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Study of antibacterial activity and bonding properties of a multimode adhesive containing tt-farnesol

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Study of antibacterial activity and bonding properties of a multimode adhesive containing tt-farnesol

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Content
Study of Antibacterial Activity and Bonding Properties of a
Multimode Adhesive Containing tt-Farnesol
By
Diana Leyva del Rio

A Thesis Presented to the
FACULTY OF THE OSTROW SCHOOL OF DENTISTRY -
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
MASTER OF SCIENCE
(CRANIOFACIAL BIOLOGY)

December 2015

TABLE OF CONTENTS

List of Tables ii
List of Figures iii
Abstract iv
Chapter 1: Introduction 1
Chapter 2: Materials and Methods 4
Chapter 3: Results 12
Chapter 4: Discussion 24
Chapter 5: Conclusion 30
Bibliography 31

ii

LIST OF TABLES

Table 1: Scotchbond Universal Adhesive composition and application 9
Table 2: Colony-Forming Units, Dry Weight and Extracellular Insoluble 14
Polysaccharides results

Table 3: Microtensile Bond Strength, pH and Degree of Conversion results 17
iii

LIST OF FIGURES

Figure 1: Schematic of biofilm growth on top of the adhesive 5
Figure 2: Schematic representation of groups for Microtensile Bond Strength 7
Figure 3: Colony-Forming Units results 12
Figure 4: Dry weight results 13
Figure 5: Extracellular Insoluble Polysaccharides results 14
Figure 6: Microtensile Bond Strength results 15
Figure 7: Degree of Conversion results 16
Figure 8: Representative SEM images of control group at day 5 18
Figure 9: Representative SEM images of 100 MIC group at day 5 19
Figure 10: Representative SEM images of 500 MIC group at day 5 20
Figure 11: Representative SEM images of 1000 MIC group at day 5 21
Figure 12: Representative CLSM images of all groups in etch-and-rinse mode 22
Figure 13: Representative CLSM images of all groups in self-etch mode 23

iv

ABSTRACT
Adhesive systems with added antibacterial properties could be a solution to reduce the
incidence of secondary caries adjacent to bonded restorations. The objective of this study was to
evaluate the antibacterial and bonding properties of experimental adhesives combining the
antibacterial agent tt-farnesol with the multimode adhesive Scotchbond Universal. The
antibacterial agent was incorporated into the adhesive at 3 different concentrations based on the
minimum inhibitory concentration against S. mutans: 100xMIC, 500xMIC and 1000xMIC. The
adhesive with no antibacterial agent served as control. Biofilm was grown on top of composite
discs coated with the adhesives. The antibacterial activity was evaluated by colony-forming units
(CFU), biofilm dry weight and production of extracellular insoluble polysaccharides for all the
groups at day 2, 3, and 5. The bonding properties of the experimental adhesives were tested with
microtensile-bond strength to human dentin in both etch-and-rinse and self-etch application
modes, pH evaluation and degree of conversion. The experimental adhesive at 1000xMIC
exhibited the lowest CFU count and amount of extracellular insoluble polysaccharides at day 5
compared with the control (P < 0.05). All experimental adhesives showed decrease in bond
strength in both application modes and degree of conversion (P < 0.05). Dry weight and pH
values did no exhibited statistical differences with its controls (P > 0.05). In conclusion, the
experimental adhesive at 1000xMIC concentration of tt-farnesol showed important antibacterial
properties with limited decrease in its bonding properties.

1

CHAPTER 1. INTRODUCTION
Cavitated dental caries is usually treated by removal and replacement of infected tooth
tissues with restorative materials, such as adhesively bonded resin composites. Nonetheless, half
of the restorations fail within 10 years caused mainly by secondary caries (Sakaguchi, 2005).
Composite restorations accumulate biofilm/plaque on the surface more than other restorative
materials (Beyth et al., 2007). Overtime, the integrity at the interface tooth/restoration can be
compromised yielding a pathway for cariogenic bacteria, mainly S. mutans (Loesche, 1986),
resulting in the formation of secondary caries (Dennison and Sarrett, 2012). Furthermore,
cariogenic bacteria not removed during cavity preparation will be entrapped in the tooth hard
tissue which might form a secondary caries lesion under the restoration.
A solution to reduce the incidence of secondary caries would be the addition of
antibacterial agents into dental materials (Chen et al., 2012), since regular adhesive systems have
little or no antibacterial properties (Imazato et al., 2002; Imazato, 2003). The use of natural
products has been a successful strategy for the discovery of new medicines (Harvey, 2000), also
proving antibacterial effects against cariogenic bacteria (Gazzani et al., 2012). One of these
sources is propolis, a resinous substance produced by Appis mellifera bees used for the
construction, maintenance and protection of the bee hive (Burdock, 1998; Ghisalberti, 1979).
The antibacterial properties of propolis against oral pathogens have been widely studied
(Bonvehí, 1994; Koo et al., 2000; Park et al., 1998), being observed also the reduction of dental
caries in rats (Ikeno et al., 1991; Koo et al., 1999). It was later determined that from the 3 distinct
chemical groups found in propolis (a) flavonoid aglycones, (b) cynamic derivatives and (c)
terpenoids, tt-farnesol (terpenoid) showed to be most effective antibacterial agent producing a
rapid decrease in S. mutans viable counts al lower concentrations (Koo et al., 2002b). The anti-
2

cariogenic properties of tt-farnesol were later confirmed in an animal study where this agent
showed a reduction of the incidence and the severity of carious lesions in rats (Koo et al., 2002a;
Koo et al., 2005), where no adverse effects in the use of tt-farnesol have been reported in these
animal studies.
Streptococcus mutans possess numerous virulence factors to initiate and develop a
carious lesion, one of them is the synthesis of extracellular insoluble polysaccharides (EIP) from
sucrose using glucosyltransferases enzymes (Gtfs) (Bowen and Koo, 2011; Hamada and Slade,
1980). These extracellular insoluble polysaccharides highly contribute to the formation of
extracellular polysaccharide matrix composition which provides structural integrity to biofilm
(Hotz et al., 1972; Paes Leme et al., 2006). These glucans also promote adhesion and
accumulation of cariogenic bacteria to tooth surfaces which is vital for the development of
carious lesions (Schilling and Bowen, 1992).
It is clear the importance of the reduction of the viability of biofilm a primary goal to
prevent secondary caries. Therefore, it can be justified the addition of tt-farnesol into an adhesive
system since it has not been previously reported in the literature. Therefore, the aim of this study
was to investigate the effect of different concentrations of tt-farnesol incorporated into a
commercially available universal adhesive on S. mutans biofilm viability determined by colony-
forming units (CFU), biomass (dry weight) and extracellular insoluble polysaccharide formation
assays at different time points. Furthermore, to test the influences of the antibacterial agent plus
the adhesive on the bonding properties, microtensile bond strength (µTBS), pH and degree of
conversion (DC) of the different concentrations were also evaluated. The first null hypothesis
tested was that the incorporation of different concentrations of tt-farnesol into the adhesive
system had no effect on the viability of S. mutans biofilm. The second null hypothesis tested was
3

that the addition of the antibacterial agent into the adhesive system had no effect on the bonding
properties evaluated.

4

CHAPTER 2: MATERIALS AND METHODS
2.1 Microorganism
The bacterial strain used in this study was S. mutans UA159 (ATCC# 700610) because is
proven to be a highly virulent pathogen in the development of dental caries and selected for
genomic sequencing (Ajdic et al., 2002).
2.2 Antimicrobial activity
Initially the minimum inhibitory concentration (MIC) of tt-farnesol (3,7,11-trimethyl-
2,6,10-dodecatrien-1ol) (Sigma-Aldrich Co, St Louis, MO, USA) against S. mutans UA159 was
determined according to the NCCLS protocols (NCCLS, 1992; 2000). A starting bacterial
inoculum at 1x10
5
was prepared, and the concentration of the applied antibacterial agent ranged
from 10-500 µM. The solutions were incubated at 37°C and 5% CO
2
for 24 hours. The MIC was
obtained by observing bacterial growth inhibition at 150 µM determined by triplicates in at least
3 different experiments.
2.3 Antibacterial agent
The commercially available adhesive Adper Scotchbond Universal (SB) (3M ESPE, St.
Paul, MN, USA) was combined with the antibacterial agent tt-farnesol. The antibacterial agent
was incorporated directly into the adhesive at different concentrations (based on the MIC) to
obtain 3 experimental adhesives: (1) 100xMIC = 0.38% (w/w); (2) 500xMIC = 1.90% (w/w); (3)
1000xMIC = 3.80% (w/w). The adhesive with no addition of the antibacterial agent was used as
control.

5

2.4 Biofilm preparation and analysis
S. mutans UA159 strain biofilm was grown on composite discs (Imazato et al., 1998)
covered with any of the three experimental adhesives or the control group. The composite discs
were made using a microhybrid resin composite (Filtek Z250, 3M ESPE, St. Paul, MN, USA)
placed on a circular stainless-steel mold to obtain smooth and uniform surfaces. The dimensions
of the discs were 13mm in diameter and a thickness of 1mm. In order to avoid any contamination
of other microorganisms, the composite discs were sterilized by ethylene oxide.
After a 48h ethylene oxide degasification period, 20µL (size of a drop) of the adhesive
formulations were applied to one surface of the composite disc, agitated 20s with a microbrush
and a gentle steam of air was applied to promote solvent evaporation. It was later light-cured for
10s with a halogen unit (Elipar 2500, 3M ESPE) with 600 mW/cm
2
light output. The curing unit
was periodically tested for ideal light intensity with a radiometer. Once treated, the discs were
immediately placed in 24-well plates fully covered in 1mL of BHI + sucrose + S. mutans (1x10
5
)
inoculum medium for biofilm growth (Fig. 1). The media was changed daily without disrupting
the biofilm on top of the discs (Murata et al., 2008).

Fig 1. Schematic of biofilm growth on top of the adhesive.
Biofilm was collected at the 2
nd
, 3
rd
, and 5
th
day and sonicated in 5mL of phosphate-
buffered saline (PBS) solution to obtain a homogenized solution. The colony-forming units assay
was performed as followed: 1mL of the homogenized solution was subjected to serial dilution
Composite disc
Adhesive layer
Biofilm
6

(1x10
-1
– 1x10
-4
) to later 20µL of each dilution be inoculated in a tryptic soy agar plate with 5%
sheep's blood (TSA 5% SB) and placed in an incubator for 24-48h at 37°C and 5% CO
2
to allow
colony growth. After this period the colonies were counted and recorded. The numbers of
colonies were normalized and converted into Log
10
values for data interpretation. The CFU assay
was performed in triplicate in at least three different experiments.
Furthermore, 2mL of the original homogenized suspension was completely dried in a
SpeedVac concentrator (Thermo Scientific, Rockford, IL, USA) to obtain a dry pellet. The dry
pellet was first weighted to obtain the biofilm biomass dry weight and later processed for
extracellular insoluble polysaccharide assay. Extracellular insoluble polysaccharides were
extracted using 0.05mL of 1M NaOH per 1mg of biofilm biomass dry weight (Koo et al., 2003).
The resulting supernatant was collected via centrifugation and quantified by colorimetric assay
as described by Dubois et al. (Dubois, 1956).
2.5 Scanning Electron Microscopy (SEM) analysis
Discs with grown biofilms were subjected to fixation for SEM analysis as followed: the
samples were washed with 0.2% formaldehyde/0.1% chromic acid in 0.1M sodium cacodylate
buffer (Electron Microscopy Sciences, Hatfield, PA, USA) and sodium cacodylate 0.19M buffer
(Electron Microscopy Sciences, Hatfield, PA, USA). Subsequently, the samples were serially
dehydrated with ethanol (25%, 40%, 50%, 70%, 95% and 100%). The samples were completely
dried with hexamethyldisilizane (HMDS) to be later gold sputter coated and observed under a
scanning electron microscope (JSM-7001F, JEOL, Tokyo, Japan). Representative images were
taken for all groups at day 5.
7

2.6 Microtensile bond strength
Twenty-four caries-free human third molars where collected in accordance of school
regulations (IRB #APP-13-05027). The external surface of the teeth was cleaned of any
remaining organic debris and stored in 0.5% chloramine-t solution for no more than 2 months
before being processed for microtensile bond strength test (μTBS). Teeth were divided into 8
groups: 4 in etch-and-rinse and 4 in self-etch modes for all of the concentrations (n=3) Fig 2.

Fig 2. Schematic representation of groups for μTBS.
Each tooth was sectioned perpendicular to the long axis with a low speed diamond saw
(Isomet 1000, Buehler Ltd., Lake Bluff, IL, USA) and water-cooling to remove the coronal
enamel and root. The dentinal surface of the mid-coronal segment was polished with 600 grit
aluminum oxide sandpaper for 60s to create a uniform standardized smear layer (Oliveira et al.,
2003). Two different modes for dentin bonding approaches were used: Two-step etch-and-rinse
and One-step self-etch, strictly following the manufacturers’ instructions in regard to application
layers and time (see Table 1). Light-curing was performed with the halogen unit for 10s. Resin
Groups
Etch-and-
rinse
Control 100xMIC
0.38% (w/w)
500xMIC
1.90% (w/w)
1000xMIC
3.80% (w/w)
Self-etch
Control
100xMIC
0.38% (w/w)
500xMIC
1.90% (w/w)
1000xMIC
3.80% (w/w)
8

composite build-ups were made with the micro-hybrid composite resin on top of the bonded
surfaces in 3 successive increments of 2mm, each light-cured for 20s.
After 24h of storage in distilled water at room temperature, the samples were cut under
water-cooling in both X/Y axis parallel of the long axis of the tooth to obtain untrimmed sticks
with a cross-sectional surface area of 0.8±0.2 mm
2
. All sticks were measured with a digital
caliper (Mitutoyo digital caliper, Mitutoyo Corp., Tokyo, Japan) to obtain the area of the stick
and the values were recorded. The sticks were fixed with cyanoacrylate glue (Zapit, Dental
Ventures of America, Corona, CA, USA) in a testing jig (Perdigao et al., 2002) . The jigs were
placed in a universal testing machine (Instron Model 4400, Instron Corp., Canton, MA, USA)
and loaded in tensile force at a cross-head speed of 1 mm/min until fracture. The failure modes
were evaluated at 32x magnification in a stereoscopic microscope to be classified as cohesive
(failure entirely within dentin substrate or resin composite), mixed (failure at dentin/resin
interface including cohesive failure of one of the substrates), or adhesive (failure at the
dentin/resin interface). The load (Newtons) and the bonding surface area of the specimen were
registered using the software TestWorks 4 (MTS Nano Instruments, Eden Prairie, MN, USA),
and microtensile bond strengths were calculated in mega Pascals (MPa).

9

Table 1. Adhesive system composition and its application, according to the manufacturer recommendations.
Adhesive Composition Application modes
Adper Scothbond Universal
(3M ESPE)

10-MDP Phosphate monomer
Dimethacrylate resins
HEMA
Methacrylate-modified polyalkenoic acid
copolymer
Fillers
Ethanol
Water
Initiators
Silane
A. Two-step etch-and-rinse
Apply etchant for 15s
Rinse
Blot dry
Apply adhesive for 20s rubbing it against
the tooth surface
Air dry for 5s until the adhesive doesn’t
move
Light-cure for 10s

B. One-step self-etch
Apply adhesive for 20s rubbing it against
the surface
Air dry for 5s until the adhesive doesn’t
move
Light-cure for 10s

2.7 pH evaluation
The pH values of each of the experimental groups and the control were measured with a
pH meter (Mettler Toledo, Columbus, OH, USA). Before the measurement, the probe of the
meter was calibrated with a buffer standard solution. To obtain pH values, the tip of the probe
was submerged in 2mL of the experimental solution for 2 minutes and the value was recorded.
After every measurement, the tip of the meter was thoroughly rinsed with ethyl alcohol and
distilled water to remove any remnants of previous solution (Elsaka, 2012). The measurements
were performed under minimum light to avoid premature polymerization of the solution. Five
different readings for each individual adhesive were performed, and the mean pH value was
calculated.

10

2.8 Degree of conversion
Using micro-Raman spectroscopy (InVia, Spectrometer, Renishaw, New Mills, UK) with
a laser wavelength of 532nm, power output at 750mW, microscope objective of 50X and with a
pin-hole aperture of 370μm the degree of conversion was evaluated. The spectra range obtained
was from 400-2000 cm
-1
, with an integration time of 20s using a RenCam CCD detector with a
1024x256 pixel resolution. Data was analyzed using Origin2015 software (OriginLab
Corporation, Northampton, MA, USA).
Three different samples were fabricated for each of the experimental adhesives and the
control. After spectrometer calibration with a silicon sample, one drop (20μL) of fresh mixture of
adhesive was placed on top of a glass slide and spectra of the uncured adhesive was obtained and
recorded. The sample was agitated for 20s with a microbrush and then air was gently blown for
5s to promote solvent evaporation. A thin glass slide was placed on top of the drop to both create
a uniform layer and prevent an oxygen inhibition layer. The surface was light cured for 10s with
the curing light. The thin glass slide was removed and additional spectra of the sample was taken
and recorded. At least 5 spectra per sample were taken. The DC was calculated according the
Rueggeberg’s formula:
[

]
where R is the ratio of areas under the aliphatic 1639 cm
-1
peak and aromatic 1609 cm
-1
peak in
cured and uncured material.

11

2.9 Confocal Laser Scanning Microscopy (CLSM)
Two specimens per group were evaluated under a Confocal Scanning Electron
Microscopy with a Zeiss confocal microscope (LSM 5 PASCAL, Jena, Germany). The samples
were prepared by incorporating rhodamine B (Sigma-Aldrich Co, St Louis, MO, USA) into the
adhesive interface to trace the adhesive penetration as well as its ability to seal the dentin
surface. After 24h of storage in distilled water, fluorescein dye (Sigma-Aldrich Co, St Louis,
MO, USA) was applied in the pulpal chamber for 4h. The samples were finally cut at low speed
with a diamond saw in 1mm slabs perpendicular to the bonded surface. The samples were then
fixed in glass slides to be observed under CLSM. Representative images of each group were
taken.
2.10 Statistical analysis
In order to detect differences between all the groups triplicates of at least 3 separate
experiments were performed for the colony-forming units, dry weight and extracellular insoluble
polysaccharide assays. This data was checked for normality with Shapiro-Wilk test. Whereas
normal distribution was detected, data was analyzed with ONE-way ANOVA followed by Tukey
HSD post-hoc test. When no normal distribution was detected, Nonparametric Multiple
Comparisons test with Dunn post hoc-test was used. All the days were independently analyzed in
each of the assay. For microtensile bond strength, pH and degree of conversion evaluation, data
was checked for normality with Kolmogorov-Smirnov test. Microtensile bond strength data was
analyzed using TWO-way ANOVA to detect differences between both application modes in all
the groups, meanwhile degree of conversion and pH values were analyzed using one-way
ANOVA. These ANOVA analyses were followed by Tukey HSD and Dunnett post-hoc tests
(α=0.05).
12

CHAPTER 3. RESULTS
3.1 Colony Forming Units
The effects of the different concentrations of tt-farnesol on the CFU activity are shown in
Fig. 3 and Table 2. Similar values were observed for all experimental adhesives and the control
at day 2. At day 3, our results show statistical differences (P < 0.05) between both the control
and the 100xMIC group with the 1000xMIC group (1.41 Log
10
and 1.51 Log
10
respectively). At
day 5, an evident

difference was observed between the control group and the 1000xMIC group
with a value of 1.98 Log
10
. Additionally, the 500xMIC group showed a significant reduction (P
< 0.05) with the control, resulting in a 1.45 Log
10
difference between them.

Finally, a less evident
reduction, but statistically different (P < 0.05) was shown between the 100xMIC and the
1000xMIC groups with a 1.12 Log
10
difference.
Fig 3. Mean and Standard deviation of CFU analysis of the different concentrations of tt-farnesol in combination
with the Scotchbond Universal adhesive at day 2, 3, and 5. Values linked with a bracket are statistically different (P
< 0.05) from the other groups evaluated at the same day.
0
1
2
3
4
5
6
7
8
9
Day 2 Day 3 Day 5
CFU (Log10)
Control
100xMIC
500xMIC
1000xMIC
13

3.2 Biofilm biomass (dry weight)
Biofilm biomass of the groups tested is shown in Fig. 4 and Table 2. There was no
significant difference (P > 0.05) between all the groups tested at any of the time points evaluated.
Fig 4. Mean and Standard deviation of Dry Weight analysis of the different concentrations of tt-farnesol in
combination with the Scotchbond Universal adhesive at day 2, 3, and 5. There was no significant difference (P >
0.05) of the concentrations at the 3 days independently evaluated.

3.3 Extracellular insoluble polysaccharides
In the extracellular insoluble polysaccharides assay, there was no statistical difference (P
> 0.05) between any of the groups in both days 2 and 3. At day 5, the 1000xMIC group showed a
significant difference (P < 0.05) with the other two experimental and control groups as shown in
Fig. 5 and Table 2.
0
5
10
15
20
25
Day 2 Day 3 Day 5
Dry weight (mg)
Control
100xMIC
500xMIC
1000xMIC
14

Fig 5. Mean and Standard deviation of extracellular insoluble polysaccaryde analysis in relationship with dry weight
of the different concentrations of tt-farnesol in combination with Scotchbond Universal adhesive at day 2, 3, and 5.
The value at day 5 with an asterisk is statistical different (P < 0.05) from the other groups evaluated at the same
day.

Table 2 - CFU, Dry weight and Dry weight/Extracellular insoluble polysaccharide (DW/EIP)
values (means ± standard deviations) of the different groups tested at the days evaluated.
CFU (Log
10
) Dry weight (mg) DW/EIP (mg)
Groups/
Days
2 3 5 2 3 5 2 3 5
Control 5.72±0.41
a
5.80±0.61
a
6.56±0.65
a
19.79±1.18
a
19.13±0.48
a
18.94±0.79
a
0.47±0.27
a
0.31±0.06
a
1.46±0.56
a

100x
MIC
5.64±0.37
a
5.90±0.34
a
5.70±1.18
ab
19.39±0.66
a
18.76±2.00
a
18.17±1.62
a
0.34±0.29
a
0.48±0.20
a
1.41±0.52
a

500x
MIC
5.59±0.50
a
5.30±0.50
ab
5.11±0.33
bc
18.93±0.43
a
18.62±1.21
a
18.43±1.64
a
0.29±0.21
a
0.46±0.22
a
0.85±0.47
a

1000x
MIC
5.42±0.44
a
4.39±0.35
b
4.58±0.45
c
19.38±0.39
a
18.03±1.36
a
16.99±1.88
a
0.26±0.12
a
0.45±0.26
a
0.48±0.33
b
Within the same vertical column, means with the same superscript lower letter are not significantly
different (P > .05)

0
0.5
1
1.5
2
2.5
Day 2 Day 3 Day 5
Extracellular insoluble
polysacharydes (mg)
Control
100xMIC
500xMIC
1000xMIC
*
15

3.4 Microtensile bond strength test
For the microtensile bond strength test results (Fig. 6 and Table 3), all experimental
groups showed a statistical difference (P < 0.05) with the control group in both application
modes. The control groups in both bonding approaches yielded the highest bond strength means
of 74.34±26.1 MPa for the etch-and-rinse and 66.90±16.4 MPa for the self-etch mode. In both
application modes, all experimental groups showed statistical difference (P < 0.05) between their
control groups. When comparing bonding approaches, there was only statistical difference (P <
0.05) in the experimental adhesive with 1000xMIC concentration of tt-farnesol.

Fig 6. Mean and Standard deviation of µTBS analysis of the different concentrations of tt-farnesol in combination
with the Scotchbond Universal adhesive at the different application modes. Values linked by a bracket are
statisitically different (P < 0.05). Values with an asterik represent statistical difference with the control group (P <
0.05).

0
10
20
30
40
50
60
70
80
90
100
110
Control 100xMIC 500xMIC 1000xMIC
µTBS (MPa)
Etch-and-rinse mode
Self-etch mode
*
* *
16

3.5 pH measurement
Regarding mean pH values, there was no statistical difference (P > 0.05) on the mean
values of any of the experimental adhesives compared between each other or the control group
(Table 3).
3.6 Degree of conversion
The control group obtained the highest mean DC of 73.11% and it is statistically different
(P < 0.05) form all the experimental groups. The mean DC values of the experimental groups
ranged between 49.23% and 45.48% showing no statistical difference (P > 0.05) between each
other (Fig.7 and Table 3).

Fig 7. Mean and Standard deviation of Degree of Conversion (DC) analysis of the different concentrations of tt-
farnesol in combination with the Scotchbond Universal adhesive. Values with an asterik represent statistical
difference with the control group (P < 0.05).

0
10
20
30
40
50
60
70
80
90
100
Control 100xMIC 500xMIC 1000xMIC
DC%
*
*
*
17

Table 3 - µTBS (etch-and-rinse and self-etch modes), pH and DC values (means ±
standard deviations) of the different groups tested
µTBS (MPa) pH DC (%)
Groups
Etch-and-rinse
mode
Self-etch mode
Control 74.34±26.1
aA
66.90±16.4
aA
2.978±0.037
a
73.11±8.54
a
100xMIC 64.29±17.8
bA
54.35±20.9
bB
3.010±0.031
a
49.23±6.06
b
500xMIC 59.80±13.5
bA
55.71±19.9
bA
2.990±0.041
a
48.78±18.38
b
1000xMIC 55.69±16.0
bA
57.69±19.1
bA
2.994±0.029
a
45.48±10.04
b
Within the same vertical column, means with same superscript lower-case letters (comparing
different concentrations of tt-farnesol) are not statistically different (P > .05). Within the same
horizontal row, means with the same superscript upper-case letters (comparing application mode)
are not statistically different (P > 0.05).

18

3.7 Scanning Electron Microscopy images

Fig. 8. Representative Scanning Electron
Micrograph images of biofilm grown on
top of a composite disc covered with the
control adhesive at day 5.

A
x 100 100µm
19

D
x 100 100µm

Fig. 9. Representative Scanning Electron
Micrograph images of biofilm grown on
top of a composite disc covered with the
100xMIC experimental adhesive at day 5.
20

x 100 100µm
G
Fig. 10. Representative ScanningElectron
Micrograph images of biofilm grown on
top of a composite disc covered with the
500xMIC experimental adhesive at day 5.
21

J
x 100 100µm

Fig. 11. Representative Scanning Electron
Micrograph images of biofilm grown on
top of a composite disc covered with the
1000xMIC experimental adhesive at day 5.
22

3.8 Confocal Laser Scanning Microscopy images

Fig. 12. Representative CLSM images of the adhesive interfaces of the different experimental
groups tested in the etch-and-rinse mode. (A) Control. (B) Experimental adhesive containing tt-
farnesol at 100xMIC. (C) Experimental adhesive containing tt-farnesol at 500xMIC. (D)
Experimental adhesive containing tt-farnesol at 1000xMIC. A: Adhesive layer; D: Dentin; T:
Resin tags; HL: Hybrid layer.

D
T
HL HL
T
D
D
T
T
D
HL
A
A
HL
A
A
23

Fig. 13. Representative CLSM images of the adhesive interfaces of the different experimental
groups tested in the self-etch mode. (A) Control. (B) Experimental adhesive containing tt-
farnesol at 100xMIC. (C) Experimental adhesive containing tt-farnesol at 500xMIC. (D)
Experimental adhesive containing tt-farnesol at 1000xMIC. A: Adhesive layer; D: Dentin; T:
Resin tags; HL: Hybrid layer.

A A
A A
HL
HL
HL
HL
T
D
D
D
D
24

CHAPTER 4: DISCUSSION
The addition of antibacterial agents incorporated into restorative materials has
been widely reported in the literature (Chen et al., 2012). Adhesive systems with added
antibacterial properties become a potential vehicle for the delivery of these microbicidals since
they are in direct contact with the tooth substrate and adjacent restoration. Current anticariogenic
therapy focuses on targeting specific bacterial virulence factors which affect the formation,
development and maintenance of biofilms on tooth substrate rather than the use of broad-
spectrum microbicides, such as clorhexidine, which can cause an indiscriminate eradication of
beneficial oral microflora (Jeon et al., 2011b). Following this philosophy, the present study
aimed to evaluate the effect of different concentrations of tt-farnesol incorporated into a multi-
mode or universal adhesive on S. mutans biofilm viability and mechanical properties. With the
results obtained, it was demonstrated the ability of these experimental adhesives to affect
important virulence factors such as cell viability and extracellular insoluble polysaccharide
production. Additionally, bond strength and degree of conversion showed a decrease without
fully compromising adhesion to dentin.
One proven virulence factor of S. mutans affected by tt-farnesol is the production of
extracellular insoluble polysaccharides. The results in this study demonstrate a clear 67%
reduction on the production of these glucans using the highest dose of tt-farnesol (1000xMIC)
evaluated over an extended period of time (5 days). With this substantial decrease of
extracellular insoluble polysaccharides, it would be expected for the biofilms to have less
adhesion to tooth/restoration substrate. Furthermore, since extracellular insoluble
polysaccharides also serve as a protective and diffusion-limiting physical barrier, its disruption
will result in biofilm cells exposed to inimical influences (Flemming and Wingender, 2010;
25

Lewis, 2001; Stoodley et al., 2002). Our results coincide with previous studies where it was also
found a reduction of extracellular insoluble polysaccharides in biofilms treated with tt-farnesol
(Koo et al., 2003; Koo et al., 2005). Additionally the viability of cells (CFU) in the treated
biofilms is an important indicator of the antibacterial effect of the agent tested. Our data shows a
significant reduction (P < 0.05) of recoverable viable cells starting at day 3 between the control
and the highest concentration of the agent. This trend becomes more evident at day 5 showing a
≈2 log
10
difference. Our results displayed a more efficient antibacterial effect of the agent against
S. mutans cells than previous studies (Koo et al., 2002a; Koo et al., 2002b; Koo et al., 2003; Koo
et al., 2005) where it was observed a reduction between 0.5 – 1 log
10
of live recoverable cells
when biofilms where treated with tt-farnesol.
Another feature evaluated in S. mutans biofilm in this study was biomass (dry weight).
Cariogenic biofilm is not only conformed of communities of microbial cells; these cells are
enmeshed in a 3D extracellular matrix which contains mainly extracellular polysaccharides
(insoluble & soluble) with smaller amounts of proteins, nucleic acids and other sugars (Branda et
al., 2005; Flemming and Wingender, 2010). All these components conforms the total biofilm
biomass. Our results show a decrease, however not statistically significant (P > 0.05), of biofilm
biomass in the experimental adhesives throughout the days evaluated. These results are in
contrast with previous data (Jeon et al., 2011a; Koo et al., 2003; Koo et al., 2005) were there was
a significant biomass reduction in biofilms treated with tt-farnesol. Although, we did not observe
a substantial decrease in dry weight, it was noteworthy the reduced amount of viable cells and
presence of extracellular insoluble polysaccharides at day 5.
Previous studies have proven the ability of tt-farnesol to affect the acidogenicity,
acidurity (pH drop) and the production of intracellular polysaccharides of S. mutans biofilm.
26

With the decrease of the acidic environment, S. mutans capacity to survive and carry out
glycolysis can lead to the reduction in demineralization of the adjacent dental substrate (Jeon et
al., 2011a). Moreover, it was also demonstrated the ability of tt-farnesol to reduce the amount of
intracellular polysaccharides, which are an important source for acid production when exogenous
carbohydrates are absent resulting in an indirect reduction of the acidogenicity of biofilms (Koo
et al., 2003; Koo et al., 2005). The suggested mechanism of action of tt-farnesol against
important S. mutans virulence factors is the result of damage in the cell-membrane function
causing a reduction in Gtfs production and subsequent extracellular/intracellular polysaccharide
production, reduction in acid production and also reduction in the ability to tolerate acids (Jeon et
al., 2011a). Our data supports this hypothesis since the experimental adhesive with tt-farnesol
resulted in an important reduction in production of extracellular polysaccharides and cell
viability of S. mutans biofilm.
With the use of scanning electron microscopy, the surface morphology and structure of S.
mutans biofilms can be observed in detail. At high magnification it can be observed the S.
mutans chain-like cell arrangement, surrounded by fibrous hair-like structures which represent
extracellular polysaccharides. This morphology can be observed similar like in all the groups. At
low magnification, it can be observed an overall view of the bacterial colonies coating the
surface of the disc. The thick structure observed in the surface of the disc of the control group, is
consistent of a mature biofilm (Fig. 8). The effect of tt-farnesol on S. mutans biofilm is evident in
the group at 1000xMIC due to of a noteworthy reduction of bacterial colonies (Fig. 11). The
antibacterial agent tested successfully altered bacterial growth and accumulation on the surface
of the disc. This observation is consistent with our results where the experimental adhesive at the
highest concentration yield the lowest CFU count at day 5 of the experiment. Therefore, the first
27

null hypothesis was rejected since the experimental adhesive at 1000xMIC of tt-farnesol, did
show strong antibacterial properties by reducing significantly the presence of viable cell counts
and production of extracellular insoluble polysaccharides.
With the confirmed antibacterial properties of the experimental adhesives, it was unclear
if the addition of an antibacterial agent would affect the adhesive system’s bonding properties.
The experimental groups and the control were evaluated for microtensile bond strength, pH and
degree of conversion. Scotchbond Universal is one of the latest generations of adhesives so
called “Multi-mode” or “Universal adhesives”. These adhesives can be used in two application
modes: etch-and-rinse and self-etch without compromising the bonding effectiveness to dentin
(Hanabusa et al., 2012; Perdigao et al., 2012). Therefore, both application modes were used to
evaluate the bonding performance of the experimental adhesives to human dentin.
Our results show similar bonding performance in both application modes for all the
experimental groups, except for the 100xMIC, where it showed significantly higher value for its
etch-and-rinse group. This data overall coincides with previous studies where Scothbond
Universal is capable of producing similar µTBS values in either application mode at immediate
bonding (24h) to human dentin (Chen et al., 2015; Marchesi et al., 2014; Munoz et al., 2013;
Wagner et al., 2014). Universal adhesives such as Scothbond Universal, have the acidic
monomers found in self-etch adhesives. Self-etch adhesives can be classified according to the pH
and interaction depth with dentine in ultra-mild (pH >2.5; 0.2-0.5 µm interaction depth), mild
(pH ≈ 2; 0.5-1 µm interaction depth), intermediate (pH, 1-2; 1-2 µm interaction depth) and strong
(pH ≤1, ≥5 µm interaction depth) (Van Meerbeek et al., 2011). Self-etch adhesives bonding
mechanism relies on the demineralization of the dental substrate to further be replaced with resin
monomers getting interlocked upon polymerization. All experimental adhesives tested yielded
28

pH values of ≈ 3, similar to the control, therefore getting classified as ultra-mild adhesives. The
fact that the pH of the adhesives was not modified with the addition of different concentrations
of tt-farnesol, suggests that its intrinsic acidity of the self-etching properties of the adhesives
remained unaltered. Additionally, the acidic monomer 10-MDP found in Scothbond Universal
and other universal adhesives, has proven to add a chemical bonding to the dental substrate due
to its high affinity to hydroxyapatite found in the smear layer (Yoshida et al., 2004; Yoshihara et
al., 2010).
When evaluating the different concentrations of tt-farnesol incorporated into the adhesive
and the control group within application modes, there was a reduction in bond strength in the
experimental groups in both application modes. Moreover, similar values were observed between
the different concentrations of tt-farnesol within each application mode. This decrease in the
bond strength irrespective of the concentration of tt-farnesol used, is directly associated with the
reduction in the degree of conversion values obtained for all experimental adhesives. In this
study, DC was assessed with micro-Raman spectroscopy. This method is considered to be more
accurate in evaluating DC in light-cured dental materials due to its ability to measure the resin
component inside the material, meanwhile the other commonly used technique (Fourier
Transform Infrared spectroscopy), only measures at the surface and is significantly more time
consuming (Pianelli et al., 1999). The importance of evaluating the DC relies on a quantitative
evaluation of the monomer conversion after polymerization of light-cured dental materials,
which directly affects the mechanical properties of these materials (Ferracane and Greener,
1986). In our study, it was observed a direct relationship between DC and bond strength
effectiveness as reported by Bae et al. (Bae et al., 2005). The addition of tt-farnesol at different
concentrations resulted in a decrease of reacted monomers of all experimental adhesives,
29

resulting in a decrease bonding performance to dentin in both application modes. Therefore, the
second null hypothesis is also rejected since the addition of the agent did affect the adhesion to
dentin and degree of conversion, still, pH values remained unaltered.
The interaction of the experimental adhesives at the hybrid/adhesive layer can be
evaluated with the use of Confocal Scanning Laser Microscopy. In the etch-and-rinse mode (Fig.
12), resin tag formation was noted in all groups. Furthermore, all the experimental groups reveal
similar dentin hybrid layer to that of the control group. Additionally, small droplets of adhesive
far into the dentinal tubules were observed for all the experimental adhesives, most likely due to
the reduced monomer conversion of the adhesives. In the self-etch mode (Fig. 13), a hybrid layer
is also observed in all the groups, thus demonstrating the ability of the adhesives to partially
dissolve and increase the permeability of the smear layer. This characteristic mechanism of the
self-etch adhesives results in chemical bonding to tooth substrate. Furthermore, it can be
observed the incoming water from the dentinal tubules being limited by the smear layer.
This is the first study that evaluates the antibacterial and bonding properties of an
adhesive system incorporated with tt-farnesol. Our study follows the current trend of combining
natural antibacterial agents into bonding agents to ultimately reduce the incidence of secondary
caries adjacent to bonded restorations. It was evident the antibacterial properties obtained with
the experimental adhesive at the highest concentration (1000xMIC). Moreover, this study is
limited to the use of tt-farnesol into Scotchbond Universal, so our findings may not extend to
other adhesive systems since it may have a different chemical interaction, therefore yielding
different results. Additionally, this study only evaluated the short-term adhesion to dentin, thus
further experiments are needed in order to evaluate long term adhesion.
30

CHAPTER 5. CONCLUSION
The experimental adhesive at 1000xMIC concentration of tt-farnesol, showed an
important reduction in the production of extracellular insoluble polysaccharides and also a
reduction in the bacterial viability of S. mutans biofilm. From the bonding properties tested,
microtensile bond strength and degree of conversion exhibited some reduction in all the
experimental adhesives; nonetheless, pH values remained unmodified irrespective of the
concentration of tt-farnesol.

A
31

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Abstract (if available)

Abstract Adhesive systems with added antibacterial properties could be a solution to reduce the incidence of secondary caries adjacent to bonded restorations. The objective of this study was to evaluate the antibacterial and bonding properties of experimental adhesives combining the antibacterial agent tt-farnesol with the multimode adhesive Scotchbond Universal. The antibacterial agent was incorporated into the adhesive at 3 different concentrations based on the minimum inhibitory concentration against S. mutans: 100xMIC, 500xMIC and 1000xMIC. The adhesive with no antibacterial agent served as control. Biofilm was grown on top of composite discs coated with the adhesives. The antibacterial activity was evaluated by colony-forming units (CFU), biofilm dry weight and production of extracellular insoluble polysaccharides for all the groups at day 2, 3, and 5. The bonding properties of the experimental adhesives were tested with microtensile-bond strength to human dentin in both etch-and-rinse and self-etch application modes, pH evaluation and degree of conversion. The experimental adhesive at 1000xMIC exhibited the lowest CFU count and amount of extracellular insoluble polysaccharides at day 5 compared with the control (P < 0.05). All experimental adhesives showed decrease in bond strength in both application modes and degree of conversion (P < 0.05). Dry weight and pH values did no exhibited statistical differences with its controls (P > 0.05). In conclusion, the experimental adhesive at 1000xMIC concentration of tt-farnesol showed important antibacterial properties with limited decrease in its bonding properties.

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Creator Leyva del Rio, Diana (author)

Core Title Study of antibacterial activity and bonding properties of a multimode adhesive containing tt-farnesol

School School of Dentistry

Degree Master of Science

Degree Program Craniofacial Biology

Publication Date 09/03/2015

Defense Date 08/18/2015

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag adhesive systems,antibacterial properties,bonding agents,bonding properties,micro-Raman,microtensile bond strength,OAI-PMH Harvest,tt-farnesol

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Contributor Electronically uploaded by the author (provenance)

Advisor Duarte, Sillas (committee chair), Murata, Ramiro (committee member), Paine, Michael L. (committee member)

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