Effect of nanomicelle curcumin-based photodynamic therapy on the dynamics of white spot lesions and virulence of Streptococcus mutans in patients undergoing fixed orthodontic treatment: A randomized double-blind clinical trial

Fixed orthodontic appliances cause white spot lesions (WSLs) and tooth decay due to food impaction, restrictions on oral hygiene, and microbial plaque accumulation. WSLs of enamel adjacent to orthodontic brackets, known as precursors to the cavity, with a prevalence of 4.9% to 84%, continues to be a common side effect associated with fixed orthodontic treatment [1,2].

Streptococcus mutans is a facultatively anaerobic, Gram-positive coccus commonly found in the human oral cavity and has a significant role in tooth demineralization and dental caries via great acid production from fermentable carbohydrates. It is also found that the streptococcal glucosyltransferases (Gtfs) play critical roles in the synthesis of extracellular polysaccharides, providing sites on dental surfaces for glucan-dependent microbial biofilm formation [3,4]. Levels of S. mutans, present in saliva and dental plaques, notably are higher in patients with fixed orthodontic appliances compared to non-orthodontic patients. It has been found that the level of S. mutans is a reliable index for the risk of WSLs formation and dental caries [5]. S. mutans has been used to determine the effectiveness of the anti-caries of the different antimicrobial agents [5], [6], [7], [8], [9].

Many approaches such as the use of fluoride varnish have been suggested to prevent enamel demineralization and dental caries via the reduction of cariogenic bacteria and induction the enamel remineralization in patients with fixed orthodontic appliances [10]. However, topical applications of fluoride varnishes, have been proven to be beneficial but need frequent visits to the clinician. There are pieces of evidence of the effectiveness of the use of oral antiseptics in the control of cariogenic plaque and WSLs in patients with fixed orthodontic appliances. The application of chlorhexidine mouthwash may lead to discoloration of the tooth and causes reduced esthetics [11], [12], [13]. The use of antimicrobial mouthwashes has been used to suppress dental caries, but it never completely eliminates it, and these agents must be used at regular intervals to attain long-term effects [14].

Antimicrobial photodynamic therapy (aPDT) is a relatively modern and non-invasive therapy that has been used in the reduction of cariogenic bacteria in patients with fixed orthodontic appliances [8]. It is based on the interaction between a photoactive compound (photosensitizer), light source, and oxygen. Activation of the photosensitizer following the light irradiation leads to the production of reactive oxygen species (ROS) including superoxide, hydroxyl radical, and singlet oxygen. ROS result in significant irreversible damage to microbial cell structures. Several advantages have been reported for aPDT over conventional antimicrobial agents, such as minimal adverse effects on adjacent tissue and low risk of developing resistant bacterial strains [15,16].

Curcumin (Cur) is an organic and phenolic bioactive substance of turmeric (Curcuma longa). Cur acts as a photosensitizer due to its ability to the production of ROS following the absorption of blue light [17], [18], [19], [20]. However, the clinical application of Cur in aPDT is limited due to its poor bioavailability, hydrophobicity, and low stability. As a result, different modifications including nanomicelle curcumin (NMCur) have been designed and evaluated to overcome these limitations [21].

The advantages of the current study contain the fact that it included a new formulation of Cur, NMCur, as a photosensitizer, an RCT study, an evaluation of the changes in population and virulence of S. mutans over time, a clinical examination of the WSLs over time, and a proper consideration of statistical analysis methods. So, the current study aims to investigate the anti-virulence, and antimicrobial activities against S. mutans, as well as anti-WSLs potency of aPDT mediated NMCur in patients with fixed orthodontic appliances. The null hypothesis is that NMCur-aPDT has no in vivo antimicrobial and anti-WSLs effects in patients with fixed orthodontic appliances.

The consent form and all experimental protocols were approved by the Human Research Ethics Committee of the Tehran University of Medical Sciences (TUMS ethical code: N258.461). Written informed consent was obtained from all selected participants.

In the current RCT, subjects were randomly allocated into one of the following treatments (13 patients/group) using block randomization procedures: Group I: NMCur and no light (PS+L−; PS and L stand for photosensitizer and light, respectively), which was treated with NMCur alone; Group II: LED, light and no NMCur (PS−L+), which was treated only with LED irradiation; Group III: NMCur and light (NMCur-aPDT; PS+L+), which was treated with NMCur and LED; Group IV: VITIS® anti-caries mouthwash.

According to Fleiss Statistical Methods using Open Epi software with a confidence level of 95, and a power of 80 the required participant for each group was 12. Considering the possibility of losing some subjects, n = 13 was established [22].

The criteria for inclusion of individuals were as follows: ages between 15 and 30 years, absence of periodontal disease, having not used systemic Cur, antibiotics, and anti-inflammatory drugs 30 days before the study, having not used fluoride mouthwashes, and being under fixed orthodontic treatment. Subjects with the following conditions were excluded: patients who had the upper and lower anterior teeth with WLSs on clinical examination, patients with the edentulous prosthesis, smokers, alcoholics, presence of uncontrolled underlying and systemic diseases (e.g., hypertension, diabetes, heart diseases, and cancer), pregnant women or breastfeeding, persons with active dental caries or allergy history to Cur. Clinical examinations for detection of WSLs were performed using visual inspection according to International Caries Detection and Assessment System II (ICDAS II) guideline [22,23] (Table 1).

In the current study, the 52 patients were randomly divided into four experimental groups with 13 samples in each group by Random Allocation Software based on the selected type of blocking.

Patients were coded before sample collection at each time point so that treatment groups could not be identified before completing the analysis. During the counting of S. mutans and expression assessment of gtfB mRNA in each treatment group, an expert microbiologist was blinded to the treatment each patient received, and which patients were grouped. The clinical examinations were conducted by a well-trained orthodontist, who was blind to the types of treatment. During the data analysis, an epidemiologist analyzing the obtained data was blinded to the treatment each group received.

The counting of S. mutans (CFU/mL), gtfB mRNA expression (fold change), and the number and dynamics of WSLs were measured as outcomes.

A total of 71 patients were recruited from the Dental School of Shahed University in Iran, 11 were excluded based on the study criteria, and 8 of which refused to participate, totaling 52 patients. Two patients refused orthodontic treatment after bonding the lower and upper arches, with 48 participants (12/each group) analyzed. The CONSORTguide [24] for RCT was followed for the study design (Fig. 1).

One month before the start of the treatment, all participants received oral hygiene instructions per the correct toothbrushing with non-fluoridated toothpaste. Patients in the NMCur group received one gelatin capsule of NMCur (Exir Nano Sina Company, Tehran, Iran) containing 80 mg of 10 nm curcumin orally daily 10–15 min after breakfast for four months. In patients of LED and NMCur-aPDT groups (Group II and Group III, respectively), after opening the mouth with a cheek retractor the labial surfaces of the upper and lower anterior teeth were dried with compressed air for 10 s, and then the blue light was applied as follows: for the LED group, buccal surfaces of teeth were irradiated for 180 s using commercial bleaching LED device with four blue LEDs (450±10 nm; optical power output of 200 mW; and power intensity of 80 mW/cm2) for 180 s (energy dose of 14 J/cm2). Patients of the NMCur-aPDT group received LED following NMCur as described in NMCur and LED groups, respectively. Patients of the VITIS® group used 5 ml of VITIS® anti-caries mouthwash (Dent-O-Care, London, UK) for one minute twice daily for four months. VITIS® anti-caries mouthwash contains hydroxyapatite (0.0125%), xylitol (3.33%), and sodium monofluorophospate (0.172% [226 ppm]).

At predetermined time intervals (on days 0 [T1; before bonding as a control], 30 [T2], 60 [T3], 90 [T4], and 120 [T5]), samples were collected using a sterile swab drawn on the area of the brackets and the tooth of the upper and lower anterior teeth.

Following sampling, the two fold-serial dilutions were prepared, and 10 μL of each dilution was inoculated onto brain heart infusion (BHI) agar (Merck, Germany). The plates were incubated at 37 °C under 5% CO2 for 24 h and CFUs/mL were calculated based on the previous study [25].

The total RNA of collected S. mutans from treated patients was obtained using a super RNA extraction Kit (AnaCell, Iran). 150 ng of total RNA was reverse transcribed to cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific GmbH, Germany) based on instructions of the manufacturer. qRT-PCR was then down on Line-GeneK Real-Time PCR Detection System (Bioer Technology, Hangzhou, China) using SYBR® Premix Ex Raq™ II (TliRNaseH Plus; Takara, Korea). The gtfB and 16S rRNA (as housekeeping) forward and reverse primers sequences were as follows: gtfB-forward 5́-TGTTGTTACTGCTAATGAAGAA-3́ and gtfB-reverse 5́-GCTACTGATTGTCGTTACTG-3́; 16S rRNA-forward 5́-GCAGAAGGGGAGAGTGGAAT -3́ and 16S rRNA-reverse 5́-GGCCTAACACCTAGCACTCA-3́. The conditions of thermal cycling were one cycle at 95 °C (5 min) followed by 40 cycles at 95 °C (10 s), 54 °C (10 s), and 72 °C (30 s).

In the current study, clinical examinations were conducted by a well-trained orthodontist, who was blind to the types of treatment. The naked eye method (visual inspection) was used for the detection of WSLs on the labial surface in four areas (i.e., gingival, incisal, mesial, and distal) of the upper and lower anterior teeth, which is clinically relevant, more feasible and used in most of the studies [26]. Identified WSLs with visual inspection using magnifying loupes and the aid of a dental mirror, triplex syringe, artificial light, and a ball‐ended probe on wet and dried debris‐free teeth, were recorded [27]. Since visual inspection has a limited ability to assess the dynamics of WSLs, laser fluorescence (LF) detection using a DIAGNOdent device was used as a method for the monitoring of the dynamics of WSLs, as was used in vivo by Ferreira et al. [28]. Briefly, before scanning, each surface was blow-dried using compressed air for 5 s. The LF scanning device was calibrated according to the manufacturer's instructions. The device was disinfected between the scans. The LF values in every scan were registered on a computer program designed for this study. The LF scanning was repeated in the same manner in each follow-up period.

The data were analyzed through SPSS version 26 software and the significant P-value was set to less than 0.05. The normality assumption of the obtained data was tested using the Shapiro-Wilk test. Since the data was normally distributed the repeated measures ANOVA was used to compare the mean of CFU/mL of S. mutans in the four treatment groups through the four subsequent measurement time points. Mauchly's test was used to evaluate the sphericity of the dependent variable (CFU/mL of S. mutans) and the Bonferroni test was applied for pairwise comparisons. The gtfB mRNA expression analysis raw Cp values following each treatment were normalized to the 16S rRNA as housekeeper gene using an analysis of covariance method before relative fold changes over baseline (T0) were calculated using 2−ΔΔCT (Livak and Schmittgen) method [29]. The WSLs data of surfaces including gingival, incisal, mesial, and distal in each treatment group during the different follow-up times were analyzed using Fisher's exact test.

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