Effects of cleaning methods on the removal efficacy of Streptococcus mutans biofilm and material properties of thermoplastic aligner materials

Abstract

Background

Clear aligner therapy is widely used in modern orthodontics, but maintaining adequate appliance hygiene remains challenging. Aligners are prone to biofilm accumulation due to their surface characteristics and material wear during clinical use. However, evidence remains limited regarding the effectiveness of various cleaning methods and their effects on aligner material properties. Therefore, this study aimed to evaluate the efficacy of various cleaning methods in removing Streptococcus mutans biofilm from thermoplastic aligner materials and to assess their effects on surface morphology and mechanical properties.

Methods

S. mutans biofilm was cultivated on specimens excised from thermoplastic aligners. Six cleaning methods were applied: toothbrushing (TB), cleaning solution soaking (CS), ultrasonic cleaning (US), and combined methods (CSTB, CSUS), as well as no treatment (CTR). Biofilm removal efficacy was then evaluated using crystal violet (CV) staining, colony-forming unit (CFU) enumeration, and scanning electron microscopy (SEM). After 14 days of repeated cleaning, surface morphology, surface roughness, elastic modulus and ultimate tensile strength (UTS) were analyzed. For statistical analysis, depending on variance homogeneity, either one-way ANOVA with Tukey’s post-hoc test or Welch ANOVA with Dunnett’s T3 test was used. Statistical significance was set at p < 0.05.

Results

The TB and CSTB groups showed the lowest S. mutans biofilm retention, while the TB, CSTB, and CSUS groups exhibited the greatest reductions in viable bacterial counts. After 14 days of repeated cleaning, surface roughness increased in the TB and CSTB groups (p < 0.05), whereas no significant differences in elastic modulus or UTS were observed among the six groups (p > 0.05).

Conclusions

Toothbrushing, especially when combined with chemical solution, effectively removes S. mutans biofilm from thermoplastic aligner materials without compromising mechanical integrity, though some surface roughening may occur. Ultrasonic cleaning demonstrated partial efficacy, indicating the need for further optimization before clinical application. In the absence of more advanced alternatives, mechanical brushing, ideally supplemented by chemical disinfectants, remains the most reliable strategy for maintaining aligner hygiene without compromising material performance. The findings of this study provide experimental data and scientific evidence to guide the optimization of future cleaning methods for clear aligners.

Background

With advancements in thermoplastic materials and digital orthodontic technologies, clear aligner therapy has become increasingly popular in clinical orthodontics [1]. Despite their aesthetic and removable advantages, clear aligners are not exempt from adverse effects on oral health. Like other orthodontic devices, clear aligners can induce shifts in the oral microbiome [2], and microecological changes have been observed on their inner surfaces even after short-term use [3]. These alterations often promote plaque accumulation and increase the risk of enamel demineralization, which may manifest as larger yet shallower white spot lesions [4]. Furthermore, inadequate hygiene during aligner wear can exacerbate these effects, potentially resulting in extensive decay—even at the incisal edges and cusp tips—and marked gingival inflammation [5], thereby compromising the clinical effectiveness of orthodontic treatment.

Even newly manufactured aligners tend to exhibit a corrugated surface, which reflects the corrugations of the 3D-printed models on which the aligners were manufactured [6], along with scratch marks, microabrasions and ridges that facilitate microbial adhesion [7]. Bacterial colonization initially concentrates on the raised edges or textured surfaces of the appliance, whereas recessed regions on the inner surface of the aligners, particularly those corresponding to cusp tips and attachment sites, tend to harbor more biofilm than flat surfaces [7]. After clinical use, clear aligners undergo surface morphology alterations, manifesting microcracks, surface abrasions, and delaminated areas, all of which further promote bacterial colonization [8, 9]. Such changes can be attributed to intraoral aging processes, as both short-term exposure and simulated conditions such as thermocycling have been shown to increase surface roughness and alter the elastic and stress relaxation properties of thermoplastic materials [10, 11]. Aging patterns may also vary according to manufacturing technique, as thermoformed and directly printed aligners differ in surface morphology, water absorption–desorption behavior, and long-term mechanical stability [12, 13]. In addition to aging effects, hygiene procedures may further contribute to material degradation, potentially impairing aligner fit, tooth movement, and the overall therapeutic outcome. Studies suggest that different cleaning methods may exert distinct effects on surface roughness, translucency, and mechanical properties [14–16]. These findings suggest potential interactions between cleaning protocols and material characteristics. Collectively, these factors highlight the importance of developing cleaning protocols that effectively control biofilm formation while preserving the structural integrity of thermoplastic aligner materials.

Current cleaning strategies for removable thermoplastic dental appliances can be broadly categorized as mechanical and chemical [17]. Mechanical approaches include manual brushing (with water, soap, toothpaste, or abrasives) and ultrasonic cleaning [18]. Brushing remains the most commonly recommended method for both teeth and removable appliances, with proven efficacy in reducing surface biofilm [19, 20]. However, brushing alone may fail to adequately clean hard-to-reach areas such as cusp tips. Ultrasonic baths offer a useful adjunct, operating via fluid agitation and cavitation induced by sound wave vibrations to disrupt and remove adherent biofilms [21]. Studies have demonstrated that combining vibrating baths with cleaning solutions can further reduce bacterial biofilm adherence [22, 23]. Chemical cleansers offer a convenient alternative, requiring minimal patient dexterity. These solutions typically contain active ingredients such as sodium bicarbonate, acids, chelators and quaternary ammonium compounds [22–25]. Quaternary ammonium salts are cationic surfactants with broad-spectrum antibacterial activity, effective at low concentrations and widely employed in disinfectants, personal hygiene products, cosmetics, and antiseptics due to their potent efficacy and low toxicity [26].

Despite the availability of various cleaning techniques, their relative effectiveness on clear aligners and potential effects on material properties remain insufficiently studied within an integrated evaluation framework. Previous studies have often focused either on biofilm removal efficacy or on changes in material surface and mechanical characteristics, but few have assessed both antimicrobial performance and material integrity simultaneously. Therefore, the present study aimed to evaluate the efficacy of different cleaning methods, including brushing, ultrasonic baths, and chemical soaking on biofilm removal from thermoplastic orthodontic materials. Additionally, the impact of these cleaning methods on the surface morphology and mechanical properties of the materials was also assessed. The null hypotheses were: (i) there is no significant difference among the six cleaning methods in removing biofilm from thermoplastic aligner materials; (ii) the surface morphology of the materials remains unchanged after cleaning; (iii) the mechanical properties of the materials remain unaffected after cleaning.

Methods

Specimen preparation

For subsequent experiments, two types of specimens were prepared from the same batch of unused Invisalign^® aligners (Align Technology, San Jose, CA, USA). Specimen A comprised 150 uniform segments excised from the left mandibular second molar region with buccal-surface attachment cavities. This region was selected because its cuspal morphology and attachment cavities present complex surface features that are highly susceptible to biofilm accumulation. Specimen B comprised 120 rectangular segments (2.5 mm × 9 mm) excised from the middle one-third of the facial surface of the maxillary central incisors [8]. These relatively flat surfaces were chosen to facilitate more accurate tensile testing compared to curved aligner segments. All aligners used in this study were designed based on three-dimensional data derived from standardized dental arch models and subsequently fabricated by the manufacturer through a digital workflow, ensuring uniformity in geometry and material properties.

In vitro S. mutans biofilm model

A schematic overview of the experimental workflow is presented in Fig. 1. All specimens were UV-irradiated on both surfaces for 15 min each for disinfection [24]. For biofilm formation, S. mutans UA159 (ATCC 25175), as a virulent cariogenic pathogen and well-characterized biofilm-forming strain, was cultured in Brain Heart Infusion (BHI) broth (Land Bridge Technology Co., Ltd., Beijing, China) at 37℃ under 5% CO₂ for 16–18 h until the mid-exponential growth phase [27]. For S. mutans biofilm growth, each well of a 24-well plate (Nest Biotechnology Co., Ltd., Wuxi, China) contained 40 µL bacterial suspension (1 × 10⁹ CFU/mL) and 2 mL BHI supplemented with 1% sucrose. One sterilized specimen was then immersed per well and incubated at 37℃ for 48 h to establish mature S. mutans biofilm.

Fig. 1.

Schematic illustration of the experimental workflow

Cleaning methods

Following S. mutans biofilm formation, all specimens were gently rinsed three times with phosphate-buffered saline (PBS; G4202, Servicebio Technology Co., Ltd., Wuhan, China) to remove non-adherent cells. Specimen A (mandibular molar segments; n = 150) and Specimen B (maxillary incisor segments; n = 120) were randomly allocated to each of six experimental groups. Each group was then subjected to a distinct cleaning method. For groups involving the cleaning solution, Aligner Cleaner^® (Angelalign Technology Inc., Shanghai, China) was used. This quaternary ammonium-based cleaning solution primarily contains benzalkonium bromide (120 g/L) and hexamethylenetetramine (331 g/L).

• Group 1 (CTR): No cleaning treatment applied (negative control).

• Group 2 (TB): Brushing with a GC ruscello P-20 S toothbrush (TAIYO BRUSH Co., Ltd., Hyogo, Japan) for 2 min under running distilled water.

• Group 3 (CS): Immersion in the cleaning solution for 5 min according to the product instructions.

• Group 4 (US): Ultrasonic cleaning in distilled water at 45 kHz and 24 W for 5 min using an Ultrasonic Cleaner® (Angelalign Technology Inc., Shanghai, China) according to the product instructions.

• Group 5 (CSTB): Combined treatment of immersion in cleaning solution for 5 min and then brushing for 2 min.

• Group 6 (CSUS): Ultrasonic cleaning in cleaning solution for 5 min.

To simulate routine patient cleaning, manual toothbrushing was employed. Brushing was performed for 2 min under continuous distilled water flow, consistent with previous in vitro studies [17, 28]. Although manual brushing introduces some variability in applied force, which is a known limitation, this method was chosen for its clinical relevance. All brushing procedures were performed by a single trained operator following a predefined protocol with back-and-forth motions under light pressure. The ultrasonic cleaning parameters were set at a frequency of 45 kHz and a power of 24 W, based on relevant literature [29, 30] on aligner cleaning and in accordance with the manufacturer’s recommendations, with a cleaning duration of 5 min as specified in the product instructions (Table 1).

Table 1.

Summary of cleaning protocols applied in the six experimental groups

Group name	Cleaning device/material	Cleaning method
CTR	N/A	No cleaning treatment applied
TB	● GC ruscello P−20 S toothbrush (TAIYO BRUSH Co., Ltd., Hyogo, Japan)	Brushing with a toothbrush for 2 min
CS	● Aligner Cleaner® (Angelalign Technology Inc., Shanghai, China)	Immersion in the cleaning solution for 5 min
US	● Ultrasonic Cleaner® (Angelalign Technology Inc., Shanghai, China; 45 kHz, 24 W )	Ultrasonic cleaning in distilled water for 5 min
CSTB	● GC ruscello P−20 S toothbrush (TAIYO BRUSH Co., Ltd., Hyogo, Japan) ● Aligner Cleaner® (Angelalign Technology Inc., Shanghai, China)	Combined treatment of immersion in cleaning solution for 5 min and then brushing for 2 min
CSUS	● Aligner Cleaner® (Angelalign Technology Inc., Shanghai, China) ● Ultrasonic Cleaner® (Angelalign Technology Inc., Shanghai, China; 45 kHz, 24 W )	Ultrasonic cleaning in cleaning solution for 5 min

S. mutans biofilm removal assay

After cleaning, six A specimens from each group were gently rinsed with PBS, fixed in 4% paraformaldehyde (G1101, Servicebio Technology Co., Ltd., Wuhan, China) and stained with 0.1% crystal violet (G1014, Servicebio Technology Co., Ltd., Wuhan, China) for 20 min [31]. Excess dye was removed by rinsing with PBS, and the specimens were air-dried. Subsequently, 1 mL of 95% ethanol was added to solubilize the bound dye, and the optical density (OD) was measured at 595 nm using a microplate reader (Synergy™ H1, BioTek Instruments, Inc., Vermont, USA) to quantify the residual biofilm biomass [31].

In a parallel experiment, adherent S. mutans biofilm from three additional A specimens from each group, collected after cleaning, were detached via sonication (30 s pulse; SB-120D, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) [24]. The resulting suspensions were serially diluted and plated in triplicate on BHI agar to quantify viable colonies (CFU/mL).

Scanning Electron Microscopy (SEM) evaluation

After cleaning, three A specimens from each group were fixed in 4% paraformaldehyde, dehydrated through a graded ethanol series, and sputter-coated with gold. The internal mesio-lingual surfaces were examined using a scanning electron microscope (SEM; Zeiss SIGMA, Carl Zeiss Ltd., Cambridge, UK). Images were acquired at 5 kV under both low and high magnifications to evaluate the residual S. mutans biofilm morphology. To simulate clinical use, another set of three A specimens from each group were immersed in an artificial saliva (ISO/TR10271, Yuanye Bio-Technology Co., Ltd., Shanghai, China) at 37 ℃ and subjected to their respective cleaning methods daily for 14 consecutive days. After the final treatment, the specimens were carefully wiped with sterile cotton swabs to remove any remaining biofilm and avoid interference with subsequent imaging. They were then sputter-coated and the internal mesio-lingual surfaces were examined under SEM to assess potential surface alterations.

Surface roughness measurements

After 14 consecutive days of cleaning, ten A specimens from each group were wiped with sterile cotton swabs to remove any residual biofilm that might affect surface profilometry measurements. Surface roughness (Sa) was evaluated using a white-light interferometry 3D surface profiler (NewView™ 9000, ZYGO Corp., USA). A square area of 874.627 × 874.627 μm on the internal lingual surface of each specimen was analyzed.

Mechanical properties measurements

To simulate intraoral conditions, twenty B specimens per group were immersed in artificial saliva at 37 ℃ and subjected to their assigned cleaning method once daily for 14 consecutive days. Before testing, all specimens were gently wiped with sterile cotton swabs to remove any adhering biofilm and minimize the potential impact on subsequent mechanical measurements. Subsequently, tensile tests were performed using a universal testing machine (Instron^® E1000, High Wycombe, UK). Ten specimens per group were stretched at a strain rate of 2% per minute up to 1% strain to determine the elastic modulus from the linear portion of the stress–strain curves [8], while the remaining ten specimens were tested to fracture at a rate of 2 mm per minute, and ultimate tensile strength (UTS) was calculated as the maximum load divided by the original cross-sectional area [32].

Statistical analysis

For the biofilm removal assays, sample size selection followed established in vitro biofilm quantification protocols [33] and previously published studies [27, 34]. CV staining was performed with six specimens per group (n = 6), while CFU enumeration was conducted with three specimens per group (n = 3). For the evaluation of material properties after 14 days of cleaning across six experimental groups, an a priori power analysis was performed using G*Power software (version 3.1; Heinrich Heine University Düsseldorf, Germany). Based on an effect size of 0.5, α = 0.05, and 80% power [11, 13], a sample size of n = 10 per group was required for surface roughness measurements and mechanical testing (elastic modulus and UTS). SEM analysis was performed with three specimens per group (n = 3) as a qualitative morphological evaluation.

All data were expressed as means ± standard deviations. For statistical analysis with SPSS 22.0 (IBM, USA), the result of the Shapiro–Wilk test showed that the data were normally distributed (p > 0.05). Homogeneity of variances was evaluated using the Brown–Forsythe test. For data meeting the assumption of equal variances, one-way analysis of variance (ANOVA) was utilized to assess the intergroup differences, followed by Tukey’s post hoc test for multiple comparisons. For data with unequal variances, Welch ANOVA was applied, followed by Dunnett’s T3 post hoc test. Statistical significance was defined as the value of p < 0.05.

Results

S. mutans biofilm removal evaluation

SEM observation confirmed the successful formation of mature, structured S. mutans biofilms on the specimen surfaces (Fig. 2A). Stereomicroscopic images (Fig. 2B-G) revealed varying degrees of residual S. mutans biofilm following different cleaning methods. In the control group, material surface was covered by a uniform and dense bacterial biofilm. The CS group exhibited thinner and uneven biofilm coverage, whereas the US and CSUS groups showed patchy deposits (arrows). No visible biofilm was observed in the TB and CSTB groups.

Fig. 2.

Morphological evaluation of S. mutans biofilm formation and residual biofilm after cleaning. A SEM images confirming mature S. mutans biofilm formation on the internal surface of thermoplastic aligner material at increasing magnifications. B-G Stereomicroscopic images of residual biofilm on aligner specimens after six different cleaning methods

Crystal violet staining (Fig. 3A) revealed consistent results. The control group showed a continuous dense purple layer, whereas the CS group showed an uneven purple biofilm. Patchy purple biofilms were present in the US and CSUS groups. No purple staining was visible in the TB or CSTB groups. Quantitative analysis using optical density (OD) measurements in Fig. 3B revealed a statistically significant difference among the six groups (F = 51.92, p < 0.0001). The TB and CSTB groups exhibited the lowest OD values, indicating significantly less residual biofilm compared to the control (p < 0.0001), with no significant difference between the TB and CSTB groups (p > 0.05). The US and CSUS groups also demonstrated significantly lower OD values than the control (p < 0.01 and p < 0.05, respectively). There was no statistically significant difference between the CS and control groups (p > 0.05), suggesting that soaking alone in disinfectant solution was ineffective in removing adherent biofilm.

In the colony-forming unit (CFU) assay, S. mutans colonies from each group are displayed in Fig. 3C. The control group showed abundant, densely packed colonies. In contrast, the CS, US and CSUS groups showed a moderate reduction, while the TB and CSTB groups exhibited only sparse, scattered colonies. Quantitative CFU analysis (Fig. 3D) revealed statistically significant differences among groups (F = 86.94, p < 0.0001), with the TB, CSTB and CSUS groups containing significantly fewer viable bacteria than the CS and US groups (p < 0.05).

Fig. 3.

Quantitative and qualitative evaluation of biofilm removal after six cleaning methods. A Crystal violet staining of residual S. mutans biofilm on aligner specimens. B Quantification of residual biofilm biomass by measuring optical density (OD) at 595 nm (n = 6 per group). C Representative photographs of S. mutans colonies on BHI agar plates after cleaning. D Colony-forming unit (CFU) counts of viable bacteria on aligner specimens (n = 3 per group). Groups with the same lowercase letter are not significantly different (p > 0.05)

SEM evaluations

Low-magnification SEM images (Fig. 4A) showed dense biofilm coverage across the surface in the control group. In contrast, the surfaces of the TB and CSTB groups were free of visible biofilm. Specimens in the US and CSUS groups exhibited residual flaky biofilm. The CS group still showed a substantial amount of biofilm; however, compared to the control, the biofilm appeared thinner and more fragmented. High-magnification SEM images (Fig. 4B) revealed densely packed bacterial clusters embedded in abundant extracellular polymeric substance in the control group. In the CS, US, and CSUS groups, biofilm structures were sparse, with loosely arranged cells and short chains remaining in localized regions. No discernible bacterial cells were observed in the TB or CSTB groups.

Fig. 4.

SEM images of residual biofilm on thermoplastic aligner surfaces after six cleaning methods. A Low-magnification images showing the overall distribution of biofilm. B High-magnification images illustrating detailed bacterial morphology and extracellular matrix structure

After 14 days of treatment, low-magnification SEM images (Fig. 5A) revealed the typical corrugated surface topography of Invisalign® aligner materials. No noticeable differences in surface morphology were observed among the groups under high magnification (Fig. 5B), indicating that none of the cleaning methods visibly affected the surface structure of the thermoplastic material.

Fig. 5.

Surface morphology and topographical changes of thermoplastic aligner materials after 14 consecutive days of repeated cleaning. A Low-magnification SEM images. B High-magnification SEM images. C Representative 3D surface profilometry images showing surface height distribution patterns

Surface roughness

Three-dimensional surface profilometry using white light interferometry (Fig. 5C) provided a visual representation of height distribution on the measured surfaces. Quantitative analysis (Fig. 6A) reflected the surface roughness (Sa) of the thermoplastic materials after cleaning for 14 days. Compared to the control group, the TB and CSTB groups showed a statistically significant increase in surface roughness (p < 0.05), whereas no significant changes were observed in the other groups.

Mechanical properties

Mechanical properties measurements after 14 days of repeated cleaning are shown in Fig. 6B and C. No statistically significant differences in elastic modulus (F = 0.21, p > 0.05) or UTS (F = 0.11, p > 0.05) were observed among the six groups.

Fig. 6.

Surface and mechanical properties of thermoplastic aligner materials after 14 consecutive days of repeated cleaning under simulated intraoral conditions (37 ℃ artificial saliva). A Surface roughness (Sa) measured by a white-light 3D surface profiler over a square area of 874.627 × 874.627 μm (n = 10 per group). B Elastic modulus determined from the linear region of the stress–strain curve at a strain rate of 2% per minute up to 1% strain (n = 10 per group). C Ultimate tensile strength (UTS) calculated as the maximum load divided by the original cross-sectional area at 2 mm per minute until fracture (n = 10 per group). Groups with the same lowercase letter are not significantly different (p > 0.05)

Discussion

Maintaining thermoplastic aligners in a clean, odor-free, and visually acceptable state is essential for both patient comfort and treatment success. This study evaluated six commonly used cleaning methods, in which biofilm removal was quantitatively assessed using CV staining and CFU counting, and qualitatively confirmed by SEM. The results demonstrated that toothbrushing, either alone or combined with cleaning soaking, was the most effective in removing S. mutans biofilms from aligner materials. Accordingly, the first null hypothesis was rejected.

Toothbrushing remains the most accessible and widely recommended cleaning method for removable dental appliances. In our study, brushing significantly reduced both residual biomass and viable bacteria, confirming its efficacy. However, in clinical settings, thoroughly cleaning aligners remains challenging due to complex geometries such as incisal edges, cusp tips, and attachment hollows, which are difficult to access. In addition, brushing technique itself plays a key role in biofilm control [35], yet achieving thorough cleaning requires time and precision. Patients may prefer more convenient alternatives such as chemical soaking or ultrasonic cleaning. Chemical cleaning, a convenient alternative, commonly uses disinfectants (e.g., sodium hypochlorite, bicarbonate, quaternary ammonium compounds, and chlorhexidine) and commercial products (e.g., Polident^®, Retainer Brite^®, NitrAdine^®) to clean removable dental devices [31, 36–38]. The Aligner Cleaner^® used in this study contains quaternary ammonium salts, which are cationic surfactants with broad-spectrum antimicrobial activity through interaction with negatively charged bacterial membranes [39]. They have demonstrated potential in bacterial inhibition and caries prevention [40]. In this study, chemical soaking alone (CS) significantly reduced viable bacterial counts, demonstrating its antibacterial activity and potential to reduce microbial load on aligner surfaces. This effect highlights the clinical contribution of chemical cleaning to microbial control. However, substantial residual biofilm biomass remained, indicating limited efficacy in mechanical biofilm removal. This finding underscores the distinction between reducing bacterial viability and removing biofilm biomass. Although certain chemical cleaners like alkaline peroxide tablets can aid mechanical removal through effervescence [41], evidence suggests they are only partially effective against mature biofilms [37]. Residual biofilm of sufficient thickness in attachment cavities or at the incisal edge may impair aligner fit and compromise tooth movement [24], underscoring the necessity of mechanical brushing for effective biomass removal. Combining soaking with subsequent brushing (CSTB) further improved cleaning efficiency, as chemical agents can reduce cellular metabolism, weaken biofilm structure, and enhance subsequent removal [36, 37, 42, 43]. In contrast, applying chemical cleaners after brushing shows limited additional benefit [44], highlighting the importance of cleaning sequence. Although combining soaking with brushing was not statistically superior to brushing alone in this in vitro model, clinical biofilms are multispecies and exhibit complex interbacterial synergy that may enhance resistance and biomass formation [31], suggesting that combined protocols may be advantageous in practice.

Ultrasonic cleaning provides an alternative non-manual approach, which has been widely used in dentistry for its proven efficacy in instrument disinfection and plaque disruption via acoustic cavitation [23, 38, 45]. In this study, ultrasonic cleaning provided moderate biofilm reduction, an effect attributable exclusively to ultrasonic cavitation since no chemical substance was incorporated into the apparatus [46]. Notably, the addition of cleaning solution in the CSUS group further reduced viable bacterial counts compared to US or CS alone, indicating that cavitation enhances the antibacterial effect of the cleaning solution by loosening adherent biofilms. However, residual biofilm was still visible in both US and CSUS groups, demonstrating that ultrasonic bath treatment, with or without cleaning solution, did not achieve complete eradication. Unlike our findings, a previous study reported complete biofilm removal from PMMA surfaces after 30 s of ultrasonic cleaning at 26 kHz and 200 W [45]. This discrepancy may be attributed to the higher ultrasonic power, which likely enhanced biofilm removal. In addition to device power, the effectiveness of ultrasonic cleaning also depends on the cleaning medium. Some studies report enhanced biofilm removal with agents like benzalkonium chloride [23], while others found no added benefit from chemical additives compared to water [38]. Taken together, these discrepancies suggest that ultrasonic cleaning efficacy is highly dependent on multiple factors, including device power, ultrasound frequency, and the properties of the cleaning medium. While ultrasonic cleaning has potential as an adjunctive method for aligner hygiene, further optimization is essential before it can match the efficacy of mechanical brushing in clinical practice.

An effective cleaning method must not only remove biofilm but also preserve the structural and mechanical integrity of aligner materials. In this study, surface morphology was evaluated using optical profilometry and SEM, which, in combination with mechanical testing, provided a comprehensive assessment of the changes in material properties. SEM images showed that the corrugated surface topography of the material reflected the stereolithographic fabrication process of the original models used for aligner production [6]. After 14 days of repeated cleaning, no noticeable morphological changes were observed in any group, confirming that none of the methods visibly compromised the macroscopic surface integrity. However, surface roughness increased significantly in the TB and CSTB groups, likely due to microabrasions caused by brushing. Accordingly, the second null hypothesis was rejected. In this study, brushing was performed under running distilled water without toothpaste or detergent. Thermoplastic materials are susceptible to surface degradation when exposed to simulated oral environments, primarily due to water absorption and hydrolytic processes that increase surface roughness [12, 47]. Under such conditions, mechanical cleaning with a toothbrush may further exacerbate material wear, with bristle stiffness playing a notable role in determining the extent of surface alteration [48]. In addition, the choice of cleaning medium affects the surface integrity. Mild, surfactant-based solutions without abrasive or oxidising agents cause less surface roughness alteration than harsher formulations, highlighting the importance of the cleaning medium’s composition [15]. The magnitude of roughness increase observed in this study might lead to increased bacterial adhesion and biofilm formation on these aligners. Consistent with previous long-term studies on polyurethane and copolyester retainer materials, the present study found no statistically significant changes in surface roughness following exposure to the cleaning solution [49, 50]. In contrast, another study found that prolonged exposure (360 days) to chemical disinfectants increases the surface roughness of acrylic materials, which in turn promotes microbial adhesion [51]. This suggests that the 14-day cleaning simulation reflects typical clinical use for a single aligner, but cannot assess cumulative effects from repeated daily cleaning over the full course of treatment. Longer-term studies are needed to understand how routine cleaning affects aligner materials. Interestingly, previous studies have also suggested that the mechanical turbulence generated by acoustic waves and cavitation could damage both bacterial cells and thermoplastic polyurethane surfaces [23]. However, when compared with direct brushing cleaning, ultrasonic cleaning induces less mechanical damage to PETG surfaces [47]. Ultrasonic cleaning in this study did not increase surface roughness, possibly due to the relatively lower power output (24 W). This discrepancy highlights that the surface effects of ultrasonic cleaning are highly dependent on device parameters such as power and frequency, and optimization of these conditions is necessary to balance biofilm removal efficacy with material preservation.

The modulus of elasticity reflects the stiffness of a material. Aligners with a higher elastic modulus are expected to be stiffer, potentially making insertion and removal more difficult, whereas those with a lower modulus may produce insufficient force for effective orthodontic tooth movement [52]. UTS, defined as the maximum stress a material can withstand before failure, tearing, or rupture [52], together with the elastic modulus, showed no significant differences among groups after 14 days of cleaning, indicating that none of the cleaning methods compromised either the material’s resistance to elastic deformation or the ability to withstand fracture. Thus, the third null hypothesis was accepted. The finding of “no significant change” is clinically meaningful, as it demonstrates that the bulk mechanical integrity of the tested thermoplastic materials remains stable under 14-day simulated clinical use and supports the safety of commonly used cleaning methods. This stability is consistent with previous research, which reported that repeated brushing or soaking in common disinfectants did not significantly alter the flexural modulus of polyurethane materials over six months [53]. Nevertheless, stable bulk mechanical properties do not imply that the material is entirely unchanged under extended clinical use. Previous investigations [12] have shown that hydrophilicity, polymer network structure, and moisture absorption–desorption dynamics can influence dimensional stability, surface integrity, and clinical fit during use. Materials with high hydrophilicity and open polymer networks have been reported to exhibit faster moisture uptake, potentially leading to gradual dimensional changes, whereas densely cross-linked structures may restrict water ingress but show delayed desorption. These properties may also modulate the effects of chemical exposure, as materials prone to water uptake may be more susceptible to surface alterations from staining solutions or common cleaning agents. Chemical exposure can independently disrupt hydrogen bonding, alter intermolecular spacing, or promote microcrack formation [54]. In addition, surface defects have been reported to progressively worsen with material aging, presenting as increased scratches or porosities, which may facilitate bacterial retention and fluid penetration [10]. Although not evaluated in this 14-day simulation, these changes may become clinically relevant over extended use, highlighting the need for longer-term cleaning studies with surface characterization and fit assessments to balance biofilm control and material durability.

This study employed a once-daily cleaning protocol to maintain consistency with previous in vitro and clinical research [22, 35, 41, 44]. In practice, however, patients may clean their aligners more frequently. Increased cleaning frequency may enhance bacterial control but could also accelerate surface wear, and the effects of repeated chemical exposure on material properties remain unclear. Further studies are needed to determine optimal cleaning frequencies that balance biofilm removal with material preservation. In addition, this study was conducted under in vitro conditions using only S. mutans biofilms, which do not fully represent the multispecies and dynamic microbial communities found in the oral cavity. Multispecies biofilms exhibit complex interbacterial interactions and enhanced resistance mechanisms, and may therefore respond differently to mechanical or chemical cleaning methods compared with single-species models. Moreover, only Invisalign^® aligners were tested. Given the known differences in material composition, polymer structure, manufacturing processes, and multilayer configurations among aligner systems, caution should be exercised when extrapolating these findings to other thermoplastic aligner materials. Further research under clinical conditions is required, including multispecies biofilms and different aligner materials.

Conclusions

The present in vitro study compared six cleaning methods for thermoplastic aligner materials with respect to S. mutans biofilm removal, surface changes, and mechanical integrity. Toothbrushing, alone or combined with chemical soaking, was the most effective approach, significantly reducing both biofilm biomass and viable bacteria. Ultrasonic cleaning offered partial efficacy, particularly when combined with disinfectants, but failed to achieve complete eradication. After 14 days of repeated cleaning, no significant alterations in elastic modulus or tensile strength were observed, indicating preserved mechanical integrity, although brushing slightly increased surface roughness.

Overall, in the absence of more advanced alternatives, mechanical brushing, ideally supplemented by chemical disinfectants, remains the most reliable strategy for maintaining aligner hygiene without compromising material performance. Ultrasonic cleaning shows potential but requires further optimization before clinical adoption.

Abbreviations

CV

crystal violet

CFU

colony-forming unit

SEM

scanning electron microscopy

UTS

ultimate tensile strength

OD

optical density

ANOVA

analysis of variance

CTR

No cleaning treatment applied

TB

Brushing with a toothbrush for 2 min

CS

Immersion in the cleaning solution for 5 min

US

Ultrasonic cleaning in distilled water for 5 min

CSTB

Immersion in cleaning solution for 5 min and then brushing for 2 min

CSUS

Ultrasonic cleaning in cleaning solution for 5 min

Authors’ contributions

**Jun Hu** and **Jinmei Lei** conducted the entire experiment, analyzed the data and wrote the main manuscript text. **Jian Yu** designed the methodology of the study and reviewed the manuscript. **Chunxiao Jin** and **Shengjie Liang** contributed to study design and data analysis. **Jingmei Guo** contributed to the conceptual design and reviewed the manuscript. **Cui Huang** contributed to the conceptual design and provided funding support. The final manuscript was reviewed and approved by all authors.

Funding

This work was financially supported by National Natural Science Foundation of China (82271010), and Special Project of Benxi High-tech Zone, Liaoning Province for the Development of the Biomedical Industry (2023JH1/11500003).

Data availability

The data of this study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.