Enhancing the Antibacterial Effect of Erythrosine-Mediated Photodynamic Therapy with Ethylenediamine Tetraacetic Acid

At a Glance

Metadata	Details
Publication Date	2024-02-26
Journal	THE JOURNAL OF THE KOREAN ACADEMY OF PEDTATRIC DENTISTRY
Authors	MinKi Choi, Haeni Kim, Si Young Lee, Juhyun Lee
Institutions	Gangneung–Wonju National University
Analysis	Full AI Review Included

Executive Summary

This study investigates the synergistic effect of Ethylenediamine Tetraacetic Acid (EDTA) on Erythrosine-mediated Photodynamic Therapy (PDT) for eliminating Streptococcus mutans (S. mutans) biofilm, a key cariogenic microorganism.

Core Achievement: The combination of EDTA and PDT resulted in a significantly enhanced antibacterial effect compared to PDT alone, achieving the lowest bacterial count (1.41 Log₁₀ CFU/mL).
Synergistic Mechanism: EDTA, acting as a chelating agent, removes the dentin smear layer, thereby increasing dentin permeability and allowing the Erythrosine photosensitizer to penetrate deeper into the S. mutans biofilm.
Efficacy Improvement: The EDTA + PDT group reduced bacterial counts by approximately 2.7 Log units compared to the PDT-only group (4.10 Log₁₀ CFU/mL).
Low Concentration Viability: The combined approach demonstrated significant antibacterial efficacy even when using a low concentration (20 µM) of the FDA-approved photosensitizer, Erythrosine.
Treatment Parameters: The protocol utilized 17% EDTA for 1 min, followed by 20 µM Erythrosine application for 3 min, and subsequent irradiation with a high-output LED (2000 mW/cm²) for 17 s.
Clinical Relevance: This method offers a less invasive alternative for treating dental caries, particularly beneficial for pediatric patients or individuals with cooperation difficulties.

Technical Specifications

Parameter	Value	Unit	Context
Specimen Material	Bovine Incisors	N/A	Dentin specimens (6.0 x 3.0 x 2.0 mm)
Target Microorganism	Streptococcus mutans	ATCC 25175	Biofilm model
Initial Biofilm Concentration	1.0 x 10⁶	CFU/mL	Concentration used for inoculation
EDTA Concentration	17	%	Applied for 1 min
Photosensitizer	Erythrosine	20 µM	Minimum effective concentration used
Light Source Type	LED (VALO^TM)	385 - 515 nm	Wavelength range
Light Output Power Density	2000	mW/cm²	Extra power mode setting
Light Irradiation Time	17	s	Duration of PDT application
Irradiation Energy (Maximum)	34	J/cm²	Calculated energy dose
Bacterial Count (PDT only)	4.10 ± 0.74	Log₁₀ CFU/mL	Post-treatment count
Bacterial Count (EDTA + PDT)	1.41 ± 1.49	Log₁₀ CFU/mL	Highest efficacy achieved

Key Methodologies

The study utilized an in vitro model on bovine dentin specimens to evaluate the antimicrobial efficacy of combined EDTA and Erythrosine-mediated PDT.

Specimen Preparation: Bovine incisors were sectioned into 6.0 mm x 3.0 mm x 2.0 mm dentin blocks, polished with 1000 grit sandpaper, and sterilized using ethylene oxide gas.
Bacterial Culture and Biofilm Establishment: S. mutans ATCC 25175 was cultured in BHI broth (37°C, 5% CO₂). Specimens were inoculated in BHI broth containing 1% sucrose and the bacterial suspension (1.0 x 10⁶ CFU/mL final concentration) and incubated for 24 h to form biofilm.
Photosensitizer Preparation: Erythrosine powder was dissolved in phosphate-buffered saline (PBS) to achieve a 20 µM concentration. The solution was freshly prepared and shielded from light.
Treatment Application (EDTA + PDT Group):
- Chelation: 40 µL of 17% EDTA solution was applied to the specimen surface for 1 min.
- Rinsing: Specimens were washed with sterilized saline solution and dried with sterile gauze.
- Photosensitizer Loading: 40 µL of 20 µM Erythrosine was applied for 3 min.
- Irradiation: Specimens were exposed to the LED light source (385 - 515 nm) at 2000 mW/cm² for 17 s (total dose 34 J/cm²).
Bacterial Quantification (CFU): Treated specimens were washed, subjected to 20 s sonication to detach biofilm, diluted (1/1000), plated on blood agar, and incubated for 3 days (37°C, 5% CO₂). Counts were converted to a Log scale.
Visualization (CLSM): Confocal Laser Scanning Microscopy was performed using the LIVE/DEAD KIT to differentiate live (green fluorescence) and dead (red fluorescence) bacteria within the biofilm structure.

Commercial Applications

The enhanced Erythrosine-PDT technique, leveraging EDTA for improved photosensitizer penetration, has immediate relevance in clinical and sterilization fields:

Pediatric Dentistry: Non-invasive treatment of early childhood dental caries, offering an alternative to mechanical removal, especially in uncooperative patients.
Endodontics and Restorative Dentistry: Sterilization of carious dentin and root canal systems, where EDTA is already used to remove the smear layer, allowing deeper antimicrobial penetration.
Biofilm Management: Development of enhanced photodynamic antimicrobial chemotherapy (PACT) protocols for managing persistent biofilms in various medical settings (e.g., chronic wounds, implant surfaces).
Infection Control Products: Formulation of two-step antimicrobial systems (chelating agent pre-treatment followed by photosensitizer/light activation) for high-efficacy sterilization of surfaces or tissues.
Cosmetic/Oral Hygiene: Potential application in advanced plaque control or periodontal disease treatment, utilizing erythrosine (a common tooth colorant) as a safe photosensitizer.

View Original Abstract

This study evaluated the additive impact of ethylenediamine tetraacetic acid (EDTA) on erythrosine-mediated photodynamic therapy (PDT) against Streptococcus mutans (S. mutans) biofilm by measuring colony-forming units and applying confocal laser scanning microscopy. Fifty-six bovine incisors, free from dental caries or structural defects, were utilized in this study. Dentin specimens were created by cutting with a low-speed diamond disk under a continuous flow of water, resulting in dimensions of 6.0 mm × 3.0 mm × 2.0 mm. The specimens were categorized into 4 groups: Control, EDTA, PDT, and EDTA + PDT. S. mutans ATCC 25175 was employed to establish biofilm on the dentin specimens. A 17% EDTA solution was applied for 1 min. For PDT, erythrosine served as the photosensitizer. Finally, a light-emitting diode source (385 - 515 nm) was employed in this study. The PDT group exhibited a significantly lower bacterial count than both the control and EDTA groups (p < 0.001). The EDTA + PDT group demonstrated a significantly reduced bacterial count compared to the other 3 groups (p < 0.001). This study demonstrated that EDTA enhances the antimicrobial efficacy of PDT on S. mutans biofilm. Even at a low concentration of photosensitizer, the combination of EDTA and PDT yields a significant antibacterial effect.

Tech Support

Original Source

DOI: https://doi.org/10.5933/jkapd.2024.51.1.32

References

2007 - Dental caries: from in-fection to prevention
2015 - Topical PDT in the treatment of benign skin disease: Principles and new applications