Comparative SEM analysis of nine F22 aligner cleaning strategies
Lombardo et al. Progress in Orthodontics
Comparative SEM analysis of nine F22 aligner cleaning strategies
Luca Lombardo 0
Marco Martini 0
Francesca Cervinara 2
Giorgio Alfredo Spedicato 1
Teresa Oliverio 0
Giuseppe Siciliani 0
0 Postgraduate School of Orthodontics, University of Ferrara , Via Montebello 31, Ferrara 44100 , Italy
1 School of Economics, Management and Statistics, University of Bologna , Piazza Scaravilli 2, Bologna 40121 , Italy
2 School of Orthodontics, University of Ferrara , Ferrara , Italy
Background: The orthodontics industry has paid great attention to the aesthetics of orthodontic appliances, seeking to make them as invisible as possible. There are several advantages to clear aligner systems, including aesthetics, comfort, chairside time reduction, and the fact that they can be removed for meals and oral hygiene procedures. Methods: Five patients were each given a series of F22 aligners, each to be worn for 14 days and nights, with the exception of meal and brushing times. Patients were instructed to clean each aligner using a prescribed strategy, and sections of the used aligners were observed under SEM. One grey-scale SEM image was saved per aligner in JPEG format with an 8-bit colour depth, and a total of 45 measurements on the grey scale (“Value” variable) were made. This dataset was analysed statistically via repeated measures ANOVA to determine the effect of each of the nine cleaning strategies in each of the five patients. Results: A statistically significant difference in the efficacy of the cleaning strategies was detected. Specifically, rinsing with water alone was significantly less efficacious, and a combination of cationic detergent solution and ultrasonication was significantly more efficacious than the other methods (p < 0.05). Conclusions: Of the nine cleaning strategies examined, only that involving 5 min of ultrasonication at 42 k Hz combined with a 0.3% germicidal cationic detergent was observed to be statistically effective at removing the bacterial biofilm from the surface of F22 aligners.
A recent survey by YouGov estimated that 45% of adults
are unsatisfied with their smile and that 20% would like
to undergo orthodontic treatment to improve their
tooth alignment and appearance (www.bos.org.uk/news/
NOWYouGovSurvey). Hence, the orthodontics industry
has paid great attention to the aesthetics of orthodontic
appliances, seeking to make them as invisible as possible
]. In this context, in 1997, Zia Chishti and Kelsey
Wirth developed a barely noticeable aligner system,
which they called Invisalign [
]. This system involves the
use of a series of customised aligners made of
transparent plastic; if worn for a minimum of 20 h per day and
replaced every 2 weeks, this system can achieve dental
movements of approximately 0.25–0.33 mm per tooth,
or group of teeth, per aligner. Dental movement and
malocclusion correction stages are planned using virtual
planning software 3D (ClinCheck), based on CAD-CAM
technology, which is also used to view the final result before
treatment is begun [
There are several other advantages to clear aligner
systems, including aesthetics, comfort, chairside time
reduction, and the fact that they can be removed for meals
and oral hygiene procedures [
]. However, like any
orthodontic device, aligners do contribute to a
worsening of oral health due to the accumulation of biofilm.
Indeed, biofilm deposition on aligner surfaces has been
clearly documented and its impact on oral health
requires careful investigation [
]. That being said, there
have thus far been very few studies to analyse the impact
of clear orthodontic aligners on the oral ecosystem, and
their consequent influence on caries formation and
decalcification. Nevertheless, it has been shown that adults
in active treatment with clear aligners have better
periodontal health than those undergoing fixed
multibracket appliance treatment, who show worse plaque
index, bleeding on probing and periodontal pocketing .
This is likely due to the fact that clear aligners can be
removed and, therefore, cleaned more effectively to remove
dental plaque—a complex community of microorganisms
existing on the surface of the teeth in a polymeric matrix of
bacterial origin. This biofilm is the cause of many oral
diseases, and it has been estimated that roughly 60% of human
infections can be ascribed to microbial biofilms [
Recent research has shown that a minimal dose of
chlorhexidine (0.06%) has no beneficial effect on the oral
health of aligner wearers [
], who must keep their
appliances in place for 22 h per day in order to ensure good
orthodontic outcomes (though it should be mentioned
that many patients also keep their aligners on while
eating and drinking). While they are being worn, aligners
accumulate plaque and a bacterial biofilm forms around
the teeth [
]. Aligners also limit the buffering,
detergent and remineralising effects of saliva.
The biofilm on the enamel is not disturbed by the
mechanical action of the lips, cheeks and tongue if an
aligner is in place. In fact, one of the main characteristics
of the biofilm is the cellular adhesion that occurs
between microorganisms and non-exfoliative surfaces. The
structure of the biofilm changes according to the
bacterial species it is composed of [
], but the way in
which it is organised protects against the action of
chemical and pharmaceutical agents. Indeed, all
infections closely linked to the development of the biofilm
are highly resistant to non-invasive treatments (i.e.
12, 15, 16
However, it has been demonstrated that oral bacteria
can be destroyed by ultrasonication, via a mechanical
phenomenon known as cavitation (bubble formation)
]. Hence, in a recent study, published by the Journal
of Clinical Orthodontics in 2013, Moshiri et al. [
included this method of cleaning in a list of
recommendations they drew up for orthodontists. They also included
it in the suggested home hygiene protocol to be given to
patients in order to ensure optimal outcomes in aligner
therapy. In particular, they advised that patients should
refrain from eating with their aligners in, remove any
white deposits from their aligners, brush their teeth with
a soft brush for two minutes, use dental floss and rinse
with fluoride mouthwash in the evening, and always put
clean aligners into a clean mouth. For aligner cleaning,
they advised the use of either an ultrasonic bath or the
Invisalign Cleaning System detergent. However, they
cited no specific information regarding the efficacy of
Hence, in order to provide more information on the
topic, we set out to conduct a comparative SEM (scanning
electron microscopy) study of various appliance cleaning
strategies in real-world patients being treated using F22
Five patients were scheduled for orthodontic treatment
via clear aligners. Silicon impressions were taken of their
upper and lower teeth using the dual-impression
method. Class 4 plaster (Ortotypo, Lascod®) was used to
make the casts, which were then scanned in the lab
using an extraoral scanner (SMART, Open
Technologies), to obtain STL files and 3D renderings of the
patients’ dentition. These were then converted into resin
models using a laser printer (EnvisionTEC ULTRA 3SP),
which were in turn used as moulds for a series of nine
F22 aligners [
], formed out of thermoplastic
polyurethane (TPU), after isolating them with Isofolan
Each aligner in the series was worn by the patient for
14 days, being removed only at meals and during oral
hygiene procedures. Patients were instructed to clean the
nine aligners using the following strategies, respectively:
1. Rinsing with water
2. Immersion in water in a sonic bath
3. Immersion in water in an ultrasonic bath
4. Immersion in water and anionic detergent
5. Immersion in water and anionic detergent in a
6. Immersion in water and anionic detergent in an
7. Immersion in water and cationic detergent
8. Immersion in water and cationic detergent in a
9. Immersion in water and cationic detergent in an
The sonic device used (TCS Fresh®) for aligners 2, 5
and 8 in the series features a bath in which to immerse
the aligner and vibrates at a frequency 5800 Hz, while
the ultrasonic device (iSonic F3900®) used for aligners 3,
6 and 9, though very similar in structure, vibrates at a
frequency of 42,000 Hz. According to the manufacturer’s
instructions, the anionic detergent used (TCS Fresh®) for
aligners 4, 5 and 6 should not be used directly in the
mouth or for more than 7 days. It is sold in powder
form in 1.75-g packets, each to be dissolved in 100 ml of
water. Similarly, direct contact with the cationic
detergent (benzalkonium chloride, Caelo®) used with aligners
7, 8 and 9, sold in a 0.3% aqueous solution, should also
be avoided, as it is toxic to the skin and upon ingestion,
and can irritate the skin and eyes. Before replacing in
the mouth, therefore, patients were instructed to
thoroughly rinse their aligners under running water to
remove any trace of detergent. Aligner cleaning times for
each strategy were standardised at 5 min, and each
disinfection procedure was performed by the patient at home
twice a day.
After being worn for 14 days, the aligners were returned
to the orthodontist for analysis. Specifically, a 6 × 6-mm
section of each aligner was cut from the vestibular surface
of the upper right first premolar area (the transition zone
between anterior and posterior sectors). Each section was
immediately conserved in glutaraldehyde, and the aligner
sealed in a plastic bag; both the container of
glutaraldehyde were labelled with the same ID (Fig. 1). The
preserved aligner sections were dried and gilded at the
University of Ferrara Microscopy Lab and then observed
under SEM. The scanning electron microscope was
focused on the centre of each sample, which was enlarged
10 thousand times. The resulting SEM images were saved
in JPEG format on an 8-bit colour-depth grey scale. A
histogram of the grey-scale attributes was created for each
SEM image to provide a graphical representation of the
number of pixels in the image for each grey on the scale.
The dataset composed of the 45 observations,
representing the measures on the grey scale (Value variable)
of the nine different cleaning methods (one per aligner)
used by the five patients, was then subjected to statistical
analysis. Repeated-measures ANOVA was used to
determine whether the Value variable was influenced by the
different aligner/cleaning strategy combinations. The
software R nlme [
] was used for ANOVA modelling,
specifying “cleaning strategy” as a “within subject”
variable, and the patient as a random factor. In order to
identify statistically significant differences between aligner and
cleaning strategy combinations, a post hoc analysis was
performed using Multcomp software [
]. This analysis
showed that the differences between the effects of distinct
cleaning strategy pairs were statistically equal to zero.
The SEM image analysis (Table 1) and statistical analysis
summarising the Value measurements per
aligner/cleaning strategy (Table 2) show that the cleaning strategy
variable does have a statistically significant effect on the
Value (p value zero), i.e. the “cleanliness” of the aligner.
However, as shown in Table 3 and Fig. 2, only the first
and ninth cleaning strategies (rinsing with water and
immersion in water and cationic detergent in an
ultrasonic bath, respectively) were significantly different from
the others, the former being significantly less efficacious,
and the latter being significantly more efficacious.
All aligner cleaning strategies did reduce the bacterial
biofilm on the surface of our aligner samples, with the
exception of rinsing under tap-water alone. However,
our data also show that the most efficacious strategy
tested was cationic detergent combined with
ultrasonication. Our comparative results also appear to confirm the
literature reports that the only way to kill bacteria via
low-intensity ultrasound is by combining it with a
bactericidal agent [
]. Indeed, we clearly show that a
cationic detergent alone is not sufficient to eliminate the
bacterial biofilm from the surface of aligners, despite its
bactericidal properties, which have been demonstrated
in other studies [
]. Similarly, 5 min of 42,000 Hz
ultrasound alone was not sufficient to remove the
], and only a combination of the two methods
proved useful in this regard according to our statistical
Indeed, even a brief glance at the SEM images for each
of the aligners reveals evident bacterial colonisation of
each, with the exception of those cleaned by the ninth
strategy, which combined an ultrasonic bath with a
cationic bactericide. These compare very favourably not
only with those rinsed with water alone, which show
visible layers of abundant bacteria organised into a biofilm,
but also those cleaned using the other strategies (2–8),
which show variable levels of bacterial proliferation.
As the SEM image of the unused aligner (Fig. 3) is
much darker than that cleaned by the first method
(Fig. 4)—rinsing with water alone—we decided to
numerically quantify the visible biofilm on the aligner
sections using grey-scale analysis, taking a mean of the
levels of grey in the images of the aligners cleaned by
each method. The cleaner aligners are darker in colour
and show grey-scale levels closer to that of the unused
aligner, i.e. 61.924, which appears totally dark. Bacterial
proliferation on the surface of the aligner, on the other
hand, makes them appear lighter in colour, and the
multi-layer biofilm causes the mean level of grey to
differ considerably from that of the clean TPU of the
unused aligner. By statistically analysing this data, we were
able to confirm that there was indeed a statistically
significant relationship between the SEM grey-scale levels
and the cleanliness of the aligner sections.
Careful SEM analysis of the aligner sections cleaned
via the ninth method (Fig. 5), i.e. ultrasonic bath and
cationic detergent, reveal interesting points regarding
their surface characteristics. The first is that the TPU
material itself bears clear striations, an indication of its
hydrophilic status [
], and its absorption of water.
The second visible feature is related to the method of
cleaning used; the surface of the aligner bears signs of
pitting. This is likely an effect of ultrasonic cavitation
and is visible thanks to the lack of bacterial proliferation.
It demonstrates that the mechanical turbulence
generated by the acoustic waves, the continual formation and
rupture of tiny bubbles, releases sufficient energy to
damage not only bacterial cell walls  but also the
surface of thermoplastic polyurethane.
In order to directly compare these findings with those
in the literature in any meaningful way, we would need
to rely on studies conducted using the same methods for
analysing the aligner surfaces and counting the bacterial
colonies. However, there is great variability in the
literature in terms of these factors, and in any case, our
design prevented us from providing precise bacterial
counts for comparison. Nevertheless, our findings generally
confirm those of Torlak and Sert [
MermillodBlondin et al. [
] that the association between a germicidal
detergent and ultrasonication is able to reduce the bacterial
load on the surfaces analysed.
Of the nine cleaning strategies examined, only that
involving 5 min of ultrasound treatment at 42 kHz combined
with a 0.3% solution of the germicidal cationic detergent
benzalkonium chloride was statistically observed to be
effective at removing the bacterial biofilm from the surface
of used F22 TPU aligners [
] (p < 0.05).
There are no sponsors for this study. The research is funded by its own
means of the researchers.
Conception and design of study: SG; LL. Acquisition of data: MM, CF. Analysis
and/or interpretation of data: FC, SGA, GS. Drafting the manuscript: CF, MM, LL.
Revising the manuscript critically for important intellectual content: SG. Approval
of the version of the manuscript to be published: GS, LL, MM, FC, SGA.
Conception and design of study and acquisition of data: TO. All authors read and
approved the final manuscript.
Ethics aproval and consent to participate
The protocol of the study was approved by the Director and Chairman of
School of Orthodontics, University of Ferrara Prof., Giuseppe Siciliani.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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