A new improved protocol for in vitro intratubular dentinal bacterial contamination for antimicrobial endodontic tests: standardization and validation by confocal laser scanning microscopy

Author(s)

de Andrade, Flaviana Bombarda ^[1] ; Castro Arias, Marcela Paola ^[1] ; Alves Maliza, Amanda Garcia ^[1] ; Hungaro Duarte, Marco Antonio ^[1] ; Zardin Graeff, Marcia Sirlene ^[2] ; Amoroso-Silva, Pablo Andres ^[1] ; Midena, Raquel Zanin ^[1] ; de Moraes, Ivaldo Gomes ^[1]

Affiliation

^[1] Univ Sao Paulo, Bauru Sch Dent, Dept Operat Dent Endodont & Dent Mat, BR-17012901 Bauru, SP - Brazil

^[2] Univ Sao Paulo, Bauru Sch Dent, Integrated Res Ctr, BR-17012901 Bauru, SP - Brazil

• Document type: Journal article
• Source: Journal of Applied Oral Science; v. 23, n. 6, p. 591-598, NOV-DEC 2015.
• Web of Science Citations: 12

Abstract

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. faecalis during 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. (AU)

• FAPESP's process: 10/20186-3 - Adherence factors and biofilms generated by root canal microorganisms
• Grantee: Flaviana Bombarda de Andrade
• Support Opportunities: Regular Research Grants