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

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.

Comparing the effectivities of chitosan citrate and chitosan acetate in eradicating Enterococcus faecalis biofilm

“…2,3 To achieve a successful endodontic treatment, in addition to preparation, effective antibacterial irrigants are required to eliminate bacteria in the root canal. 4,5 Recent studies have suggested that the presence of Enterococcus faecalis in the root canal can increase the risk of endodontic infections during or after the root canal treatment. 6,7,8 Studies on NaOCl and chlorhexidine as antimicrobial irrigants have shown that both irrigants are effective to be used as antibacterial agents.…”

Exopolysaccharide dispelled by calcium hydroxide with volatile vehicles related to bactericidal effect for root canal medication

“…Recently, a dentin infection model was generated to establish standard and deep penetration of bacteria into dentinal tubules 1,14 . This visual strategy efficiently assessed the efficacy of endodontic medication against E. faecalis in dentin 20 .…”

Descontaminação intra-tubular de dentes bovinos por pastas de hidróxido de calcio associadas a diferentes veículos

“…Para promover a contaminação bacteriana por E. faecalis dentro dos túbulos, utilizou-se o protocolo de centrifugação dos espécimes utilizado por Ma et al (2011) em dias alternados e repetidos. Desta forma, aliando-se o formato dos espécimes com centrifugações repetidas e demais adaptações, pode-se comprovar por meio de MCVL, melhor proliferação intra-tubular do que os métodos de contaminação anteriormente utilizados, (ANDRADE et al, 2015).…”

“…Assim, é possível verificar se a utilização de aditivos a uma substância já bastante utilizada como o hidróxido de cálcio pode potencializar sua ação antimicrobiana sobre Enterococcus faecalis. Para isso, foi utilizado um novo protocolo de contaminação já avaliado comprovadamente eficaz na contaminação em grandes profundidades da massa dentinária (ANDRADE et al, 2015). A análise do efeito antimicrobiano das pastas foi realizada através de cultura microbiológica e microscopia confocal de varredura a laser (MCVL), possibilitando uma adequada escolha da medicação para o uso clínico.…”