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A new improved protocol for in vitro intratubular dentinal bacterial contamination for antimicrobial endodontic
Flaviana Bombarda de Andrade1, Marcela Paola Castro Arias1, Amanda Garcia Alves Maliza1
1Department of Operative Dentistry, Endodontics and Dental Materials, Bauru School of Dentistry,, University of São Paulo, Bauru, SP, Brazil.
Objectives:
To compare three methods of intratubular contamination that simulate endodontic infections using confocal laser scanning microscopy (CLSM).
Material And Methods:
Two pre-existing models of dentinal contamination were used to induce intratubular infection (groups A and B). These methods were modified in an attempt to improve the model (group C). Among the modifications it may be included: specimen contamination for five days, ultrasonic bath with BHI broth after specimen sterilization, use of E. faecalisduring the exponential growth phase, greater concentration of inoculum, and two cycles of centrifugation on alternate days with changes of culture media. All specimens were longitudinally sectioned and stained with of LIVE/DEAD for 20 min. Specimens were assessed using CLSM, which provided images of the depth of viable bacterial proliferation inside the dentinal tubules. Additionally, three examiners used scores to classify the CLSM images according to the following parameters: homogeneity, density, and depth of the bacterial contamination inside the dentinal tubules. Kruskal-Wallis and Dunn's tests were used to evaluate the live and dead cells rates, and the scores obtained.
Results:
The contamination scores revealed higher contamination levels in group C when compared with groups A and B (p<0.05). No differences were observed between group A and B (p>0.05). The volume of live cells in group C was higher than in groups A and B (p<0.05).
Conclusion:
The new protocol for intratubular infection resulted in high and uniform patterns of bacterial contamination and higher cell viability in all specimens when compared with the current methods.
Insights
A new protocol for intratubular contamination effectively simulates endodontic infections, showing higher bacterial levels and viability. This improved method offers more uniform and deeper contamination patterns in dentinal tubules.
Area of Science:
- Microbiology
- Endodontics
- Dental Research
Background:
- Intratubular contamination models are crucial for simulating endodontic infections.
- Existing models have limitations in achieving consistent and deep bacterial penetration.
- Confocal laser scanning microscopy (CLSM) is a valuable tool for visualizing bacterial growth within dentinal tubules.
Purpose of the Study:
- To compare three methods for intratubular contamination to simulate endodontic infections.
- To evaluate the effectiveness of a modified contamination protocol using CLSM.
- To assess bacterial homogeneity, density, and depth within dentinal tubules.
Main Methods:
- Two established dentinal contamination models (Groups A and B) were used.
- A modified protocol (Group C) incorporated extended contamination time, ultrasonic treatment, specific bacterial growth phase, higher inoculum concentration, and centrifugation.
- Specimens were stained with LIVE/DEAD stain and analyzed using CLSM.
Main Results:
- Group C demonstrated significantly higher contamination scores compared to Groups A and B (p<0.05).
- No significant difference in contamination was found between Group A and Group B (p>0.05).
- Group C exhibited a greater volume of live bacterial cells than Groups A and B (p<0.05).
Conclusions:
- The novel protocol (Group C) achieved superior and uniform intratubular bacterial contamination.
- The modified method resulted in higher bacterial cell viability within dentinal tubules.
- This new protocol provides a more robust model for studying endodontic infections.
