Antibacterial and antibiofilm efficacy of nanoparticle-enhanced clear aligners against Streptococcus mutans – a systematic review of in-vitro studies

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https://doi.org/10.53453/ms.2026.6.7

Antibacterial and antibiofilm efficacy of nanoparticle-enhanced
clear aligners against Streptococcus mutans: a systematic review of
in-vitro studies
Aušrinė Kuzmauskaitė
1
, Alvyda Žarovienė
2
1
Independent researcher
2
Department of Orthodontics, Faculty of Dentistry, Lithuanian University of Health Sciences, Kaunas, Lithuania.
Abstract
Introduction. Clear aligner therapy is increasingly used in orthodontics due to its aesthetic and removable nature;
however, prolonged intraoral use may promote microbial accumulation and biofilm formation, increasing the risk
of enamel demineralization and other oral health complications. Nanotechnology-based modifications of aligner
materials may enhance their antibacterial and antibiofilm properties.
Objective: to synthesize the available evidence on the antibacterial and antibiofilm efficacy of nanoparticle-
modified clear aligners.
Materials and methods. This systematic review was conducted following the Preferred Reporting Items for
Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A comprehensive search was performed in
PubMed, ScienceDirect, SpringerLink, and Web of Science databases from November 1, 2025, to January 31,
2026. Based on predefined inclusion and exclusion criteria, studies evaluating the antibacterial and antibiofilm
effects of nanoparticle-modified clear aligners against Streptococcus mutans were included.
Results. A total of 468 records were identified, and after duplicate removal and screening, 10 studies were
included. All investigated nanoparticles, including zinc oxide (ZnO), magnesium oxide (MgO), titanium dioxide
(TiO₂), silver nanoparticles (AgNPs), barium titanate (BaTiO₃), and chitosan-based nanoparticles, demonstrated
antibacterial and antibiofilm activity against Streptococcus mutans. However, these effects varied depending on
nanoparticle concentration, composition and experimental conditions.
Conclusions. Nanoparticle-modified clear aligners show promising antibacterial and antibiofilm potential.
Nevertheless, the evidence is limited by heterogeneity and the predominance of in vitro studies. Further well-
designed in vivo and clinical studies are needed to confirm their long-term safety and effectiveness and to support
clinical application.
Keywords: clear aligners, nanoparticles, nanocoating, biofilm, antibacterial.
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Medical Sciences 2026 Vol. 14 (4), p. 64-78, https://doi.org/10.53453/ms.2026.6.7
64
1. Introduction
Clear aligners are individually fabricated, transparent,
removable orthodontic appliances typically produced
from thermoplastic materials such as polyethylene
terephthalate glycol (PETG). They are designed to
achieve gradual tooth movement and have become an
increasingly preferred alternative to conventional fixed
orthodontic appliances due to their aesthetic appeal
and improved patient comfort [1]. Despite these
advantages, clear aligners are typically worn for
prolonged periods, often up to 20–22 hours per day,
and their design enables close and continuous
adaptation to the tooth surfaces, thereby promoting
bacterial adhesion and biofilm accumulation on the
enamel surface [2,3]. In addition, prolonged tray wear
and the presence of composite attachments may
modify the intraoral environment by affecting salivary
flow, buffering capacity, and overall microbial
composition [4]. Reduced salivary clearance beneath
the trays may favor localized acidogenic biofilm
formation, increasing the risk of enamel
demineralization [5]. From a clinical perspective, these
alterations are often observed as white spot lesions
(WSLs), which represent the earliest visible stage of
enamel carious lesions [6].
Streptococcus mutans is the primary microorganism
associated with the development of white spot lesions
(WSLs) and dental caries. The development of dental
caries begins with the adhesion of S. mutans to the
tooth surface. This process is facilitated by
glucosyltransferase enzymes (e.g., glucosyltransferase
B (GtfB)), which synthesize extracellular glucans from
sucrose and promote stable bacterial attachment to
enamel [7]. The risk of tooth decay increases as the
proportion of S. mutans bacteria in the oral microbiota
rises, particularly when it reaches around 50% of the
total bacterial population [8]. Furthermore, S. mutans
forms an insoluble extracellular polysaccharide (EPS)
matrix, which enhances bacterial adhesion to enamel
and promotes biofilm development [9]. The
established biofilm exhibits pronounced acidogenic
activity, as S. mutans produces short-chain organic
acids that lead to a localized decrease in pH, initiating
the demineralization of enamel hydroxyapatite
crystals—an early indicator of white spot lesion
(WSL) development [10]. Orthodontic appliances,
including clear aligners, can serve as additional
substrates for biofilm formation alongside tooth
surfaces. The extent of biofilm accumulation is largely
influenced by surface characteristics, including
morphology, chemical composition, and surface
charge [11].
Conventional caries prevention strategies, including
fluoride application, tooth brushing, and antimicrobial
mouth rinses, rely heavily on patient compliance,
which may limit their effectiveness in clinical practice
[12]. In addition, EPS matrix within S. mutans biofilms
reduces antimicrobial efficacy and promotes microbial
tolerance [13]. Consequently, increasing attention has
been directed toward antimicrobial strategies that
operate independently of patient compliance and are
less prone to microbial resistance, particularly
nanotechnology-based approaches involving the
incorporation of nanoparticles into dental materials or
their application as antimicrobial surface coatings [14].
This systematic review aims to evaluate the currently
available in vitro evidence on the antibacterial and
antibiofilm efficacy of nanoparticle-modified clear
aligners against S. mutans, with additional
consideration of their cytotoxic effects.
2. Materials and Methods
This systematic review was designed and reported in
compliance with the Preferred Reporting Items for
Systematic Reviews and Meta-Analyses (PRISMA)
guidelines [15]. The study protocol was prospectively
registered with the International Prospective Register
of Systematic Reviews (PROSPERO) under the
identifier CRD420251186452.
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2.1 Focus Question
The focus question of this review was formulated in
accordance with the Population, Intervention,
Comparison, Outcome, and Study design (PICOS)
framework [16]: does nanoparticle incorporation or
nanoparticle-based surface coating of clear aligner
thermoplastic materials enhance antibacterial efficacy
against Streptococcus mutans compared with
unmodified aligners? (Table 1).
Table 1. Focus question development according to the
PICOS model.
PICOS
Method
Description of the Components of
Question Formulation
P—
population
Clear aligner thermoplastic
materials
I—
intervention
Nanoparticle incorporation or
nanocoating applied to aligner
materials
C—control
Unmodified clear aligners without
nanoparticles or coatings
O—
outcome
Primary outcome: antibacterial
efficacy against Streptococcus
mutans (bacterial growth inhibition,
CFU reduction, biofilm reduction).
Secondary outcome: cytotoxicity of
nanoparticle-enhanced aligners
S—Study
Design
In vitro experimental studies
2.2 Eligibility Criteria
The inclusion criteria included:
• Studies evaluating clear aligner thermoplastic
materials;
• Studies incorporating nanoparticles into clear
aligner materials or applying nanoparticle-based
surface coatings;
• Antibacterial and/or antibiofilm against
Streptococcus mutans;
• Articles published in English;
• Original experimental studies published in peer-
reviewed journals;
• In vitro experimental studies.
The exclusion criteria included:
• Studies evaluating orthodontic materials other
than clear aligners (e.g., brackets, wires,
composites, adhesives);
• Studies using antimicrobial agents not based on
nanoparticles;
• Studies that do not report antibacterial outcomes
specifically against Streptococcus mutans, or
studies assessing antibacterial effects on other
bacteria;
• Reviews, systematic reviews, meta-analyses,
case reports.
2.3 Search strategy and study selection
A comprehensive literature search was performed
across four electronic databases: PubMed,
ScienceDirect, SpringerLink, and Web of Science. The
search was conducted between November 1st, 2025,
and January 31, 2026. In PubMed, the following search
strategy was used: (clear aligners OR aligners OR
invisible aligners OR orthodontic aligners) AND
(nanoparticles OR coating OR coated) AND
(antibacterial OR antibiofilm OR biofilm). This
strategy was adapted as necessary to meet the specific
search requirements of the other databases included in
the review.
Study selection was carried out independently by two
reviewers. Initially, titles and abstracts were screened
for relevance, followed by full-text evaluation of
articles deemed potentially eligible. Duplicate records
were identified and removed using Zotero reference
management software (version 7.0.24; Corporation for
Digital Scholarship, USA). Any discrepancies between
reviewers were resolved through discussion until a
consensus was reached.
2.4 Data extraction
Data from studies meeting the eligibility criteria were
independently extracted by two reviewers. Extracted
data from each study included: (1) authors and year of
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publication, (2) aligner or sample material, (3) sample
size and experimental groups, (4) nanoparticle type,
(5) mode of nanoparticle integration, (6) outcomes
measured, (7) evaluation time points, and (8) main
findings. Data on cytotoxicity were extracted as
secondary outcomes when reported.
2.5 Risk of bias assessment
To assess the methodological quality of the included
studies, a modified Joanna Briggs Institute (JBI)
critical appraisal checklist adapted for in vitro
experimental studies was used [17]. Given the absence
of a universally accepted and standardized risk of bias
assessment tool for in vitro studies, a modified Joanna
Briggs Institute (JBI) critical appraisal checklist was
used to evaluate key methodological aspects and
potential sources of bias. Each criterion was assessed
using the response options “Yes”, “No”, “Unclear”, or
“Not applicable”. The overall risk of bias for each
study was determined based on the proportion of “Yes”
responses in the checklist. Studies with ≥70% “Yes”
answers were considered to have a low risk of bias,
those with 50–69% “Yes” answers were classified as
having a moderate risk of bias, and studies with <50%
“Yes” answers were considered to have a high risk of
bias. The assessment was performed independently by
two reviewers, and disagreements were resolved
through discussion until consensus was reached.
3. Results
3.1 Study selection
The initial database search identified a total of 468
records. After removal of duplicate entries (n = 39)
using Zotero reference management software (version
7.0.24; Corporation for Digital Scholarship, USA),
429 unique records remained for screening. Titles and
abstracts of these records were independently
reviewed, leading to the exclusion of 410 publications
that did not meet the eligibility criteria. Nineteen
articles were subsequently assessed for full-text
eligibility. Of these, one article was excluded due to
the unavailability of the full text, as only the abstract
was accessible. Following full-text evaluation of the
remaining articles and application of the predefined
inclusion and exclusion criteria, a total of 10 studies
were included in the final qualitative synthesis.
3.2 Study charasteristics
A total of 10 in vitro experimental studies were
included in this systematic review. The studies
investigated a range of clear aligner and aligner-related
polymeric materials, including Invisalign® aligners
[18,19], thermoplastic polymers such as polyethylene
terephthalate glycol (PETG)—including both
conventionally processed [20–22] and 3D-printed
PETG [23]—and thermoplastic polyurethane (TPU)
[24,25], as well as 3D-printed clear photopolymer
resin materials [26,27].
Sample sizes varied across studies and were not
consistently reported; however, most investigations
employed multiple experimental and control groups. A
variety of nanoparticle types were evaluated across the
included studies, including zinc oxide (ZnO)
[18,22,25,27], silver nanoparticles (AgNPs) , titanium
dioxide (TiO₂) [19,21], copper-doped TiO₂ (TiO₂–Cu)
[21], barium titanate (BaTiO₃) [20], magnesium oxide
(MgO) [22], and chitosan nanoparticles (Chs) [26].
Nanoparticles were integrated either into the aligner
material matrix [20,23,24,26] or applied as surface
coatings [18,19,21,22,25,27], depending on the study
design. For cytotoxicity assessment, different cell lines
were used across studies, including mouse fibroblasts
(L929) [20,21,25,26], preadipocyte-derived osteoblast
precursor cells (2T3-L1) [26], and human gingival
fibroblasts (HGFs) [23,24,27]. Detailed characteristics
of the studies included in the review are presented in
Table 1 and Table 2.
3.3 Results of individual studies
The included studies evaluated the biological
properties of nanoparticle-modified aligner materials,
primarily focusing on antibacterial and anti-biofilm
activity, with several studies additionally assessing
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cytotoxicity. The main antibacterial and anti-biofilm
outcomes are summarized in Table 3, while
cytotoxicity findings are presented in Table 4.
3.3.1 Antibacterial effects (primary outcome)
Eight studies evaluated the antibacterial activity of
nanoparticle-modified aligner materials against S.
mutans [18,20–25,27]. Antibacterial activity was
primarily assessed using colony-forming unit (CFU)
based assays [18,20–25], while one study employed
the zone of inhibition method [27]. Across all studies,
nanoparticle modification was associated with reduced
bacterial counts compared with uncoated controls.
The magnitude of antibacterial efficacy varied across
studies and experimental conditions. Variations in
antibacterial efficacy were reported across studies,
including differences related to nanoparticle
concentration, material polarization, and visible-light
irradiation. Shi et al. reported reductions of up to 67%
in viable bacterial counts in the highest nanoparticle
concentration, with polarized samples exhibiting
greater antibacterial activity than unpolarized
materials [20]. Ye et al. observed a 38.49% reduction
in S. mutans in the highest concentration group [25].
Zhang et al. reported a 5–6 log reduction in bacterial
counts in the highest concentration group [24], while
Ha et al. demonstrated bacterial reductions exceeding
99% under visible-light irradiation [21].
Time-dependent antibacterial activity was evaluated in
two studies. Anita et al. assessed bacterial counts from
6 hours to 7 days and reported significantly reduced
CFU counts at all time points, with the strongest
antibacterial effect observed within the first two days
and persistence of activity throughout the 7-day period
[18]. Gharibnavaz et al. compared results at 24 hours
and 1 week; although reductions in S. mutans were
observed, these differences were not statistically
significant over time [22]. Teramoto-Iida et al.
evaluated antibacterial activity only after 7 days of
incubation and reported significant inhibition
compared with uncoated controls [27].
3.3.2 Antibiofilm effects (primary outcome)
Seven studies evaluated the anti-biofilm activity of
nanoparticle-modified aligner materials [19,20,22–
26]. Biofilm formation was assessed using different
methodologies, including crystal violet (CV) assays
[19,20], CFU-based assays [25,26], 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assays [23,24], scanning electron
microscopy (SEM) analysis [22], and combined CFU
and SEM evaluation [25]. Across all studies,
nanoparticle modification was associated with reduced
biofilm formation or bacterial adhesion compared with
uncoated controls.
In most investigations, biofilm suppression was
concentration-dependent, with greater inhibition
observed at higher nanoparticle concentrations
[19,20,23–26]. In addition, certain experimental
conditions, including material polarization [20] and
ultraviolet A (UVA) irradiation [19], were associated
with enhanced anti-biofilm effects.
3.3.3 Cytotoxicity (secondary outcome)
Cytotoxicity was evaluated in seven of the ten included
studies [20,21,23–27]. Across all studies, high levels
of cell viability were reported for nanoparticle-
modified aligner materials. Reported cell viability
values generally exceeded 84%, frequently reaching
>90–95% at different exposure times (24–96 hours and
up to 14 days), with no statistically significant
differences between test and control groups. None of
the included studies reported cytotoxic effects under
the tested conditions (Table 4).
3.4 Quality assessment
The results of the risk of bias assessment for the
included studies are summarized in Table 5. The
overall methodological scores ranged from 72.7% to
100%, and according to the predefined criteria, all
included studies were classified as having a low risk of
bias
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Table 1. Characteristics of included studies reporting primary outcomes.
Authors, Year
Aligner /
sample
material
Sample size, groups
NP type
Integration
type
Anita et al.
2023 [18]
Invisalign
Sample size – 26
13 – ZnO
13 – control (no coated)
ZnO
Surface
coating
Shi et al. 2023
[20]
PETG
Sample size – not reported
PETG/ BaTiO
3
(10 wt%)
PETG/ BaTiO
3
(20 wt%)
PETG/ BaTiO
3
(30 wt%)
Control – PETG (0 wt%)
Each tested in unpolarized and polarized
conditions
BaTiO
3
Incorporation
Taher et al.
2023 [26]
3D-
printed
CR
Sample size – 50
10 – CR + 2% Chs
10 – CR + 3% Chs
10 – CR + 5% Chs
10 – CR control (no coated)
10 – Thr control (no coated)
Chs
Incorporation
Ha et al. 2024
[21]
PETG
Sample size – not reported
TiO₂–Cu (3 wt%)
TiO₂–Cu (5 wt%)
TiO₂–Cu (9 wt%)
Control (control / pristine TiO₂ / PETG /
PETG dark)
TiO₂
TiO₂–Cu
Surface
coating
Gharibnavaz et
al. 2025 [22]
PETG
Sample size – 20
5 – ZnO
5 – MgO
5 – ZnO+MgO
5 – control (no coated)
ZnO
MgO
ZnO+MgO
Surface
coating
Huang et al.
2025 [23]
3D-
printed
PETG
Sample size – not reported
PETG/0.1% Ag
PETG/1% Ag
PETG/10% Ag
Control (0% Ag)
Control (no coated)
AgNP
Incorporation
Zhang et al.
2025 [24]
TPU
Sample size – not reported
TPU/Ag-1 (1 wt% AgNP) TPU/Ag-2 (2
wt% AgNP) TPU/Ag-5 (5 wt% AgNP)
CAP control (no coated)
TPU control (no coated)
AgNP
Incorporation
Ye et al. 2025
[25]
TPU
Sample size – not reported
T (TPU only) – control
TP (only PVDF coating)
TZ (Only ZnO NPs coating)
10% TPZ (PVDF+ZnO NPs)
20% TPZ (PVDF+ZnO NPs)
30% TPZ (PVDF+ZnO NPs)
ZnO
Surface
coating
Teramoto-lida
et al. 2025 [27]
3D-
printed
resin
Sample size – not reported
10% ZnO
20% ZnO
30% ZnO
Negative control (no coated)
Positive control (CHX)
ZnO
Surface
coating
Pourhajibagher
et al. 2025 [19]
Invisalign
Sample size - 20
5 – 1% TiO2 + UVA
5 – 2% TiO2 + UVA
5 – 4% TiO2 + UVA
5 – control (no coated, no UVA)
TiO2
Surface
coating
Ag – silver; BaTiO₃ – barium titanate; Chs – chitosan; Chx – chlorhexidine; CR – clear resin; Cu – copper; FDM – fused
deposition modeling; MgO – magnesium oxide; NP – nanoparticle; PETG – polyethylene terephthalate glycol; PVDF –
polyvinylidene fluoride; Thr – thermoplastic resin; TiO₂ – titanium dioxide; TPU – thermoplastic polyurethane; UVA –
ultraviolet A; wt% – weight percent; ZnO – zinc oxide.
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Table 2. Characteristics of included studies reporting secondary outcomes.
Authors, Year
Cell
Line
Test Groups
Control Group
Cytotoxicity
Assessment
Method
Shi et al. 2023
[18]
L926
PETG/ BaTiO
3
(30 wt%) (unpolarized
and polarized conditions)
PETG
Live/Dead
staining
(Calcein-AM /
PI);
CCK-8 assay
Taher et al. 2023
[26]
L926
2T3-L1
CR + 2% Chs
CR + 3% Chs
CR + 5% Chs
CR
Thr
Distilled water (negative)
Polyethylene (negative)
Ethanol 70% (positive)
MTT assay
Ha et al. 2024
[21]
L926
TiO₂–Cu (9 wt%)
0% extract (pure medium)
MTT assay
Huang et al.
2025 [23]
HGFs
PETG/0.1% Ag
PETG/1% Ag
PETG/10% Ag
Virgin PETG
αMEM (0% extract; pure
medium)
CCK-8 assay
Zhang et al.
2025 [24]
HGFs
TPU/Ag-1 (1 wt% AgNP) TPU/Ag-2 (2
wt% AgNP) TPU/Ag-5 (5 wt% AgNP)
αMEM (0% extract; pure
medium)
Live/Dead
staining
(Calcein-AM /
PI);
CCK-8 assay
Ye et al. 2025
[25]
L926
TP (only PVDF coating)
TZ (only ZnO NPs coating)
10% TPZ (PVDF+ZnO NPs)
20% TPZ (PVDF+ZnO NPs)
30% TPZ (PVDF+ZnO NPs)
Pure medium; no material
Live/Dead
staining
(Calcein-AM /
PI);
CCK-8 assay
Teramoto-lida et
al. 2025 [27]
HGFs
10% ZnO NPs
20% ZnO NPs
30% ZnO NPs
Uncoated resin discs
WST-1 assay
2T3-L1 – mouse preadipocyte cell line; Ag – silver; BaTiO₃ – barium titanate; Calcein-AM / PI – calcein-acetoxymethyl ester
/ propidium iodide staining; CCK-8 – Cell Counting Kit-8 assay; Chs – chitosan; CR – clear resin; Cu – copper; HGFs –
human gingival fibroblasts; L926 – mouse fibroblast cell line; MTT – 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide assay; NP – nanoparticle; PETG – polyethylene terephthalate glycol; PVDF – polyvinylidene fluoride; Thr –
thermoplastic resin; TiO₂ – titanium dioxide; WST-1 assay – water-soluble tetrazolium-1 assay; wt% – weight percent; ZnO
– zinc oxide; αMEM – alpha minimum essential medium.
Table 3. Primary outcomes.
Authors, Year
NP
composition
and
concentration
Outcome(s)
measured
(method(s))
Time
points
Main findings
Anita et al.
2023 [18]
ZnO
Antibacterial
(CFU assay)
6 hours
12
hours
24
hours
2 days
4 days
1 week
Coated aligners showed consistently lower bacterial
growth across all time points, with delayed and slower
S.M accumulation compared with uncoated aligners.
The reduction of S.M count for the coated aligner was
statistically significant at the time points tested.
Shi et al. 2023
[20]
BaTiO
3
(10-
30 wt%)
(unpolarized
and polarized
conditions)
Antibacterial
(CFU assay)+
antibiofilm (CV
assay)
N/A
Polarized PETG/BaTiO₃ nanocomposites
demonstrated significantly stronger antibacterial
activity against S.M compared to unpolarized samples
resulting in a reduction of both viable bacteria (up to
~67% CFU reduction) and surface associated biofilm
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biomass, with stronger effects observed at higher
BaTiO
3
nanoparticle concentrations.
Taher et al.
2023 [26]
Chs (2-5%)
Antibiofilm
(CFU assay)
24
hours
The addition of 3% and 5% chitosan nanoparticles into
the resin significantly reduced bacterial adhesion, as
evidenced by a marked decrease in colony-forming
uints.
Ha et al. 2024
[21]
TiO₂–Cu (3-9
wt%)
Antibacterial
(CFU assay)
N/A
Clear aligners coated with TiO₂–Cu films demonstrated
markedly enhanced antibacterial activity against S.M
under visible-light irradiation, with all Cu
concentrations (3, 5, and 9 wt%) achieving consistently
high inactivation efficiencies (>99%) across different
bacterial dilutions.
Gharibnavaz et
al. 2025 [22]
ZnO
MgO
ZnO+MgO
Antibacterial
(CFU assay)+
antibiofilm
(SEM,
qualitative)
24
hours
1 week
The combination of ZnO and MgO particles provided
the highest antibacterial activity. Reduced bacterial
colonisation and less developed biofilm structures on
coated surfaces, with more pronounced differences
observed at later time points.
Huang et al.
2025 [23]
AgNPs (0.1-
10%)
Antibacterial
(CFU assay) +
antibiofilm
(MTT assay)
N/A
1 wt% and 10 wt% AgNP samples showing strong
antibacterial activity and pronounced reductions in
bacterial viability, whereas lower concentrations
exhibited only minimal effects. The 1 wt% and 10 wt%
AgNP sheets also exhibited significant anti-biofilm
activity, effectively suppressing biofilm formation.
Biofilm inhibition increased with AgNP concentration.
Zhang et al.
2025 [24]
AgNPs (1-
5wt%)
Antibacterial
(CFU assay) +
antibiofilm
(MTT assay)
N/A
Sheets showed significantly reduced S.M
concentrations versus controls, with Ag-5 being the
most effective and Ag-1 and Ag-2 showing comparable
activity. Higher AgNP concentrations resulted in
greater biofilm suppression.
Ye et al. 2025
[25]
TP (only
PVDF
coating)
TZ (Only
ZnO)
TPZ (ZnO
NPs (10-
30wt%) in
PVDF )
Antibacterial
(CFU assay) +
antibiofilm (CFU
assay and SEM,
qualitative)
N/A
The 30% TPZ coating demonstrated the strongest
antibacterial activity among all tested groups. It
achieved a 38.49% reduction of S.M The TZ group also
showed notable antibacterial effects, confirming the
instrinsic bactericidal properties of ZnO nanoparticles.
Biofilm formation decreased stepwise across all coated
groups, with 30% TPZ showing the strongest
antibiofilm effect.
Teramoto-lida
et al. 2025 [27]
ZnO (10-30%)
Antibacterial
(zone of
inhibition)
1 week
ZnO NPs–coated samples showed statistically
significant inhibition of S.M compared with uncoated
controls, although no significant differences were
observed between concentrations.
Pourhajibagher
et al. 2025 [19]
TiO2 (1-4%) +
UVA
Antibiofilm (CV
assay)
N/A
All treatment groups showed reduced biofilm
formation compared to the control; however, a
statistically significant reduction was observed only in
the group with 4% TiO₂-coated clear aligners combined
with UVA.
Ag – silver; BaTiO₃ – barium titanate; CFU – colony-forming units; Chs – chitosan; Cu – copper; CV assay – crystal violet
assay; MgO – magnesium oxide; MTT – 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; N/A – not
applicable; NP – nanoparticlePETG – polyethylene terephthalate glycol; PVDF – polyvinylidene fluoride; SEM – scanning
electron microscopy; S.M. – Streptococcus mutans; TiO₂ – titanium dioxide; UVA – ultraviolet A; wt% – weight percent; ZnO
– zinc oxide.
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Table 4. Secondary outcomes.
Authors, Year
NP composition
and concentration
Exposure
Duration
Main Findings
Shi et al. 2023
[18]
PETG/ BaTiO
3
(30
wt%) (unpolarized
and polarized
conditions)
24h
48h
72h
High cell viability across all groups (>95%) at all time points, with
no statistically significant differences. Live/Dead staining
confirmed predominantly viable cells. No cytotoxicity observed for
polarized or unpolarized PETG/BaTiO₃NPs (30 wt%).
Taher et al.
2023 [26]
Chs (2-5%)
14 days
High cell viability across all groups (>85%), with no statistically
significant differences, indicating no cytotoxicity.
Ha et al. 2024
[21]
TiO₂–Cu (9 wt%)
24h
High cell viability remained >84% across all extract concentrations
with no statistically significant differences, indicating no
cytotoxicity.
Huang et al.
2025 [23]
AgNPs (0.1-
10wt%)
24h
High cell viability across all groups (>92%) with no statistically
significant differences, indicating no cytotoxicity.
Zhang et al.
2025 [24]
AgNPs (1-5wt%)
24h
High cell viability across all groups (>90%) with no statistically
significant differences. Live/Dead staining confirmed
predominantly viable cells and minimal cell death. No cytotoxicity
observed.
Ye et al. 2025
[25]
TP (only PVDF
coating)
TZ (Only ZnO)
TPZ (ZnO NPs (10-
30wt%) in PVDF )
24h
48h
96h
Viability remained >90% for 10% and 20% TPZ and >85% for 30%
TPZ at all time points, with no statistically significant differences.
Live/Dead staining confirmed predominantly viable cells. No
cytotoxicity observed.
Teramoto-lida
et al. 2025 [27]
ZnO NPs (10-30%)
48h
72h
ZnO-coated discs showed good biocompatibility, with significantly
increased cell viability at 72 h compared with 48 h, and the 20–30%
coatings demonstrating the highest viability. No cytotoxicity
observed.
Ag – silver; Chs – chitosan; Cu – copper; NP – nanoparticle; PVDF – polyvinylidene fluoride; TiO₂ – titanium dioxide; wt%
– weight percent ;ZnO – zinc oxide.
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72
Table 5. RISK OF BIAS (Modified Joanna Briggs Institute Checklist for in vitro studies).
Y – Yes; U – Unclear.
Study
Was the
rationale
for the
chosen
methodolog
y
adequately
explained?
Was the
sample
size or
number of
replicates
adequatel
y
justified?
Was the
aligner
material
and
specimen
preparatio
n described
in sufficient
detail?
Was the
nanoparticl
e or coating
compositio
n clearly
described?
Was the
coating or
nanoparticle
application
method
adequately
described and
reproducible?
Were the
methods for
measuring the
study outcomes
appropriate
and clearly
described?
Were
appropriat
e control
groups
included?
Were
experiment
s
adequately
replicated?
Was
contaminatio
n or cross-
contaminatio
n prevented
or
controlled?
Were
quantitativ
e outcomes
clearly and
completely
reported?
Was
appropriat
e statistical
analysis
performed?
Score
out of
11
(100%
)
Anita et al.
2023 [3]
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
100%
Shi et al. 2023
[4]
Y
U
Y
Y
Y
Y
Y
U
Y
Y
U
72.7%
Taher et al.
2023 [5]
Y
U
Y
Y
Y
Y
Y
Y
Y
Y
Y
90.9%
Ha et al. 2024
[6]
Y
U
Y
Y
Y
Y
Y
U
Y
Y
U
72.7%
Gharibnavaz
et al. 2025 [7]
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
100%
Huang et al.
2025 [8]
Y
U
Y
Y
Y
Y
Y
Y
Y
Y
Y
90.9%
Zhang et al.
2025 [9]
Y
U
Y
Y
Y
Y
Y
Y
Y
Y
Y
90.9%
Ye et al. 2025
[10]
Y
U
Y
Y
Y
Y
Y
U
Y
Y
Y
81.8%
Teramoto-
lida et al.
2025 [11]
Y
U
Y
Y
Y
Y
Y
U
Y
Y
Y
81.8%
Pourhajibagh
er et al. 2025
[12]
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
100%
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73
4. Discussion
Microbial colonization and subsequent enamel
demineralization remain prevalent challenges in
orthodontic patients. Although various preventive
and therapeutic approaches have been proposed, their
effectiveness is largely dependent on patient
compliance, which may limit their clinical impact
[28]. Nanotechnology-based modifications have been
successfully applied in fixed orthodontic appliances
to reduce bacterial colonization [29]. However, oral
hygiene levels and colonization by Streptococcus
mutans have been reported to be comparable between
clear aligners and fixed appliances, suggesting that
aligners are not exempt from biofilm-related
complications [30]. Therefore, there is a need for
alternative strategies to reduce bacterial adhesion and
biofilm formation in clear aligner systems. This
systematic review aimed to synthesize the available
evidence on the antibacterial and antibiofilm efficacy
of nanoparticle-modified clear aligners.
A majority of the included studies focused on metal
oxide nanoparticles, such as zinc oxide (ZnO)
[18,22,25,27], magnesium oxide (MgO) [22], and
titanium dioxide (TiO₂) [19,21], all of which were
applied as surface coatings. The antibacterial effect of
these materials is mainly due to their ability to interact
with and penetrate bacterial cell walls of both Gram-
positive and Gram-negative bacteria. In addition, the
release of metal ions helps inhibit bacterial growth
and prevent biofilm formation [31]. Enhanced
antibacterial activity was also observed when
nanoparticles were used in combination. For
example, Gharibnavaz et al. reported that the
combination of ZnO and MgO demonstrated greater
antibacterial efficacy compared to either material
used alone, suggesting a potential synergistic effect
[22]. A similar approach was reported by Ye et al.,
where ZnO nanoparticles were incorporated into
polyvinylidene difluoride (PVDF), a piezoelectric
polymer [25]. This strategy improves nanoparticle
dispersion and limits aggregation, while the
piezoelectric response generated under functional
conditions, such as occlusion, may further contribute
to antibacterial effects and reduce the risk of enamel
demineralization [25]. Importantly, the antibacterial
efficacy of these materials is influenced not only by
their composition but also by external conditions.
TiO₂-based systems, in particular, showed improved
performance under UVA irradiation, as reported by
Pourhajibagher et al., due to their ability to generate
reactive oxygen species (ROS), which disrupt
bacterial cells and biofilm [19,32,33].
Silver nanoparticles (AgNPs) were investigated in
two studies, both utilizing incorporation into
polymer-based materials [23,24]. These studies
consistently demonstrated significant antibacterial
and antibiofilm effects against S. mutans. The
antimicrobial activity of AgNPs is attributed to their
broad-spectrum mechanisms of action, including the
continuous release of silver ions (Ag⁺), which interact
with bacterial cell walls and membranes, increasing
membrane permeability and leading to structural
disruption. Following cellular uptake, silver ions can
induce the generation of reactive oxygen species
(ROS) and interfere with essential cellular processes,
including deoxyribonucleic acid (DNA) replication
and protein synthesis [34]. Zhang et al. reported that
silver ion release was initially high but decreased
sharply after 48 hours, followed by a slower and
sustained release over time, suggesting a strong initial
antibacterial effect followed by prolonged, lower-
level antimicrobial activity [24]. Notably, the
antibacterial efficacy appeared to be concentration-
dependent, with higher AgNP concentrations
associated with greater reductions in bacterial
viability and biofilm formation [23,24].
In addition to metal-based nanoparticles, other
materials such as barium titanate (BaTiO₃) and
chitosan-based (Chs) nanoparticles were also
investigated [20,26]. BaTiO₃, a piezoelectric
Journal of Medical Sciences. 18 Jun, 2026 - Volume 14 | Issue 4. Electronic - ISSN: 2345-0592
74
material, showed enhanced antibacterial activity
under polarized conditions, suggesting that its
electrical properties may influence bacterial adhesion
and viability [20]. It has been proposed that the
negatively charged surface of BaTiO₃ nanoparticles
may inhibit the adhesion of negatively charged S.
mutans [35]. Furthermore, under mechanical
stimulation, such as that occurring during normal oral
activities, BaTiO₃ can generate surface charges that
may contribute to bacterial disruption and the
production of reactive oxygen species (ROS) [36].
Consistent with this mechanism, polarized BaTiO₃-
containing materials demonstrated stronger
antibacterial and antibiofilm effects compared to
unpolarized systems, with enhanced efficacy
observed at higher nanoparticle concentrations [20].
In contrast, chitosan-based nanoparticles exhibited
antibiofilm activity primarily through electrostatic
interactions with bacterial cell membranes, which
interfere with bacterial adhesion and essential cellular
functions [26]. Furthermore, higher chitosan
concentrations were associated with greater
reductions in bacterial adhesion, indicating a
concentration-dependent effect, consistent with
trends observed in other nanoparticle systems [19–
21,23–25,27].
While nanoparticle-modified aligners demonstrated
promising antibacterial and antibiofilm effects,
concerns regarding their potential cytotoxicity should
be considered. Several factors may influence the
cytotoxicity of nanoparticle-modified materials,
including nanoparticle type, concentration, particle
size [37]. Among the studies that evaluated
cytotoxicity, high cell viability was consistently
reported, suggesting that these modifications do not
appear to compromise biocompatibility under the
tested conditions [20,21,23–27]. Despite favorable
biocompatibility observed in the included studies, the
long-term effects of nanoparticle accumulation in oral
tissues and potential systemic implications remain
unclear and require further investigation.
This review has several limitations that should be
considered when interpreting the findings. A high
degree of variability was observed among the
included studies, particularly in terms of nanoparticle
types, material composition, concentration, and
methodological approaches used to assess
antibacterial and antibiofilm activity. Such
heterogeneity limits direct comparison between
studies and may affect the overall reliability of the
conclusions. In addition, the available evidence is
derived from in vitro studies, which do not fully
reflect the complex biological conditions of the oral
environment. Factors such as saliva, mechanical
forces, and the diversity of oral microbiota may
influence the actual clinical performance of these
materials. Moreover, the limited number of studies
and the absence of well-designed clinical trials further
restrict the strength of the evidence. Finally, the short
duration of most experimental studies makes it
difficult to draw conclusions regarding the long-term
effectiveness and safety of nanoparticle-modified
aligners.
5. Conclusion
Based on the current evidence, nanoparticle-modified
clear aligners demonstrate promising antibacterial
and antibiofilm potential. However, the available
studies are characterized by considerable
heterogeneity and are largely limited to in vitro
designs. Therefore, further well-designed in vivo and
clinical studies are required to confirm their long-
term safety and effectiveness and to support their
translation into clinical practice.
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