Dental Materials Journal 2015; 34(5): 585–594

The influence of surface roughness and surface dynamics on the attachment of

Methicillin-Resistant Staphylococcus aureus onto orthodontic retainer materials
Dheaa H. AL GROOSH1,2, Laurent BOZEC2, Jonathan PRATTEN2 and Nigel P. HUNT2

1
College of Dentistry, University of Baghdad, Iraq
2
UCL Eastman Dental Institute, 256 Gray’s Inn Road, London, UK
Corresponding author, Dheaa H. Abd Awn AL GROOSH; E-mail: d.al-groosh@ucl.ac.uk; dheaaha73@yahoo.com

Staphylococci species have been isolated from removable orthodontic retainers. The aims of this study were to determine the most
suitable device to analyze surface roughness of autopolymerized acrylic and thermoplastic materials and whether the surface dynamics
of these materials influences the attachment of Methicillin-Resistant Staphylococcus aureus (MRSA). Clinically simulated samples
of autopolymerized acrylic and thermoplastic material were first evaluated using laser non-contact, stylus mechanical profilometries
and atomic force microscopy (AFM) followed by contact angle measurement to characterize their surface dynamics. Finally, an
in vitro biofilm assay was carried out using a constant depth film fermentor to assess biofilm attachment. The results showed
a significant difference between the roughness values obtained from the tested profilometers with the AFM exhibiting the most
consistent roughness values. MRSA tended to accumulate initially within the microscopic irregularities of autopolymerized acrylic
samples whereas acid-base and electron donor interactions influenced the bacterial attachment onto the thermoplastic samples.

Keywords: Orthodontic retainers, MRSA biofilms, Atomic force microscope, Surface properties

biofilms represent a substantial issue for the success of

INTRODUCTION
Orthodontic retainers are devices that are used to
retain the new position of teeth after active orthodontic
therapy has been completed. These appliances
allow for the adjustment and adaptation of the
surrounding periodontal tissue and alveolar bone1).
Orthodontic retainers can be divided into two types;
fixed and removable. The removable retainers can
be detached from the teeth in accordance with the
clinician’s recommendation and are either composed
of thermoplastic polymer or polymethylmethacrylate
acrylic (PMMA) base2) (Fig. 1).
Removable orthodontic retainers confer the same
problems as other implants in the oral cavity. For
instance, the prevalence of staphylococci cultivated on
orthodontic retainers is approximately 50% and can
comprise almost 10% of the recovered viable microbiota.
Furthermore, of these about 8% were found to be
MRSA3).
Biomaterials are prone to microbial accumulation
during and after implantation. The microorganisms
adhere to these materials by either specific
interactions, where specific surface associated
appendages recognize a wide range of organic
molecules that cover the implanted materials soon
after implantation, or non-specific interactions, where
bacterial adhesion is brought by physicochemical
interaction between a substratum and bacteria4,5).
After the initial adhesion, bacteria will aggregate
and form a biofilm that is enclosed in an extracellular
matrix of polymeric substances such as plaque. These
Color figures can be viewed in the online issue, which is avail-
able at J-STAGE.
Received Feb 10, 2014: Accepted Feb 27, 2015
doi:10.4012/dmj.2014-045 JOI JST.JSTAGE/dmj/2014-045
the implants and may require implant removal, since
bacteria embedded in biofilms resist both antibiotic
treatment regimens and the host immune response6).
Additionally, the detached bacteria from those biofilms
may cause an infection at distant sites7). Biofilms form
on many medical devices and implants and can be
formed if at least two properties are available: the ability
of cells to adhere and the accumulation to form a cluster
of multilayer cells8,9).
In most natural environments biofilms consist of
multispecies. However with biomaterial associated
infections, Staphylococcus epidermidis comprise about
80%. This bacterial species have the ability to adhere
and accumulate with the help of a slime material that
envelop and protect the cells, although this substance
is not a true capsule and loosely bound to the cells9,10).
However, Staphylococcus aureus differs from S.
epidermidis in that it doesn’t have slime material but
possess a capsule polymer of polysaccharide9.11.12).
Staphylococci biofilm formation appears to be a
bacterial survival mechanism against unfavorable
environmental conditions regulated by the production
of certain proteins. There is little information available
to understand the mechanism of staphylococci
adhesion to a substratum and the application of
surface physicochemical properties i.e. dynamics
may explain some experimental observations13).
These dynamics, involving surface roughness, surface
hydrophobicity and surface free energy, occur in the
interfacial region between the substratum and the
adhering microorganism and cause strengthening of
the bond shortly after initial contact. Quirynen et
al.14) suggested that surface roughness is important in
586 Dent Mater J 2015; 34(5): 585–594

Fig. 1 The most widely used removable orthodontic retainers; autopolymerized acrylic based
and thermoplastic, Essix, retainers.
SEM images show the differences in their surface topography.

biofilm development and that roughness is important

in early stages of Candida albicans adhesion. However,
Radford et al.15) showed that surface hydrophobicity
influences bacterial adherence to a surface and when
the surface become more hydrophobic the number
of adherent microorganisms is less. Croes et al.16)
suggested that biofilm formation in Methicillin-
susceptible Staphylococcus aureus (MSSA) is dependent
of cell adhesion enhanced by the production of poly-N-
acetylglucosamine (PNAG) or slime, whereas biofilm
formation of MRSA may be regulated by surface
adhesions17).
The aims of this study were firstly, to determine
the best protocol to study surface roughness for
autopolymerized acrylic and thermoplastic materials
appropriate for microbiology studies and secondly, to
ascertain whether the surface dynamics of removable
orthodontic retainer materials influences biofilm
formation. To our knowledge, this is the first study
to involve the effect of surface interface dynamics on
MRSA biofilm formation on autopolymerized acrylic and
thermoplastic materials.
MATERIALS AND METHODS
Samples preparation
1. Preparation of autopolymerized acrylic and
thermoplastic samples mimicking the clinical situation
Two moulds of silicone (Rema® Sil silicone duplicating
material, Dentaurum, Ispringen, Germany) were
made from a smooth glass block 100×80 mm of known
roughness value (Ra which referred to the average
Fig. 2 Preparation of silicone mould for the
autopolymerized acrylic.
surface roughness). A type IV stone (Crystacal R, BPB
Formula, Newark, UK) was vacuum mixed according
to the manufacturer’s instructions (P:L ratio=2.8:1)
and poured to produce a positive replica for the
reference block. To make room for the fabrication of the
autopolymerized acrylic sheet, a thickness of 1.5 mm
sheet of modelling wax (Auntex, Kemdent, Associated
Dental Product, Wiltshire, UK) was added to the glass
block as shown in Fig. 2.
The stone blocks were immersed in water for 2 min
and then reinserted into the mould with the fitting
surface facing outward. Sheet of autopolymerized
PMMA (Forestacryl, Forestadent, Pforzheim, Germany)
of 1.5 mm thickness was constructed using the addition
technique in which the polymer is saturated with
monomer18).
Simultaneously, thermoplastic sheets (Essix
ACE plastic, Raintree Essix, New Orleans, LA,
USA), which is polycarbonate and polyethylene, were
fabricated from the stone block (flat template) using a
 Dent Mater J 2015; 34(5): 585–594
Table 1 Retainer materials and their composition
Materials
Powder
Autopolymerized Lot #401-0010
PMMA liquid
Lot #403-0020
0.7 mm sheet Lot
Essix ACE Plastic
#70125
thermoforming machine (Biostar, Scheu-Dental,
Iserlohn, Germany) according to the manufacturers’
instructions (Biostar code, 113; Vacuum heat time, 25
s; Vacuum, 1:25, Druformat, 0:55). These samples were
used for later comparison. Table 1 shows the retainer
materials composition used in this study.
2. Construction of thermoplastic samples which simulate
the clinical situation
Thermoplastic, Essix, retainers are three dimensional
structures designed to cap the teeth and extend beyond
the gingival margin i.e. covering the bucco- and liguo-
gingival area, Fig. 1; therefore, in order to simulate
the clinical situation i.e. the vertical extensions of its
flanges, an initial laboratory based study was designed
to investigate and compare the possible variation in the
fitting surface topography of these flanges.
Firstly, the heights of the labial, posterior and
lingual flanges of 10 Essix retainers, constructed for the
lower and upper arches, were measured as well as the
heights of the corresponding flanges of their dental casts
using a digital caliper (0.01 mm accuracy; Mitutoyo®
digimatic sliding caliper, Mitutoyo, Andover, UK).
Following this, a template of four stone blocks (Crystacal
R) of different heights was constructed using wax blocks
(Kemdent) as shown in Fig. 3a. The wax blocks were
cut using a wax knife, polished and glazed by flaming.
Moulds were made for the wax models using silicone
duplicating material (Dentaurum) which was mixed
with a ratio of 1:1 and poured using the addition
technique. The negative replica was then poured with
type IV stone. A template with 16 mm stone block was
chosen according to the measurements obtained above.
Essix samples were processed using
thermoforming machine (Biostar) into which the stone
template was placed. The temperature and compression
were adjusted according to the manufacturer’s
recommendation. The constructed Essix sheet is shown
in Fig. 3b.
3. Preparation of autopolymerized acrylic samples with
conventional laboratory polished surfaces
The autopolymerized acrylic sheets were divided into
two halves of identical surface topography. One of
them was finished with a tungsten bur (Bracon,
Etchingham, UK) at 15,000 rpm and polished using
587
Composition
Polymethylmethacrylate, Accelerator, Color agent,
Ferrous oxide, Titan oxide.
Methylmethacrylate, Accelerator, Vemetzer, Dispersion of
fatty acid ester, Color pigments, Amorphous silicate.
Polycarbonate, Polyethylene.
Fig. 3 Construction of thermoplastic samples simulate the
clinical situation: (a) stone template with blocks of
different heights; (b) a cross-sectional view shows
Essix sheet, in red, made on the stone blocks.
a conventional laboratory technique consisting of a
slurry of pumice (Pumex, Lipari, Italy), water and lathe
bristle brush (C&L E. Attenborough, Nottingham,
UK). The surface of the other half was kept without
modification to simulate the clinical condition.
Surface characteristics
1. Surface roughness
Five specimens of 5 mm diameter autopolymerized
acrylic (polished and unpolished surface) and
thermoplastic Essix material (made on flat template,
Essix H, and on a template with 16 mm height
a block, Essix V) were analyzed using three different
profilometers. Firstly, laser profilometry (Proscan
1000, Scantron Industrial Products, Taunton, UK) to
measure an area of 9 mm2 with 0.02 µm resolution of the
displaced probe, with the laser beam diameter of 25 µm.
Stylus profilometry (Dektak 150, Veeco instrument,
NewYork, USA) in which measurements were obtained
with the radius tip of 5 µm under a measuring force
of 15 mg and 0.1 µm resolution. The cutoff length was
1 mm with a 30 s scan duration. Finally, atomic force
microscopy (AFM; XE 100 Park Instruments, Suwon,
Korea) equipped with sharp silicon nitride tips of 10
nm in radius (NSC nanoscience center, MikroMasch,
588 Dent Mater J 2015; 34(5): 585–594
Tallinn, Estonia). The bending constant of the probe
was 0.3 N/m. The scan scale of the image was 1 Hz with
an area of 45×45 µm and consisted of a 512×512 pixel
scan. The surface roughness values were expressed by
Ra (arithmetic roughness).
2. Hydrophobicity and surface free energy
Five samples of 10×15 mm of each material described
above were tested with regard to their hydrophobicity
and surface free energy. The contact angle of distilled
water (for hydrophobicity), glycerol (Sigma-Aldrich,
MO, USA) and hexadecane (Sigma, Poole, UK) were
performed according to a sessile drop method. A 5 µL
drop of each liquid was delivered from a distance of 2
mm onto the surface of each sample, which was
horizontally levelled, at a temperature of 23±0.5°C and
humidity condition of 40%19,20).
The drop profile was photographed using an optical
contact angle meter (Cam 200, KSV Instruments,
Helsinki, Finland) after 2 s spreading time. The
contact angle was calculated between the surface of the
substrata and the surface tangent of the drop20).
The total surface free energy γ TOT consists of two
components:
γ TOT = γ LW+ γ AB (1)
γ LW is the apolar component of the surface free energy
associated with Lifshitz-Van der Waals interactions,
whilst γ AB is the acid-base component of surface free
energy and results from the electron-donor (γ− ) and
electron-acceptor ( γ+) molecular interactions (i.e. Lewis
acid-base interactions). The acid-base term is expressed
as the product of the electron donor and electron
acceptor parameters:
γ AB =2√ γ+ γ− (2)
The surface energy parameters for the three probe
liquids used in this work are measured as shown in
Perni et al.20).
In vitro evaluation of biofilm formation
In vitro studies were performed to assess the effects of
different retainer materials and their surface properties
on biofilm growth. For this purpose the constant depth
film fermenter (CDFF; AC Service Group, Poole, UK)
was used as described previously21).
Briefly, the CDFF consisted of a glass vessel with
a rotating stainless steel turntable. The vessel was
contained within two stainless steel plates forming a
roof and a base and retained by polytetrafluoroethylene
(PTFE) seals. The turntable held 15 PTFE pans located
flush around the rim. Each of these pans had five
cylindrical holes of 5 mm in diameter containing PTFE
plugs upon which a disc of substrata, the tested samples,
was placed and recessed to a depth of 300 µm. The pans
rotated under PTFE scraper blades which smeared the
medium over the 15 pans and therefore maintained the
biofilm at a constant depth i.e. thickness.
Artificial saliva was used as a growth medium
according to Pratten et al.22); lab lemco 1 g/L, yeast
extract 2 g/L, protease peptone 5 g/L, Type III hog
gastric mucin 2.5 g/L, sodium chloride 0.2 g/L, potassium
chloride 0.2 g/L, calcium chloride 0.3 g/L, 40% urea
1.25 mL/L (all Sigma). The medium was delivered
using the peristaltic pump at a flow rate of 0.72 L per
day which corresponded to the resting salivary flow
rate in humans23). The experiment was maintained for
48 h under an aerobic atmosphere and at a constant
temperature of 36°C in an incubator (Philip Harris,
Shenstone, UK).
For each experiment, 10 mL of an overnight (16 h)
culture (adjusted OD600=0.95) of EMRSA-16 (Epidemic
Methicillin-Resistant Staphylococcus aureus, a common
strain of MRSA) in brain heart infusion (BHI) was
inoculated into 250 mL of sterile artificial saliva. To
inoculate the CDFF, the inoculum was pumped for 8 h at
a rate of 0.72 L per day via a peristaltic pump (Watson-
Marlow, Falmouth, UK).
Discs of 5 mm in diameter were cut from the
autopolymerized PMMA and thermoplastic, Essix,
sheets using a trepanning tool. The discs were then
sealed in transparent plastic bags and sterilized under
ultra-violet radiation for 20 min. The samples were then
inserted into the CDFF as described above.
At the appropriate time, the turntable was stopped
and the required pans were aseptically removed from
the CDFF. The pans were then immersed gently into
20 mL tubes containing 5 mL of PBS to remove the
planktonic bacteria. Sterile forceps were used to remove
the disc from the pan by forcing the PTFE plugs upward;
once the discs were exposed they were removed and
placed separately into a bijou bottle containing 1 mL
of PBS with 5 sterile glass beads (Sigma). The disc was
vortex-mixed for 1 min to disrupt the biofilm then ten-
fold serial dilutions were prepared and 20 µL aliquots of
each dilution plated onto blood agar (Oxoid, Basingstoke,
UK). The plates were then incubated aerobically for 24
h at 37°C.
1. Scanning electron microscopy (SEM) and Confocal
laser scanning microscopy (CLSM)
1) SEM: Aseptically removed autopolymerized acrylic
and Essix discs were carefully immersed in
3% gluteraldehyde in 0.1 M sodium cacodylate
buffer to fix the cells and were stored at 4°C
for 24 h. The samples were then dehydrated
in a series of graded ethanol (20%, 50%, 70%
and 90%). The specimens were left in each
concentration for 15 min before immersing
three times in 100% ethanol for 10 min each at
room temperature. The specimens were then
transferred into hexadimethylsilane for 2 min
then placed in a desciccator and left to dry.
The discs were mounted onto aluminum stubs
by using Araldite (Sigma) and were sputter
coated with gold palladium in a Polaron
E5000 sputter coater and imaged using a
Cambridge 90B scanning electron microscope
 Dent Mater J 2015; 34(5): 585–594 589

(Zeiss, Cambridge scientific instruments, Ely,

UK) operating at 15 Kv.
2) CLSM: Biofilms were visualized using methods
described previously by Hope and Wilson24).
Briefly, autopolymerized acrylic and Essix
discs were placed into small petri dishes (5 cm
diameter) with the biofilms facing upwards
held in place using vacuum grease. 10 mL
of PBS containing 3 mL of Live/Dead stain
(Molecular probe, Eugene, OR, USA) carefully
submerged the biofilm which was then
incubated in the dark for 15 min. The biofilms
were visualized with a Radiance 3000 confocal
laser scan head (Bio-Rad, Jena, Germany) in
conjunction with a BX51 stereomicroscope
(Olympus, UK Southhall, UK) equipped
with a 40 HCX water immersion lens with a
numerical aperture of 0.8 µm using helium
neon 543 nm and argon 488 nm lasers. The Fig. 4 Box plot for the logarithmic transformation of
resultant confocal optical sections were surface roughness (Ra) values of various retainer
collected by Bio-Rad LaserSharp software materials; Autopolymerized acrylic with polished
and unpolished samples and thermoplastic Essix
as stacks of images. The images were
(H) samples made on flat template, and Essix (V)
analyzed using image J software (National
samples on a template with 16 mm stone block,
Institute of Health, Bethesda, MD, USA) to
measured by three types of profilometers: Laser
produce XY projection and three dimension
(Proscan), Stylus and AFM.
representation. Small circles represent the outliers.

2. Statistical analysis
Results from the surface roughness evaluation using
different profilometers were compared, following log
transformation of data, using one-way analysis of
variance (ANOVA) and Tukey multiple comparison
test. For the biofilm assays, independent student t-tests
were used to compare the difference in means of colony
forming units following log transformation.
smooth structures by the SEM images and the three
dimensional outcomes obtained from the AFM (Figs.
5c and 5f). A similar outcome was obtained when an
anatomically flat sample, Mica, was scanned (data not
included).

RESULTS Hydrophobicity and surface free energy

Surface roughness measurement
Mean surface roughness values of the tested removable
orthodontic materials measured by the three
profilometers are shown in Fig. 4.
There were significant differences in surface
roughness values presented by the three methods. The
laser profilometer measurements did not conform to
those of the contact profilometer i.e. the apparent
smoother surface had the highest roughness value
and the highest scattered value among all the tested
materials, whereas, the stylus and AFM showed
comparable results.
However, regarding the thermoplastic Essix
samples made from a template with a 16 mm stone block
(Essix V), there was a significant difference between
the surface roughness measurements obtained by the
stylus prophylometry and the AFM (p<0.001). The
stylus profilometer recognized the surface irregularities
occurring in the fitting surface of the samples as
‘roughness’ features (1.86±0.94 µm; N=15), whereas
the AFM interpreted them as a smooth structures
(0.37±0.30 µm; N=15). Those features appeared as
The contact angle of the water and the surface of a
substratum can be used as a quantitative method to
assess the surface hydrophobicity of a material. It
can be seen from Table 2 that all the tested materials
showed hydrophilic surfaces <90° with values ranged
between 57.7° and 74.1° where the thermoplastic
samples showed low average contact angle values
(60.7°±5.7°; N=5) i.e. greater wetting ability. The total
surface free energies and Van der Waals forces of
the tested materials were not significantly different.
However, the thermoplastic materials showed higher
electron donor properties (55 mJ/m2) and acid-
base interactions (3.5 mJ/m2) when compared to
autopolymerized acrylic materials.
In vitro biofilm evaluation
The number of bacteria attached to the surface of the
tested materials increased over the 48 h time period
regardless of the material chemistry and surface
topography (Fig. 6). There was no significant difference
in MRSA biofilm formed on the surface of these
materials. However, the SEM and the CLSM showed
that the distribution of bacterial aggregates increased
590 Dent Mater J 2015; 34(5): 585–594

Fig. 5 SEM and AFM outcome images of autopolymerized acrylic and thermoplastic samples; (a, d) Unpolished
acrylic sample made to simulate the clinical condition, (b, e) Polished acrylic samples using conventional
laboratory polishing procedures. The arrow refers to the unpolished areas left during the polishing
procedure; and (c, f) Thermoplastic, Essix V, samples made on template of 16 mm block.
The arrow refers to surface irregularities recognized as smooth structures by the AFM.

Table 2 Contact angle (in degrees) and surface free energy parameters (in mJ/cm2) of autopolymerized acrylic and
thermoplastic samples

Contact angle Surface Free Energy (mJ/cm2)

Materials
(degree) LW (+) (−) AB Total
Essix (on flat template; Essix H) 57.7±10.7 17.93 0.06 55.09* 3.54 21.47
Essix (on template with 16 mm height block; Essix V) 64.5±12.3 22.48 0.11 31.40 3.63 26.11
Autopolymerized acrylic (polished) 74.1±5.9 16.44 0.41 22.96 1.06 22.95
Autopolymerized acrylic (unpolished) 72.7±5.5 22.80 0.00 26.21 0.77 22.00

(LW) Lifshitz-Van der Waals; (−) electron donor; (+) electron acceptor; (AB) acid-base component; (total) total free energy
* p=0.037

on the rougher surface of the autopolymerized acrylic

samples whereas the pattern of bacterial attachment
was more uniformly distributed on the thermoplastic
samples (Fig. 7).

Fig. 6 Viable count of EMRSA-16 deposit on
autopolymerized acrylic and thermoplastic samples;
Unpolished samples made to simulate the clinical
condition, thermoplastic Essix V samples, made on
template of 16 mm block, Polished samples using
laboratory polishing procedures.
Bars represent the standard deviation (p>0.05).
DISCUSSION
Removable orthodontic retainers are intraoral
appliances and subjected to the same problems as other
medical implants in that they are susceptible to biofilm
formation. It is well known that surface roughness
increases the physical surface area of a material and
may provide protected niches where the bacteria are
sheltered against the dislodgement forces such as
mechanical brushing. A threshold surface roughness
value for microbial aggregation of 0.2 µm has been
suggested by some in vivo studies that below this value,
no further reduction in microbial accumulation could
be detected25). However, to investigate this further, it
 Dent Mater J 2015; 34(5): 585–594 591

Fig. 7 SEM and CLSM images with three dimensional representation of MRSA deposition
on autopolymerized acrylic and thermoplastic Essix samples; starting from the top
right; (a, d and g) Unpolished acrylic sample made to simulate the clinical condition.
Arrows in (a) refer to bacteria attached within the microscopic surface irregularities;
(b, e and h) Thermoplastic Essix sample; (c, f and i) Polished acrylic sample using
conventional laboratory polishing procedures. The arrows refer to the distribution of
bacterial attachment. All bacteria are viable.

is important to investigate which type of profilometry
provides the most reproducible and representative
surface roughness measurement of autopolymerized
acrylic and thermoplastic materials appropriate for
microbiology studies, as both contact and optical
profilometers have been reported.
Laser profilometry has previously been used to
measure the surface topography and roughness of
autopolymerized acrylic materials26). It was suggested
that laser light can resolve features smaller than the
diameter of the light spot (25 µm) and it has a vertical
resolution of 0.1 µm. Rodriguez et al.27) reported that
the non-contact profilometer can accurately calculate
the Ra from a vertical reference tool. Additionally, the
non-contact characteristics of the laser profilometry
overcome the possibility of distorting or abrading certain
surfaces. Furthermore, previous work on an optical
light scanner by DeLong et al.28) showed no relationship
between the actual surface roughness of a colored
impression materials and its digitized form.
The results obtained from the current study
have shown that there was a significant difference
in roughness data recorded by the different types of
profilometers. Laser profilometry produced the most
inconsistent, highest and most scattered values (Ra
range from 4.46 µm to 37.17 µm). The data were highly
affected by the sample color, transparency and laser
reflectivity on the surface. When the polished surfaces
were tested, the laser tended to be reflected providing a
false reading of the actual roughness. Similarly, when
the samples were transparent, as in thermoplastic Essix
samples, the laser could penetrate through the sample
and thus also give a false reading. It is worth noting that
laser profilometry is a technique that suffers from light
absorption of the laser radiation as it scans the sample.
Contact profilometers have been used in several
studies on autopolumerized acrylic and non-flexible
materials to scan and quantify surface roughness. Stylus
profilometry has been widely used to quantify surface
topography of acrylic materials18,29,30). The resolution
of stylus profilometry is 0.1 µm and is governed by
the diameter of the diamond stylus probe (5 µm in our
case) which is appropriate for most studies31). AFM has
been used to measure surface topography of composite
materials and the film layer of some materials with high
atomic resolution32). It can detect very small structures
592 Dent Mater J 2015; 34(5): 585–594
on an anatomically flat surface with high resolution
and the information obtained is more descriptive and
comprehensive than the two dimensional measurements
obtained from the stylus profilometry33).
The roughness data from the current study
showed no significant difference between the stylus
profilometry and the AFM in quantifying the polished
autopolymerized acrylic and the thermoplastic Essix
samples made on a flat template (Essix H). However,
the clinically simulated samples made of PMMA and
thermoplastic Essix materials (made on 16 mm stone
block; Essix V) have been interpreted differently in such
a way that relatively smooth structures, detected by
SEM images, were recognized as roughness data by the
stylus profilometry during the scan process. In contrast,
the AFM recognized them as smooth irregularities and
have low effect on Ra outcome.
Several factors may contribute to the inconsistency
and misinterpretation of the roughness value (Ra)
obtained by stylus profilometry. These include the
presence of voids and irregularities which make it
difficult to measure accurately the surface roughness
of the samples as their surface appears in a multiple
roughness scale. Additionally, the scan length may
impact on the surface morphology and when it is
changed the surface roughness value will be changed
too34). The Ra results obtained from the AFM showed
the least scattered data throughout the measured
samples. Moreover, a single data outcome obtained from
the AFM represents a mean roughness value for an
area of 45×45 µm which can be represented as a three
dimensional high resolution image.
However, there are certain drawbacks limit the use
of the AFM in PMMA based appliances. These include
firstly, the use of this device is limited to measure flat
surfaces without irregularities larger than the z-range
of the instrument’s probe. In ‘real life’ practice the
fitting surface of the acrylic based retainers characterized
by multiple roughness scale with sharp uneven
structures. This could challenge the practicality of the
use of profilometers, especially the AFM. Secondly, the
time needed to get a representative measurement from
scanning a large object is much longer than the other
two profilometers
For the current study, the AFM represents the most
appropriate device to describe the surface roughness
of autopolymerized PMMA acrylic and thermoplastic
samples with a more consistent way. It can image
delicate structures on surfaces with greater resolution
than stylus profilometry, because of the small radius
of probe being only 10 nm and therefore smaller
than the diamond stylus probe. This high resolution
measurement gives a clearer idea when biofilm
attachment and growth is considered. Thus, AFM is
suitable for microbiological applications. However, the
representativity of AFM for large surfaces needs further
investigations using a probe with higher z-range i.e. 225
µm and larger scan length i.e. 100×100 µm.
In vitro biofilm evaluation
To ensure clinical relevance, the biofilm assay should
be undertaken using materials that are already
applied in orthodontics; therefore, the substratum
was fabricated following the manufacturer’s
recommendations and finished using the same laboratory
procedure. Furthermore, a CDFF was used which
generates large numbers of reproducible biofilms with
conditions similar to that of the oral cavity35).
The use of artificial saliva demonstrates several
advantages compared to the natural saliva for this in
vitro investigation; firstly each experiment required
large amount of saliva which reached up to 6 L;
therefore, artificial saliva demonstrates a standardized
mean compared to the inconsistent and instable nature
of natural saliva. Secondly, it was suggested that the
way the artificial saliva reacts with microorganisms is
similar to that of natural saliva. Finally artificial saliva
allows us to specify the mode of bacterial adhesion
without considering the variability of the natural saliva
such as the chemical composition, buffering status and
viscosity21,22). For the above mentioned reasons, artificial
saliva was considered as an appropriate substitute.
Several studies have been conducted to find the
effect of surface roughness of denture acrylic materials
on Streptococcus spp. or Candida spp. attachment15,26).
However, studies involved staphylococci biofilm have
been carried out using bone cement as substrata36).
All the tested substrata revealed hydrophilic
surfaces with different wetting properties. When the
surface roughness increased the contact angle decreased
and thus the wetting characteristics increased. These
findings are in accordance with Eliades et al.19) and Crick
and Parkin37). The current study showed that there were
no marked differences in the total surface free energy
and Van der Waals force’s regardless of the materials’
chemistry and surface roughness. However the Essix
samples exhibited higher acid-base and electron donor
properties.
The surface free energy is influenced by the
chemical composition of a substratum and the
fabrication procedure which in turn affects the surface
properties of that material38). The low surface energy
of the autopolymerized PMMA acrylic samples might
be due to the fact that the autopolymerized acrylic
material is made of methylmethacrylate monomers.
A similar finding has been reported by Ahn et al.39)
who showed that composite adhesives containing this
monomer showed a low surface free energy compared
to resin modified glass ionomer cement. Furthermore,
the diffused water molecules within the polymeric
structure may contribute to the low surface energy.
Glantz et al.40) suggested that the higher the
surface energy of a material the more favorable the
effect on microbial adherence. However, Busscher et
al.41) and Sardine et al.42) showed that strong initial
adhesion between a material and oral bacteria tends
to be influenced by a great acid-base polar interaction.
The current study showed that the MRSA was able to
adhere and form biofilms on all of the surfaces of the
 Dent Mater J 2015; 34(5): 585–594
tested substrata with little difference in the number of
bacteria recovered.
Autopolymerized acrylic is a rough material when
considering bacterial attachment and its surface
irregularities increase the physical surface area and
provide protected niches that encourage bacterial
adhesion43). The result of this study showed that
MRSA was detected within the microscopic surface
irregularities of the unpolished samples and accumulated
initially in the rough areas within the polished
autopolymerized acrylic samples. The surface free
energy parameters did not have an obvious effect on
bacterial attachment. In contrast, the Essix samples
exhibited a smooth surface compared to autopolymerized
acrylic, and were more hydrophilic, with higher electron
donor properties and acid-base interactions. These
surface properties may explain the initial bacterial
attachment of the MRSA and the uniform distribution
of the bacteria in the Essix samples as seen by SEM
and CLSM images. This result is in agreement with
Eliades et al.19) and de Morais et al.44) who suggested
that surface physico-chemical properties are crucial
when considering initial bacterial adhesion. Additional,
Van Oss et al.45) claimed that electron donor/electron
acceptor interactions of the acid-base element of surface
free energy are more important than other interactions.
CONCLUSION
This study was undertaken to determine the influence
of surface roughness and surface dynamics of two
commonly used retainer materials i.e. autopolymerized
PMMA acrylic and thermoplastic Essix materials on
MRSA biofilm formation.
The data obtained from the current study showed
that surface roughness, interpreted by AFM which is
the most appropriate devise to measure the surface
roughness of autopolymerized acrylic and thermoplastic
samples with a consistent and representative way
compared to the other tested devices, may influence
MRSA biofilm formation on autopolymerized acrylic
samples. Moreover, the interfacial surface dynamics may
play an important role in the early phases of bacterial
attachment onto the thermoplastic Essix material.
Both autopolymerized acrylic and thermoplastic Essix
materials have shown favorable surface characteristics
for MRSA adhesion and biofilm formation. Further work
on the effect of surface treatment of autopolymerized
acrylic materials on MRSA biofilm is therefore
required.
ACKNOWLEDGMENTS
Many thanks to Mr Ed PAYNE for his help and advice
in the laboratory work. This project is part of a PhD
study at the UCL/ Eastman Dental Institute sponsored
by the Ministry of Higher Education and Scientific
Research-Iraq.
593
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