J. Stomat. Occ. Med. (2010) 3: 171–176
Printed in Austria
© Springer-Verlag 2010
Bioﬁlm formation on oral piercings
, Christoph Steiner
*, Ulrike Beier
, Natalia Schiefermeier
, Frederik Klauser
Department of Operative Dentistry, Dental School, Innsbruck Medical University, Innsbruck, Austria
Division of Cell Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria
Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria
Institute of Physical Chemistry, University of Innsbruck, Innsbruck, Austria
Received July 21, 2010; accepted after revision September 29, 2010
Purpose: Bioﬁlms on oral piercings might serve as a bacterial
reservoir in the host and lead to bacteraemia and even septic
complications. The use of piercing materials less susceptible
to bioﬁlm accumulation could contribute to alleviation of
problems. The present study aimed to assess bioﬁlm forma-
tion on four commercially available, surface characterized
piercing materials in vitro (polytetraﬂuoroethylene, titanium,
stainless steel, and polypropylene).
Material and methods: Autoclave-sterilized piercings
were surface characterized by X-ray photoelectron spectros-
copy, contact angle measurements, and atomic force micros-
copy. Bioﬁlms were grown for 1, 4, or 20 h on the piercing
surfaces by immersion in pooled human whole saliva. Colony
forming units (CFUs) were determined. For ﬂuorescence
microscopy, samples were stained with DAPI.
Results: All four piercing materials included showed
signiﬁcant amounts of bioﬁlm after 20 h of incubation. Bio-
ﬁlm formation was signiﬁcantly lowest on polytetraﬂuoro-
ethylene piercings (p< 0.001), and was mainly determined by
wettability –which was signiﬁcantly lowest for polytetraﬂuor-
oethylene (p< 0.001) –and the prevalence of carbon- and
oxygen-rich components. Surface roughness measurements
showed no statistically signiﬁcant differences, but standard
deviations were rather high. High standard deviations are
caused by substantial pits and elevations and are due to poor
machining quality in the manufacture of the piercings.
Conclusion: Bioﬁlm formation on oral piercings was
mainly determined by surface free energy and the prevalence
of carbon- and oxygen-rich components, and was signiﬁcant-
ly lowest on polytetraﬂuoroethylene piercings. The ﬁndings
indicate that oral piercings might serve as a reservoir of
potentially pathogenic bacterial species.
The trend in oral piercing has grown in popularity over the
past decade. Oral piercing mostly involves lips, tongue, and
cheeks, with the tongue being the most commonly pierced
intraoral site . A variety of complications have been
reported and can be categorized as early (acute) and late
(chronic) . Early complications include bacterial infec-
tion, pain, swelling, prolonged bleeding, and difﬁculties in
swallowing, speech, and mastication . Late complica-
tions include recurrent infections, gingival trauma, local-
ized periodontitis, chipped and fractured teeth, persistent
difﬁculties in oral functions, and swallowing of the device
. A number of studies have been conducted to assess the
prevalence of these complications [7, 16–18, 23, 25, 27].
However, many biologic questions on these foreign bodies
remain unaddressed, one of which being bioﬁlm formation.
Bioﬁlms on oral piercing may serve as a bacterial reservoir
and lead to systemic bacteraemia and even septic compli-
cations. Several case reports have been published on life-
threatening systemic infections after oral piercing: Infective
endocarditis , breast abscess , epidural abscess ,
chorioamnionitis , herpes simplex virus hepatitis ,
hepatitis C virus infection , toxic shock syndrome due to
Staphylococcus aureus infection , and cerebellar brain
abscess  are rare, but dangerous complications. Addi-
tionally, bioﬁlms on oral piercings might serve as reservoirs
for bacteria associated with periodontitis, due to the anaer-
obic condition in the piercing channel. The ﬁrst microbio-
logical analysis of piercing bioﬁlms was conducted by
Ziebolz et al. . The microbiological analysis of tongue
piercing sites showed that jewellery can provide a reservoir
for periodontopathogenic bacteria. The incidence and the
could depend on the extent and the bacterial composition of
the bioﬁlm formed on the piercing’s surface. The use of
piercing materials less susceptible to bioﬁlm accumulation
could hence contribute to alleviation or even prevention of
problems. However, at the time being, there is no reference
available regarding the surface plaque retention of different
materials used as oral jewellery.
*Author contributed equally to this work.
Correspondence: Ines Kapferer, Reichenauerstraße 46, 6020 Innsbruck,
Austria. E-mail: firstname.lastname@example.org
J. Stomat. Occ. Med. ©Springer-Verlag Bioﬁlm formation on oral piercings 3/2010 171
Oral piercings are made of different materials, usually
metals such as stainless steel or titanium. Steel as well as
titanium is well known for good mechanical properties, high
corrosion resistance and excellent biocompatibility . To-
day these desirable properties are also achievable by Teﬂon
(PTFE) and polypropylene (Bormed
), which are heat and
radiation sterilizable polymers designed for medical applica-
tions [1, 2]. Materials such as gold and silver –popular in
other body regions –are only of scarce utilization in the oral
cavity, and thus have not been taken into consideration in
It has been demonstrated that initial adhesion of bacte-
ria to a biomaterial surface is inﬂuenced by the surface
physico-chemical properties, such as surface roughness,
surface free energy (SFE) and elemental surface composition
[8, 13, 22, 30], of both the bacterial cell and the substratum.
Rough surfaces promote bioﬁlm formation and maturation,
and high-energy surfaces are known to collect more oral
bioﬁlm and to bind it more strongly . The charge of the
surface inﬂuences electrostatic interactions and bacterial
adhesion, too . In the oral cavity, natural and artiﬁcial
surfaces are immediately covered with a so-called acquired
pellicle consisting mainly of saliva-derived proteins. The
pellicle serves as adhesion site for the pioneering bacteria.
The adsorption of pellicle proteins is a selective process and
is largely affected by the material characteristics. Therefore,
the bacterial adhesion is –via the selectivity of molecule
adsorption –indirectly inﬂuenced by the material
The objectives of the present investigation were (i) to
characterize the surface physico-chemical properties of four
commercially available piercing systems in their sterilized
state and (ii) to quantitatively assess bacterial adhesion on
these piercing surfaces.
Materials and methods
Commercially available lip piercings of four commonly used
materials (titanium, PTFE, Bormed
, and stainless steel)
were selected for the present study, three samples for each
material. The intraoral disc (diameter: 5 mm, gauge: 1.2 mm)
of each piercing was cut off and used for further experiments.
All discs were packaged individually and sterilized (121C,
20 min, steam autoclaving machine) prior to experiments.
Surface roughness, surface chemistry, and surface
For atomic force microscopy (AFM) (Nanoscope IVa Dimen-
sion 3100 AFM, Digital Instruments, Santa Barbara, CA, USA),
three samples of each piercing material were analyzed.
Surface plots were made of each measurement to provide
a three-dimensional perspective of the surface, from which
the mean surface roughness (R
) was computed. R
sents the average distance of the roughness proﬁle to the
center plane of the proﬁle. The surface area ratio expresses
the ratio between the surface area (taking the z height into
account) and the area of the ﬂat x, y plane.
The surface elemental and chemical compositions of
sterilized piercing discs were determined by X-ray photoelec-
tron spectroscopy (XPS) (Thermo MultiLab 2000 spectrome-
ter equipped with a monochromatic Al X-ray source
1486.6 eV). To remove the ﬁrst few atomic layers, the samples
were analyzed as received and after a 5 min Argon sputter
For surface free energy, 1 ml of double distilled water was
dispensed manually on the surfaces of the discs and photo-
graphed immediately using a Moticam 2000 CCD camera
) mounted on a Wild Heerbrugg stereomicroscope.
Contact angles were measured between a tangent to the
water drop and the substratum surface.
Bioﬁlm formation and analysis
Human whole saliva from ﬁve healthy volunteers of both
sexes was collected into ice-chilled beakers after stimula-
tion by chewing parafﬁn. After the saliva was pooled,
phenylmethylsulfonylﬂuoride as a protease inhibitor was
added to a concentration of 1 mM to reduce protein
breakdown. Each piercing disc was individually immersed
in 2 ml of freshly pooled saliva, and bioﬁlms were allowed
to grow on the piercing surfaces at 37C for 1, 4, or 20 h in
an aerobic incubator. Three independent experiments
The piercing discs with adhering bioﬁlms were sonicat-
ed for 15 min in individual Eppendorf tubes containing 1 ml
PBS to disperse adhering bacteria. Aliquots of 50 ml were
spread onto tryptic soy agar plates in duplicate. Plates were
incubated at 37C under aerobic conditions, and CFUs were
counted after 48 h.
For ﬂuorescence microscopy, samples were ﬁxed in 4%
paraformaldehyde (Sigma-Aldrich) for 10 min, brieﬂywashed
with distilled water and stained with DAPI (40,6-Diamidino-2-
phenylindole dihydrochloride, Sigma-Aldrich; concentration
1:50000) for 20 min, followed by washing in distilled water.
Images were taken with an AxioImager M1.2 microscope (Carl
Zeiss Micro-Imaging, Inc.) equipped with a CoolSnapHQ2
CCD camera (Photometrics) and the AxioVision Release4.5
SP1 software (Carl Zeiss Micro-Imaging, Inc.).
Statistical analysis was carried out using one-way analysis
of variance (ANOVA) with Turkey post-test analysis.
Signiﬁcance was set at p< 0.05 (VassarStats
). Linear regres-
sion analysis was performed to identify the surface properties
most predictive for bioﬁlm formation on sterilized piercings.
Probability levels of 0.05 or less were considered to indicate
statistical signiﬁcance (VassarStats
Contact angle measurements were signiﬁcantly different
between all tested materials (p< 0.01). Surface free energy
(wettability) was the lowest for PTFE (88.78±0.40), followed
172 3/2010 Bioﬁlm formation on oral piercings ©Springer-Verlag J. Stomat. Occ. Med.
(81.26±0.16), titanium (78.56±0.15), and
stainless steel (61.90±1.09).
The mean surface roughness was the highest for titani-
um (range 84–148 nm), followed by Bormed
194 nm), PTFE (range 62–448 nm), and stainless steel (range
56–163 nm). The proﬁles obtained from AFM revealed dif-
ferences in surface morphology (Fig. 1).
The results of XPS analysis are displayed in Fig. 2.
Small amounts of surface contaminants, referred to as
‘other’in the image, were detected on all as received
surfaces except on PTFE. All these contaminants vanished
after removing the ﬁrst few atomic layers with argon
After 1h of incubation, Bormed
and titanium piercings had
signiﬁcantly higher numbers of CFU attached to the surface
(5.20 ±0.25 a nd 5 .19 ±0.29, respectively) than steel and PTFE
(4.90 ±0.11 and 4.89 ±0.11, respectively; p¼0.01). After 4 and
20 h of incubation, Bormed
continuously revealed the high-
est numbers of CFU. However, the differences between Bor-
(5.20 ±0.26 and 5.34 ±0.26, respectively), titanium
(5.19 ±0.10 and 4.77 ±0.26, respectively), and steel piercings
(5.11 ±0.31 and 5.11 ±0.20, respectively) were only small. At
both time points, bioﬁlm formation was highly signiﬁcantly
lower on PTFE piercings (4.88 ±0.30 and 4.65 ±0.26; p<0.001).
X (µm) X (µm)
Fig. 1: Atomic force microscopy (AFM), large area scans (50 mm50 mm) of Ti, steel, PTFE, and Bormed
. All materials showed clear surface
roughening and prominent pittings of variable pore sizes. The surfaces of Ti and steel piercings were characterized by clearly visible striations, likely
to be the result of machining. Bormed
and PTFE piercings are produced by a casting process. For Bormed
, this results in a ﬁbrillar surface
Elemental composition (%)
Fig. 2: X-ray photoelectron spectroscopy (XPS). The surface elemental and chemical compositions of samples were analyzed as received and after a
5 min Argon sputter cleaning treatment, sufﬁcient to remove the ﬁrst few atomic layers. The oxygen content was lower for Bormed
samples than for steel and Ti. Small amounts of surface contaminants, referred to as ‘other’in the image, were detected on all untreated surfaces
except on PTFE. All these contaminants were removed after the argon sputter treatment
J. Stomat. Occ. Med. ©Springer-Verlag Bioﬁlm formation on oral piercings 3/2010 173
After 20 h incubation, differences in bioﬁlm formation were
clearly visible in ﬂuorescence microscopy (Fig. 3).
Effects of surface physico-chemical properties
on bioﬁlm formation
Effects of the investigated physico-chemical properties on the
number of CFU were investigated by linear regression ana-
lysis. CFU showed a negative correlation to surface free
energy after exclusion of Bormed
from analyses: the lower
the contact angle, the higher the bacterial adhesion (R
0.999, p¼0.014). CFU correlated to surface roughness
¼0.998, p¼0.020), again after exclusion of Bormed
¼0.8461, p¼0.8). Bormed
, titanium, and steel pier-
cings showed a linear increase in the number of adhering
bacteria with increasing roughness, whereas for PTFE there
was no such trend observed. Surface ﬂuorine content had a
negative correlation with CFU (R
¼0.977, p¼0.011). Sur-
face oxygen content correlated signiﬁcantly with SFE (R
With infections being one of the most frequent complications
, bioﬁlm formation on oral piercing is a fundamental
issue. Additionally, bioﬁlms on oral piercings might serve as a
bacterial reservoir for systemic infections. The piercing pro-
cedure exposes the piercee to a high risk of infection because
the oral cavity harbours a huge amount of bacteria . The
high vascularity of the area is a further aspect to be consid-
ered. The present study was designed to quantify bacterial
colonization and visualize bioﬁlm formation on surface
characterized piercings. Freshly pooled human whole saliva
was used as a source of bacteria for bioﬁlm formation to
closely simulate in vivo conditions.
One pivotal outcome of this study is that all four piercing
materials included showed in general signiﬁcant amounts of
bioﬁlm after 20 h of incubation, conﬁrmed by ﬂuorescence
microscopy (Fig. 3) as well as bacterial cultivation. Bioﬁlm
formation was signiﬁcantly lower on commercially available
PTFE piercings compared to all other materials. These ﬁnd-
ings indicate that piercings might serve as an important
reservoir of potentially pathogenic bacterial species when
placed in the oral cavity. Piercees should be informed about
the microbiological contamination of oral piercings, and
about possible side effects such as recurrent infections or
There is a general agreement that rough surfaces accumulate
considerably more bioﬁlm in relation to the number of
bacteria, the thickness, the area covered, and the maturity
–that is more rods, spirochetes, and motile organisms –as
smooth surfaces [30, 32]. On a rough surface, bacteria are
better protected against shear forces, facilitating the estab-
lishment of an irreversible attachment . Moreover, the
surface area for attachment is increased .
Surface roughness measurements of the investigated
piercings showed no statistically signiﬁcant differences, but
standard deviations were rather high. Roughness differ-
ences between the piercings of one material group and
consequently the high standard deviations are partially
caused by several substantial pits and elevations on the
surface (Fig. 1) and are due to poor machining quality in the
manufacture of the piercings, where optimization of rough-
ness does not seem to be a crucial procedure. With dimen-
sions of 0.5 to 1 mm, streptococci –common oral bacteria –
can easily hide in these pits. Oral piercing is a personal
decision, therefore, if patients cannot be convinced of
removing their piercings, they should at least choose studs
of high quality.
Surface free energy
The surface free energy (wettability) directly inﬂuences pro-
tein adsorption on biomaterial surfaces . The impact of
the substratum surface free energy remains on the whole
after the pellicle formation . Thus, the surface free energy
properties are transferred through the adsorbed protein layer
. Bacteria that have a high surface free energy themselves
selectively adhere better to high-energy (hydrophilic) sur-
faces, whereas bacteria with a low surface free energy adhere
better to low-energy (hydrophobic) surfaces [29, 33]. The
majority of the oral bacteria possess a high surface free
energy (Streptococcus mitis and some Streptococcus milleri
strains for example are an exception with a low surface free
energy) . Hydrophobic materials thus are less prone to
Fig. 3: Fluorescence microscopy after 20h incubation. The piercing
discs with adhering bioﬁlms were stained with DAPI. PTFE showed the
lowest number of adhering bacteria, which was highly signiﬁcant
174 3/2010 Bioﬁlm formation on oral piercings ©Springer-Verlag J. Stomat. Occ. Med.
bioﬁlm accumulation in the oral cavity, which explains the
reduced bacterial growth on the hydrophobic, low-
energy PTFE piercings in our study (88.78±0.40). Steel
(61.90±1.09), titanium (78.56±0.15), and Bormed
(81.28±0.16) in contrast showed smaller contact angles,
that is a higher surface free energy (hydrophilic), and conse-
quently more bioﬁlm formation.
Surface elemental composition
The surface elemental composition of the four materials was
determined by X-ray photoelectron spectroscopy. The high
sensitivity of this technique reveals the elemental composi-
tion of the outermost few atomic layers of the piercings,
which determine the interaction of the material with the
environment. This surface composition can differ from the
material’s bulk. Therefore the samples were analyzed ﬁrstly
as received and secondly after a short argon sputter treat-
ment, sufﬁcient to remove layers eventually belonging to
physisorbed or chemisorbed species. Elements completely
removed after sputter etching can be regarded as surface
contaminants. Several of these were detected on as received
surfaces with the exception of PTFE piercings. The oxygen
content was lower for Bormed
and PTFE samples (13.50%
and 9.98%, respectively) than for steel (23.07%) and titani-
um (28.29%). After sputter cleaning, the oxygen signal
vanishes on Bormed
and PTFE, whereas it remains al-
most unchanged for steel and decreases to 13.34% for
titanium. This elucidates that oxygen extends to several
atomic layers on the metals, indicative of a distinctive oxide
coating, whereas it is limited to the ﬁrst layer on Bormed
and PTFE. There it belongs either to an oxide coating too or
to physisorbed oxygen containing molecular species. In any
case, the presence of oxygen on a surface is expected to
enhance the hydrophilic interactions with the environment
because of the high polarity of heterogeneous oxygen
The lower number of attached oral bacteria with PTFE is
often explained by its low wettability . Additionally, one of
its major components is ﬂuorine (39.63%). The effect of
ﬂuorine on the inhibition of initial bacterial attachment was
recently investigated . The attachment of both hydrophil-
ic and hydrophobic bacterial strains was dramatically re-
duced when introducing ﬂuorine into a polydimethylsiloxane
The outstanding bacterial colonization of Bormed
piercings cannot be explained by surface roughness (the
linear regression analysis for R
and CFU was only signiﬁ-
cant after exclusion of this material) or surface free energy.
The adhesion mechanism appears to lie rather in a chemi-
cal attraction between the microorganisms and the sub-
stratum. The surface of Bormed
in water (or saliva) is
altered through oxidation, resulting in free carbonyl and
portunity to attach . The relative surface carbon content
piercings (82.39%) was higher than of the
other investigated materials, and was signiﬁcantly related to
CFU in our study. Additionally, the surface of Bormed
contains sulfur, which is an essential element for bacterial
metabolism and could thus act as a chemical cue to attract
All piercing materials included showed in general signiﬁcant
amounts of bioﬁlm. However, bioﬁlm formation was signiﬁ-
cantly lowest on commercially available PTFE piercings. The
results of this study afﬁrm the importance of good oral
hygiene to prevent postpiercing complications. Patients need
better information on the microbiological contamination of
oral piercings and potential complications such as recurrent
infections or systemic bacteriaemia. Additionally, they
should attend regular dental checks and receive professional
advice on cleaning their piercing jewellery regularly with a
toothbrush and/or a disinfectant.
Patients should be informed about the microbiological con-
tamination of oral piercings and possible side effects such as
recurrent infections or systemic bacteriaemia. If patients
cannot be convinced of removing their oral piercings, it is
important that they are fully aware of potential complications
and they should at least choose highly polished PTFE studs
instead of cheap custom jewellery.
Conﬂict of interest
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