DOI

10.17077/etd.6tk7-u1jj

Document Type

Thesis

Date of Degree

Spring 2016

Degree Name

MS (Master of Science)

Degree In

Oral Science

First Advisor

Drake, David G

First Committee Member

Teixeira, Fabricio B

Second Committee Member

Williamson, Anne E

Third Committee Member

Dawson, Deborah V

Abstract

Bacterial disinfection of the root-canal system is the goal in achieving a successful root canal treatment. Currently, this is accomplished by mechanical and chemical debridement. However, limitations continue to exist with these processes and bacteria can still remain, resulting in potential failure of treatment. The aims of this study were to develop a stable, reproducible polymicrobial biofilm in human teeth and utilize this model to compare the bactericidal activity of the UV light disinfection to current root canal chemo-mechanical debridement techniques. We hypothesized that use of UV light at known bactericidal wavelengths were lethal to bacterial cells. Our pilot studies have revealed strong bactericidal capabilities of the UV light against four bacterial species which have been previously isolated in failed root canal procedures; Enterococcus facealis, Actinomyces viscosus, Porphyromonas gingivalis and Fusobacterium nucleatum. We then developed a novel stable reproducible human tooth multi-species biofilm model for UV light disinfection testing. Studies were conducted to study the treatment effects of NaOCl only, UV only and NaOCl+UV. The data revealed strong evidence of initial treatment effect with NaOCl and NaOCl+UV (p

Objectives: Bacterial disinfection of the root-canal (RC) system is critical for successful RC treatment. The aim of this study was to develop a stable reproducible polymicrobial biofilm in human teeth using four bacterial species previously isolated from failed RCs (Enterococcus facaelis (EF), Actinomyces viscosus (AV), Porphyromonas gingivalis (PG), and Fusobacterium nucleatum (FN) that can be used to compare new methods with established root canal disinfection techniques.

Methods: The four bacterial species were cultured individually according to species requirements. A polymicrobial bacterial suspension containing all 4 species was prepared when cultures reached the desired optical density. The polymicrobial suspension was first tested on enriched blood agar to confirm that all species would grow well together. Optimal concentrations of each bacteria for the tooth model system were determined through a series of experiments growing polymicrobial biofilms in 12-well plates. Following successful growth in plates, the multi-species biofilm was grown in human premolar teeth under anaerobic conditions. Biofilms were assessed by physical removal of the biofilms and spiral-plating resulting multi-species suspensions onto selective and differential agars for incubation.

Results: All bacteria have been consistently recovered from multi-species biofilms colonizing root canals of human teeth in our model system. Our studies to date have shown that we can achieve stable and reproducible microbial communities established in the root canals of human teeth.

Conclusions: We have developed a stable reproducible human tooth multi-species biofilm model. Our immediate future studies will focus on development of a prolonged, multi-species biofilms maintained over a period of several weeks. We believe this novel biofilm model will allow for more accurate determination of the efficacy of new and innovative canal disinfection techniques; such as cold-plasma disinfection and/or laser-light therapy.

Public Abstract

Bacterial decontamination of infected root-canal (RC) systems is critical for either the prevention or for healing of apical periodontitis. The aim of this study was to develop a stable polymicrobial biofilm with four commonly isolated bacteria in previously treated teeth with persistent infection (Enterococcus facaelis (EF), Actinomyces viscosus (AV), Porphyromonas gingivalis (PG), and Fusobacterium nucleatum (FN). Our initial studies in the development of a tooth model system involved biofilm on agar plates. The species of bacteria were grown as single cultures for 24-48hrs (incubated according to species requirements), spiral-plated onto selective/differential agar plates. Multispecies biofilms were also prepared on enriched blood agar plates. Polymicrobial biofilms and single-species biofilms were subjected to our Ultra-violet (UV) light disinfection protocol. Agar plates were incubated according to species requirements for 48-72 hrs and zones of inhibition were measured. UV light was placed 83-89 mm over the agar surfaces with different exposure times ranging from 15-240 seconds. Treatment of single species and multi-species biofilms with the UV light exhibited strong bactericidal activity. Exposure to different times (15-240 seconds) was compared by one-way (ANOVA, p<0.05). No statistical significant differences were found. These results suggest potential clinical significance for this disinfection protocol. A 15-second exposure regimen could easily be incorporated into endodontic treatment standards. In conclusion, a stable reproducible polymicrobial biofilm model in human mandibular teeth was developed. We will use this model in our next investigation using a new microfiber optic system as additional disinfection protocol.

Pages

xiii, 81 pages

Bibliography

Includes bibliographical references (pages 76-81).

Copyright

Copyright © 2016 Kimberly Ann Morio

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