TY - JOUR

T1 - Development of an ex vivo coculture system to model pulpal infection by Streptococcus anginosus group bacteria

AU - Roberts, Jessica L

AU - Maillard, Jean-Yves

AU - Waddington, Rachel J

AU - Denyer, Stephen P

AU - Lynch, Christopher D

AU - Sloan, Alastair J

N1 - Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

PY - 2013/1

Y1 - 2013/1

N2 - INTRODUCTION: Streptococcus anginosus group (SAG) bacteria are opportunistic pathogens and a major cause of pulpal infection and subsequent abscess formation. Understanding of the processes involved in SAG oral infections has been limited by the lack of an appropriate model system.METHODS: Cocultures of SAG bacteria and mammalian tooth slices were maintained using a combination of Dulbecco modified eagle medium and brain-heart infusion broth at 60 rpm, 37°C, 5% CO(2) for 4, 8, or 24 hours before histologic examination or staining with acridine orange/ethidium bromide. Tooth slices were also incubated as described with SAG bacteria stained with fluorescein diacetate. Pulps were extirpated from infected and sterile cultured tooth slices, messenger RNA was extracted and converted to complementary DNA, and polymerase chain reaction were performed for genes encoding tumor necrosis factor α, interleukin 1β, and interleukin-6.RESULTS: SAG bacteria were able to adhere directly to the central region of the pulpal matrix in small foci that were associated with a localized matrix breakdown. Acridine orange-ethidium bromide staining and cell counts indicated a decrease in mammalian cell viability with increasing incubation times in the presence of SAG bacteria. The increased expression of tumor necrosis factor α and interleukin 1β was detected in infected tooth slices.CONCLUSIONS: A novel ex vivo model system has been developed that allows coculture of SAG bacteria with a 3-dimensional organotypic tooth slice. The model allows observation of bacterial growth patterns and subsequent responses from host tissues. Therefore, it may be of future use in testing the efficacy of both antimicrobial and anti-inflammatory treatments for use in endodontic therapy.

AB - INTRODUCTION: Streptococcus anginosus group (SAG) bacteria are opportunistic pathogens and a major cause of pulpal infection and subsequent abscess formation. Understanding of the processes involved in SAG oral infections has been limited by the lack of an appropriate model system.METHODS: Cocultures of SAG bacteria and mammalian tooth slices were maintained using a combination of Dulbecco modified eagle medium and brain-heart infusion broth at 60 rpm, 37°C, 5% CO(2) for 4, 8, or 24 hours before histologic examination or staining with acridine orange/ethidium bromide. Tooth slices were also incubated as described with SAG bacteria stained with fluorescein diacetate. Pulps were extirpated from infected and sterile cultured tooth slices, messenger RNA was extracted and converted to complementary DNA, and polymerase chain reaction were performed for genes encoding tumor necrosis factor α, interleukin 1β, and interleukin-6.RESULTS: SAG bacteria were able to adhere directly to the central region of the pulpal matrix in small foci that were associated with a localized matrix breakdown. Acridine orange-ethidium bromide staining and cell counts indicated a decrease in mammalian cell viability with increasing incubation times in the presence of SAG bacteria. The increased expression of tumor necrosis factor α and interleukin 1β was detected in infected tooth slices.CONCLUSIONS: A novel ex vivo model system has been developed that allows coculture of SAG bacteria with a 3-dimensional organotypic tooth slice. The model allows observation of bacterial growth patterns and subsequent responses from host tissues. Therefore, it may be of future use in testing the efficacy of both antimicrobial and anti-inflammatory treatments for use in endodontic therapy.

KW - Acridine Orange

KW - Animals

KW - Bacterial Adhesion

KW - Bacterial Load

KW - Bacteriological Techniques

KW - Cell Death

KW - Cell Survival

KW - Coculture Techniques

KW - Culture Media

KW - Dental Pulp

KW - Dental Pulp Diseases

KW - Dentin

KW - Ethidium

KW - Fibroblasts

KW - Fluoresceins

KW - Fluorescent Dyes

KW - Incisor

KW - Interleukin-1beta

KW - Interleukin-6

KW - Male

KW - Odontoblasts

KW - Organ Culture Techniques

KW - Rats

KW - Rats, Wistar

KW - Streptococcal Infections

KW - Streptococcus anginosus

KW - Streptococcus constellatus

KW - Time Factors

KW - Tumor Necrosis Factor-alpha

KW - Comparative Study

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1016/j.joen.2012.09.005

DO - 10.1016/j.joen.2012.09.005

M3 - Article

C2 - 23228257

VL - 39

SP - 49

EP - 56

JO - Journal of Endodontics

JF - Journal of Endodontics

SN - 0099-2399

IS - 1

ER -