The complex of tannic acid and cetylpyridinium chloride: An antibacterial and stain-removal cleaner for aligners

Introduction

Effective aligner hygiene is recognized as an important part of orthodontic treatments and oral hygiene. However, there is no effective cleansing method for removable aligners.

Methods

In this study, we incorporated tannic acid (TA) with cetylpyridinium chloride (CPC) to develop the TA-CPC complex. The antibacterial properties of 15.8 mg/mL TA-CPC against Escherichia coli and Staphylococcus aureus were evaluated in vitro, which were compared with 5.1 mg/mL TA, 10.7 mg/mL CPC, a commercial denture cleansing solution (YA; 15 mg/mL), and water. As for the assessment of stain-removal ability, the aligners stained by coffee were soaked in cleansing solutions, and the color changes (ΔE∗) were calculated on the basis of the CIE L∗a∗b∗ color system, and the National Bureau of Standards system was used for the clinical interpretation of the color change. Atomic force microscope examination, tensile property assessment, and wavelength dispersive x-ray fluorescence analysis were performed to investigate the material compatibility of TA-CPC, and Cell Counting Kit-8 assay and live/dead assay were used to test the cytotoxicity of TA-CPC.

Results

The results showed that TA-CPC had a positive zeta-potential, and cation-π interaction changed the chemical environments of the phenyl group in TA-CPC, resulting in greater inhibition zones of S. aureus and E. coli than other cleaners. The quantification of the biofilm biomass and the fluorescent intensities also reflected that the TA-CPC solution exhibited better antibacterial ability. As for the ability of stain removal, ΔE∗ value of group TA-CPC was 2.84 ± 0.55, whereas those of stained aligners immersed with deionized distilled water, TA, YA, and CPC were 10.26 ± 0.04, 9.54 ± 0.24, 5.93 ± 0.36, and 4.69 ± 0.35, respectively. The visual inspection and National Bureau of Standards ratings also showed that the color of stained aligners cleansed by TA-CPC was much lighter than those of the other groups. Meanwhile, TA-CPC had good compatibility with the aligner material and cells.

Conclusions

TA-CPC is a promising strategy to inhibit the formation of biofilms and remove the stains on the aligners safely, which may disinfect the aligners to improve oral health and help keep the transparent appearances of aligners without impacting the morphology and mechanical properties.

Graphical abstract

Highlights

•

A new cleansing solution for aligners is developed by TA-CPC.
•

It is a promising strategy to inhibit the formation of biofilms and remove the stains on the aligners.
•

Aligners can maintain transparency with normal morphology and mechanical properties.

Clear aligners for treating malocclusion were initially invented by Kesling in 1946 and developed rapidly because of the innovation and breakthrough of digital design and biocompatible materials in the last 20 years. ^, These thermoplastic orthodontic appliances are made entirely of transparent plastic and are removable for meals and brushing, which offer advantages of esthetics and comfort. ^, Consequently, clear aligners attracted more adult and teenage patients with malocclusion globally.

Although these thermoplastic orthodontic appliances proved less noticeable changes to the appearance of patients, it has been reported that some potential downsides come during their use. Discoloration and stains of the aligners are typical occurrences over time, which are important clinical esthetic considerations. Some studies have reported patients are in insufficient compliance with the recommendation that clear aligners should be removed before eating and drinking staining food, such as curry, tea, and coffee. As a result, during orthodontic treatments, the staining agents from food accumulate on the aligners, changing their color and making them less aesthetically pleasing. This has become a serious clinical concern. ^,

In addition, many studies presented bacterial biofilms are localized on the surfaces of the thermoplastic aligners, especially on attachment dimples and cusp tips, which are more recessed and sheltered. ^, Increased plaque levels not only affect the esthetic aspect of clear aligners but are also a strong risk factor for developing bacteria-related diseases, such as dental caries and periodontal disease. ^, Therefore, aligner hygiene is recognized as an important part of orthodontic treatments and oral hygiene.

An appropriate method of thoroughly cleansing aligners can retard or impede the accumulation of bacteria and staining agents on the surfaces of aligners, thus keeping the color stability and transparency of aligners and reducing the risks of spreading cariogenic and periodontal pathogens in the oral cavity. According to the dental health professional recommendation, brushes are widely accepted as a cleansing method for removable appliances. However, it is difficult to remove bacterial biofilms with mechanical cleansing alone, and mechanical cleaning could increase the surface roughness of these removable appliances, which might subsequently be more prone to pigment accumulation and microbial colonization. Some commercial cleansing tablets are specifically designed to cleanse removable oral appliances. The antimicrobial action of cleansing tablets is typically based on peroxide-generating chemistry, and they also usually contain surfactants to aid cleansing. However, their stain-removal potentials are less than clinically satisfactory. Some wearers also use household bleach (sodium hypochlorite) to cleanse the removable oral appliances, which may risk damaging denture materials chemically and change the flexibility of the material. Therefore, developing an effective cleansing method to clean and disinfect the removable aligners is important.

Cetylpyridinium chloride (CPC) is a kind of quaternary ammonium compound and a cationic surface-active agent. In clinical dental practice, CPC is mainly used as an antimicrobial ingredient in products marketed for diminishing plaque accumulation, including hard-surface cleaners, mouthwashes, and toothpaste. ^, However, it has not been used for stain removal. Tannic acid (TA) is a natural antibacterial polyphenol ubiquitous in various plants, such as Chinese nutgall and unripe fruit, which is widely applied to adsorption materials, coatings, biomedical materials, and food additives. Because TA is a compound with multiple phenolic hydroxyl groups, it can chemically and physically interact with other materials, such as Michael addition, hydrogen bonds, coordination with metals, and Schiff-base reactions. In addition, TA are excellent candidates to produce biosorbents, and it is reported that TA-based adsorbents have a natural affinity to various types of dyes in wastewater.

Thus, we incorporated TA with CPC to develop a new cleansing solution and investigated its antibacterial activity, ability of bacteria and stain removal, and effects on morphology and mechanical property of thermoplastic material in the present study. It was hypothesized that TA-CPC could remove the biofilm and staining on the thermoplastic aligners more effectively and had good antibacterial activity, material compatibility, and cytocompatibility. Our results suggest a promising strategy to remove bacterial biofilms and stains on the aligners, which may reduce the bacterial infection of the aligners to improve oral health and help keep their transparent appearances.

Material and methods

TA was obtained from Aladdin Chemistry (Shanghai, China). CPC and Tris(hydroxymethyl)aminomethane (Tris) were purchased from Solarbio Life Sciences (Beijing, China). A commercial denture cleansing tablet (YA) was obtained from Yakelin (Anhui, China). The Biolon polyethylene terephthalate glycol (PET-G) sheets (Φ120 mm, δ0.75 mm) were obtained from Dreve Dentamid GmbH (Belgium, Germany), and 9-mm-diameter discs were punched from these thermoplastic material sheets in this study. The aligners made of thermoplastic materials were manufactured by Invisalign (Align Technology, Santa Clara, Calif). The aligners were split, and canine, first premolar, and second premolar were chosen as the representative teeth.

Staphylococcus aureus was a gift from the First Affiliated Hospital of Ningbo University, and Escherichia coli (ATCC 25922) was purchased from the American Type Culture Collection. Tryptic Soy Broth (TSB) and Luria-Bertani medium (LB) were obtained from Solarbio Life Sciences (Beijing, China). Phosphate-buffered saline was purchased from Phygene Life Sciences (Fujian, China). Coffee power (Pure Black Instant Coffee; UCC Coffee, Co, Ltd, Osaka, Japan) was used in this study. LIVE/DEAD BacLight Bacterial Viability Kits L13152 was obtained from Thermo Fisher Scientific (Waltham, Mass).

Bone marrow mesenchymal stem cells (BMSCs) were obtained from Cyagen Biosciences (Guangzhou, China) and cultured in Minimum Essential Media α basic culture medium (Gibco, Grand Island, NY) supplemented with 1% antibiotic solution (penicillin and streptomycin) and 10% fetal bovine serum (Gibco). Cells from passages 3-4 were used for subsequent experiments. Cell viability was detected by Cell Counting Kit-8 (CCK-8) Cell Proliferation and Cytotoxicity Assay Kit (Solarbio Life Sciences, China) and Live/Dead kit (Calcein AM/PI; KeyGEN BioTECH, Nanjing, China)

TA-CPC was synthesized via a 1-step electrostatic assembly between TA and CPC. Fifty-one mg TA and 107 mg CPC were dissolved in 10 mL of 10 mM Tris buffer (pH 8.5) at room temperature. Fifteen minutes after the reaction, the TA-CPC complex formed in the solution was collected by lyophilization. TA, CPC, and TA-CPC powders were analyzed using ¹ H nuclear magnetic resonance (NMR) (AVANCE NEO, 400 MHz, Bruker; Kontich, Belgium), using dimethyl sulfoxide-d6 as the solvent. Fourier transformation infrared (FT-IR) analysis of TA, CPC, and TA-CPC powders was performed using a Nicolet iS50 Spectrometer (Thermo Fisher Scientific). The 5.1 mg/mL TA, 10.7 mg/mL CPC, and 15.8 mg/mL TA-CPC powders were suspended in 10 mM Tris buffer (pH 8.5). The particle zeta-potential distribution of these samples and coffee solution (3 g coffee powder per 100 mL deionized distilled water [dH ₂ O]) was determined by the dynamic light scattering method (Zetasizer Nano ZS 90; Malvern Instruments Ltd, Worcestershire, United Kingdom) at 25°C.

The PET-G sheets, immersed in 10 mL of water and 15.8 mg/mL TA-CPC solution for 12 hours, were fixed on the stage respectively, and the surface topography of the samples was analyzed by Atomic Force Microscope (AFM) (Bruker Fast Analyst; Bruker) with tapping mode in air using a FastScan-b probe at 0.999 Hz scan rate for the measurements.

The mechanical property of the PET-G samples after being immersed in 10 mL of water and 15.8 mg/mL TA-CPC solution for 12 h was also tested by the Universal Testing Machine (CMT-1104; SUST, Xi’an China) as reported. Rectangular specimens (5.00 mm × 40.00 mm × 0.75 mm) of PET-G sheets were prepared for tensile tests according to International Organization for Standardization standard 527-2. Each sample was stretched until ruptured at a speed of 5 mm/min at room temperature by the Universal Testing Machine in accordance with guideline GB/T1040.3-2006. The tensile stress-strain curve was recorded, and the elastic modulus was calculated. The mean value and standard deviation of 6 test specimens were calculated for each group.

Nitrogen in the TA-CPC complex and that remaining on the surface of PET-G discs after the cleansing process was measured by wavelength dispersive x-ray fluorescence (WD-XRF) spectrometer (S8 TIGER; Bruker). The discs were immersed in TA-CPC solution (10 mL, 15.8 mg/mL) for 12 hours, and then the discs were washed with deionized dH ₂ O and dried in air. The WD-XRF spectrometry used a 4 kW Rh anode x-ray tube with a proportional flow counter and scintillation counter detectors as the energy source.

CCK-8 and live/dead assays were used to test the cytotoxicity of TA, CPC, and TA-CPC according to the manufacturer’s instructions. Briefly, BMSCs were seeded in 96-well plates (cell density of 3000 cells/well) and treated with 100 μL culture medium containing 5.1 mg/mL TA, 10.7 mg/mL CPC, and 15.8 mg/mL TA-CPC solutions, respectively, for 10 minutes. Then, 10 μL CCK-8 solution was added to each well and incubated in the dark under 5% carbon dioxide and 37°C environment for 2 hours. Cell viability was assessed by a microplate reader (Multiskan GO Microplate Spectrophotometer, Thermo Scientific) at 450 nm. Furthermore, BMSCs were seeded in 24-well plates (cell density of 5 × 10 ⁴ cells/well) and treated with 1 mL culture medium containing 15.8 mg/mL TA-CPC solutions for 10 minutes, and then the solution was removed, and cell viability was detected by the Live/Dead kit (Calcein AM/PI; KeyGEN BioTECH). The cell images were visualized and taken with a fluorescence microscope (Leica, Wetzlar, Germany). The cells treated with a culture medium were set as the controls.

S. aureus and E. coli were cultured in an appropriate medium (TSB for S. aureus and LB for E. coli ) at 37°C, shaking at 100 rpm overnight to reach the midexponential phase (optical density at 600 nm of the bacterial culture reached 0.5). Fifty microliters of the culture were transferred onto an agar culture plate to produce a bacteria lawn. Filter paper discs (diameter of 8 mm) were fully swelled in solutions of TA (5.1 mg/mL), CPC (10.7 mg/mL), TA-CPC (15.8 mg/mL), and YA (15 mg/mL), and placed onto the agar plates. The plates were incubated at 37°C for 24 hours; then, the photographs of the plates were taken, and the diameters of bacterial growth inhibition zones were measured by a Vernier digital caliper. All the measurements were performed in triplicate by the same operator. Representative graphs were shown, and the results were expressed as the mean ± standard deviation of the triplicates.

The overnight bacterial culture described above was collected and diluted 100 times using the appropriate culture medium (TSB for S. aureus and LB for E. coli ). PET-G discs (diameter of 9 mm) were sterilized by ultraviolet light irradiation for 60 minutes and co-cultured with 5 mL bacterial suspension at 37°C for 24 hours with shaking at 100 rpm. The samples were then washed with phosphate-buffered saline 3 times to remove any planktonic bacteria and then immersed in 10 mL of aqueous solutions of TA (5.1 mg/mL), CPC (10.7 mg/mL), TA-CPC (15.8 mg/mL), YA (15 mg/mL), and deionized water for 10 minutes at room temperature under static condition, respectively. Discs without intervention (pristine group) were set as control.

Biofilm was observed and quantified by crystal violet (CV) assay and scanning electron microscopy (SEM). Briefly, the samples after the biofilm formation experiment were washed with deionized dH ₂ O and fixed by 2.5% glutaraldehyde solution, followed by stepwise dehydration using 50%, 70%, 80%, 90%, 95%, and 100% ethanol for each step. For the CV assay, each sample was immersed in 1 mL of 0.5% (w/v) CV solution for staining for 15 minutes. The samples with stained biofilms were gently rinsed with water again, air-dried, and observed under microscopy (Olympus, Japan). In addition, 1 mL of 33% (v/v) acetic acid was added to each sample to solubilize the dye of CV. Optical densities of the CV-dissolved acetic acid solution were tested using a SpectraMax 190 microplate reader at 570 nm. As for SEM observation, the samples were dried in air and coated with Au by ion sputtering (MC1000; Hitachi High-tech, Tokyo, Japan). The bacteria on the surface of the discs were observed by SEM (Regulus 8230; Hitachi), and the biofilm coverage was quantified by ImageJ software (version 2.1.0; National Institutes of Health and the Laboratory for Optical and Computational Instrumentation, University of Wisconsin, Madison, Wis).

Three-dimensional (3D) structures of the biofilms and live/dead bacteria were observed by confocal laser scanning microscopy (TCS SP8, Leica). The combined dye (LIVE/DEAD BacLight Bacterial Viability Kits, L13152; Thermo Fisher Scientific) was used to stain bacteria on the surface of discs as per the manufacturer’s instruction. The samples were incubated with dye solutions in the dark for 15 minutes and washed with sterilized dH ₂ O. Microscopic observations were performed with confocal laser scanning microscopy with a 488 nm laser source and SYTO Green and propidium iodide dye channels. Three-dimensional images were reconstructed, and the means of fluorescence intensity in the SYTO Green channel and propidium iodide dye channel were quantified by Leica Application Suite X. Three replicates were assessed for each group. Representative graphs were shown, and the results were expressed as the mean ± standard deviation of the triplicates.

Liu et al reported that coffee can lead to severe stains on aligners made of thermoplastic materials. According to previous studies, coffee solution (3 g coffee powder per 100 mL boiling deionized dH ₂ O) was used. The lower aligners were immersed in 100 mL coffee solution in a water bath at 37°C for 1 day. The aligners were cleansed in water by ultrasound (40 kHz) for 5 minutes and dried in air. Then they were immersed in 100 mL solutions of TA (5.1 mg/mL), CPC (10.7 mg/mL), TA-CPC (15.8 mg/mL), YA (15.0 mg/mL), and deionized water for 10 minutes at room temperature with shaking at 200 rpm, respectively, and the stained aligners without intervention (named as pristine group) were as controls.

The color changes (ΔΕ) were characterized via the Commission Internationale de I’Eclairage L∗a∗b∗ color system (CIE L∗a∗b∗). ^, The parameters L∗, a∗, and b∗ of aligners were measured with NR110 Precision Colorimeter (3nh, Shenzhen, China). The optical sensor tip of the Precision Colorimeter contacted firmly and vertically to the labial surface of the mandibular lateral incisors of each aligner, and white paper was used as the background reference. Standard measurements were performed in the same room and were conducted by one investigator blind to the arrangement of the group. The ΔΕ value, which represents the color change, was calculated according to the formula: ΔΕ = [(ΔL∗) ² +(Δa∗) ² +(Δb∗) ² ] ^1/2 . ΔL∗, Δa∗, and Δb∗ are the subtractions of the L∗, a∗, and b∗ color parameters measured before staining (as-received aligners) and after cleansing (staining in coffee solutions for 1 day and then immersing in cleansing solutions for 10 minutes), respectively.

The National Bureau of Standards (NBS) system was used to describe the perceptible color change (a clinical interpretation) ^, :

NBS = ΔE∗ × 0.92 (Trace: NBS <0.5, the color change is extremely slight; Slight: 0.5≤ NBS <1.5, the color change is slight; Noticeable: 1.5≤ NBS <3.0, the color change is perceivable; Appreciable: 3.0≤ NBS <6.0, the color change is marked; Much: 6.0≤ NBS <12.0, the color change is extremely marked; Very much: 12≤ NBS, the color changes to other color).

Representative graphs were shown, and the results were expressed as the mean ± standard deviation of the triplicates.

Data analysis

All data were displayed as the mean ± standard deviation. Statistical analysis was evaluated using SPSS (version 15.0; SPSS, Chicago, Ill). Analysis of 2 groups was performed via a 2-tailed Student t test, and analysis of ≥3 groups was conducted by One-way analysis of variance followed by Student–Newman–Keuls post-hoc tests for multiple comparisons. P values of <0.05 were considered significant statistically.

Results

In this study, TA-CPC was synthesized via the assembly of TA and CPC ( Fig 1 , A ). Zeta potential is an important surface property of nanoparticles. It can influence the interactions between nanoparticles and other substances by electrostatic repulsion or attraction. The results of zeta-potential measurement in this study showed that TA-CPC (31.3 ± 6.1 mV) and CPC (52.2 ± 13.6 mV) had positive zeta-potential, whereas TA (−52.7 ± 9.3 mV) and coffee (−10.3 ± 0.3 mV) had negative zeta-potential ( Fig 1 , B ).