R. OVSENIK et al.: EVALUATION OF MICROBIAL FLORA IN PATIENTS WITH GINGIVAL ENLARGEMENT ...
51–57
EVALUATION OF MICROBIAL FLORA IN PATIENTS WITH
GINGIVAL ENLARGEMENT DURING THE TREATMENT AND
SURFACE CHEMISTRY OF FIXED ORTHODONTIC APPLIANCE
ARCHWIRE
OVREDNOTENJE MIKROBNE FLORE PRI PACIENTIH S
POVE^ANJEM DLESNI MED OBRAVNAVO IN ANALIZA
POVR[INE @I^NEGA LOKA NESNEMNEGA ORTODONTSKEGA
APARATA
Rok Ovsenik
1*,
Miha Pirc
2*
, Jasmina Primo`i~
1
, Rok Schara
2
, Janez Kova~
3
,
Monika Jenko
4,5
, Boris Ga{pirc
2
1
Department of Orthodontics and Dentofacial Orthopaedics, Faculty of Medicine, University of Ljubljana,
Hrvatski trg 6, 1000 Ljubljana, Slovenia
2
Department of Oral Medicine and Periodontology, Faculty of Medicine, University of Ljubljana, Hrvatski trg 6, 1000 Ljubljana, Slovenia
3
Jo`ef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia
4
Institute of Metals and Technology, Lepi pot 11, 1000 Ljubljana Slovenia
5
MD-RI Institute for Materials Research in Medicine, Bohori~eva 5, 1000 Ljubljana Slovenia
Prejem rokopisa – received: 2020-07-15; sprejem za objavo – accepted for publication: 2020-08-13
doi:10.17222/mit.2020.137
Gingival enlargement is a common complication of treatment with a fixed orthodontic appliance, biocompatible stainless-steel
archwires AISI 304L (SS). Due to the retention areas, dental plaque accumulation is increased, which results in anaerobic condi-
tions, favourable for periodontopathogenic bacteria. Therefore, it is reasonable to assume that periodontopathogenic bacteria can
be found in patients with fixed orthodontic appliances. Organic deposits on the surface of archwires were analyzed. Twenty-one
patients with fixed orthodontic appliances and gingival enlargement in the upper dental arch were included in the study. For a
determination of the periodontopathogenic bacteria Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis,
Prevotella intermedia, Tannerella forsythia and Treponema denticola the molecular microbiological method GenoType Test
System was used. For the surface analysis of new and in-vivo exposed SS archwires X-ray Photoelectron Spectroscopy was
used. The average probing depth at the baseline was 3.76±0.85 mm. Three types of periodontopathogenic bacteria, namely A.
actinomycetemcomitans, T. forsythia and T. denticola, were found to be present. The thickeness of the thin oxide film on new
archwires was 8 nm, while organic deposits on the in-vivo exposed archwires was estimated to 60–80 nm. Anaerobic
periodontopathogenic bacteria can be found in patients with fixed orthodontic appliances. Therefore, special care is recom-
mended during this kind of treatment.
Keywords: biocompatible stainless steel, AISI 304L, archwires, periodontopathogenic bacteria, gingival enlargement, fixed
orthodontic appliance, X-ray photoelectron spectroscopy (XPS)
Pove~anje dlesni je pogost zaplet zdravljenja z nesnemnim ortodontskim aparatom. Pove~anje je posledica velikega {tevila
zastojnih mest in posledi~no kopi~enja zobnih oblog, kar privede do vzpostavitve anaerobnih pogojev, primernih za naselitev
parodontopatogenih bakterij. Analizirali smo organske obloge na kovinskih lokih iz nerjavnega jekla (SS) AISI 3014L. V
preiskavo smo vklju~ili 21 pacientov s pove~anjem dlesni v zgornjem zobnem loku, ki so imeli name{~en nesnemni ortodontski
aparat; kovinski loki, ki so bili vstavljeni, pa so bili iz biokompatibilnega nerjavnega jekla, AISI 304 L (SS). Pri vseh smo
preverjali prisotnost parodontopatogenih bakterij Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis,
Prevotella intermedia, Tannerella forsythia in Treponema denticola z molekularno mikrobiolo{ko metodo GenoType Test Sys-
tem. Z rentgensko fotoelektronsko spektroskopijo (XPS) smo dolo~ili oksidno plast na novih in organske obloge na in-vivo
izpostavljenih `i~nih lokov. V preiskavi smo ugotovili prisotnost treh (3) vrst parodontopatogenih bakterij, in sicer: A.
actinomycetemcomitans, T. forsythia in T. denticola. Debelina oksidne plasti na novih lokih je bila okrog 8 nm, debelina
organskih oblog na povr{ini in vivo izpostavljenih lokov je bila ocenjena na 60–80 nm. Pri pacientih z nesnemnim ortodontskim
aparatom je potrebna posebna pozornost, saj tak{no okolje spodbuja rast parodontopatogenih bakterij.
Klju~ne besede: parodontopatogene bakterije, pove~anje dlesni, nesnemni ortodontski aparat, biokompatibilni SS-loki, XPS
1 INTRODUCTION
Gingival enlargement is one of the possible compli-
cations during orthodontic treatment with fixed orth-
odontic appliances, with biocompatible archwires of
stainless steal, AISI 304L (SS). It usually occurs 1–2
months after the insertion of the appliance and is present
in 10 % of patients.
1
Gingival enlargement forms due to
inflammatory factors, pharmacological substances or in
connection with neoplastic formations. However, the
most common cause for gingival enlargement is chronic
inflammation, which is caused by the deposition of den-
tal plaque.
2–4
Materiali in tehnologije / Materials and technology 55 (2021) 1, 51–57 51
UDK 579.61:616-052:616.31:620.1:617.3 ISSN 1580-2949
Original scientific article/Izvirni znanstveni ~lanek MTAEC9, 55(1)51(2021)
*Corresponding author's e-mail:
rok.ovsenik@mf.uni-lj.si (Rok Ovsenik)

Different mechanical factors effect friction forces
during orthodontic treatment and tooth movement, in
particular the brackets, SS (stainless steel) and NiTi
(nickel-titanium) archwires.
5
Fixed orthodontic appli-
ances allow the colonization of important periodonto-
pathogenic bacteria.
6,7
Etiological factors for the occur-
rence of gingival enlargement in patients with fixed
orthodontic appliances are: i) mechanical irritation of the
appliance, ii) chemical irritation of substances with
which brackets are attached to the teeth and iii) food
jamming as perfect oral hygiene is much harder to main-
tain.
8
The composition of oral flora depends on different
factors, but mostly on eating habits and oral hygiene. Ac-
cording to the literature, there are more than 80 % of fac-
ultative anaerobic microbes, which can grow either in the
presence or absence of oxygen, streptococci and bacilli
(such as Actinomyces) and 15 % of anaerobic bacilli
(such as Fusobacterium and Bacteroides), in a healthy
gingiva with periodontal pockets up to 2 mm. With the
presence of inflammation, the share of aerobic bacteria
decreases, while the share of anaerobic bacteria in-
creases.
9,10
The colonization of bacteria on hard surfaces of the
teeth and their metabolism is the most common cause for
caries, gingival inflammation, periodontal disease,
periimplantitis, stomatitis and candidiasis.
11–13
In order
for gingival inflammation to occur, three criteria must be
fulfilled:
1) the presence of a significant number and species of
bacteria in the oral flora,
2) the presence of retention places, which enable growth
of anaerobic bacteria and
3) a weakened immune system of the host.
9
The number
of retention places greatly increases in patients with
fixed orthodontic appliances and therefore the in-
flammatory response of the organism is more fre-
quent.
7,14,15
Most bacteria have the ability to attach themselves on
hard surfaces of the teeth, which enables the accumula-
tion of dental plaque. With the growing accumulation of
dental plaque the need for nutrients traveling by diffu-
sion increases. The inner structure of the dental plaque
becomes a perfect environment for the colonization of
anaerobic bacteria, while most nutrients for bacteria in
plaque come from saliva and ingested food. However,
this is not the case when it comes to bacteria forming
subgingival plaque. Since access for nutrients coming
from saliva and ingested food is limited, they get nutri-
ents from deeper periodontal pockets, mostly from blood
and periodontal tissue.
9
There are 10
8
bacteria per mm
3
of dental plaque, producing acids, endotoxins and anti-
gens, which results in irritation of the periodontal tissue,
its inflammation and with prolonged impact even in
periodontal tissue damage.
10
Therefore, the aim of this study was to determine the
change in probing depth and the presence of period-
ontopathogenic bacteria in patients with gingival
enlargement during orthodontic treatment with fixed
orthodontic appliances and surface chemistry of new and
in-vivo-exposed biocompatible SS archwires.
2 MATERIALS AND METHODS
This prospective study was performed at the Depart-
ment of Orthodontics and Dentofacial Orthopaedics at
the University Medical Centre Ljubljana and Orthos over
a period of 3 months. A signed, written consent was ac-
quired before a patient was included to the study. This
study was approved by the local ethics committee (KME
79/08/13).
Patients treated at the Department of Orthodontics
and Dentofacial Orthopaedics, University Medical Cen-
tre Ljubljana and Orthos, were included in the study. All
the patients were wearing fixed orthodontic appliances
for at least 2 years and were therefore on rectangular
biocompatible stainless steel AISI 304L (SS) archwires.
They had gingival enlargement in the upper dental arch
bilaterally (Figure 1). Patients with allergies, cranio-
facial syndromes and systemically unhealthy patients
were excluded from the study.
Medical history and clinical examination were car-
ried out at the beginning of the study. Periodontal pocket
depth was measured at 6 points and two microbiological
R. OVSENIK et al.: EVALUATION OF MICROBIAL FLORA IN PATIENTS WITH GINGIVAL ENLARGEMENT ...
52 Materiali in tehnologije / Materials and technology 55 (2021) 1, 51–57
Figure 1: Intraoral photograph of a patient on SS archwires and
gingival enlargement in the upper dental arch bilaterally

samples from each patient were obtained. The microbio-
logical sample was taken from the deepest periodontal
pocket on the left and right sides of the upper dental
arch.
Before microbiological samples were taken, supra-
gingival plaque was removed with ultrasound. A sterile
paper point was inserted into the periodontal pocket for
10 s, using sterile tweezers without the interference of
saliva, in order to guarantee only subgingival bacteria in
the sample (Figures 2a and 2b). The paper point was re-
moved after 10 seconds and was then inserted into the
appropriate sterile test tube. The samples were analysed
by the molecular microbiological method GenoType Test
System usingmicro-IDent
®
(Hain Lifescience GmbH,
Nehren, Germany).
For the evaluation of microbial flora in the
periodontal pocket the micro-IDent
®
(Hain Lifescience
GmbH, Nehren, Germany) was used. It makes it possible
to determine the presence and concentration of the five
most common and most aggressive species of perio-
dontopathogenic bacteria, namely: Aggregatibacter-
actinomycetemcomitans, Porphyromonasgingivalis,
Prevotella intermedia, Tannerella forsythia and Trepo-
nemadenticola, which are all Gram-negative bacteria.
The samples were analysed by the molecular micro-
biological method GenoType Test System (Figures 3a
and 3b), where bacterial DNA was isolated using
QiaAmp DNA Mini Kit (QIAGEN, Hilden, Germany)
and the addition of a buffer. The isolated DNA was kept
at -20 °C until it was mixed together with two pre-pre-
pared reagents and multiplied by the PCR method. PCR
(polymerase chain reaction) consists of three phases. In
phase I the double-screw molecular sample of DNA is
separated into two single-screw DNA molecules at
90–96 °C. Phase II is where nucleotides start to bond to
the single-screw DNA molecule at 40–75 °C, which we
call hybridization. Phase III is where, at 65––75 °C and
by the help of polymerase, the synthesis of DNA is fin-
ished. Each temperature cycle that follows doubles the
amount of the target DNA part. PCR usually consists of
25–40 consecutive repetitions of the temperature cycle
resulting in exponential accumulation of typical target
parts of the DNA.
2.1 Statistical analysis
The SPSS program (The Statistical Package for So-
cial Science SPSS Inc., Chicago, Illinois, USA) was used
for statistical data analysis. In case of a normal distribu-
tion, parametric statistical tests were used, otherwise we
used nonparametric statistical tests. The McNemar test
was used to determine differences in the proportion of
periodontopathogenic bacteria. Five patients were ran-
domly selected to determine the inter- and intra-exam-
iner variability, which were then assessed using ICC (In-
terclass Correlation Coefficient) and interpreted after
Landis and Koch (1977).
16
To determine the statistical
significance, the standard value for the confidence inter-
val of at least 95 % (p < 0.05) was used.
2.2 XPS surface analysis
The XPS measurements of the thin oxide film on new
and organic deposit – dental plaque on in-vivo exposed
(SS) archwires were performed using non-monochro-
matic Al–K
 radiation (1486.6 eV) with an anode voltage
of 12.5 kV and an emission current of 16 mA (200 W).
For the XPS depth profiling, a 3-keV Ar-ion beam
scanned overa4mm×5mmarea was used, and this
corresponded to a 0.1 nm/min sputtering rate.
XPS measurements were made at an emission angle
of 0°. The analysed area is about 1 mm × 2 mm, so the
results represent a laterally averaged chemical composi-
tion over this area. Survey scans covering the bind-
ing-energy range 0–1200 eV were taken for each sample
R. OVSENIK et al.: EVALUATION OF MICROBIAL FLORA IN PATIENTS WITH GINGIVAL ENLARGEMENT ...
Materiali in tehnologije / Materials and technology 55 (2021) 1, 51–57 53
Figure 3: a) The result of bacterial proof using molecular microbio-
logical method and b) schematic view of the bacterial proof result
Figure 2: a) Procedure for obtaining microbiological sample and b)
schematic view of inserting paper points into periodontal pockets

with a constant analyser pass energy of 50 eV. The
high-resolution XPS measurements were taken at a pass
energy of 25 eV to allow for a determination of the oxi-
dation state of the elements.
3 RESULTS
3.1 Evaluation of microbial flora in patients with
gingival enlargement and statistical analysis
The average probing depth among all subjects in the
study was 3.76±0.85mm.
The proportion of individual bacteria in the subjects
is presented in Table 2. Three species of periodonto-
pathologic bacteria were detected, namely A. actinomy-
cetemcomitans, T. forsythia and T. denticola (Table 1).
Table 1: Number of subjects with the presence of individual bacteria
(%); Aggregatibacteractinomycetemcomitans (A.a.), Porphyromonas
gingivalis (P.g.), Prevotella intermedia (P.i.), Tannerella forsythia (T.f.)
and Treponema denticola (T.d.) in periodontal pockets on both sides of
the arch
3.2 XPS analysis results of new and in-vivo exposed SS
archwires
Figure 4 shows an XPS survey spectrum from the
surface of a new SS sample. The elements C, O, Fe and
Si are present on the surface. The Fe 2p
3/2
spectrum has a
maximum at 711 eV, which means that Fe is present as
Fe-oxide on the surface. Figure 5 shows an XPS depth
profile of a new SS sample. We decomposed the Fe 2p
and Cr 2p spectra into metallic peaks (Fe(0) at 707.0 eV
and Cr(0) at 574.4 eV) and oxide peaks (Fe-oxide at
711 eV and Cr-oxide at 576.8 eV). We found that the
surface is covered with double oxide layer. The top-most
layer consists mainly of Fe-oxide and it is about 4 nm
thick. Beneath this layer is a Cr-oxide/Fe-oxide mixed
layer of thickness about 8 nm. This mixed Fe-Cr-oxide
layer was grown on the SS matrix consisting of
R. OVSENIK et al.: EVALUATION OF MICROBIAL FLORA IN PATIENTS WITH GINGIVAL ENLARGEMENT ...
54 Materiali in tehnologije / Materials and technology 55 (2021) 1, 51–57
Figure 6: XPS spectrum from the surface of in-vivo exposed SS sam-
ple. Elements C, O, Fe, Si, Na, Cl, Ca are present on the surface
Figure 4: XPS spectrum from the surface of a new SS sample. Ele-
ments C, O, Fe and Si are present on the surface
Figure 5: XPS depth profile of a new SS sample
Figure 7: XPS depth profile of an exposed SS sample

Fe-Cr-Ni. The surface of the exposed sample is covered
with a very thin C-rich layer (1–2 nm) of contamination,
where Si is also detected. Figure 6 shows an XPS spec-
trum from the surface of exposed SS sample. Elements
C, O, Fe, Si, Na, Cl and Ca are present on the surface.
Figure 7 shows an XPS depth profile of elements of the
subsurface region on the exposed SS sample. The SS
sample is covered with 60–80-nm-thick layer rich in car-
bon, wherein also O, N, Ca, K, Na and Si are present.
This layer was formed during exposure of the SS sample.
4 DISCUSSION
The study shows that in a certain percentage of sub-
jects during treatment with fixed orthodontic appliances,
periodontopathogenic bacteria can be found. The highest
percentage had T. forsythia (9.52 %), following with
A. actinomycetemcomitansin 7.14 % of subjects and
T. denticolain 4.76 % of subjects.
A. actinomycetemcomitans, a facultative anaerobic
Gram-negative bacteria, is one of the first organisms
connected with the early stage of the periodontal disease.
17,18
It was found that there is a connection between an
increased pocket depth and quantity of A. actino-
mycetemcomitans subgingivally.
19
The larger the quantity
of A. actinomycetemcomitans subgingivally, the lower
the success of a classic periodontal treatment and the
lesser pocket-depth reduction. A. actinomycetem-
comitans is the only anaerobic species, which is present
already at an early age, between 5–7 years, and persists
in the oral flora even later.
20,21
A. actinomycetemcomitans
was present in 7.14 % of the subjects. Paolantonio et. al
22
reported that the increase in quantity of A. actino-
mycetemcomitans occurs after the bonding of fixed orth-
odontic appliances, since the increased plaque accumula-
tion positively induces the colonization of A. actino-
mycetemcomitans.
22
Changes in the oral flora of
subgingival plaque in general after the bonding of fixed
orthodontic appliances is well reported in the litera-
ture.
23–25
However, the change in microbial flora soon af-
ter the bonding of fixed orthodontic appliances only per-
sists for a short period of time, since the immune system
recovers again after three months and a balanced state is
established.
1
Orthodontic treatment with fixed orthodontic appli-
ances causes increased plaque accumulation and de-
creases the ability for a mechanical removal of plaque,
since the number of retention surfaces increases se-
verely.
26
All non-linear parts of the appliance greatly de-
crease the ability of mastication and salivary flow to re-
move bacteria from these surfaces, this is why a larger
accumulation of plaque on the surfaces is more probable,
which can lead to a severe periodontal tissue inflamma-
tion.
27
The presence of periodontal tissue inflammation
additionally increases plaque accumulation.
14,28,29
Stain-
less steel rectangular archwires, when exposed to the
intraoral environment, showed a significant increase in
the amount of debris accumulation,
30
while a significant
correlation between the amount of debris and friction
was also observed.
31
On the other hand, some investiga-
tors have concluded that in order to lower the presence of
bacteria it is beneficial to carefully clean brackets and
archwires at each visit as the amount of debris accumula-
tion increases significantly with exposure.
5
In the present study P. gingivalis and P. intermedia
are not the present microbial flora of subgingival plaque
in patients with gingival enlargement during treatment
with fixed orthodontic appliances. The results coincide
with the study of W. E. Moore et al.,
32
who found high
concentrations of P. gingivalis in the supragingival, but
not in the subgingival plaque. The presence of P.
gingivalis is usually associated with the occurrence of
advanced periodontitis in adult patients and with early
stages of a localised periodontal disease, although its
presence is not as pathogenic as that of A. actino-
mycetemcomitans.
21
Assuming that all of our subjects
have taken good care of their oral hygiene and that be-
fore every microbiological sample was taken and the
supragingival plaque removed, the absence of
P.gingivalisis was expected. According to the literature
A. actinomycetemcomitans and P. gingivalis are only
rarely present at the same time.
32–34
P. gingivalis was not present in patients with gingival
enlargement, while its share in the Slovenian patients
with chronic periodontitis was equally high (93.3 %).
35
The quantitative alteration of the oral microbiota is
related to an increase in clinical parameters, plaque index
and bleeding on probing, which are risk indicators for
periodontal pathologies.
36
Together with the quantitative
change, there is also a qualitative variation; indeed, there
is an increase in more aggressive gram-positive and
gram-negative bacteria, such as: S. mutans and
Lactobacillus spp. (gram-positive) and P. gingivalis,
T. forsythia, and T. denticola (gram-negative). These bac-
teria are closely associated with, respectively, enamel
and dentin pathologies and with periodontal disease.
37
Oral microbiota alterations detected in orthodontic pa-
tients appear to be consistent with the alterations occur-
ring in patients with poor oral hygiene presenting gingi-
vitis and/or periodontal diseases.
23,38,39
However, the
susceptibility of each subject as well as other factors that
may alter the biofilm balance, can play a key role in de-
termining the entity of periodontal consequences.
The results of in-vivo exposed SS archwire XPS sur-
face analysis confirmed elements C, O, Fe, Si, Na, Cl,
Ca on the surface. An XPS depth profile of the elements
of the subsurface region on the exposed SS sample
showed that the SS archwire sample is covered with a
60–80 nm thick layer rich in carbon, wherein also O, N,
Ca, K, Na and Si are present.
41
This layer was formed
during in-vivo exposure of the SS sample.
The most important result is the determination of the
deposit thickness of an in-vivo exposed SS archwire,
where a 60–80-nm-thick organic film is formed contain-
R. OVSENIK et al.: EVALUATION OF MICROBIAL FLORA IN PATIENTS WITH GINGIVAL ENLARGEMENT ...
Materiali in tehnologije / Materials and technology 55 (2021) 1, 51–57 55

ing O, N, Fe Si, Na, Cl and Ca. This layer growth during
in-vivo exposure is dental plaque, since it is of organic
origin. FTIR results of our previous studies showed that
the presence of the minerals apatite Ca
5
(PO
4
)
3
(F,Cl,OH),
calcite (CaCO
3
), halite (NaCl) and sylvite (KCl)
42
5 CONCLUSIONS
This study proved the presence of periodontopatho-
genic bacteria in the periodontal pockets of subjects with
gingival enlargement during a treatment with fixed orth-
odontic appliances. However, the proportion was low in
comparison to the patients with chronic periodontitis.
Considering the results, it is reasonable to conclude that
even though the share of periodontopathogenic bacteria
is low in patients with gingival enlargement during treat-
ment with fixed orthodontic appliances, additional hy-
giene measures are mandatory. Special precautions have
to be made in order to prevent a greater increase in the
quantity of periodontopathogenic bacteria, which can
lead to a severe periodontal tissue inflammation and con-
sequently periodontal attachment loss. A passive oxide
film of 6–8 nm in thickness and a 60–80-nm-thick or-
ganic film containing O, N, Fe, Si, Na, Cl and Ca, where
confirmed on in-vivo exposed SS archwires. This layer
growth during in-vivo exposure is dental plaque, since it
is of organic origin. Therefore, it would be appropriate
for patients undergoing dedicated hygiene protocols to
keep the oral bacterial charge under control and then to
reduce the risk of the carious process and periodontal
disease.
Acknowledgment
We would like to thank dr Bo{tjan Lavri{a, Nina
@agar, Petra Vidmar Nikoli~ and Jura [tok for their kind
collaboration during the research performed at the De-
partment of Orthodontics and Dentofacial Orthopaedics,
Faculty of Medicine, Univeristy of Ljubljana, and to
Orthos specialists dr. Toma` Ko{orok, dr. Ma{a Farka{
and dr. Martina Zore Albreht for their kind collaboration.
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