Efficacy of peptide-based enamel coatings in the prevention of demineralization using fixed orthodontic brackets in a rat model

Introduction

White spot lesions (WSLs) represent a prominent pathology encountered during orthodontic treatment, originating from enamel demineralization induced by the accumulation of bacterial biofilms. The previously developed bioinspired enamel coating form of self-assembling antimicrobial peptide D-GL13K exhibited antimicrobial activity and enhanced acid impermeability, offering a potential solution to prevent demineralization. The primary aim of this investigation is to assess the in vivo anti-demineralization properties and biocompatibility of the D-GL13K coating.

Methods

A rat model was developed to assess the antimicrobial enamel coating during fixed orthodontic treatment. The anti-demineralization efficacy attributed to the D-GL13K coating was evaluated by employing optical coherence tomography, Vickers microhardness testing, and scanning electron microscopy. The biocompatibility of the D-GL13K coating was investigated through histologic observations of vital organs and tissues using hematoxylin and eosin.

Results

The D-GL13K coating demonstrated significant anti-demineralization effects, evidenced by reduced demineralization depth analyzed through optical coherence tomography and enhanced Vickers hardness than in the noncoated control group, showcasing the coating’s potential to protect teeth from WSLs. Scanning electron microscopy analysis further elucidated the diminished enamel damage observed in the group treated with D-GL13K. Importantly, histologic examination of vital organs and tissues using hematoxylin and eosin staining revealed no overt disparities between the D-GL13K coated group and the noncoated control group.

Conclusions

The D-GL13K enamel coating demonstrated promising anti-demineralization and biocompatibility properties in a rat model, thereby suggesting its potential for averting WSLs after orthodontic interventions. Further research in human clinical settings is needed to evaluate the coating’s long-term efficacy.

Highlights

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A rat model was developed to assess the demineralization in the presence of fixed orthodontic brackets.
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The enamel coating exhibits excellent anti-demineralization properties and biocompatibility.
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The coating provides a new strategy for preventing white spot lesions after orthodontic treatment.

With the improvement of people’s living standards and the development of dentistry, orthodontic treatment has progressively gained widespread acceptance. Since the invention of the edgewise appliance by Dr Angle in 1928, orthodontists have been continuing to improve the appliances to accurately align teeth with good orthodontic results and long-term stability. Nevertheless, the use of fixed orthodontic appliances poses a challenge to achieving optimal oral hygiene, as it introduces impediments to standard oral care procedures and fosters conditions favorable for bacterial growth, ^, which subsequently leads to demineralization, manifesting as white spot lesions (WSLs). Patients undergoing orthodontic treatment exhibit a significantly higher incidence of WSLs than those without orthodontic treatment. Without preventive measures, enamel demineralization may affect 50%-80% of orthodontic patients with fixed appliances. WSLs not only compromise the esthetic integrity of anterior teeth but also elevate the susceptibility to dental caries.

An array of studies has focused on preventing demineralization during orthodontic treatment. One effective countermeasure against WSL formation is the use of fluoride because of its ability to integrate into the enamel and transform hydroxyapatite into fluorapatite. Various fluoride-containing orthodontic bonding agents have been reported to reduce demineralization. However, the presence of fluoride in the bonding agents may adversely affect the bonding strength of the brackets. Brackets with antimicrobial nanocoatings or coatings that reduce surface roughness have been shown to minimize bacterial build-up and enamel demineralization, but these coatings are unstable or not cost-effective, rendering their clinical applications. ^, Thus, we have proposed an alternative strategy by direct coating tooth surfaces to protect the enamel from demineralization during orthodontic treatment.

In our previous work, we formulated an enamel coating employing the self-assembled nanofibers of amphiphilic peptide D-GL13K and demonstrated the capabilities of inhibiting demineralization caused by orthodontic treatment in vitro. The anti-demineralization effect was attributed to the coating’s antimicrobial properties and its ability to mitigate acid permeability. Nonetheless, the in vitro study imposed limitations regarding the peptide’s efficacy within the intricate oral milieu. Factors such as saliva composition, flow rate, pH, and the presence of multispecies bacteria could potentially influence the outcomes of the anti-demineralization coating. Therefore, before the evaluation of the D-GL13K coating in clinical trials, a rigorous evaluation of its efficacy and safety in an animal model is imperative. Notably, there is currently a dearth of research pertaining to the anti-demineralization effect within an animal model throughout the fixed orthodontic treatment period.

The primary objectives of this study encompassed (1) establishing an animal model for assessing the peptide enamel coating during fixed orthodontic treatment, (2) investigating the anti-demineralization properties of the D-GL13K coating, and (3) evaluating the biocompatibility of the D-GL13K coating. We hypothesized that the D-GL13K coating can confer anti-demineralization properties to enamel while exhibiting good biocompatibility.

Material and methods

D-GL13K with all D-amino acids (Gkiiklkaslkll-NH2, purity >98%; Bankpeptide Biological Technology Co, Ltd) was initially dissolved at a concentration of 100 mg/mL in deionized water and subsequently diluted to 5 mg/mL using a 0.1 M sodium carbonate buffer solution (pH = 9.8).

Streptococcus mutans (CGMCC 2500) was inoculated onto a sterile brain-heart infusion (BHI) (HB8297-5; Hopebio, Qingdao, China) agar plate and incubated in an anaerobic chamber containing 5% carbon dioxide at 37°C for 48 hours. Subsequently, 3-5 colonies were picked and cultivated in BHI broth in the anaerobic incubator at a speed of 150 rpm until an OD ₆₀₀ of 0.20. The S mutans solution was then diluted to an OD ₆₀₀ of 0.02 in BHI.

The animal procedures employed in this study underwent thorough review and received approval from the Ethics Committee of The Affiliated Stomatological Hospital of Nanchang University, China. Specific pathogen-free, 7-week-old female Sprague Dawley rats (Hunan Slacker Kingda Experimental Laboratory Animal Co, Hunan, China) were housed individually, and the experiment commenced after a 3-day acclimatization period of feeding.

Rats were randomly divided into 2 groups of 4 rats each, consistent with previous study. In addition, they were given ad libitum access to a high carbohydrate diet, Keyes 2000 (sucrose, 56%; skim milk powder, 28%; whole wheat flour, 6%; brewer’s yeast, 4%; powdered alfalfa, 3%; whole liver powder, 1%; sodium chloride, 2%), along with drinking water supplemented with sucrose (8%, w/v). After administering 4000 μg/mL of penicillin in the drinking water for 3 days, the rats underwent off antibiotics for 1 day. Then, the rats’ mouths were swabbed with sterile cotton swabs and cultured on mitis salivarius-bacitracin agar plates anaerobically to confirm the absence of endogenous S mutans . After that, the animals were infected with S mutans using individual cotton swabs immersed in 1 mL of bacterial inoculum twice a day. This infection procedure was repeated on the after 2 days.

Human premolars extracted for orthodontic purposes were promptly rinsed with normal saline after extraction. Teeth exhibiting smooth enamel surfaces, normal color, no cracks, no demineralization signs, no chalky spots, and no visible defects were selected for experimentation. Enamel specimens approximately 1 mm thick were obtained from the buccal surfaces of the teeth, and a small orifice was drilled near the tip of each tooth using a high-speed tungsten steel ball drill. The samples were categorically assigned into 2 distinct groups: the noncoated enamel group (denoted as the control group) and the D-GL13K coated enamel group. All samples were etched with 35% phosphoric acid for 20 seconds (Gluma Etch; Kulzer GmbH, Hanau, Germany), rinsed with an air and water spray for 20 seconds, and gently air-dried for 20 s before use. For the control group, standard straight-wire metal brackets (MBT; Shinye, Zhejiang, China) were bonded to the center of the crown using a hydrophobic Transbond XT primer and adhesive bonding system (3M Unitek, Monrovia, Calif) and light cured (ZMN/WI-10-605 V1.1; Woodpecker) for 20 seconds. For the experimental group, before bonding the metal brackets, the samples were coated with the meticulously prepared D-GL13K solution. This coating application was performed using a brush after a thorough dehydration step with 75% ethanol and air-drying for 20 seconds.

The rat model is delineated in Figure 1 . The rats were fully anesthetized using 7% chloral hydrate at a standard intraperitoneal injection of 1 mL/100 g body weight. After securing the anesthetized rat in a surgical splint, the oral cavity was accessed using a custom-designed rat mouth opener (Chinese Utility Model Patent, ZL 202222163326.9; Figs 2 , A and B ). After sterilization with iodine, a meticulous procedure ensued involving the use of a low-speed ball drill to create a perforation in the basal bone situated between the 2 mandibular central incisors of the rat under saline. Subsequently, a 0.025-mm ligature wire was threaded through the perforation to secure the enamel samples and the basal bone together, and then the wire was anchored to the lingual side of the mandibular anterior teeth using resin (RelyX Ultimate; 3M) and cured with light for 60 seconds. Bleeding was noted during the procedure, and obstruction of the airway should be avoided ( Fig 2 , C ). Then, the D-GL13K solution was added to the rat’s molar by the cotton swabs in the D-GL13K coating group for the after cytotoxicity test. After completion of the procedure, Keyes 2000 cariogenic food and sucrose water were fed for 1 month. The samples were extracted for subsequent experiments, and the rats were killed via an overdose of anesthetic. Their hearts, livers, spleens, lungs and kidneys, and submandibular glands were used to assess the biocompatibility of D-GL13K.

The enamel demineralization was evaluated with a spectral domain optical coherence tomography (OCT) system (Telesto-II; Thorlabs, Newton, NJ) characterized by a central wavelength of 1300 nm and an A-scan rate ranging 5.5-76 kHz. The axial resolution, imaging depth, and signal-to-noise ratio were 5.5 μm, 3.5 mm, and 111 dB, respectively. Each sample was analyzed by 2-dimensional OCT scans in 3 randomly selected regions around the brackets and 3 randomly selected regions in the bonding area to calculate the average demineralization depth.

The hardness of the enamel samples was executed with a Vickers microhardness tester (Wilson VH1202; Buehler, Lake Bluff, Ill) at a weight of 50 g and a dwell time of 15 seconds. For each sample, the average value of Vickers hardness was calculated from 5 randomly oriented indentations on the enamel surface.

Samples after Vickers microhardness testing were sputter-coated with gold and examined using scanning electron microscopy (SEM) (SU8100; Hitachi, Tokyo, Japan). SEM images were obtained at 2-10 μm from the adhesive area.

To evaluate the potential cytotoxicity of the D-GL13K coating, histology was conducted on the hearts, livers, spleens, lungs, kidneys, and submandibular glands of the rats. The harvested organs underwent fixation in 4% buffered paraformaldehyde, followed by embedding in paraffin and subsequently stained with hematoxylin and eosin (HE). The processed samples were scrutinized using an inverted microscope (Leica Microsystems, Wetzlar, Germany).

Statistical analysis

Statistical analysis was conducted using SPSS (version 19.0; IBM, Armonk, NY). The normality and homogeneity of variances were assessed through the Kolmogorov-Smirnov test and Levene’s test, respectively. Analysis of variance with the Dunnett T3 test was employed to analyze the demineralization depth values from the OCT tests. Analysis of variance with the Tukey test was performed for the Vickers microhardness values. The statistical significance level was set at 0.05.

Results

Throughout the treatment period, the body weights of rats from both groups exhibited stability. There were no fatalities during the study; however, instances of lethargy were observed in rats from both groups, potentially associated with a chronic craving for sweet foods and the presence of a foreign body affecting their feeding process ( Fig 2 , D ). The absence of swelling or other oral mucosal diseases indicated that the peptide under investigation did not cause any apparent toxicity.

Figure 3 ( A and B ) shows the D-GL13K coatings significantly inhibited enamel demineralization after 1 month of in vivo incubation as compared with the noncoated control group, as evidenced by the significantly reduced demineralization depths ( Table ).