Preparation of microbial suspension.

A. actinomycetemcomitans ATCC 29,523 was cultured in brain–heart infusion (BHI) broth and incubated under microaerophilic conditions at 37 °C for 48 h in a 15 mL plastic tube. For bacterial growth, a bacteriological incubator maintaining an atmosphere enriched with 5–10% CO2 was employed, achieved using the candle flame technique. A candle was fixed at the bottom of a hermetically sealed container, and the flame was lit and monitored until extinguished. The operation of this system involves the removal of oxygen (O2) in the flame, producing carbon dioxide (CO2) [19, 21].

After the incubation period, the bacterial suspension was centrifuged at 876 ×g for 5 min to remove the supernatant and subsequently resuspended in 8 mL of phosphate-buffered saline (PBS). The suspension was homogenized by vortexing. To reduce the bacterial concentration, 150 µL of the resulting inoculum was transferred to another 15 mL plastic tube containing 8 mL of PBS, thereby producing the standard suspension. The bacterial concentration was visually adjusted in PBS to approximately 2 × 10⁸ CFU/mL, corresponding to the 0.5 McFarland standard [4, 19, 21, 22].

2.1 Preparation of bovine samples

This study was approved by the Animal Ethics Committee (CEUA) of Universidade Nove de Julho (UNINOVE), under protocol number 3,956,031,022. All animal procedures were conducted in accordance with the guidelines of the National Council for the Control of Animal Experimentation (CONCEA), Brazil [23].

A total of twenty-four roots of bovine incisors, obtained from a certified slaughterhouse (CNPJ 33.837.015/0001–68, 1147 Doutor Jose Ranieri Street, São Paulo, Brazil), were used. The samples were accompanied by an official invoice (No. 000.000.109), which certifies their legal origin and compliance with animal welfare and ethical sourcing regulations. Each root was sectioned into 2 × 4 mm segments using a carborundum disc. After the preparation of these samples, they were placed in a jar containing 250 mL of distilled water in a 5% CO₂ atmosphere at 37 °C for 48 h. Although this study does not involve live animals, it was conducted in accordance with the principles outlined in the ARRIVE guidelines [24], ensuring transparency and ethical standards in the use of biological samples.

2.2 Preparation of the photosensitizer

Pure BuTBO was produced as in previous work [22]. The stock solution was made by dissolving the powder in deionized water to achieve a concentration of 10,000 µM. This solution was filtered through a sterile 0.22 μm membrane (Millipore, SP, Brazil) and stored in plastic tubes, protected from light. For the experiment, a plastic tube was prepared containing 1 mL of PBS and 10 µL of butyl toluidine blue O, resulting in a final concentration of 100 µM, based on BuTBO concentrations previously reported in the literature [19, 21].

2.3 Irradiation source

The light source used to excite the photosensitizer was a diode laser (Photon Lase III, DMC, São Carlos, Brazil) with the parameters outlined in Table 1 [19, 21].

Table 1 Dosimetry of the laser light source used in the experiments2.4 Growth of microbial biofilm on dental samples

Sterile and hydrated bovine root samples were placed on a 48-well plate, with each specimen allocated to an individual well. The surfaces were conditioned with Kin Hidrat® artificial saliva spray (Laboratorios Kin S.A., Diagonal 200, Barcelona, Spain) for 2 h, which resulted in the formation of a conditioning film, consistent with in vitro conditions rather than an in vivo acquired pellicle. After removing the artificial saliva, the samples were incubated with the bacterial inoculum prepared in BHI broth (HiMedia Laboratories Pvt. Ltd., Mumbai, India), which provided the protein content necessary for biofilm formation and A. actinomycetemcomitans adhesion and [25,26,27]. The inoculum contained approximately 10⁵ CFU/mL of the ATCC 29,523 strain, standardized using the McFarland scale. The incubation was conducted for 48 h under microaerophilic conditions, generated using the candle flame technique, creating an atmosphere with 5 to 10% CO₂.

Although this approach does not reproduce an in vivo salivary acquired pellicle, pilot stereomicroscopy and SEM analyses unequivocally demonstrated that the method reliably enabled A. actinomycetemcomitans adhesion and early biofilm formation, confirming the feasibility and validity of this in vitro biofilm model.

2.5 Sample groups

The samples were divided into six groups:

Ctrl - samples with biofilm that received no treatment;

Light – treated with 5 min of irradiation without a photosensitizer;

0 – exposure to BuTBO at 100 µM for 1 min without light irradiation, representing the pre-irradiation period;

PDT1 - exposure to BuTBO at 100 µM for 1 min followed by 1 min of irradiation;

PDT3 - exposure to BuTBO at 100 µM for 1 min followed by 3 min of irradiation;

PDT5 - exposure to BuTBO at 100 µM for 1 min followed by 5 min of irradiation.

2.6 Microbiological assessment

After 48 h of biofilm formation on the bovine samples, they were washed three times with 1 mL of PBS to remove floating microorganisms, ensuring suitable conditions for evaluating treatment efficacy and minimizing interference in result analysis.

The samples were placed on a 24-well plate with 1 mL of PBS, each well corresponding to an experimental group: Ctrl, Light, 0, PDT 1, PDT 3, and PDT 5, performed in quadruplicate.

After each intervention, the samples were placed into plastic tubes and homogenized using a vortex for 30 s.

A 96-well U-bottom microplate was prepared by adding 180 µL of PBS to each of 30 wells. Then, 20 µL from each experimental group tube was transferred to the corresponding wells. Next, 20 µL from the first well was pipetted and transferred to the next well, repeating the process to achieve serial dilutions from 10⁻¹ to 10⁻⁵.

Aliquots of 10 µL from each dilution were plated in duplicate on BHI agar plates, which were incubated anaerobically at 37 °C for 48 h to allow colony-forming unit (CFU) formation. CFUs were then counted for analysis. The experiment was repeated three times to ensure reproducibility.

2.7 Scanning electron microscopy

For SEM analysis, 24 bovine dental samples were prepared as described previously. After each group intervention, the specimens were fixed in 2.5% glutaraldehyde for 1 h at 25 °C, followed by secondary fixation in 1% osmium tetroxide and dehydration in sequential ethanol concentrations (50%, 60%, 70%, 80%, 90%, and 100%). Microscopic evaluations were performed on the Control, PDT3, and PDT5 groups, where statistically significant results were observed following microbiological assessment [4]. The cells were then coated with a thin layer of gold and analyzed using scanning electron microscope SEM (FEI Quanta FEG 250, Hillsboro, Oregon, United States). Images were obtained at magnifications of 4,000× and 8,000×, with a voltage of 15 kV, focusing on areas of higher biofilm density. For qualitative evaluation of the biofilm structure, three specimens per group were examined.

2.8 Statistical analysis

Analyses were performed using OriginPro 8 software. The Kolmogorov-Smirnov test was used to assess data distribution. Variance analysis was conducted with one-way ANOVA, followed by Tukey’s test for mean comparisons. Groups were considered statistically different when p < 0.05. Additionally, the statistical power of the test was calculated, resulting in a value of 1.