an hour at 37 0C to allow evaporation of liquid and establishment of direct contact between
bacteria and test material. Then 300 µl of fresh suitable broth was added to the tube and incubated
for 48 hours in 5 % CO2 Incubator (Thermo Scientific USA). Then the bacterial suspension was
serially diluted prior to colony forming unit counting via miles and misra technique (Tin-Oo MM
et al, 2007). Preparation of serial dilutions for colony forming unit (CFU) count comprised of
transfer of 100 µl bacterial suspension from original specimen containing eppendorf tube to
subsequent seven 1.5 ml eppendorf tube each consisting of 900 µl plain PBS. The original
eppendorf tube containing specimen with bacteria and each dilution eppendorf tube were
thoroughly mixed before sampling and a separate pipette was used for each transfer step to prevent
carry-over of bacteria.
72
Figure 3.20: RBCs specimen fit into bottom of sterile eppendorf tube for DCT
73
3.7.2.1 Miles-Misra technique
This technique is easy and economical for bacterial enumeration. An inoculum of 20 µl from each
serial dilution was pipetted as a drop on the suitable agar (Tin-Oo MM et al, 2007). At least 4
separate drops per sample dilution were pipetted on each agar containing petri dish. The bacterial
inoculums were allowed to dry and the petri dishes were incubated at 37°C for 24 hours in 5 %
CO2 Incubator ( Thermo Scientific USA). A sample dilution yielding about thirty (30) colonies per
drop was selected (Figure 3.21). An average count from at least 4 drops was obtained. The bacterial
CFU were calculated using following formula
Number of CFU/ml =N x 10n x 50
N = number of colonies on the petri dish at the selected dilution n.
74
Figure 3.21: The CFU of Lactobacilli casei evident on MRS agar via miles and misra technique
75
3.8 Water sorption and solubility
Total five disc-shaped specimens (15 mm diameter, 2 mm thickness) of each experimental and
control RBCs group were prepared using aluminum mold as stated in section 3.2.2. The water
sorption and solubility of each RBCs group (n = 5) was assessed following 1 week immersion in
distilled water (Figure 3.22) which is the commonly employed specimen storage period according
to ISO 4049, 2000. Initially, the specimens were stored in a desiccator (Humboldt Mfg. Co. USA)
(Figure 3.23). Then the same desiccator containing specimens was placed in an incubator at 37°C
for 22 hours. Subsequently, the desiccator was removed from the incubator and kept at 23°C for 2
hours. Then specimens were removed and weighed in a weighing boat using an analytical balance
(Ay-Z20 Japan) accurate to 0.001 gram. This conditioning cycle was repeated until a constant mass
(m1) was achieved. The diameter and thickness of each specimen was measured with a micrometer
screw gauge (Aiishil International Thane West, Thane) accurate to 10µm to calculate the
specimen volume (V; in mm3) (Figure 3.24). The diameter of specimen was measured at two points
and mean was taken. Thickness was measured at five points and mean was taken for analysis. The
volume of specimen was calculated by using following formula
V= π×r2×h
Subsequently, specimens were immersed in distilled water for 1week at 37°C. Following storage
regime, specimens were taken out. The excess water was removed using paper towel and the
specimen were waived in the air at 23°C for 15 seconds and reweighed (m2). The immersed
specimens were reconditioned using the above-said conditioning cycle until a constant mass was
obtained (m3). The mean water sorption and solubility of each specimen was calculated according
to equations (1) and (2).
76
Water sorption = m1- m2 (1)
V
Water solubility = m1- m3 (2)
V
77
Figure 3.22: RBCs specimen in distilled water
Figure 3.23: RBCs specimen in desiccator
78
Figure 3.24: Micro meter screw gauge
79
3.9 Hardness Test
For determination of vicker hardness, total five disc-shaped specimens (15 mm diameter, 2 mm
thickness) of each experimental and control RBCs group were prepared as stated in section 3.2.2.
All specimens were light polymerized from top and bottom surface by Elipar LED curing unit (3M
ESPE, Germany) for total 160 seconds for each surface in four overlapping shots. Prior to hardness
test specimens were finished, polished and then mounted. Three Vickers indentations (load: 200g;
dwell time: 10 seconds) were performed on either top or bottom surface of each specimen, using
a digital vicker hardness tester (Wolpert 402MVD USA). The mean of these three individual indent
measurements were taken as the hardness value (Hv) for each specimen.
3.10 Statistical Analysis
One-way AOVA is used to compare the means of three or more study groups having one
independent variable and one measurement/dependent variable. The independent variable has two
or more levels. One way ANOVA determines whether the means of the measurement variable are
the same for the different levels of independent variable. The independent variable in this study
was chitosan based dental composites with three levels i.e. 0.25, 0.5 and 1 % of chitosan. The
measurement variable/ dependent variable in this study included antibacterial activity, water
sorption, water solubility and hardness, total five observations for each measurement variable were
made in this study.
The limitation of one way ANOVA test is that it determines the difference in means as a whole.
This test alone does not tell which independent variable level differed. Hence post hoc tukey were
also run to confirm where the differences occurred between groups. The advantage of ANOVA
80
test is that it avoids the type 1 (False positive) error by comparing means of all sample groups at
the same time in contrast to T test. The level of significance was set at P ≤ 0.05.
81
CHAPTER 4
Results
82
4.1 Antibacterial Activity test Results
According to agar disc diffusion test method disc shaped specimens of control and experimental
flowable as well as micro hybrid RBCs supplemented with 0.25, 0.5 or 1 % w/w chitosan did not
inhibit the growth of test bacteria, hence no growth inhibition zone was evident in the lawn growth
of Streptococcus mutans (Fig 4.1 A and B), Lactobacilli casei (Fig 4.2 A and B) and Actinomyces
viscous (Fig 4.3 A and B).
The antibacterial activity of control and experimental RBCs was determined via direct contact test
method also. It was found that colony forming unit (CFU) count of Streptococcus mutans,
Lactobacilli casei and Actinomyces viscous were comparable/ similar among the experimental and
control RBCs as shown in table 1-6. There was statistically no significant difference (P≥0.05)
among control and experimental RBCs.
The experimental RBCs failed to exhibit antibacterial activity in this study.
83
Figure 4.1 A: No inhibition zone is evident around experimental flowable RBCs against
Streptococcus mutans
Figure 4.1 B: No inhibition zone is evident around experimental micro hybrid RBCs against
Streptococcus mutans
84
Figure 4.2 A: No inhibition zone is evident around experimental flowable RBCs against
Lactobacilli casei
Figure 4.2 B: No inhibition zone is evident around experimental micro hybrid RBCs against
Lactobacilli casei
85
Figure 4.3 A: No inhibition zone is evident around experimental flowable RBCs against
Actinomyces viscous
Figure 4.3 B: No inhibition zone is evident around experimental micro hybrid RBCs against
Actinomyces viscous
86
Table 4. 1: Colony forming unit (CFU) count of Streptococcus mutans bacteria per 20 µl after 48
hours of contact with Flowable RBCs
P value= 0.668 (One way ANOVA with post hoc Tukey test)
Material N=specimen Minimum
CFU
Maximum
CFU
Mean SD Dilution
Control Flowable
RBC
5 21 23 21.400
0.894 10-7
Flowable RBC +
0.25 % CS
5 21 22 21.400
0.548 10-7
Flowable RBC +
0.50 % CS
5 20 23 21.400
1.34 10-7
Flowable RBC + 1 %
CS
5 21 23 22.000
0.707 10-7
87
Table 4. 2: Colony forming unit (CFU) count of Streptococcus mutans bacteria per 20 µl after
48hours of contact with micro hybrid RBCs
P value= 0.329 (One way ANOVA with post hoc Tukey test)
Material N=specimen Minimum
CFU
Maximum
CFU
Mean SD Dilution
Control Micro hybrid
RBC
5 21 26 23.40
2.30 10-7
Micro hybrid RBC +
0.25 % CS
5 21 24 22.200
1.095 10-7
Micro hybrid RBC +
0.50 % CS
5 21 25 23.000
1.871 10-7
Micro hybrid RBC +
1 % CS
5 23 26 24.200
1.095 10-7
88
Table 4.3: Colony forming unit (CFU) count of Lactobacilli casei bacteria per 20 µl after 48 hours
of contact with flowable RBCs
P value= 0.58 (One way ANOVA with post hoc Tukey test)
Material N=specimen Minimum
CFU
Maximum
CFU
Mean SD Dilution
Control Flowable
RBC
5 23 28 25.400
1.817 10-7
Flowable RBC +
0.25 % CS
5 30 30 30.00
0.00 10-7
Flowable RBC +
0.50 % CS
5 20 28 24.80
3.27 10-7
Flowable RBC + 1 %
CS
5 18 30 26.00
4.69 10-7
89
Table 4.4: Colony forming unit (CFU) count of Lactobacilli casei bacteria per 20 µl after 48 hours
of contact with micro hybrid RBCs
P value= 0.409 (One way ANOVA with post hoc Tukey test)
Material N=specimen Minimum
CFU
Maximum
CFU
Mean SD Dilution
Control Micro hybrid
RBC
5 19 22 21.200
1.30 10-7
Micro hybrid RBC +
0.25 % CS
5 15 22 19.40
2.88 10-7
Micro hybrid RBC +
0.50 % CS
5 18 24 20.60
2.41 10-7
Micro hybrid RBC + 1
% CS
5 16 22 18.80
2.77 10-7
90
Table 4.5: Colony forming unit (CFU) count of Actinomyces viscous bacteria per 20 µl after 48
hours of contact with micro hybrid RBCs
P value= 0.615 (One way ANOVA with post hoc Tukey test)
Material N=specimen Minimum
CFU
Maximum
CFU
Mean SD Dilution
Control Micro hybrid
RBC
5 20 23 21.400
1.342 10-7
Micro hybrid RBC +
0.25 % CS
5 20 24 21.800
1.483 10-7
Micro hybrid RBC +
0.50 % CS
5 21 24 22.200
1.304 10-7
Micro hybrid RBC +
1 % CS
5 20 22 21.200
0.837 10-7
91
Table 4.6: Colony forming unit (CFU) count of Actinomyces viscous bacteria per 20 µl after
48hours of contact with flowable RBCs
P value= 0.153 (One way ANOVA with post hoc Tukey test)
Material N=specimen Minimum
CFU
Maximum
CFU
Mean SD Dilution
Control Flowable
RBC
5 22 23 22.200
0.837 10-7
Flowable RBC +
0.25 % CS
5 20 22 20.200
2.049 10-7
Flowable RBC +
0.50 % CS
5 17 22 21.400
0.894 10-7
Flowable RBC + 1 %
CS
5 20 22 21.600
1.140 10-7
92
4.2 Water sorption and solubility
The mean water sorption and solubility of control and experimental flowable RBC groups are
presented in table 4.7. The water sorption values of flowable RBCs in this study ranged from 3.3
to 5 µg/mm3 which is within the acceptable range of 40 µg/mm3 as per ISO 4049 standard. When
comparing mean water sorption among control and experimental flowable RBCs, the results follow
this order: control flowable RBCs < flowable RBCs + 0.50 % CS< flowable RBCs + 0.25 % CS <
flowable RBCs + 1 % CS. The difference in water sorption of control and experimental flowable
RBCs were found statistically non-significant (P>0.05). Moreover, solubility of experimental
flowable RBC groups ranged from -2.2 to -3.8 𝜇g/mm3 which remained in ISO acceptable range
i.e. <7.5 𝜇g/mm3.
The mean water sorption and solubility values of control and experimental micro hybrid RBC
groups are listed in table 4.8. The control and experimental micro hybrid RBCs water sorption
values ranged from 4.8 to 7 𝜇g/mm3. The increase in CS content into micro hybrid RBCs led to
greater water sorption in current investigation. The results follow this order: control micro hybrid
RBCs < micro hybrid RBCs + 0.25 % CS< micro hybrid RBCs + 0.50 % CS < micro hybrid RBCs
+ 1 % CS. However, water sorption of control and experimental micro hybrid RBCs was found
to be within the ISO accepted range i.e. 40 𝜇g/mm3.
The water sorption and solubility values of experimental RBCs remained within ISO acceptable
range indicating that there is no negative effect of up to 1 % CS addition into RBCs.
93
Table 4.7: Mean water sorption and solubility values (µg/mm3) of experimental and control
flowable RBCs, same alphabet letters in P value column indicate that data are not statistically
significant.
Material Mean Water
sorption
(Standard deviation)
P value=
0.236
Mean Water
Solubility
( Standard
deviation)
P
value=0.429
Control Flowable
RBC
3.3 (1.3) A -3.8 (2.5) A
Flowable RBC +
0.25 % CS
5.0 (1.2) A -3.7 (1.5) A
Flowable RBC +
0.50 % CS
4.1 (1.4) A -2.7 (3.2) A
Flowable RBC + 1
% CS
4.6 (1.1) A -2.2 (3.1) A
One way ANOVA with post hoc Tukey test was used
94
Table 4.8: Mean Water sorption and solubility values of experimental micro hybrid RBCs
(µg/mm3), same alphabet letters in P value column indicate that data are not statistically
significant.
Material Mean Water
sorption
(Standard deviation)
P value=
0.091
Mean Water
Solubility
( Standard deviation)
P value=
0.261
Control Micro hybrid
RBC
4.8 (0.3) A -6.5 (1.6) A
Micro hybrid RBC +
0.25 % CS
5.4 (2.2) A -2.5 (2.7) A
Micro hybrid RBC +
0.50 % CS
6.3 (1.0) A -3.5 (5.8) A
Micro hybrid RBC + 1 %
CS
7.0 (1.2) A -5.5 (1.0) A
One way ANOVA with post hoc Tukey test was used
95
4.3 Hardness
The mean Vickers hardness values for control and experimental flowable as well as micro hybrid
RBCs are listed in table 4.8 and 4.9 respectively. The control and experimental flowable RBCs
hardness values ranged from 47.5 to 52.2 HV. The flowable RBCs containing 1 % CS had
significantly higher hardness values compared to control and other experimental RBC groups (P
value= 0.002).
The control and experimental micro hybrid RBCs hardness number varied from 75 to 82.5 HV.
The micro hybrid RBCs consisting of 0.50 % CS exhibited significantly higher hardness values
compared to experimental micro hybrid RBC group containing 1 % CS (P value=0.015). The
addition of CS into RBCs increased its hardness indicating positive effect of CS incorporation into
RBCs.
There was a statistically significant difference among the mean hardness values of control and
experimental flowable as well as control and experimental micro hybrid RBCs.
96
Table 4.9: Mean Vickers hardness values and standard deviation (SD) of experimental and control
flowable RBCs. The different alphabet letters in P value column indicate statistically significant
difference.
Material Vickers Hardness number (HV) SD P value=
0.002
Control Flowable RBC 48.764
1.940 B
Flowable RBC + 0.25 % CS 47.514
1.847 B
Flowable RBC + 0.50 % CS 49.940
1.244 A,B
Flowable RBC + 1 % CS 52.218
1.040 A
97
Table 4.10: Mean Vickers hardness values and standard deviation (SD) of experimental and
control micro hybrid RBCs. The different alphabet letters in P value column indicate statistically
significant difference.
Material Vickers Hardness number
(HV)
SD P value=
0.015
Control Micro hybrid RBC 82.10
4.44 A, B
Micro hybrid RBC + 0.25 % CS 76.720
0.836 A,B
Micro hybrid RBC + 0.50 % CS 82.57
3.36 A
Micro hybrid RBC + 1 % CS 75.16
5.31 B
98
CHAPTER 5
Discussion
99
5. Discussion
It was discovered that addition of up to 1 % CS into flowable and micro hybrid RBCs had no
negative effect on its water sorption and solubility properties. The water sorption and solubility of
experimental RBCs remained within ISO acceptable range. These water sorption and solubility
results of experimental RBCs indicates over all satisfactory dispersion and interaction of CS with
content of commercial RBCs. Moreover, the Vickers hardness of some experimental RBCs also
increased, indicating no negative effect of addition of CS on degree of polymerization of
experimental RBCs. Many previous studies investigating antibacterial activity of experimental
RBCs reported negative effect of antimicrobial agent on RBCs physical properties (Sevinc BA and
Hanley, 2010). On the contrary, in this study it was discovered that CS incorporation into RBCs
had no negative effect on its physical properties.
5.1 Antibacterial activity
The Streptococcus mutans, Lactobacilli casei and Actinomyces viscous bacteria are often reported
to cause dental caries in human beings (Mjör, 2005; Wang et al. 2014). These bacteria accumulate
on the surface of dental restorations as well as at interface of tooth and restoration leading to
secondary caries. One of the major reasons for restoration replacement is secondary caries; this
problem has attracted great attention of clinicians as well as researchers. Since the RBCs has
tendency to allow more accumulation of cariogenic microorganisms compared to other restorative
materials resulting in secondary caries and clinical failure (Song et al, 2015). Therefore,
investigators incorporated various antimicrobial agents into RBCs to address this problem. Many
of these antibacterial agents had negative effect on physical properties of RBCs (Weng Y et al.
2012).
100
The chitosan a versatile and safe biopolymer has been used in many commercial products due to
its antimicrobial activity. There is ample evidence that addition of CS in dental restorative
materials inhibit the attachment of cariogenic bacteria without compromising its mechanical and
physical characteristics (Tavaria FK et al, 2013). Therefore, present study made an attempt to
determine the antibacterial activity of RBCs containing CS coarse ground flakes and powder. The
ADT method has been frequently used by researchers for evaluation of antimicrobial activity of
commercial and experimental RBCs. Therefore, to allow comparison of findings of current study
with previous studies ADT method was used. The CS was added at 0, 0.25, 0.5, and 1 % into
flowable and micro hybrid RBCs. The antibacterial activity test results revealed in present study
that control and experimental groups of RBCs exhibited no inhibition zone in lawn growth of
Streptococcus mutans, Lactobacilli casei and Actinomyces viscous after 48 hours of incubation.
The lack of inhibition zone in lawn growth of test bacteria in current investigation indicates no or
low diffusion of CS from experimental RBCs in surrounding agar media. Moreover, lack of
inhibition zone may be attributed to probable copolymerization of CS biopolymer with matrix of
RBCs and non-releasing behavior. Since, graft copolymerization of chitosan (CS) with methyl
methacrylate, meth acrylic acid, 2 hydroxy ethyl methacrylate (HEMA) and N,N- dim ethyl amino
ethyl methacrylate (DMAEMA) has been reported in the literature (Singh DK and Ray AR. 1994
and 1997).
The previous studies reported no growth inhibition zone around control and experimental RBCs
containing polyethylenimine (PEI) nanoparticles and MDPB antibacterial agents (Beyth N et al,
2010; Imazato S. 2003). Since ADT is capable of determining antibacterial activity of only soluble
agents which diffuse into agar growth media and inhibit the bacterial growth. This method is not
reliable for testing non soluble antibacterial agents which exhibit bacterial growth inhibition via
101
surface contact. Therefore, in current study direct contact test (DCT) test was used as well to
quantitatively determine antibacterial activity of control and experimental RBCs following
established methodology reported in the literature.
In DCT method bacteria responsible for dental decay are allowed to come in direct close contact
with experimental and control RBCs and are incubated at appropriate temperature for specific
duration. Many past studies used spectrophotometer to determine antibacterial activity of RBCs
following direct contact between test material and bacteria. This method is based on the turbidity
measurement of the inoculated culture media without taking into account viability of test bacteria
(Beyth N et al. 2007). The metabolically active and vital cariogenic bacteria play pivotal role in
development of dental decay. Hence, determination of bacterial count via spectrophotometer or
direct imaging of bacteria after staining on the surface of RBCs specimen under a fluorescent
microscope may not be a suitable method (Farrugia C and Camilleri J, 2015). Therefore, in the
present study only living bacterial colonies were enumerated via miles and misra technique
following direct contact between bacteria and test material. The colony forming unit (CFU) count
of Streptococcus mutans, Lactobacilli casei and Actinomyces viscous were comparable/ similar
among the control and experimental RBCs in the current investigation. There was statistically no
significant difference in CFU count of control and experimental RBCs. In contrast to the present
study, Kim (2013) reported antibacterial effect of CS when incorporated into RBCs at a
concentration of 2 % w/w. The difference in results may be due to lower concentration of CS used
in present study. More over the composition of RBCs, type of CS and antibacterial activity testing
method used in the two studies are not consistent. This study attempted to determine antibacterial
activity of CS, therefore, CS coarse ground flakes and powder was simply mixed manually via
glass rod into the commercial RBCs without a use of solvent acetic acid to avoid antibacterial
102
effect of solvent. Since, in this investigation ATCC pure cultures of three cariogenic bacteria were
used in plank tonic form only, it is recommended to determine antibacterial activity of RBCs
against bacterial biofilm form. Addition of increased CS weight percent to RBCs and its effect on
antimicrobial activity warrants further investigation in order to determine optimum concentration
of CS.
5.2 Water sorption and solubility
Since oral cavity environment is most often wet due to saliva and ingestion of drinks and food.
Therefore, to better understand behavior of CS based RBCs in similar situations and predict its
survival clinically. The water sorption and solubility properties of control and experimental
flowable as well as micro hybrid RBCs were evaluated in current study using gravimetric analysis.
Although several studies for water sorption and solubility of RBCs have been published, but it is
difficult to compare the findings due to lack of consistency among researchers. The results reported
vary a lot in water immersion time periods, measurement units, specimen size and curing. Since
specimen of variable sizes will take different time periods for water to thoroughly diffuse into the
polymer matrix. The specimen of small size may take the short time for equilibration with water
(Toledao M et al. 2003).
The water molecules diffuse into the polymer matrix of RBCs and occupy any free space between
polymer chains. This causes polymer chains separation and monomer elution leading to
degradation of material (Zhang Y and Xu J 2008; Ferracane. 2006). It is reported that water may
react with the filler at the filler resin matrix interface and break the siloxane bonds between silanol
103
groups of the silica surface and the silane coupling agent resulting in debonding of fillers (Zhang
Y and Xu J 2008). The water sorption of RBCs may compensate effects of shrinkage to some
extent by expansion and plasticizing of the resin content therefore minimizing deleterious impact
of shrinkage (Domingo C et al, 2003). However, degree of expansion that actually compensates
polymerization shrinkage is unknown and requires detailed investigation (Darvell, 2002). In
addition, increased water sorption may compromise hardness, strength, wear resistance and
modulus of elasticity of RBCs (Rahim TNA et al 2012). Therefore, development of new RBCs
requires water sorption values with in ISO accepted range. The highly filled RBCs are most likely
to exhibit lower water sorption due to more closely cross linked polymer structure (Ferracane.
2006). The least water sorption of RBCs indicates most effectively coupled resin matrix and the
filler (Kalachandra S and Wilson TW, 1992). The method of mixing used for experimental RBCs
may cause porosity and increase water uptake and solubility (Oysaed H and Ruyter IE 1986; Lygre
H et al. 1999).
This study aimed to determine effect of CS on sorption and solubility of commercial flowable and
micro hybrid RBCs by keeping the all other components of RBCs such as fillers, initiators and
resin chemistry consistent except percentage of CS.
The present investigation indicates slight increase in water sorption of experimental micro hybrid
RBCs compared to control group. However, mean water sorption values of experimental micro
hybrid RBCs were found to be acceptable as per ISO 4049 standard. The comparison of water
sorption values of experimental and control micro hybrid RBCs revealed statistically no significant
difference (p ≥ 0.05). The slightly increased water sorption of experimental flowable and micro
hybrid RBCs in current investigation may be attributed to incorporation of porosity into
experimental material during mixing (Toledao M et al. 2003). Moreover, CS may have
104
agglomerated into resin matrix of RBCs and provided pathways for diffusion of water. Hence,
dispersion pattern of CS into RBCs matrix requires further analysis via scanning electron
microscope (SEM). The water sorption and solubility results of experimental RBCs containing
antimicrobial agents other than CS may not be comparable with current investigation because of
variation in nature and chemical composition of antimicrobial compounds. Therefore, the findings
of this study require further scrutiny and follow up studies to rule out any negative effect of CS on
physical characteristics of commercial RBCs.
The dental RBCs often exhibit a solubility, means organic/inorganic components are released from
the material in oral cavity. The solubility of RBCs is usually reported to be highest during the
initial seven days. Increased solubility of RBCs negatively influences surface wear resistance and
marginal integrity (Ferracane. 1994). The solubility of experimental flowable RBCs in this study
varied from -2.2 to -3.8 𝜇g/mm3; these values are lower than the maximum acceptable value
established by the ISO 4049 standard. In addition, experimental micro hybrid RBCs solubility
ranged from -2.5 to -6.5𝜇g/mm3. The observed slight differences in solubility values of control
and experimental micro hybrid as well as flowable RBCs were statistically found non-significant
(𝑃>0.05). Negative solubility of the experimental flowable and micro hybrid RBCs in present
study may be related to its distinctive polymerization characteristics and ability to keep the water
within the resin matrix. Moreover, chemical reactions may have taken place that led to increased
weight of the material (Gopferich A, 1996; Ørtengren, U et al. 2000). Furthermore, insoluble
nature of CS in water and its probable copolymerization within resin matrix and fillers of RBCs
may be responsible for negative solubility of experimental RBCs in current study.
No any negative effect of CS on water sorption and solubility of RBCs are known to the author of
the present study, as no relevant research in this area was found. This study followed ISO 4049
105
standard to large extent. The present study reflects that RBCs containing up to 1 % CS exhibit
water sorption and solubility within ISO acceptable range. Within the limitations of current study
it may be concluded that addition of CS up to 1 % as antimicrobial additive to flowable and micro
hybrid RBCs had no negative effect on its water sorption and solubility.
5.3 Vickers hardness
Hardness of restorative materials including RBCs is an important characteristic and a valuable
indicator for their clinical performance. The hardness of RBCs indirectly reveals its extent of
polymerization. Hardness of RBCs depend on many factors such as type of resin, filler type its
proportion, resin shade, curing protocol, light intensity and spectrum of the curing device (Moore
et al. 2008). Many researchers used Vickers hardness test to evaluate level of polymerization of
experimental and commercial RBCs due to its ease and accuracy (Bouschlicher et al. 2004; Jain
and Pershing, 2003; Toledao, M et al. 1999). Because of these merits, Vickers hardness test was
chosen in the current investigation.
RBCs must undergo adequate polymerization to demonstrate desired physical and mechanical
properties so as to withstand chewing forces without degradation. Moreover, polymerization of
RBCs does influence its biocompatibility. Since, the inadequately polymerized resins may release
certain harmful radicals. This may irritate soft tissues, dental pulp and promote the growth of
bacteria. It has been reported that antimicrobial additives in RBCs may influence translucence and
the dispersion of light and consequently its degree of polymerization. This may negatively affect
RBCs hardness values (Conti C et al. 2005; Sideridou and Achilias, 2005).
106
The results of present study revealed that flowable RBCs containing 0.50 % and 1 % CS had higher
hardness values compared to control group. Whereas RBCs containing 0.25 % CS exhibited
slightly decreased hardness compared to control specimens. Hence, addition of 0.50 and 1 % CS
into flowable RBCs improved the hardness. The hardness values of flowable RBCs are
significantly lower than those for human enamel and dentin. Therefore, these materials are not
suitable for clinical use in high stress areas such as posterior teeth (Wilkerson MD et al. 1998).
However, to obtain superior adaptation to tooth cavity and reduced micro leakage flowable RBCs
can be used in conjunction with packable RBCs by virtue of easy handling and flowable nature
(Bertolotti RL and Laamanen H, 1999). The nature of resin monomers and their cross-linking
degree influence hardness (Mobarak E et al. 2009; Knobloch LA et al. 2004). In current
investigation, hardness values of experimental flowable RBCs were found similar to control or
significantly improved. These findings may be attributed to chemical interaction of CS with resin
matrix and better curing capacity of CS modified flowable RBCs.
The micro hybrid RBCs containing 0.25 % and 1 % CS exhibited lower hardness values in
comparison to control specimens. However, micro hybrid RBCs consisting of 0.50 % CS exhibited
hardness values similar to control group. Due to lack of homogeneity in the hardness results of
micro hybrid RBCs in current study, the direct association between the percentage of CS in these
RBCs and its impact on surface hardness could not be established. Author of this study does not
know what really differs among the experimental micro hybrid RBCs that lead to inconsistent
results. However, it is probable that the nature of interaction of CS with organic and inorganic
component of the RBCs play a decisive role. Although every effort was made to remain consistent
during specimen preparation and vickers hardness test. Nevertheless, slight variation in curing,
finishing and polishing of specimens may be one reason for inconsistent results.
107
The assumption that CS would be a determinant factor in RBCs hardness value was not confirmed.
In literature review, author did not find any research regarding the Vickers hardness of CS based
RBCs. Therefore, for better understanding the hardness of CS based RBCs needs further study. It
would be interesting if the further studies are conducted on Vickers hardness of CS based RBCs.
In addition, effect of CS incorporation on RBCs degree of conversion need to be evaluated.
Moreover, radio opacity, color stability, strength and surface properties including roughness needs
to be evaluated for further understanding the nature of CS based RBCs. In addition elution of CS
from RBCs should be determined; this could predict clinical stability of this new material in oral
cavity.
108
5.4 Conclusions
It may be concluded from this in-vitro antimicrobial activity analysis that the
addition of up to 1 % chitosan into RBCs failed to equip RBCs with antibacterial
activity against three common cariogenic bacteria.
It may be concluded that incorporation of 1% chitosan into RBCs had no negative
effect on its water sorption and solubility properties.
The addition of up to 1 % chitosan into RBCs led to increase in its vicker hardness
value.
109
5.5 Future Work
The antimicrobial activity of RBCs containing greater than 1 CS % w/w warrants further
investigation in order to determine optimum concentration of CS.
In this investigation ATCC pure cultures of three cariogenic bacteria were used in plank tonic form
only, it is recommended to determine antibacterial activity of CS based RBCs against bacterial
biofilm form.
The CS coarse ground flakes and powder was simply mixed manually via glass rod into RBCs in
current study, the dispersion pattern of CS into resin matrix needs to be evaluated via scanning
electron microscope.
The assumption that CS would be a determinant factor in RBCs hardness was not confirmed. It
would be interesting if the further studies are conducted on Vickers hardness of CS based RBCs.
In addition, effect of CS incorporation on RBCs degree of conversion need to be evaluated via
FTIR.
Moreover, radio opacity, colour stability, strength and surface properties including roughness
needs to be evaluated for further understanding the nature of CS based RBCs. In addition elution
of CS from RBCs should be determined; this could predict clinical stability of this new material
in oral cavity.
110
REFERENCE
Adams LK, Lyon DY, McIntosh A, Alvarez PJ (2006), Comparative toxicity of nano-scale TiO2,
SiO2 and ZnO water suspensions. Water Sci Technol;54:327–334.
Addy M (1981), In vitro studies into the use of denture base and soft liner materials as carriers for
drugs in the mouth. J Oral Rehabil;8:131–42.
Addy M, Thaw M (1982), In vitro studies into the release of chlorhexidine acetate, predonisolone
sodium phosphate, and predonisolone alcohol from cold cure denture base acrylic. J Biomed Mater
Res;16:145–57.
Al-Musallam T.A, Evans C, Drummond J.L. Matasa C.Wu C.D. (2006), Antimicrobial properties
of an orthodontic adhesive combined with cetylpyridinium chloride.J.Am Ortho & Dentof; 2;245-
251.
Ando M, González-Cabezas C, Isaacs R.L, Eckert G. J, and Stookey G.K. (2004). Evaluation of
Several Techniques for the Detection of Secondary Caries Adjacent to Amalgam Restorations.
Caries Res, Vol. 38(4), pp. 350-356.
Antonucci JM, Dickens SH, Fowler BO, Xu HHK, McDonough WG (2005), Chemistry of Silanes:
Interfaces in Dental Polymers and Composites, J. Res. Natl. Inst. Stand. Technol. 110, 541-558.
Anusavice K.J, Zhang N.Z, Shen C., (2006), Controlled Release of Chlorhexidine from UDMA-
TEGDMA Resin. J Dent. Res. 85 950.
Anusavice, K.J. and Brantley, W.A. (2003). Physical properties of dental materials. In: Phillip’s
science of dental materials, (11th edn) Anusavice, K.J. (Ed.). pp. 41-71, WB Saunders,
Philladelphia.
111
Apel C, Barg A, Rheinberg A, Conrads G, Wagner-Döblerd I, (2013) Dental composite materials
containing carolacton inhibit biofilm growth of Streptococcus mutans. Dent Mater. 29;1188–1199.
Arends J, Dijkman, T and Christoffersen J. (1987). Average mineral loss in dental enamel during
demineralization. Caries Res, Vol. 21(3), pp. 249-254.
Arends J, Ruben J (1988). Fluoride-releasing from a composite resin. Quintess Int 19:513-514.
Attin T, Zirkel C, Pelz K (2001), Antibacterial properties of electron beam-sterilized Gutta–Percha
cones. J Endo ;27:172–4.
Auschill TM, Arweiler NB, Brecx M, Reich E, Sculean A, Netuschil L (2002), The effect of dental
restorative materials on dental biofilm. Eur J Oral Sci;110:48–53.
Aw TC and Nicholls JI (2001), Polymerization Shrinkage of Densely-Filled Resin Composites,
Oper Dent, 26, 498-504.
Awan MA (2010), A study investigating the mechanical testing of a novel dental restorative
material and its biocompatibility, M Phil thesis, University of Birmingham UK.
Bae K, Jun EJ, Lee SM, Paik DI, Kim JB (2006), Effect of water-soluble reduced chitosan on
Streptococcus mutans, plaque regrowth and biofilm vitality, Clin Oral Invest.;10:102-7.
Bayne SC and Thompson JY (2013), Biomaterials online chapter 18.Sturdevants Art and Science
of operative Dentistry 6th Edition Mosby, Elsevier Inc. e53-e67.
Bayne SC, Thompson JY, Swift EJ Jr, et al. (1998), A characterization of first-generation flowable
RBCs. J Am Dent Assoc 129:567–577.
Beigi Burujeny S, Atai M, Yeganeh H, (2015), Assessments of antibacterial and physico-
mechanical properties for dental materials with chemically anchored quaternary ammonium
moieties: Thiolene methacrylate vs. conventional methacrylate system. Dent Mater
http://dx.doi.org/10.1016/j.dental.2014.12.014
112
Bernardo M, Luis H, Martin M.D, Leroux B.G, Rue T, Leitão J and DeRouen T.A (2007). Survival
and reasons for failure of amalgam versus composite posterior restorations placed in a randomized
clinical trial. J Am Dent Assoc. Vol. 138(6), pp. 775-783.
Bertolotti RL and Laamanen H. (1999), Bite-formed posterior resin composite restorations, placed
with a self etching primer and a novel matrix. Quintessence Int; 30(6):419-422.
Beyth N , Yudovin-Farberb I, Perez-Davidia M, Dombb AJ, Weiss EI (2010), Polyethyleneimine
nanoparticles incorporated into resin composite cause cell death and trigger biofilm stress in vivo
PNAS, vol. 107 no. 51 www.pnas.org/lookup/suppl/.
Beyth N, Bahir R, Matalon S, Domb AJ, Weiss EI (2008), Streptococcus mutans biofilm changes
surface-topography of resin composites. Dent Mater; 24:732–6.
Beyth N, Domb AJ, Weiss E.I. (2007), An in vitro quantitative antibacterial analysis of amalgam
and composite resins. J Dent;35:201–206.
Beyth N, Farah S, Domb AJ, Weiss E.I. (2014), Review Antibacterial dental composites. J React
& Func Poly; 75 81–88.
Beyth N, Farber I, Bahir R, Domb AJ, Weiss E.I.( 2006), Antibacterial activity of dental
composites containing quaternary ammonium polyethylenimine nanoparticles against
Streptococcus mutans. Biomat; 27 :3995–4002.
Black GV (1908), A work on operative dentistry. Chicago: Medico-Dental Publishing;. The
technical procedures in filling teeth; Vol. 2.
Blalock JS, Chan DCN, Browning WD, Callan R and Hackman S. (2006), Measurement of clinical
wear of two packable composites after 6 months in service. J Oral Rehab, 33: 59–63.
113
Blum, I R, Schriever A, Heidemann D, Mjör I A and Wilson N H F. (2003). The repair of direct
composite restorations: an international survey of the teaching of operative techniques and
materials. Eur J Dental Ed, 7(1), pp. 41-48.
Boulden JE, Cramer NB, Shreck KM, Couch CL, Bracho-Troconis C, Stansbury JW and Bowman
CN (2010). Thiol-ene-methacrylate RBCs as dental restorative materials. Dent Mater, DOI:
10.1016/j.dental.2010.11.001.
Bouschlicher MR, Rueggeberg FA, Wilson BM. (2004), Correlation of bottom to top surface
microhardness and conversion ratios for a variety of resin composite compositions. Oper Dent
29:698–704.
Bowen R.L (1956), Use of epoxy resins in restorative materials. J Dent Res 35:360.
Bowen R.L (1962), Dental filling materials comprising vinyl silane treated fused silica and a
binder consisting of the reaction product of Bisphenol and glycidyl methacrylate. U.S. Patent
Office 3,066,012.
Bowen, R. L ( 1963), Properties of a silica reinforced polymer for dental restorations, J Amer
Dent Assoc, Vol. 66, No. 1, 57-64
Braconnot H, Sue la natrue ces champignons. Ann Chim Phys, 1811;79:265.
Braga RR, Ferracane JL (2002), Contraction stress related to degree of conversion and reaction
kinetics. J Dent Res; 81: 114-118.
Bruno Sarmento, José das Neves (2012), Chitosan-Based Systems for Biopharmaceuticals:
Delivery, Targeting and Polymer Therapeutics. Bruno Sarmento, José das Neves (eds). John Wiley
& Sons. ISBN 111996296X, 9781119962960.
Bundy KJ, Butler MF, Hochman RF (1980), An investigation of the bacteriostatic properties of
pure metals. J Biomed Mater Res;14:653–63.
114
Buonocore MG (1955), A simple method of increasing the adhesion of acrylic filling materials to
enamel surfaces. J Dent Res; 34(6):849-53.
Bürgers R, Eidt A, Frankenberg R, Rosentritt M, Schweiki H, Handel G et al. (2009). The ant-
adherence activity and bactericidal effect of microparticulate silver additives in composite resin
materials. Arch Oral Biol;54(6):595-601.
Burgess J.O, Walker R, Davidson JM (2002), Posterior resin-based composite: review of the
literature, Pediatric Dentistry – 24:5.
Burgess, J.O. (2001). Fluoride-releasing materials, In: Fundamentals of operative dentistry: A
contemporary approach, (2nd Edn). Summitt, J.B.; Robbins, J.W. & Schwartz, R.S.(Eds), pp. 377-
385, Quintessence Publishing Co. Inc., ISBN: 0-86715-382-2, Illinois.
Burke FJ, Crisp RJ, Bell TJ, Healy A, Mark B, McBirnie R, Osborne-Smith KL (2001), One-year
retrospective clinical evaluation of hybrid composite restorations placed in United Kingdom
general practices. Quintess Int;32:293–298.
Burke F.J, Cheung, S.W, Mjör I A and Wilson N.H. (1999), Restoration longevity and analysis of
reasons for the placement and replacement of restorations provided by vocational dental
practitioners and their trainers in the United Kingdom. Quintes Inter, Vol. 30(4), pp. 234-242.
Buschang, J. Cai, A.L. Honeyman, Spencer CG, Campbell PM (2009), Antimicrobial effects of
zinc oxide in an orthodontic bonding agent. Angle Orthod;79(2):317-22.
Busscher HJ, Engels E, Dijkstra RJ, van der Mei HC. (2008), Influence of a chitosan on oral
bacterial adhesion and growth in vitro. Eur J Oral Sci; 116:493-495.
Busscher HJ, Rinastiti M, Siswomihardjo W, van der Mei HC(2010), Biofilm formation on dental
restorative and implant materials. J Dent Res; 89:657–65.
115
Cakmak I., Z. Ulukanli, M. Tuzcu, S. Karabuga, K. Genctav(2004), Emerging Nanotechnologies
in Dentistry: Materials, Processes, and Applications. Eu. Polym. J; 40; 23-73.
Calais JG, Soderholm K-JM(1988), Influence of filler type and water exposure on flexural strength
of experimental composite resins. J Dent Res; 67(5):836-840.
Carlen A, Nikdel K, Wennerberg A, Holmberg K, Olsson J (2001),Surface characteristics and in
vitro biofilm formation on glass ionomer and composite resin. Biomater ; 22:467–81.
Chang RWH, (1969), Dental restorative material, US Patent 3,452,437.
Chen MH, Chen CR, Hsu SH, Sun SP, Su WF (2006), Low shrinkage light curable nanocomposite
for dental restorative material.Dent Mater;22:138–45.
Chen X, Schluesener HJ. (2008), Nanosilver: a nanoproduct in medical application. Toxicol
Lett;176:1-12.
Cheng L,Weir MD, Xu HKK, Antonuccie JM, Kraigsleye A et al. (2012), Antibacterial
amorphous calcium phosphate nano composites with a quaternary ammonium dimethacrylate and
silver nanoparticles Dent Mater 28; 561–572.
Cheng L,Weir MD, Xua HKK, Antonuccie JM, Kraigsleye A et al. (2012), Antibacterial and
physical properties of calcium–phosphate and calcium–fluoride nanocomposites with
chlorhexidine. Dent mater 28 ; 573–583.
Cook PA, Youngson CC (1989). A fluoride-containing composite resin—an in vitro study of a
new material for orthodontic bonding. Br J Orthod, 16:207-212.
Coma, V, Martial-Gros, A, Garreau, S, Copinet, A, Salin, F. & Deschamps, A. (2002). J. Food
Sci., 67, p.1162-1169.
Combe EC , Burke FJ(2000), Contemporary resin-based composite materials for direct placement
restorations: packable, flowable and others. Dental Update, 27(7):326-32, 334-6.
116
Conti C, Giorgini E, Landi L, Putignano A, Tosi G. (2005), Spectroscopic and mechanical
properties of dental resin composites cured with different light sources. J Mol Struct, 744–
747:641–646.
Cowan MM (1999), Plant products as antimicrobial agents. Clin Microbiol Rev;12:564-582.
Cramer NB and Bowman CN (2001), Kinetics of thiol-ene and thiol-acryl ate photo
polymerizations with real time Fourier transform infrared. J Polym Scien Part A- Poly Chem; 19:
3311-3319.
Cummins D (1991), Zinc citrate/Triclosan: a new anti-plaque system for the control of plaque and
the prevention of gingivitis: short-term clinical and mode of action studies. J Clin
Periodontol;18:455-461.
Curtis A.R, Palin W.M, Fleming G.J, Shortall A.C, Marquis P.M (2009a), the mechanical property
of nanofilled resin-based RBCs: The impact of dry and wet cyclic preloading on bi-axial flexure
strength. Dent. Mater, 25, 188–197.
Curtis A.R, Palin W.M, Flemming G.J, Shortal A.C, Marquis P.M (2009b), The mechanical
properties of nanofilled resin-based composites: characterizing discrete filler particles and
agglomerates using a micromanipulation technique. Dent. Mater, 25, 180–187.
Curtis A.R, Shortall A.C, Marquis P.M, Palin W.M (2008), Water uptake and strength
characteristics of a nanofilled resin-based composites. J. Dent, 36, 186–193.
Curtis AR, Palin WM, Fleming GJP, Shortall ACC, Marquis PM (2009), The mechanical
properties of nanofilled resin-based composites: Characterizing discrete filler particles and
agglomerates using a micromanipulation technique. Dent Mater; 25(2):180-187.
Dahl BL (1978), Antibacterial effect of two luting cements on prepared dentin in vitro and in vivo.
Acta Odontol Scand;36:363-9.
117
Dart EC, Cantwell JB, Traynor JR, Taworzyn JF, Nemeck J.(1978), Method of repairing teeth
using composition which is curable by irradiation by visible light, US Patent 4,089,763.
Darvell BW, (2009), Materials Science for Dentistry, Ninth Edition (Woodhead Publishing Series
in Biomaterials)
Deligeorgi V, Mjor I.A, Wilson NH (2001), An overview of reasons for the placement and
replacement of restorations. Prim. Dent. Care, 8, 5–11.
Deng CM, He LZ, Zhao M, Yang D. Liu Y (2007), Biological properties of the chitosan gelatin
sponge wound dressing. Carbohyd. Polym. 69: 583-589.
Domingo C, Arc RW, Osorio E, Osorio R., et al., (2003), Hydrolytic stability of experimental
hydroxyapatite-filled dental composite materials Dent. Mater; 19; 478–486.
Dutta PK (2005), Chitin and chitosan opportunities and challenges. P.K. Dutta (Ed.) SSM
Eakle WS (1986), Fracture resistance of teeth restored with Class II bonded RBCs resin. J Dent
Res. 65(2):149-53.
Ebi N, Imazato S, Noiri Y, Ebisu S. (2001), Inhibitory effect of resin composites containing
bactericide-immobilized filler on plaque accumulation. Dent Mater;17:485–91.
Eick JD, Robinson SJ, Byerley TJ, Chappelow CC (1993), Adhesives and non-shrinking dental
resins of the future. Quintessence Int; 24:632–640.
Elsaka S.E. (2012), Antibacterial activity and adhesive properties of a chitosan-containing dental
adhesive. Quintessence Int; 3;43 -60.
Ernst C, Meyer G, Klocker K and Willershausen B (2004), Determination of polymerization
shrinkage stress by means of photo elastic investigation. Dent Mater; 20: 313-321.
Fan C, Chu L, Rawls HR, Norling BK, Cardenas HL, Whang K (2011), Development of an
antimicrobial resin—a pilot study. Dent Mater; 27:322–8.
118
Fang M, Chen JH, Xu X-L, Yang P-H, Hildebrand HF (2006), Antibacterial activities of inorganic
agents on six bacteria associated with oral infections by two susceptibility tests. Inter J Antimicrob
Agents; 27:513–517
Farrugia C, Camilleri J (2015), Antimicrobial properties of conventional restorative filling
materials and advances in antimicrobial properties of composite resins and glass ionomer
cements—A literature review. Dent Mater http://dx.doi.org/10.1016/j.dental.2014.12.005.
Fédération Dentaire Internationale (1962), Special commission on oral and dental statistics:
General principles concerning the international standardization of dental caries statistics. Int Dent
J, Vol. 12 pp. 15.
Felt O, Furrer, P, Mayer J.M, Plazonnet B, Buri P, and Gurny R. (1999), Topical use of chitosan
in ophthalmology: tolerance assessment and evaluation of precorneal retention. Int J Pharm 180:
185–193.
Ferracane JL (2010), Resin composite State of the art. Dent Mater, vol 27, no. 1, pp. 29-38.
Ferracane JL (2006), “Hygroscopic and hydrolytic effects in dental polymer networks,” Dent
Mater, vol. 22, no. 3, pp. 211–222.
Ferracane JL (1994), Elution of leachable components from composites. J Oral Rehabil, 21(4): p.
441-52.
Floyd CJ, Dickens SH (2006), Network structure of Bis-GMA- and UDMA-based resin systems.
Dent Mater 22 (12):1143-9.
Fontana M, Li Y, Dunipace A.J, Noblitt T.W, Fischer G, Katz B.P, and Stookey GK (1996),
Measurement of enamel demineralization using microradiography and confocal microscopy. A
correlation study. Caries Res, Vol. 30(5), 317-325.
119
Forss, Helena and Widström, E. (2004), Reasons for restorative therapy and the longevity
ofrestorations in adults. Acta Odontologica Scandinavica, Vol. 62(2), 82-86.
Frost, P.M. (2002), An audit on the placement and replacement of restorations in a general dental
practice. Prim. Dent. Care, 9, 31–36.
Fusayama T (1988), Clinical guide for removing caries using a caries-detecting solution.
Quintessence Int, Vol. 19(6), pp. 397-401.
Gajewski ves, Pfeifer CS, Fróes-salgado NRG, Boaro LCC, BragaRR(2012), monomers used
in resin composites: degree of conversion, mechanical properties and water sorption/solubility,
Braz Dent J, 23(5): 508-514
Ge Y, Wang S, Zhou X, Wang H, Xu HHK, Cheng L (2015), The Use of Quaternary Ammonium
to Combat Dental Caries, Mater, 8, 3532-3549.
Gilbert P, Moore LE (2005), Cationic antiseptics: diversity of action under a common epithet. J
Appl Microbiol; 99:703—15.
Gladwin, Marcia, Bagby, Michael (2009), Direct Polymeric Restorative Materials. In Gladwin,
Marcia; Bagby, Michael, Clinical Aspects of Dental Materials: Theory, Practice, and Cases, 3rd
Edition, Lippincott Williams & Wilkins; 59-75.
González-Cabezas, Carlos Li, Yiming Gregory Richard L, and Stookey, George K. (2002),
Distribution of cariogenic bacteria in carious lesions around tooth-colored restorations. Am J of
Dent, Vol. 15(4), pp. 248-251.
Gopferich, A. (1996), Mechanisms of polymer degradation and erosion. Biomater. 17(2): p. 103-
14.
Gwinnett AJ, Matsui A (1967), A study of enamel adhesives. The physical relationship between
enamel and adhesives. Arch Oral Biol; 12(12):1615-20.
120
Gyo M, Nikaido T, Okada K, Yamauchi J, Tagami J, Matin K (2008), Surface response of fluorine
polymer-incorporated resin composites to cariogenic biofilm adherence. Appl Environ
Microbiol;74:1428–35.
Hadis MA (2011), Polymerization kinetics and optical phenomena of photoactive dental resins
PhD Thesis. University of Birmingham UK.
Hadwiger L.A, Kendra D.G, Fristensky B.W & Wagoner W. (1981), Chitosan both activated genes
in plants and inhibits RNA synthesis in fungi, in: “Chitin in nature and technology”. Muzzarelli,
R. A. A., Jeuniaux, C. & Gooday, G. W. (Eds.), Plenum, New York.
Hafdani FN and Sadeghinia N (2011), A review on application of chitosan as a natural
antimicrobial, world academy of science, engineering and technology 50.
Hahn R, Weiger R, Netuschil L, Brüch M (1993), Microbial accumulation and vitality on different
restorative materials. Dent Mater; 9:312–316.
Hals E, Norderval I.T. (1973), Histopathology of experimental in vivo caries around silicate
fillings. Acta Odontologica Scandinavica, Vol. 31(6), pp. 357-367.
Hals E (1975), The structure of experimental in vitro lesions around silicate fillings in human teeth.
Arch of Oral Biolo, Vol. 20(4), pp. 283-289.
Hals E, Andreassen BH, Bie T (1974), Histopathology of natural caries around silver amalgam
fillings. Caries Research, Vol. 8(4), pp. 343-358.
Hansel C, Leyhausen G,Mai UEH, Geurtsen W(1998), Effects of various resin composites
(co)monomers and extracts on two caries-associated micro-organisms in vitro. J Dent Res;77:60-
7.
He J, Söderling E, Lassila LVJ, Vallittu PK (2012), Incorporation of an antibacterial and radio-
opaque monomer into dental resin system. Dent Mater ;28:e110–7.
121
Henriks-Eckerman ML, Suuronen K, et al. (2004), Methacrylate in dental restorative materials.
Contact Dermatitis 50(4):233-7.
Hernandez-Sierra JF, Ruiz F, Pena DC, Martinez-Gutierrez F, Martinez AE, Guillen Ade J et al.
(2008), The antimicrobial sensitivity of Streptococcus mutans to nanoparticles of silver, zinc
oxide, and gold. Nanomedicine; 4:237-240.
Hervás-García A, Martínez-Lozano MA, Cabanes-Vila J, Barjau-Escribano A, Fos-Galve P
(2006), Composite resins. A review of the materials and clinical indications. Med Oral Patol Oral
Cir Bucal;11: E215-20.
Hicks J., F. Garcia-Godoy, K. Donly, C. Flaitz, (2003), Fluoride-releasing restorative materials
and secondary caries. J Calif Dent Assoc;31(3):229-45.
Huang L , Xiao Y, Xing X , Li F, Maa S, Qi L , Chen J (2011), Antibacterial activity and
cytotoxicity of two novel crosslinking antibacterial monomers on oral pathogens. Arch of Oral
Biol 56 ; 367-373.
Ikada Y (1994), Surface modification of polymers for medical applications. Biomater.15:725–36.
Illum L (1998), Chitosan and its use as a pharmaceutical excipient. Pharm. res. 15: 1326-1331.
DOI: 10.1023/A:1011929016601.International Publication. Contai, Midnapore , India p 6.
Imazato S, Chen J , Ma S, Izutani N, Li F. (2012), Antibacterial resin monomers based on
quaternary ammonium and their benefits in restorative dentistry. Japanese Dental Science Review
48, 115—125.
Imazato S, Ebi N, Takahashi Y, Kaneko T, Ebisu S, Russell RRB (2003), Antibacterial activity of
bactericide-immobilized filler for resin based restoratives. Biomater;24:3605–9.
Imazato S, McCabe JF (1994), Influence of incorporation of antibacterial monomer on curing
behavior of a dental resin composites. J Dent Res; 73:1641–5.
122
Imazato S, Tarumi H, Kato S, Ebisu S (1999), Water sorption and color stability of RBCs
containing the antibacterial monomer MDPB. J Dent; 27:279–83.
Imazato S, Torii M, Tsuchitani Y, McCabe JF, Russell RRB (1994), Incorporation of bacterial
inhibitor into resin composites. J Dent Res ;73:1437–43.
Imazato, S. (2003), Antibacterial properties of resin composites and dentin bonding systems. Dent.
Mater., 19, 449–57.
Imazato, S, Torii M, Tsuchitani Y (1993), Immobilization of an antibacterial component in
composite resin. J Dent, 30, 63–68.
Ionescu A, Wutscher E, Brambilla E, Schneider-Feyrer S,Giessibl FJ, Hahnel S (2012), Influence
of surface properties of resin-based composites on in vitro S.mutans biofilm development. Eur J
Oral Sci;120:458–65.
Irie M, Suzuki K and Watts D.C. (2002), Marginal gap formation of light-activated restorative
materials: effects of immediate setting shrinkage and bond strength. Dent Mater, Vol. 18(3), 203-
210.
Jacquelyn A, Carioscia HL, Stansbury JW (2005), Thiol-ene oligomers as dental restorative
materials. Dent Mater, 21: 1137-1143.
Jain P, Pershing A. (2003), Depth of cure and microleakage with high intensity and ramped resin-
based composite curing lights. J Am Dent Assoc 134:1215–1223.
Jedrychowski JR, Caputo AA, Kerper S (1983), Antibacterial and mechanical properties of
restorative materials combined with chlorhexidines. J Oral Rehabil;10:373–81.
Jingwei H, Söderling E, Vallittu PK, Lassila LVJ (2013), Preparation and Evaluation of Dental
Resin with Antibacterial and Radio-Opaque Functions, Int. J. Mol. Sci. 14, 5445-5460.
123
Jingwei H, Söderling E, Lassila VJ ,Vallittu PK (2014), Synthesis of antibacterial and radio opaque
dimethacrylate monomers and their potential application in dental resin. Dent Mater 30; 968–976.
Jokstad A, Bayne S, Blunck U, Tyas M, Wilson N (2001), Quality of dental restorations. FDI
Commission Project 2–95. Int. Dent. J., 51, 117–158.
Jones N, Ray B, Ranjit KD, Manna AC (2008), Antibacterial activity of ZnO nanoparticle
suspensions on a broad spectrum of microorganisms. FEMS Microbiol Lett 279:71–76.
Kalachandra S, Wilson TW (1992), Water sorption and mechanical properties of light-cured
proprietary composite tooth restorative materials. Biomater ;13:105-9.
Karanika-Kouma A, Dionysopoulos P, Koliniotou-Koubia E,Kololotronis A (2001), Antibacterial
properties of dentin bonding systems, polyacid modified composite resins and composite resins. J
Oral Rehabil;28:157–60.
Karimzadeh A, Ayatollahi MR, AsgharzadehShirazi H (2014), Mechanical Properties of a Dental
Nano-Composite inMoist Media Determined by Nano-Scale Measurement, Inter J Mater, Mechan
and Manufactur, Vol. 2, No. 1.
Kasraei S, Sami L, Hendi S, Khani MYA, Soufi LR, Khamverdi Z (2014), Antibacterial properties
of composite resins incorporating silver and zinc oxide nanoparticles on Streptococcus mutans and
Lactobacillus. Restor Dent Endod. ;39(2):109-114.
Kawabata N., M. Nishiguchi (1988), Antimicrobial Polymers, Appl. Environ. Microbiol;54;25-32.
Kawai K (1988). Effects of eluate from composite resins on Gtase and growth of S.mutans. J
Conserv Dent;31:332–51.
Kawashita M.S, Tsuneyama, Miyaji F, Kokubo T, Kozuka H, Yamamoto K (2000), Antibacterial
silver- containing silica glass prepared by sol-gel method. Biomater; 21 393.
124
Keong LC and Halim AS (2009), In Vitro Models in Biocompatibility Assessment for Biomedical-
Grade Chitosan Derivatives in Wound Management. Int. Mol. Sci. 10:1300- 1313.
Khaled AN (2011), Physical properties of dental resin nano composites, MPhil thesis, University
of Manchester.
Khalichi P, Cvitkovitch DG, Santerre JP (2004). Effect of composite resin biodegradation products
on oral streptococcal growth. Biomater ; 25:5467–72.
Khatri CA, Stansbury JW, et al. (2003), Synthesis, characterization and evaluation of urethane
derivatives of Bis-GMA. Dent Mater 19(7):584-8.
Kidd E.A, Joyston-Bechal, S, Beighton D (1993), Microbiological validation of assessments of
caries activity during cavity preparation. Caries Res, Vol. 27(5), 402-408.
Kidd E.A. (1976), Micro leakage: a review. J Dent, Vol. 4(5), pp. 199-206.
Kidd E.A. (2010), Clinical threshold for carious tissue removal. Dental Clinics of North America,
Vol. 54(3), pp. 541-549.
Kim JS, Shin DH (2013), Inhibitory effect on streptococcus mutans and mechanical properties of
the chitosan containing composite resin. Restor Dent Endod. 38:36–42.
Knobloch LA, Kerby RE, Clelland N, Lee J. (2004), Hardness and degree of conversion of
posterior packable composites. Oper Dent; 9(6):642-49.
Konishi N, Torii Y, Kurosaki A, Takatsuka T, Itota T, Yoshiyama M. (2003), Confocal laser
scanning microscopic analysis of early plaque formed on resin composite and human enamel. J
Oral Rehabil;30:790–5.
Krämer N, Lohbauer U, García-Godoy F, Frankenberger R (2008), Light curing of resin
based composites in the LED era. Am J Dent, 21: 3.135-142.
125
Kraigsley A.M, Tang K, Lippa K.A, Howarter J.A, Gibson S.L, Lin N.J. (2012), Effect of Polymer
Degree of Conversion on Streptococcus mutans Biofilms Macromol. Biosci.DOI:
10.1002/mabi.201200214.
Kumar, M.N.V. (2000), A review of chitin and chitosan applications. Reactive Funct Polym 46:
1–27.
Kurek A, Grudniak AM, Szwed M, Klicka A, Samluk L, et al. (2010), Oleanolic acid and ursolic
acid affect peptidoglycan metabolism in Listeria monocytogenes. Antonie Van Leeuwenhoek
;97:61-68.
Labella R, Lambrechts P, Van Meerbeek B, Vanherle G.(1999), Polymerization shrinkage and
elasticity of flowable composites and filled adhesives.Dent Mater; 15:128-37.
Lahdenperä M.S, Puska M.A, Alander P.M, Waltimo T, Vallittu P.K. (2004), Release of
chlorhexidine digluconate and flexural properties of glass fibre reinforced provisional fixed partial
denture polymer. J. Mater. Sci. Mater. Med., 15, 1349–1353.
Lang BR, Jaarda M, Wang RF. (1992), Filler particle size and composite resin classification
systems. J Oral Rehabil 19:569–684.
Lattmann E, Dunn S, Niamsanit S, Sattayasai N (2005), Synthesis and antibacterial activities of 5-
hydroxy-4-amino-2(5H)-furanones. Bioorg Med Chem Lett.;15:919–21.
Lee HL, (1970) Dental filling material, US Patent 3,539,533.
Leevailoj C, Cochran M A, Matis BA, Moore BK, Platt JA (2001), Micro leakage of posterior
packable resin composites with and without flowable liners. Oper Dent;26:302-307.
Leinfelder K, Bayne C, Swift EJ (1999), Packable Composites: Overview and Technical
Considerations. J Esth and Restor Dent, 11: 234–249.
126
Leinfelder K, Prasad A (1998), A new condensable composite for the restoration of posterior
teeth. Dent Today,Feb;17(2):112-6.
Leung D, Spratt D.A, Pratten J, Gulabivala K, Mordan N.J, Young A.M. (2005), dental restorative
materials. J Dent Res; 57:171–4.
Leung D, Spratt D.A, Pratten J, Gulabivala K, Mordan N.J, Young A.M. (2005), Chlorhexidine-
releasing methacrylate dental composite materials. Biomater, 26, 7145–7153.
Lewinstein I, Matalon S, Slutzkey S, Weiss E.I. (2005), Antibacterial properties of aged dental
cements evaluated by direct-contact and agar diffusion tests. J. Prosthet. Dent., 93: 364-371
Li F, Chen J, Chai Z, Zhang L, Xiao Y, Fang M, et al. (2009), Effects of a dental adhesive
incorporating antibacterial monomer on the growth, adherence and membrane integrity of
Streptococcus mutans. J Dent ;37(4):289–96.
Li Q, Dunn ET, Grandmaison EW, Goosen MFA (1992) Applications and properties of chitosan.
J. compat. pol. 7:370–97.
Lin J, Qiu S, Lewis K, Klibanov M (2002), Bacterial properties of flat surfaces and nanoparticles
derived with alkylated polyethylenimines. Biotechnol Prog ;18:1082–6.
Ling L, Xu X, Choi Y, Billodeaux D, Guo G, Diwan RM,(2009), Novel F-releasing Composite
with Improved Mechanical Properties, J Dent Res 88(1):83-88.
Liu XF, GuanYL, Yang D Z, Li Z, Yao KD (2001), Antibacterial action of chitosan and
carboxymethylated chitosan. J Applied Poly Sci, 79, 1324–1335.
Lutz F, Phillips RW (1983), A classification and evaluation of composite resin systems. J Prosthet
Dent 50:480–488.
Lygre H, Hol PJ, Solheim E, Moe G. (1999), Organic leachables from polymer-based dental filling
materials. Euro J Oral Sci;107:378—83.
127
Matalon S, Slutzky H, Mazor Y, Weiss EI (2003), Surface antibacterial properties of fissure
sealents. Pediatr Dent; 25:43-8.
Matalon S, Slutzky H, Weiss EI (2004), Surface antibacterial properties of packable resin
composites: part I. Quintess Int;35(3): 189–193.
Mathur NK and Narang CK (1990), Chitin and chitosan, versatile polysaccharides from marine
animals. J. Chem. Educ. 67, 938-942.
McCabe JF, Walls WG (2008), Resin based materials filling materials. In McCabe J.F, Walls W,G,
Applied dental materials 9th Edition ,Blackwell Publishing;204-233.
McDonnell G, Russell AD (1999), Antiseptics and disinfectants: activity, action, and resistance.
Clin Microbiol Rev;12:147-79.
Mehdawi I et al, (2009), Development of remineralizing, antibacterial dental materials, Acta
Biomaterialia, 5: 2525–2539.
Meiers JC, Miller GA(1996), Antibacterial activity of dentin bonding systems, resin modified glass
ionomers and polyacid modified composite resins. Oper Den;21:257-64.
Melo MAS, Wua J, Weir MD , Xu HHK (2014), Novel antibacterial orthodontic cement containing
quaternary ammonium monomer dimethylaminododecyl methacrylate. J Dent. 1193 – 1201.
Mills RW, Jandt KD, Ashworth SH. (1999), Dental RBCs depth of cure with halogen and blue
light emitting diode technology. Br Dent J; 186(8):388-391.
Mitra, B S, WU D, Holmes, N B. 2003. An application of nanotechnology in advanced dental
materials. J Am DentAssoc; 134(10):1382-1390.
Mjör IA, & Toffenetti F. (2000). Secondary caries: a literature review with case reports. Quintes
Inter, Vol. 31(3), pp. 165-179.
128
Mjör IA. (2005), Clinical diagnosis of recurrent caries. J Amer Dent Assoc, Vol. 136(10), pp.
1426 -1433.
Mobarak E, Elsayad I, Ibrahim M, El-Badrawy W. (2009), Effect of LED light-curing on the
relative hardness of tooth-colored restorative materials. Oper Dent 34(1):65-71.
Mohsen NM, Craig RG. (1995), Effect of silanation of fillers on their dispersability by monomer
systems. JOral Rehab; 22(3):183-189.
Momoi Y, McCabe JF1 (1993), Fluoride release from light-activated glass ionomer restorative
cements. Dent Mater;9:151–4.
Moore BK, Platt JA, Borges G, Chu T-MG, Katsilieri I. (2008), Depth of cure of dental resin
composites: ISO 4049 depth and microhardness of types of materials and shades. Oper Dent 33–
34:408–412.
Morgana MSGC, Thayza CMS, Emerson PS, Pedro T, Fabio S. (2011). Chitosan as an oral
antimicrobial agent, Science against microbial pathogens: communicating current research and
technological advances, A. MéndezVilas (Ed.), Formatex
Moszner N and Hirt T (2012), New Polymer-Chemical Developments in Clinical Dental Polymer
Materials: Enamel–Dentin Adhesives and Restorative Composites, J of poly sci part A: poly
chemistry, 50, 4369–4402.
Moszner N, Klapdohr S (2004), Nanotechnology for dental composites. Int J Nanotechnol;1:130–
56.
Mousavinenasab SM, Jafary M (2004), Microleakage of Composite Restorations Following
Chemo-mechanical and Conventional Caries Removal. J Dent (TUMS); 1;12-17.
Murchison D.F, Roeters J, Vargas M.A, et al. (2006), Direct anterior restorations. In:
Fundamentals of operative dentistry: A contemporary approach, (3rd Edn). Summitt, J.B.;
129
Robbins, J.W, Schwartz, R.S. (Eds), pp. 261-288, Quintessence Publishing Co. Inc.,ISBN: 0-
86715-382-2, Illinois, USA.
Muzzarelli RAA Natural (1973), Polymers, Pergamon Press, London. ISBN 0080172350,
9780080172354.
Namba N, Yoshida Y, Nagaoka N, Takashima S,Matsuura-Yoshimoto K, Maeda H, et al. (2009),
Antibacterial effect of bactericide immobilized in resin matrix. Dent Mater;25:424–30.
Neuman KA (1999), Posterior esthetic dentistry—a perspective for the new century. J Can Dent
Assoc; 65:556–557.
Neumann MG, Miranda WG, Schmitt CC, Rueggeberg FA, Correa IC. (2005), Molar extinction
coefficients and the photon absorption efficiency of dental photo initiators and light curing units.
J Dent; 33:525-532.
Nganga S, Travan A, Donati I, Crosera M, Paoletti S, Vallittu P.K. (2012), Degradation of silver-
polysaccharide nanocomposite in solution and as coating on fiber-reinforced composites by
lysozyme and hydrogen peroxide. Biomacromol, 13, 2605–2608.
Niessen LC and Gibson G (1997), Oral health for a lifetime: Preventive strategies for the older
adult. Quintes Int, Vol. 28, pp. 626-630.USA.
No KH, Park NY, Lee SH and Meyers SP (2002), Antibacterial activity of chitosan and chitosan
oligomers with different molecular weights. Int J Food Micro, 74, 65–72.
Obici AC, Sinhoreti MAC, Correr-Sobrinho L, de Goes MF, Consani S (2005), Evaluation of
mechanical properties of Z250 composite resin light-cured by different methods. J Appl Oral
Sci;13(4):393-8.
Ohashi S, Yamamoto K, Shimazaki AE, Aono M, Kokubo T, Yamada I, Yamauchi J (1995), In
vitro antibacterial activity of Ag ion-implanted Orthod. Dentofac Orthop. 129-45.
130
Ono M, Nikaido T, Ikeda M, Imai S, Hanada N, Tagami J, et al. (2007), Surface properties of resin
composite materials relative to biofilm formation. Dent Mater ;26:613–22.
Orstavik D, Hensten-Pettersen A (1978), Antibacterial activity of tooth colored restorative
materials. Biomater. 26; 1051–7.
Ørtengren, U, Wellendorf H, Karlsson S, Ruyter IE. (2000), water sorption and solubility of dental
composites and identification of monomers released in an aqueous environment. J Oral Rehabil
;28(12):1106-15.
Oysaed H, Ruyter IE. (1986), Composites for use in posterior teeth: mechanical properties tested
under dry and wet conditions.J Biomed Mater Res;20:261-71.
Pallana S, Araujob MVF,Cilli R, Prakkia A, (2012), Mechanical properties and characteristics of
developmental copolymers incorporating catechin or chlorhexidine. Dent Mater, 687–694
Pandit S, Kim GR, Lee MH, Jeon JG (2011), Evaluation of S.mutans biofilms formed on fluoride
releasingand non fluoride releasing resin composites, J Dent;39:780.
Papadogiannis Y, Lakes R.S , PalaghiasG , Helvatjoglu-AntoniadesM, PapadogiannisD (2007),
Fatigue of packable dental composites, Dent Mater;23 ; 235–242.
Paula AB, Taparelli JR, Alonso R.C.B, Mei L, Puppin-Rontani RM (2013), Mechanical/physical
properties of a composite with an antimicrobial-based triclosan monomer, Dent Mater 29S;e1–
e96.
Persson A, Claesson R, Van Dijken JW (2005), Levels of mutans streptococci and lactobacilli in
plaque on aged restorations of an ion-releasing and a microhybrid hybrid composite resin. Acta
Odontol Scand; 63:21–5.
Peutzfeldt A (1997), Resin based composites in dentistry: The monomer systems. Eur J Oral Sci;
105(2):97-116.
131
Peyton FA (1943), Physical and clinical characteristics of synthetic resins used in dentistry. J Am
Dent Asso, 30:1179-1189.
Philips RW (1982), Restorative resins. In Philips RW. Skinner’s Science of dental Materials, 8th
Edition. W.B. Saunders Company, Chapter 14:216-247.
Piwowarczyk A, Lauer H, Sorensen J (2005), Microleakage of various cementingagents for full
cast crowns. J Prosth Dent, Vol. 94(6), pp. 548-548.
Powers JM, Fan PL, Raptis CN (1978), Colour stability of new composite restorative materials
under accelerated ageing, J Dent Res;59: 2071-74.
Pongprueksa P, Senawongse P. (2007), Surface roughness of nanofill and nanohybrid resin
composites after polishing and brushing. J of Esth and Restora Dent; 19(5):265-273.
Powers JM (2002), RBCs Restorative Materials. In: Craig R, Powers J, eds. Restorative Dent
Mater, 11th edition, Mosby;232-236.
Prasad, A. and Sarkar, N. (2004). Restorative composite resins. In: DhuruVirendra B.
Contemporary dental materials. Oxford University Press. p. 64.
Prati C, Fava F, Di Gioia D, Selighini M, Pashley DH(1993), Antibacterial effectiveness of dentin
bonding systems. Dent Mater;9:338-43.
Puckett A.D, Fitchie J.G, Kirk P.C. & Gamblin J. (2007). Direct composite restorative materials.
The Dental Clinics of North America, Vol. 51, pp. 659-675.
Qin CQ, Li HR, Xiao Q, Liu Y, Zhu JC, Du YM (2006),Water-solubility of chitosan and its
antimicrobial activity. Carbohydr Polym.;63: 367-74.
Rabea EI, Badawy MET, Stevens CV, Smagghe G, Steurbaut W (2003), Chitosan as antimicrobial
agent: applications and mode of action. Biomacromol 4:1457–1465.
132
Rahim TNAT, Mohamad D, Akil HM, Rahman IA. (2012), Water sorption characteristics of
restorative dental composites immersed in acidic drinks, Dent Mater; 28 ; e63–e70
Rawls, H.R, Upshaw, J.E. (2003). Restorative resins. In: Phillip’s science of dental materials, (11th
edn). Anusavice, K.J. (Ed.). pp. 399-441, WB Saunders, Philladelphia.
Rhoades, J., and Roller, S. (2000) Antimicrobial actions of degraded and native chitosan against
spoilage organisms in laboratory media and foods. Appl Environ Microbiol; 66:80–86.
Roberson TM, Heymann HO, Swift EJ (2002),Sturdevant’s Art and Science of Operative
Dentistry, 4: 237-268.
Rouget C (1859), Des substances amylacees dans le tissue des animux, specialement les Articules
(Chitine). Compt Rend;48:792.
Ruddell D.E, Maloney M.M, Thompson J.Y (1999), Effect of novel filler particles on the
mechanical and wear properties of dental composites. Dent Mater volume 18,1;72-80.
Rueggeberg FA, Caughman WF, Curtis JW, Davis HC (1993), Factors effecting cure at depths
within light-activated resin composites. Am J Dent;6:91-95.
Rupfa S, Balkenhola M, Tim O. Sahrhagea, Bauma A, Chromikb JN, Ruppertb K et al. (2012),
Biofilm inhibition by an experimental dental resin composite containing octenidine
dihydrochloride. Dent Mater, 28;974–984
Rüttermann S, Trellenkamp T, Bergmann N, Beikler T, Ritter H, et al. (2013), Bacterial Viability
and Physical Properties of Antibacterially Modified Experimental Dental Resin Composites. PLoS
ONE 8(11): e79119. doi:10.1371/journal.pone.0079119.
Sabbagh J, Ryelandt L, Bacherius L, Biebuyck JJ, Vreven J, Lambrechts P and Leloup G (2004),
Characterization of the inorganic fraction of resin composites. J Oral Rehab; 31: 1090-1101.
133
Sano H, Shibasaki K, Matsukubo T, Takaesu Y (2003), Effect of chitosan rinsing on reduction of
dental plaque formation. Bull Tokyo Dent Coll;44:9-16.
Sara TH, Alaghemand H, Hamze F, Ahmadian BF, Rajab Nia R, Rezvani MB, et al. (2013),
Antibacterial, physical and mechanical properties of flowable resin composites containing zinc
oxide nanoparticles. Dent Mater; 29(5):495-505.
Scherer W, Lippman N, Kaim J (1989), Antibacterial properties of glass-ionomer cements and
other restorative materials. Oper Dent;14:77-81.
Schmalz G (1977), The influence of various anterior tooth filling materials on the in vitro growth
of S.mutans. Dtsch Zahna¨rztl Z;32:575–9.
Schulein TM. (2005), Significant events in the history of operative dentistry. J History of Dent;
53(2):63-72.
Schwartzman BB, Caputo AA, Schein B (1980), Antimicrobial action of dental cements. J Prosthet
Dent;43:309-12.
Sehgal V, Shetty VS, Mogra S, Bhat G, Eipe M, Jacob S, et al. (2007), Evaluation of antimicrobial
and physical properties of orthodontic composite resin modified by addition of antimicrobial
agents – an in-vitro study. Am J Orthod Dentofac Orthop ;131:525–9.
Serrano-Granger C, Cerero-Lapiedra R, Campo-Trapero J, Del Rio-Highsmith J (2005), In vitro
study of the adherence of Candida albicans to acrylic resins: relationship to surface energy. Int J
Prostho;18:392–8
Sevinç BA, Hanley L (2010), Antibacterial Activity of Dental Composites Containing Zinc Oxide
Nanoparticles J Biomed Mater Res B Appl Biomater ; 94(1): 22–31
Shahal Y, Steinberg D, Hirschfeld Z, Bronshteyn M, Kopolovic K (1998), In vitro bacterial
adherence onto pellicle-coated aesthetic restorative materials. J Oral Rehabil;25:52–8.
134
Shahidi F, Arachchi J, Jeon YJ (1999), Trends Food Sci. Technol., 10, p.37-51
Shawkat ES, Shortall AC, Addison O, Palin WM (2009), Oxygen inhibition and incremental layer
bond strengths of resin composites. Dent Mater;25: 1338–1346.
Shay D.E, Allen T.J, Mantz RF (1956), The antibacterial effects of some dental restorative
materials. J. Dent. Res, 35, 25–32.
Sheng J, Nguyen PT, Marquis RE (2005), Multi-target antimicrobial actions of zinc against oral
anaerobes. Arch Oral Biol;50:747-757.
Shigemasa Y, and Minami S. (1995), Applications of chitin and chitosan for biomaterials.
Biotechnol Genet Eng Rev 13: 383–420.
Sebti I, Martial-Gros A, Carnet-Pantiez, A, Grelier S. & Coma V (2005)., - J. Food Sci., 70,
p.M100 - M104
Sudarshan NR, Hoover DG & Knorr D, (1992), Food Biotechnol., 6, p.257-272
Shirani F.A, Havaei M, Malekipour. M. Sharafi (2008), Surface Antibacterial Properties of Four
Tooth-Colored Restorative materials, J Dent Tehran, Vol: 5; No 1.
Sideridou ID, Karabela MM, Ch. Vouvoudi E (2011), Physical properties of current dental
nanohybrid andnanofill light-cured resin composites, Dent Mater; 2 7;598–607
Sideridou ID, Achilias DS. (2005), Elution study of unreacted Bis-GMA, TEGDMA, UDMA, and
Bis-EMA from light-cured dental resins and resin composites using HPLC. J Biomed Mater Res
Part B: Appl Biomater 74:617–626.
Singh D.K, Ray A.R(1997), Radiation induced grafting of N,N′ dimethylaminoethylmethacrylate
onto chitosan films, J of Appl Poly Sci, 66, 869.
Singh D.K, Ray A.R(1994), Graft copolymerization of 2 hydroxyethylmethacrylate onto chitosan
films and their blood compatibility, J Appl Poly Sci, 53, 1115.
135
Singla A.K and Chawla M (2001), Chitosan: some pharmaceutical and biological aspects – an
update. J Pharm Pharmacol 53: 1047–1067.
Slutzky H, Matalon S, Weiss EI (2004), Antibacterial surface properties of polymerized single-
bottle bonding agents:part II. Quintes Int, Apr; 35(4):275-9.
Soderpolin KJ, Mariotti A (1999), BIS-GMA-based resins in dentistry: Are they safe? J Am Dent
Assoc, 130:201-8.
Song F, Koo H, Ren1 D (2015), Effects of Material Properties on Bacterial Adhesion and Biofilm
Formation, J Dent Res, Vol. 94(8) 1027– 1034.
Sousa RP, Zanin IC, Lima JP, Vasconcelos SM, Melo MA, Beltrão HC, Rodrigues LK (2009), In
situ effects of restorative materials on dental biofilm and enamel demineralization, J Dent;37:44–
51.
Sonis AL, Snell W (1989). An evaluation of a fluoride-releasing, visible light-activated bonding
system for orthodontic bracket placement. Am J Orthod Dentofacial Orthop, 95:306-311.
Splieth C, Bernhardt O,Heinrich A, Bernhardt H and Meyer G (2003), Anaerobicmicroflora under
Class I and Class II composite and amalgam restorations. Quintes Int, Vol. 34(7), pp. 497-503.
Suh FJK and Matthew HWT (2000), Applications of chitosan-based polysaccharide biomaterials
in cartilage tissue engineering: A review. Biomater. 21: 2589-2598.
Syafiuddin T, Hisamitsu H, Toko T, Igarashi T, Goto N, Fujishima A,Miyazaki T (1997), In vitro
inhibition of caries around a resin composite restoration containing antibacterial filler.
Biomater;18:120-128.
Syafiuddin T, Igarashi T, Toko T, Hisamitsu H, Goto N (1995), Bacteriological and mechanical
evaluation of resin composites containing antibacterial filler (Apacider). J Showa Univ Dent
Soc;15:119–25.
136
Takemura K, Sakamoto Y, Staninec M, Kobayashi S, Suehiro K,Tsuchitani Y (1983),
Antibacterial activity of a bis-GMA based composite resin and antibacterial effect of chlorhexidine
incorporation. J Conserv Dent;26:540–7.
Tanagawa M, Yoshida K, Matsumoto S, Yamada T, AtsutaM (1999), Inhibitory effect of
antibacterial resin composites against S.mutans. Caries Res;33:366–71.
Tarsi R, Muzzarelli RA, Guzman CA, Pruzzo C. (1997), Inhibition of Streptococcus mutans
adsorption to hydroxyapatite by low-molecular-weight chitosans. J Dent Res;76:665-672.
Tavaria FK, Costa EM, Pina-Vaz,Carvalho MF, Pintado MM (2013), Chitosan as a dental
biomaterial: state of the art; Brazil J biomed engin, Volume 29, Número 1, p. 110-120.
Terry DA (2004), Direct applications of a nanocomposites resin system. Part 1. The evolution of
contemporary composite Materials. Pract Proced Aesthet Dent;16(6):417–22.
Temin SC, Csuros Z (1988). Long-term fluoride release from a composite resin. Dent Mater 4:184-
186.
Thomas R.Z, Ruben J.L, ten Bosch J.J, Fidler V, Huysmans M.C.D.N.J M. (2007). Approximal
secondary caries lesion progression, a 20-week in situ study. Caries Research, Vol. 41(5), pp. 399-
405.
Tobias RS. (1998), Antibacterial properties of dental restorative materials: a review. Int Endo J
;21:155–60.
Toledao M, Osorio R, Osorio E, Fuentes V, et al ( 2003), Sorption and solubility of resin-based
restorative dental materials. J Dent; 31;43–50
Toledao M, Oserio E, Oseerio R (1999), Micro hardness of selected restorative materials. J Prosth
Dent, vol. 81, pp. 610-15.
137
Tilbrook DA (2000), Photocurable epoxy-polyol matrices for use in dental composites I. Biomater;
21:1743-53.
Tin-Oo MM, Gopalakrishnan V, Samsuddin AR, Al Salihi KA, Shamsuria O (2007), Antibacterial
property of locally produced hydroxyapatite, Arch Orofac Sci, 2, 41-44.
Totiam P, Gonzaacute, lez-Cabezas C, Fontana MR, Zero DT (2007), A New in vitro Model to
Study the Relationship of Gap Size and Secondary Caries. Caries Res, Vol. 41(6), pp. 467-473.
Tung, Francis F.; Estafan, Denise; Scherer, Warren (2000), Micro leakage of a condensable resin
composite: An in vitro investigation.Quintess, IntVol. 31 Issue 6, p430-434. 5p.
Turssi CP, Ferracane JL, Serra MC (2005), Abrasive wear of resin composites as related to
finishing and polishing procedures. Dent Mater;21:641–8.
Turssi CP, Saad JR, Duarte SL, Rodrigues AL (2000), Composite surfaces after finishing and
polishing techniques. Am J Dent; 13:136–8.
Uhl A, Sigusch BW, Jandt KD (2004), Second generation LED’s for the polymerization of oral
biomaterials. Dent Mater; 20:80-87.
Van Amerongen, JP, Van Loveren C, Kidd E.A.M. (2001), Caries management: Diagnosis and
treatment strategies, In: Fundamentals of operative dentistry: A contemporary approach, (2nd
Edn). Summitt, J.B.; Robbins, J.W. & Schwartz, R.S.(Eds), pp. 70-90, Quintessence Publishing
Co. Inc., ISBN: 0-86715-382-2, Illinois, USA.
Van Houte J (1994), Role of micro-organisms in caries etiology. J Dent Res 73:672-81.
Wakefield CW, Kofford KR(2001), Advances in restorative materials. Dent Clin North Am. Jan;
45(1):7-29.
138
Waltimo T, Luo, G, Samaranayake L.P, Vallittu P.K (2004), Glass fibre-reinforced composite
laced with chlorhexidine diglucoonate and yeast adhesion. J. Mater. Sci. Mater. Med, 15, 117–
121.
Wang Z, Shena Y, Haapasalo M (2014), Dental materials with antibiofilm properties Review; Dent
Mater, 30 e1–e16.
Watts DC, Amer O, Combe EC (1984), Characteristics of visible light activated composites
systems. Brit Dent J, 156:209-215.
Weinmann W, Thalacker C and Guggenberger R (2005), Siloranes in dental composites. Dent
Mater; 21: 68-74.
Weiss EI, Shalhav M, Fuss Z (1996), Assessment of antibacterial activity of endodontic sealers by
a direct contact test. End and Dent Trauma;12:179–84.
Weng Y, Howard L, Guo X, Chong V.J, Gregory R.J, Xie D (2012), A novel antibacterial resin
composites for improved dental restoratives, J Mater Sci: Mater Med 23:1553–1561.
Wicht M.J, R. Haak, S. Kneist, M.J. Noack (2005), A triclosan-containing compomer reduces
Lactobacillus spp. predominant in advanced carious lesions. Dent Mater. 21; 8-31.
Wiegand A, Buchalla W, Attin T (2007), Review on fluoride-releasing restorative materials
fluoride release and uptake characteristics, antibacterial activity and influence on caries formation.
Dent Mater.;23:343–62.
Wilkerson MD, Thompson JY, Bayne SC and Stamatiades PJ (1998), Biaxial Flexure and fracture
toughness of flowable composites (Abstract no. 779). J Dent Res (Special Issue A); 77:203.
Willems G, Lambrechts P, Braem M, et al. (1992), A classification of dental RBCs according to
their morphological and mechanical characteristics. Dent Mater 8:310–319.
139
Wilson SJ, Wilson HJ (1993), The release of chlorhexidine from modified dental acrylic resin. J
Oral Rehabil; 20:311–9.
Wolf H, Karolin M. K, Andrea E, Norbert H, Uwe G (2012), Antimicrobial and physicochemical
properties of experimental light curing composites with alkali-substituted calcium phosphate
fillers, Dent Mater, 28 ;597–603.
Xu X, Burgess J.O. (2003), Compressive strength, fluoride release and recharge of fluoride-
releasing materials. Biomater 24(14):2451-61.
Xiao YH, Ma S, Chen JH, Chai ZG, Li F, Wang YJ (2009), Antibacterial activity and bonding
ability of an adhesive incorporating an antibacterial monomer DMAE-CB. J Biomed Mater Res
Part B Applied Biomater; 90(2):813–7.
Xu HHK (1999), Dental composite resins containing silica-fused ceramic single-crystalline
whiskers with various filler levels. J Dent Res; 78:1304-11.
Xu HHK and Jennifer LM (2010), Dental glass-reinforced composite for caries inhibition: Calcium
phosphate ion release and mechanical properties J Biomed Mater Res B Appl Biomater. 92(2):
332–340.
Yamamoto K, Ohashi S, Aono M, Kokubo T, Yamada I, Yamauchi J (1996). Antibacterial activity
of silver ions implanted in SiO2 filler on oral streptococci. Dent Mater;12:227–229.
Yap AUJ, Khor E, Foo SH (1999), Fluoride release and antibacterial properties of new-generation
tooth colored restoratives. Oper Dent ;24:297–305.
Yoshida K, Tanagawa M, Atsuta M (1999), Characterization and inhibitory effect of antibacterial
dental resin RBCs incorporating silver-supported materials. J Biomed Mater Res;47:516–22.
Young D.H, Kohle H. & Kauss H. (1982), Plant Physiol., 70, p.1449-1454).
140
Zhang M, Li XH, Gong YD, Zhao NM and Zhang XF (2002), Properties and biocompatibility of
chitosan films modified by blending with PEG. Biomater 23: 2641-2648.
Zhang N, Mac J, Melo M.AS , Weir MD, Bai Y et al. (2015), Protein-repellent and antibacterial
dental composite to inhibit biofilm and caries. J Dent, 43;225 – 234.
Zhang Y and Xu J (2008), Effect of immersion in various media on the sorption, solubility, elution
of unreacted monomers, and flexural properties of two model dental composite compositions, J
Mater Sc: Mater Med, vol. 19, no. 6, pp. 2477–2483.
Zileinski BA and Acbischer P (1994) Chitosan as a matrix for mammalian cell encapsulation.
Biomater. 15: 1049-1056.