Introduction
The combination of mechanical and chemical cleaning methods is an effective way to remove biofilms from retainers. This study aimed to evaluate the effectiveness of household ultrasonic cleaning with chlorhexidine (CHX) in reducing microbial contamination on Essix ACE orthodontic retainers; in addition, its effects on color stability, surface roughness, and microhardness were examined.
Methods
This prospective crossover study comprised 11 orthodontic patients (3 males and 8 females), each of whom sequentially wore 4 maxillary Essix ACE retainers, with each retainer subjected to a different cleaning method: brushing with fluoride toothpaste, brushing with 1% CHX gluconate, ultrasonic cleaning with 0.12% CHX mouthwash, and ultrasonic cleaning with water. Each method was used at night for 14 days, with a new retainer introduced at each phase. Microbial contamination was assessed using ATP bioluminescence and expressed in relative light units. The properties of the retainers were evaluated by measuring the color change, surface roughness, and microhardness. Data were analyzed using the Friedman test followed by Dunn’s post-hoc analysis with Bonferroni adjustment.
Results
Ultrasonic cleaning with CHX mouthwash exhibited the lowest microbial contamination, followed by brushing with CHX, brushing with toothpaste, and ultrasonic cleaning with water. Significant differences were observed among the methods ( P <0.001): ultrasonic cleaning using water differed from all others, whereas brushing with toothpaste differed from ultrasonic cleaning with CHX. No significant differences were observed in color change, surface roughness, or microhardness across all methods ( P >0.05).
Conclusions
This study is the first to demonstrate that using household ultrasonic cleaning with CHX mouthwash is effective for maintaining Essix retainer hygiene without compromising the properties of the material.
Highlights
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Ultrasonic cleaning with chlorhexidine (CHX) mouthwash showed the lowest microbial contamination.
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Brushing with CHX gel was more effective than brushing with toothpaste.
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No significant differences were observed in color, roughness, or hardness among the cleaning methods.
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Scanning electron microscopy revealed fewer surface scratches in ultrasonically cleaned retainers.
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The first clinical study to demonstrate the efficacy of CHX–ultrasonic cleaning for Essix ACE hygiene.
Retaining the tooth position is crucial for long-term orthodontic success. Lifelong use of a retainer is typically recommended after debonding to prevent relapse. Among the available options, Hawley retainers have been widely used for decades; however, they are often associated with esthetic and speech limitations that can reduce patient compliance. In contrast, clear thermoplastic vacuum-formed retainers, such as Essix retainers, have gained popularity because of their superior esthetics, ease of fabrication, and improved comfort. Studies have shown that Essix retainers are more prone to plaque accumulation than Hawley retainers, posing a risk to oral health. The presence of biofilms on retainers can contribute to oral dysbiosis and may increase the risk of both oral and systemic conditions. , Therefore, maintaining proper hygiene of thermoplastic retainers is critical.
Although the American Association of Orthodontists recommends brushing or chemical cleaning for acrylic retainers, the optimal cleaning protocol for thermoplastic appliances remains unclear. Many household ultrasonic cleaning products on the market claim to be more efficient and powerful than manual washing for dental appliances. Ultrasonic cleaners remove accumulated microorganisms by producing ultrasonic waves (20-120 kHz) and creating microbubbles in water that detach and remove microbial attachments, a process known as cavitation. Previous clinical studies have reported that using a vibrating bath with a cleaning-crystal solution and an ultrasonic cleaner with cationic detergent is one of the most effective methods for reducing microorganisms on removable thermoplastic appliances. Therefore, the combination of mechanical and chemical cleaning methods is an effective way to remove biofilms from retainers. However, no clinical studies have evaluated the use of ultrasonic cleaners in combination with disinfectants for cleaning Essix clear retainers.
ATP is the primary energy source in living cells, and its concentration correlates directly with microbial activity. ATP detection relies on the luciferin-luciferase reaction, which emits light at 580 nm as oxyluciferin returns to its ground state. This light is measured in relative light units (RLUs) using a luminometer, in which the signal intensity is proportional to the ATP concentration. The ATP bioluminescence assay is quick, straightforward, and ideal for real-time analysis. Unlike traditional culture methods, it can detect both viable and noncultivable microorganisms, offering a broader assessment of microbial contamination. ,
Alterations in the mechanical properties of clear thermoplastic retainers can substantially impact their clinical performance and patient compliance. Esthetic concerns, such as discoloration, may reduce patients’ willingness to wear the appliance as prescribed, whereas increased surface roughness can facilitate bacterial adhesion and biofilm formation, potentially compromising oral hygiene. Furthermore, reductions in microhardness may impair the long-term durability and functional integrity of the appliance. A previous in vitro study reported that prolonged exposure of Essix retainers to various cleaning agents may reduce light transmittance, affecting esthetic transparency, while having minimal impact on surface roughness or flexural properties. However, clinical data on the impact of different cleaning methods on the color stability, surface topography, and microhardness of Essix ACE retainers are limited. Therefore, this study aimed to evaluate, for the first time, the efficacy of household ultrasonic cleaners combined with disinfectants in reducing microbial contamination, as well as to assess their effects on the color stability, surface roughness, and microhardness of Essix ACE retainers after 14 days of intraoral use.
Material and methods
This prospective cohort study with a crossover design was conducted at the Department of Orthodontics, Faculty of Dentistry, Chulalongkorn University. The study received ethical approval from the Ethics Committee of the Faculty of Dentistry, Chulalongkorn University (HREC-DCU 2024-037). Informed consent was obtained from all participants.
Sample size estimation was based on ATP bioluminescence data from Levrini et al, who compared cleaning methods and reported mean RLU values of 583 for water, 188 for toothpaste, and 71 for toothpaste with sodium carbonate/sulfate. Using G∗Power (version 3.1.9.4; Franz Faul, Universität Kiel, Germany) for 1-way analysis of variance with α = 0.05 and β = 0.2, a required sample size of 20 participants per group was determined. Considering a 10% dropout rate, an initial estimate of 44 participants was calculated. However, because of the crossover design, in which each participant received all 4 interventions, the final sample size was reduced to 11. For secondary outcomes (color, surface roughness, and microhardness), a minimum of 10 specimens per method was established based on the study by Wible et al.
Participants were adults aged >18 years with good oral health, defined as having no active caries, a plaque index of <20%, and bleeding on probing of <10%. The included subjects had well-aligned or mildly crowded dentition (crowding of <0.5 mm, with no interdental spacing). The exclusion criteria included smoking habit, drug addiction, active periodontal disease or treatment within 6 months, use of medications affecting salivary flow (eg, nonsteroidal anti-inflammatory drugs, corticosteroids, and antibiotics), presence of fixed restorations, use of removable prostheses, and a history of recent chemotherapy or radiotherapy.
Before participation, all subjects received a soft-bristled toothbrush and 1500 ppm fluoride toothpaste, with instructions to brush twice daily using the Modified Bass technique. The plaque index and bleeding on probing were assessed at baseline and at the end of the study. Maxillary Essix ACE retainers (Dentsply, Charlotte, NC) were fabricated from intraoral scans (iTero; Align Technology, Tempe, Ariz).
At the second visit, the retainers were disinfected in 1.25% Hibitane solution for 10 minutes before insertion. The participants wore the retainers full-time, except during meals and oral hygiene routines, and completed 4 sequential 14-day cleaning protocols, each performed nightly before going to bed. A new retainer was provided at the start of each phase. All participants received standardized demonstrations and supervised practice of each cleaning technique.
The cleaning methods used were as follows: (1) method I: brushing with a nonabrasive fluoride toothpaste (Relative Dentin Abrasion <100) for 30 seconds; (2) method II: brushing with 1% chlorhexidine (CHX) gluconate for 30 seconds; method III: ultrasonic cleaning with 100 mL of water for 5 minutes (Xiaomi Eraclean, 45,000 Hz); method IV: ultrasonic cleaning with 100 mL of 0.12% CHX mouthwash for 5 minutes.
All methods were followed by a 30-second rinse under tap water.
The 3M Clean-Trace Surface ATP Test Kit (3M Company, St. Paul, Minn) was used to assess the cleanliness of the retainer by detecting ATP as a biomarker of microbial contamination. Validation was performed by correlating the ATP readings with bacterial colony-forming units (CFUs) from retainers worn for 14 days by 6 participants. The inner surfaces of the retainer were swabbed and vortexed in distilled water, after which aliquots were plated on nutrient agar for 24 hours of incubation at 37°C. The ATP levels were measured by applying bacterial suspensions to ATP test swabs and analyzing them using the 3M luminometer.
For each cleaning method, the retainer surfaces were swabbed after cleaning. Subsequently, the swabs were immersed in the kit solution, shaken, and the ATP levels were immediately measured as RLUs to quantify the cleanliness.
The flat labial surface of the maxillary right central incisor was selected for the evaluation of changes in mechanical properties.
Color changes in Essix ACE retainers were assessed using a spectrocolorimeter (UltraScan PRO, HunterLab, Reston, Va) with a 4-mm port. The device was calibrated using a standard white tile. Each sample was measured 3 times at the same location, and the mean values were calculated. An unused retainer served as a baseline. The color was analyzed using the CIE Lab∗ system, and the overall color change (ΔE∗) was calculated as: ΔE∗ = [(ΔL∗) + (Δa∗) + (Δb∗) 1 ᐟ 2
The clinical relevance was determined by converting the ΔE∗ to National Bureau of Standards (NBS) units using the following formula: NBS = ΔE × 0.92∗. Color changes were classified as: trace (0.0-0.5), slight (0.5-1.5), noticeable (1.5-3.0), appreciable (3.0-6.0), much (6.0-12.0), and very much (>12.0).
The surface roughness, commonly quantified by the arithmetical mean roughness (Ra), was measured using a contact-type profilometer (Talyscan 150, Taylor Hobson, United Kingdom). A 2 μm stylus scanned each specimen perpendicularly at a speed of 1500 μm/s over a 5 × 5-mm 2 area. Five parallel scans were conducted for each sample. A Gaussian filter with a 0.08 mm cutoff was applied to remove noise. The average Ra value from the 5 scans was used to represent the surface roughness.
Surface microhardness was assessed using a Vickers microhardness tester (FM-810, FUTURE-TECH, Japan) with a 200 g of force (gf) load applied for 30 seconds. The indentation size was measured under 10× magnification, and the Vickers hardness number (HV) was calculated using the following formula: HV = 1.854 × F/ d 2, in which F is the applied load (kgf) and d is the mean diagonal length (millimeters). Each specimen was tested 3 times, and the mean HV was used for analysis.
Eight used retainers (n = 2 per cleaning method) from 2 participants were randomly selected for scanning electron microscopy (SEM) analysis. The labial surface of the maxillary left central incisor area was examined. Specimens were ultrasonically cleaned in distilled water, dried, mounted on aluminum stubs, and sputter-coated with gold (3 × 40 seconds; total of 120 seconds) using a JEOL JFC-1200 coater. Imaging was conducted using a Quanta 250 SEM (FEI company, Hillsboro, Ore) at 20 kV, with magnifications of 1000×, 5000×, and 10,000×.
Statistical analysis
Data were analyzed using SPSS (version 22; IBM, Armonk, NY). Normality and homogeneity were assessed using the Shapiro-Wilk and Levene’s tests. Because of non-normal distribution, the mean RLU, color change, Ra value, and HV value were compared using the Friedman test followed by Dunn’s post-hoc analysis with Bonferroni adjustment. Pearson’s correlation test was used to assess the relationship between bacterial counts from culture and ATP bioluminescence. The statistical significance was set at P <0.05.
Results
Six clear retainers worn over 2 weeks were used to validate ATP bioluminometer readings against bacterial colony counts (cells/mL) from swab samples, showing a strong correlation (R 2 = 0.968; Table I ).
Table I
ATP bioluminescence values (RLUs) and bacterial counts (cells/mL) obtained from the inner surface of retainers worn for 2 weeks by 6 volunteers
| Volunteers | ATP bioluminescence values (RLUs) | Bacterial counts (cells/mL) |
|---|---|---|
| 1 | 8200.00 | 230,000 |
| 2 | 3562.50 | 24,000 |
| 3 | 4900.00 | 61,000 |
| 4 | 80,425.00 | 700,000 |
| 5 | 492,962.50 | 16,200,000 |
| 6 | 134,075.00 | 440,000 |
According to the ATP bioluminescence results ( Fig 1 ; Table II ), the highest contamination occurred after ultrasonic cleaning with water (mean, 109,381.45 RLUs; median, 93,580 [95% confidence interval (CI), 39,176.95-179,585.96]; interquartile range [IQR], 99,524.5). Conversely, the lowest levels were found after ultrasonic cleaning with 0.12% CHX mouthwash (mean, 2043.55 RLUs; median, 469; 95% CI, 185.55-3901.54; IQR, 2919). Intermediate contamination levels were recorded for brushing with toothpaste (mean, 20,078.27 RLUs; median, 8520; 95% CI, 4462.43-35,694.12; IQR, 32,871) and brushing with CHX gluconate (mean, 3522.09 RLUs; median, 797; 95% CI, 304.92-6739.26; IQR, 3855.5).
Box plot showing the distribution of RLU values after cleaning with 4 different methods across 11 participants. Each box represents the IQR, with the horizontal line inside the box indicating the median value. Whiskers extend to the minimum and maximum values within 1.5 times the IQR.
Table II
RLUs values after cleaning with 4 different methods
| Method | Mean ± SD | Median (IQR) | 95% CI |
|---|---|---|---|
| I | 20,078.27 ± 23,244.47 | 8520 (32,871.0) | 4462.43-35,694.12 |
| II | 3522.09 ± 4788.82 | 797 (3855.5) | 304.92-6739.26 |
| III | 109,381.45 ± 104,500.66 | 93,580 (99,524.5) | 3,9176.95-179,585.96 |
| IV | 2043.55 ± 2765.66 | 469 (2919.0) | 185.55-3901.54 |
| Comparison | Wilcoxon statistic | Raw P value | Bonferroni-corrected P value |
| I vs II | 6 | 0.013 | 0.082 |
| I vs III | 0 | <0.000 | 0.005 |
| I vs IV | 4 | 0.006 | 0.041 |
| II vs III | 1 | 0.001 | 0.011 |
| II vs IV | 22 | 0.365 | 1 |
| III vs IV | 0 | <0.000 | 0.005 |
Note. Comparisons among cleaning methods were performed using Friedman’s test followed by Dunn’s post-hoc analysis with Bonferroni adjustment.
SD , standard deviation.
Friedman’s test showed significant differences among the methods (χ 2 = 23.73, P <0.001). Post-hoc pairwise comparisons indicated that ultrasonic cleaning with water differed significantly from brushing with toothpaste ( P = 0.005), brushing with CHX gluconate ( P = 0.011), and ultrasonic cleaning with chlorhexine ( P = 0.005). Furthermore, brushing with toothpaste differed significantly from ultrasonic cleaning with CHX ( P = 0.041).
The color change parameters (ΔE∗, ΔL∗, Δa∗, and Δb∗) and NBS units of the Essix ACE retainers across different cleaning methods are summarized in Table III . Brushing with CHX gluconate resulted in the highest color change (ΔE∗: median, 4.54; NBS, 4.17), indicating an appreciable change, whereas ultrasonic cleaning with CHX mouthwash showed the lowest change in color (median, 2.66; NBS, 2.44). The Friedman test showed no statistically significant differences in any color parameter among the cleaning methods.
Table III
Descriptive statistics and Friedman test results for color change parameters (ΔE ∗ , ΔL ∗ , Δa ∗ , Δb ∗ ) and NBS units of Essix ACE retainers across different cleaning methods
| Color parameter | Method | Median (IQR) | P value | NBS |
|---|---|---|---|---|
| ΔE ∗ | I | 3.04 (4.67) | 0.591 | 2.79 |
| ΔE ∗ | II | 4.54 (10.79) | 4.17 | |
| ΔE ∗ | III | 3.10 (8.46) | 2.85 | |
| ΔE ∗ | IV | 2.66 (4.69) | 2.44 | |
| ΔL ∗ | I | 0.55 (5.88) | 0.921 | |
| ΔL ∗ | II | 3.16 (14.00) | ||
| ΔL ∗ | III | 0.88 (9.23) | ||
| ΔL ∗ | IV | 1.02 (4.13) | ||
| Δa ∗ | I | 0.01 (0.07) | 0.602 | |
| Δa ∗ | II | −0.02 (0.19) | ||
| Δa ∗ | III | 0.03 (0.18) | ||
| Δa ∗ | IV | 0.02 (0.10) | ||
| Δb ∗ | I | 0.16 (0.11) | 0.220 | |
| Δb ∗ | II | 0.06 (0.26) | ||
| Δb ∗ | III | 0.27 (0.36) | ||
| Δb ∗ | IV | 0.44 (0.41) |
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