Background This study is part of a doctoral research project at Universidad Cardenal Herrera CEU (Spain), directed by Prof. Santiago Arias de Luxán and conducted by doctoral candidate Shirli Kelmendi within the PhD program in Translational Medicine.

Fixed orthodontic appliances complicate oral hygiene by creating retention areas that favor bacterial colonization and alter microbial balance. These conditions increase plaque accumulation and Streptococcus mutans (S. mutans) proliferation in saliva and plaque. The frequent low-pH environment favors aciduric bacteria such as S. mutans and lactobacilli, promoting enamel demineralization and formation of white spot lesions (WSLs) or cavitations. WSLs appear as opaque white areas due to subsurface mineral loss, mainly in the gingival third of the crown. They may develop as early as one month after bracket placement, while in patients without appliances, progression occurs after at least six months. Increased S. mutans levels have been reported as early as six weeks after treatment start. Risk factors include poor brushing, lack of floss or rinse use, time since last cleaning, and presence of caries or lesions.

Intervention MI Paste Plus (GC, Japan) is a remineralizing cream with 0.20% sodium fluoride (900 ppm) and 10% CPP-ACP (RECALDENT™), providing calcium and phosphate stabilized by casein phosphopeptides. It has antibacterial and remineralizing effects, suitable during or after orthodontic treatment to prevent or reduce WSLs.

Objective To evaluate whether MI Paste Plus during fixed orthodontic treatment reduces S. mutans counts in saliva and/or WSL incidence.

Study Design A prospective, triple-blind, randomized clinical trial, approved by the Ethics Committee of the Ministry of Health of Albania and the Ethics Committee for Human Research of Universidad Cardenal Herrera CEU, Spain.

The study will include 200 patients (100 per group) from two orthodontic clinics in Tirana, Albania. Participants will be stratified by age, risk level, and appliance type, then randomized by third parties.

Outcome Measures Primary variables: S. mutans counts in saliva and number of WSLs after 3 months.

Standardized saliva collection, culturing, and bacterial quantification ensure consistency. Clinical assessments will be performed at 1 and 3 months using QRay Cam Pro (Inspektor Systems, Netherlands) for quantitative fluorescence and ICDAS for visual inspection.

Data will be analyzed using SPSS/R Commander software.

Fixed orthodontic appliances are among the most common treatments in contemporary dentistry, yet they significantly modify the oral environment and create plaque-retentive areas that favor bacterial colonization. Brackets, ligatures, and auxiliary components increase the difficulty of oral hygiene and shift the ecological balance of the dental biofilm, favoring aciduric and acidogenic organisms such as Streptococcus mutans (S. mutans). Repeated exposure of dental plaque to a low pH inhibits acid-sensitive species and promotes S. mutans and lactobacilli, whose metabolic activity produces lactic acid and leads to enamel demineralization. Clinically, this presents as white-spot lesions (WSLs), the earliest visible stage of dental caries. Such lesions may appear within one month of appliance placement, whereas comparable demineralization in patients without orthodontic devices usually requires six months or longer. WSLs compromise esthetics and tooth integrity and may persist after orthodontic treatment ends, making early prevention essential.

Fluoride remains the most validated anti-caries agent, and the combination of fluoride with casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) enhances remineralization by maintaining calcium and phosphate in bioavailable form. MI Paste Plus (GC Corporation, Japan) is a topical cream containing 10% CPP-ACP and 0.20% sodium fluoride (≈900 ppm F-), which releases calcium, phosphate, and fluoride ions that penetrate enamel, support remineralization, and inhibit bacterial ATPase activity. Although promising, further rigorously designed, adequately powered randomized clinical trials are needed to determine whether MI Paste Plus reduces salivary S. mutans levels and prevents WSLs during early orthodontic treatment. This doctoral research project aims to fill this gap.

The main objectives are to determine whether daily use of MI Paste Plus alters salivary S. mutans counts after one and three months of fixed orthodontic treatment and whether it prevents or reduces the incidence and severity of WSLs during the same period. Secondary objectives include assessing the relationship between oral hygiene risk status and baseline S. mutans levels or WSL prevalence, and examining whether age, hygiene risk, or appliance type modifies these outcomes. Null hypotheses state that MI Paste Plus does not significantly affect S. mutans levels or WSL development and that age, hygiene level, and appliance type do not modify these results.

This prospective, triple-blind, parallel-group randomized controlled clinical trial will take place in two private orthodontic polyclinics in Tirana, Albania. Ethical approval has been obtained from the Ministry of Health of Albania (Report No. 66/18, April 4, 2024) and from Universidad Cardenal Herrera CEU (May 12, 2025). All procedures comply with the Declaration of Helsinki, Good Clinical Practice, GDPR, the Spanish LOPD, and Albanian data protection law.

A sample of 200 patients (100 per group) will provide >90% power at α = 0.05 to detect moderate between-group differences, allowing for 10% attrition. Eligible participants will be 5-45 years old, beginning fixed orthodontic treatment, able to comply with procedures, and providing informed consent or assent. Exclusion criteria include systemic conditions affecting salivary flow, antimicrobial use within four weeks, allergies to milk proteins or fluoride, extensive restorations preventing enamel evaluation, or inability to attend follow-ups.

Participants will be stratified by age (≤15 or >15 years), oral hygiene risk (good or poor), and appliance type, then randomized 1:1 using a computer-generated list prepared by the study statistician. Allocation concealment will use sealed opaque envelopes and coded containers prepared by an independent hygienist. Test and placebo gels are identical in appearance, texture, and packaging. Participants, clinical evaluators, and microbiologists will remain blinded until data lock.

The test group will apply MI Paste Plus nightly for three months after toothbrushing. The control group will apply an inactive bioadhesive gel in the same manner. All participants will continue brushing with fluoridated toothpaste. Adherence will be monitored by daily logs and weighing returned containers.

Assessments occur at baseline (T0), one month (T1 ± 7 days), and three months (T2 ± 14 days). Clinical evaluation of WSLs will be performed using ICDAS. Teeth will be cleaned and dried for approximately five seconds using compressed air to enhance visibility of early changes. A dental operating light, mirror, and blunt probe will be used to classify surfaces from 0 to 6. Surfaces with ICDAS 0 at baseline will be considered sound; those that become ICDAS 1 or 2 at follow-up will be classified as new WSLs. For surfaces with ICDAS 1-2 at baseline, changes over time will indicate progression, stability, or improvement.

Quantitative Light-Induced Fluorescence (QLF™) imaging using the Q-Ray Cam Pro (Inspektor Systems, Netherlands) will complement ICDAS. QLF detects demineralized enamel as dark areas due to fluorescence loss and identifies porphyrin-producing bacterial activity as red/orange fluorescence. White-spot analysis will be performed in the software by activating the White Spot Analysis wizard. The system automatically generates several quantitative parameters, including ΔF (% fluorescence loss), ΔF max, ΔF Average, lesion area (WS Area), and porphyrin fluorescence metrics (ΔR, ΔR max, ΔR area). Although all parameters support clinical interpretation, only ΔF (%) will serve as a registered outcome measure. Negative ΔF values indicate demineralization, while changes in ΔF, WS Area, and ΔR patterns over time allow detection of new lesions and assessment of whether existing lesions progress, stabilize, or remineralize.

Stimulated saliva samples will be collected at least two hours after eating or brushing, refrigerated at 4 °C, and processed within 24 hours. Serial dilutions will be plated on TYCSB medium and incubated anaerobically for 72 hours. Colonies will be subcultured, identified using API 20 Strep, and quantified using standard CFU calculations. Two microbiologists will count plates independently.

Primary outcomes include salivary S. mutans counts and the presence, number, and severity of WSLs based on ICDAS and QLF ΔF values at T1 and T2. Secondary outcomes include plaque index, DMFT/DMFS, and associations with age, hygiene risk, and appliance type. Analyses will follow the intention-to-treat principle. Appropriate parametric or nonparametric tests and mixed-effects models will be used. Missing data will be addressed using multiple imputation.

All data will be recorded on paper CRFs and double-entered into a secure database. Calibration of ICDAS scoring, QLF imaging, and microbiological procedures will occur every six months. Adverse events are expected to be minor. Participants may withdraw at any time. Data confidentiality will follow GDPR, LOPD, and Albanian privacy regulations.

The preparatory phase is 90% complete, including protocol finalization, equipment procurement, staff training, and validation of all clinical and laboratory procedures. Recruitment will occur from November 2025 to June 2027, followed by analysis and dissemination. Results will be submitted to peer-reviewed journals in orthodontics, preventive dentistry, and microbiology, and presented at major conferences. A plain-language summary will be provided to participants, and anonymized datasets may be shared upon request under data-sharing agreements.

Inclusion Criteria:

• Age between 5 and 45 years.
• Indication for fixed orthodontic treatment, either with fixed auxiliary appliances (including orthopedic devices) or brackets with or without auxiliary components.
• General good health with no systemic diseases affecting oral health.

Exclusion Criteria:

• Advanced white-spot lesions with untreated dentin involvement.
• Presence of active untreated dental caries at baseline.
• Antibiotic therapy within the previous two months.
• Previous diagnosis of molar-incisor hypomineralization (MIH).
• History of immunosuppression.
• Iron-deficiency anemia or other clinically relevant hematological disorders.
• Parafunctional habits such as lip sucking or finger sucking.
• Use of any type of dental prosthesis.
• Smoking or tobacco use.
• Documented allergy to nickel.
• Requirement for orthodontic treatment using removable appliances or clear aligners.

Individual participant data will not be publicly available to protect patient privacy, but de-identified data may be provided by the corresponding author upon reasonable request after publication.

The study is conducted under a triple-blind design. Participants, investigators, and outcome assessors remain unaware of group assignments. Both MI Paste Plus and placebo gels are identical in packaging, texture, and color and are supplied in coded opaque containers prepared by a dental hygienist not involved in patient evaluation or data analysis. The randomization code will be kept sealed until the completion of data collection and statistical analysis.