Development and Characterization of an in vitro Four-species Anaerobic Dental Biofilm Model

74 Pages·2016·14.27 MB·English

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The University of Texas MD Anderson Cancer
The University of Texas MD Anderson Cancer
Center UTHealth Graduate School of
Center UTHealth Graduate School of
Biomedical Sciences Dissertations and Theses
Biomedical Sciences
(Open Access)
8-2013
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Fernando Andrade
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Biomedical Sciences Dissertations and Theses (Open Access). 399.
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DEVELOPMENT AND CHARACTERIZATION OF AN IN VITRO FOUR-
SPECIES ANEROBIC DENTAL BIOFILM MODEL
by
Fernando Andrade, BS
APPROVED:
______________________________
Heidi B. Kaplan, Ph.D.
______________________________
Catherine G. Ambrose, Ph.D.
______________________________
Millicent E. Goldschmidt, Ph.D.
______________________________
William Margolin, Ph.D.
______________________________
Hung Ton-That, Ph.D.
______________________________
Gena D. Tribble, Ph.D.

APPROVED: ____________________________
Dean, The University of Texas
Graduate School of Biomedical Sciences at Houston
Development and Characterization of an in vitro Four-species
Anaerobic Dental Biofilm Model

A
Thesis

Presented to the Faculty of
The University of Texas
Health Science Center at Houston

And
The University of Texas
M.D. Anderson Cancer Center

Graduate School of Biochemical Sciences
in Partial Fulfillment
of the Requirements
for the Degree of

MASTER OF SCIENCE

by

Fernando Andrade, BS
Houston, TX
August, 2013
ACKNOWLEDGMENTS

I would like to thank all my committee members, Catherine Ambrose, PhD,
Millicent Goldschmidt, PhD, William Margolin, PhD, Hung Ton-That, PhD, Gene
Tribble,PhD, and my advisor, Heidi Kaplan, PhD. I would also like to thank past and
present Kaplan Lab members: Zalman Vaksman, Douglas Litwin, and Carrie Graham.
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Development and Characterization of an in vitro Four-species
Anaerobic Dental Biofilm Model

Fernando Andrade, BS
Supervisory Professor: Heidi B. Kaplan, PhD

ABSTRACT
Dental caries is the most common chronic disease worldwide. It is characterized by
the demineralization of tooth enamel caused by acid produced by cariogenic dental
bacteria growing on tooth surfaces, termed bacterial biofilms. Cariogenesis is a
complex biological process that is influence by multiple factors and is not attributed
to a sole causative agent. Instead, caries is associated with multispecies microbial
biofilm communities composed of some bacterial species that directly influence the
development of a caries lesion and other species that are seemingly benign but
must contribute to the community in an uncharacterized way. Clinical analysis of
dental caries and its microbial populations is challenging due to many factors
including low sensitivity of clinical measurement tools, variability in saliva chemistry,
and variation in the microbiota. Our laboratory has developed an in vitro anaerobic
biofilm model for dental carries to facilitate both clinical and basic research-based
analyses of the multispecies dynamics and individual factors that contribute to
cariogenicity. The rational for development of this system was to improve upon the
current models that lack key elements. This model places an emphasis on
physiological relevance and ease of maintenance and reproducibility. The
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uniqueness of the model is based on integrating four critical elements: 1) a biofilm
community composed of four distinct and representative species typically
associated with dental caries, 2) a semi-defined synthetic growth medium designed
to mimic saliva, 3) physiologically relevant biofilm growth substrates, and 4) a novel
biofilm reactor device designed to facilitate the maintenance and analysis.
Specifically, human tooth sections or hydroxyapatite discs embedded into
poly(methyl methacrylate) (PMMA) discs are incubated for an initial 24 hr in a static
inverted removable substrate (SIRS) biofilm reactor at 37°C under anaerobic
conditions in artificial saliva (CAMM) without sucrose in the presence of 1 X 106
cells/ml of each Actinomyces odontolyticus, Fusobacterium nucleatum,
Streptococcus mutans, and Veillonella dispar. During days 2 and 3 the samples are
maintained continually in CAMM with various exposures to 0.2% sucrose; all of the
discs are transferred into fresh medium every 24 hr. To validate that this model is
an appropriate in vitro representation of a caries-associated multispecies biofilm,
research aims were designed to test the following overarching hypothesis: an in
vitro anaerobic biofilm composed of four species (S. mutans, V. dispar, A.
odontolyticus, and F. nucleatum) will form a stable biofilm with a community profile
that changes in response to environmental conditions and exhibits a cariogenic
potential. For these experiments the biofilms as described above were exposed on
days 2 and 3 to either CAMM lacking sucrose (no sucrose), CAMM with 0.2%
sucrose (constant sucrose), or were transferred twice a day for 1 hr each time into
0.2% sucrose (intermittent sucrose). Four types of analysis were performed: 1)
fluorescence microscopy of biofilms stained with Syto 9 and hexidium idodine to
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determine the biofilm architecture, 2) quantitative PCR (qPCR) to determine the cell
number of each species per cm2, 3) vertical scanning interferometry (VSI) to
determine the cariogenic potential of the biofilms, and 4) tomographic pH imaging
using radiometric fluorescence microscopy after exposure to pH sensitive
nanoparticles to measure the micro-environmental pH. The qualitative and
quantitative results reveal the expected dynamics of the community profile when
exposed to different sucrose conditions and the cariogenic potential of this in vitro
four-species anaerobic biofilm model, thus confirming its usefulness for future
analysis of primary and secondary dental caries.
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TABLE OF CONTENTS

Title Page …………………………………………………………………………………...ii
Acknowledgements ……………………………………………………………………….iii
Abstract …………………………………………………………………………………….iv
Table of Contents …………………………………………………………………………vii
List of Illustrations …………………………………………………………………………ix
List of Tables ……………………………………………………………………………….x
Introduction …………………………………………………………………………………1
Literature Review …………………………………………………………………………..7
Materials and Methods ……………………………………………………………………..15
1. Bacterial strains and growth conditions ………………………………………………..15
2. Media …………………………………………………………………………………..15
3. Primer Design …………….…..……………................................................................. 17
4. Bioreactors ………………………………………………………………………………..17
5. Biofilm Growth Conditions ………………………………………………………………19
6. Biofilm analysis …………………………………………………………………………...21
Results ……………………………………………………………………………………….28
1. Development of a novel static inverted substrate biofilm reactor ……………………29
2. Community profile analysis ……………………………………………………………..32
3. Analysis of biofilm cariogenic potential ………………………………………………...37
4. Analysis of biofilm micro-environmental pH ……………………………………………42
Discussion ………………………………………………………………………………….. 45
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References …………………………………………………………………………………..55
Vita……………………………………………………………………………………………63

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LIST OF ILLUSTRATIONS

Figure 1: The static inverted removable substrate (SIRS) biofilm reactor ……... 30
Figure 2: Native or artificial tooth sections embedded in poly(methyl methacrylate)
(PMMA) are the SIRS biofilm reactor substrates ………………………………… 31
Figure 3: Fluorescence microscopic images of the four-species anaerobic biofilm
model ………………………………………………………………………………….. 34
Figure 4: Community profile analysis four-species anaerobic biofilm after 72 hr of
exposure to different sucrose conditions …………………………………………….36
Figure 5: Total number of cells per biofilm substrate ……………………………….. 38
Figure 6: Vertical scanning interferometry (VSI) images native tooth substrate
exposed to in vitro model …………………………………………………………… 40
Figure 7: Quantitation of the erosion of tooth sections ……………….…………..41
Figure 8: Tomographic pH imaging of the anaerobic biofilm ….…………………43
Figure 9: Quantitation of the biofilm pH of distribution …………..……………….44

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