to open the Abstract Book for Research Day 2013

Transcription

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Acknowledgements
Research Day Organizing Committee:
Ashkan Ebrahimpour (Chair), Mohammed Zahran (Co-Chair),
Mahmoud Al-Dajani, Maher Bourbia, Gazelle Crasto, Marco Magalhaes, Dania Sabbahi,
Lida Sadeghinejad, Sonica Singhal, Irina Voronov
Leah Raz-Administrative Assistant, DRI
Dr. Dennis Cvitkovitch-Associate Dean, Research and Director, DRI
Dr. Bernhard Ganss, Vice-Associate Dean (Research)
We would like to thank the following people and corporations for their
contribution to this year’s Faculty Research Day:
Online Submission and Survey:
Dania Sabbahi, Mohammed Zahran
Posters and Cover for Abstract Book:
Marco Magalhaes
Website Support:
Maria Buda
Abstract Book:
Dania Sabbahi
Funding for Awards:
Dental Research Institute
Guest Speakers:
Dr. Lorrie A. Kirshenbaum
Dr. Christopher McCulloch
Graduate Speaker:
Nastaran Abbarin
Nilesh Talele
Selection and Nomination of Student Speakers:
Student Research Group (SRG) Committee
Judges:
Gurpreet Baht, Kyle Battiston, Jane Cheung, Yasaman Delaviz, Grace De Souza,
Delphine Dufour, Fatima Ebrahim, Ben Ganss, Bo Huang, Andrew Latos,
Lorrie Kirshenbaum, Ernest Lam, Milos Legner, Celine Levesque, Morrie Manolson,
Kirsten McKenizie, Tara Moriarty, Kamyar Motavaze, Dorrin Nilforoushan,
Getulio Nogueira, Padina Pezeshki, Andres Plazas, Maneesha Rajora, Dilani Senadheera,
Eli Sone, Quyen Su, Nilesh Talele, Michelle Visser, Aileen Zhou
Media Services – Audiovisual and Poster preparation:
James Fiege, Bruno Rakiewicz, Marisa Curmi, Jeff Comber and Andrea Cormier
Poster Boards set-up crew:
Undergraduate and Graduate Students Poster Presenters
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University of Toronto
Faculty of Dentistry’s Research Day
Tuesday, February 12, 2013
Program
12:00-12:05 p.m.
Opening Remarks
Dr. Dennis Cvitkovitch
(Room 170)
12:05-1:05 p.m.
First guest lecture
Presented by Dr. Lorrie Kirshenbaum
Professor, Director of Research Development, University of Manitoba
Departments of Physiology and Pharmacology & Therapeutics
Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Canada
Research Chair in Molecular Cardiology
Title: "Molecular Regulation of Cell Death Pathways: Where Life Meets Death "
(Room 170)
1:10-3:00 p.m.
Poster Presentation
Odd numbers: Authors present 1:30 - 2:05 p.m.
Even numbers: Authors present 2:05 - 2:45 p.m.
(Auditorium)
Complimentary Lunch will be served (Cafeteria)
3:00-5:00 p.m.
Second guest lecture
Presented By Dr. Christopher McCulloch
Professor, Matrix Dynamics Group, Faculty of Dentistry, University of Toronto
Canada Research Chair in Matrix Dynamics
Title: "Does Research Add Value to the Faculty of Dentistry?"
(Room 170)
Postgraduate student presentation
Nastaran Abbarin
PhD Candidate
Title: “The Enamel Protein Amelotin Promotes Hydroxyapatite Mineralization In Vitro”
(Room 170)
Postgraduate student presentation
Nilesh Talele
PhD Candidate
Title: “Culture On Soft Substrates Suppresses The Fibrotic Fate Of Mesenchymal Stem Cells”
(Room 170)
5:00- 5:15 p.m.
Presentation of Student Awards
(Room 170)
5:15-6:15 p.m.
Wine and Cheese
Clinical tables
Department of Anaesthesiology
Department of Dental Public Health
Department of Endodontics
Department of Pedodontics
Department of Periodontics
Department of Prosthodontics
Department of Oral Radiology
Department of Restorative Dentistry
The faculty of Dentistry Library
(Cafeteria)
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Lay-out of Auditorium for Research Day
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GUEST SPEAKERS
MOLECULAR REGULATION OF CELL DEATH PATHWAYS: WHERE
LIFE MEETS DEATH
DR. LORRIE KIRSHENBAUM
Professor, Director of Research Development, University of Manitoba
Departments of Physiology and Pharmacology & Therapeutics
Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Canada Research
Chair in Molecular Cardiology
__________
DOES RESEARCH ADD VALUE TO THE FACULTY OF DENTISTRY?
DR. CHRISTOPHER MCCULLOCH
Professor, Matrix Dynamics Group, Faculty of Dentistry, University of Toronto
Canada Research Chair in Matrix Dynamics
__________
THE ENAMEL PROTEIN AMELOTIN PROMOTES
HYDROXYAPATITE MINERALIZATION IN VITRO
N ABBARIN
Department of Biological and Diagnostic Sciences, Faculty of Dentistry, University of Toronto
_________
CULTURE ON SOFT SUBSTRATES SUPPRESSES THE FIBROTIC
FATE OF MESENCHYMAL STEM CELLS
N. TALELE
Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group, Faculty of Dentistry,
University of Toronto, Toronto, Canada
_________
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MOLECULAR REGULATION OF CELL DEATH PATHWAYS: WHERE LIFE
MEETS DEATH
LORRIE A. KIRSHENBAUM‡†
-
Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Departments of Physiology‡,
Pharmacology& Therapeutics†, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, R2H 2A6
Alternative gene splicing provides a versatile mechanism by which cells generate proteins with
different or even antagonistic properties. Previously we determined the hypoxia-inducible protein
Bnip3 is integral component of the mitochondrial death pathway that can signal apoptosis and
autophagy but the precise mechanisms that differentially regulates these divergent processes remains
cryptic. Herein, we provide novel evidence that inclusion or skipping of exon3 of Bnip3 mRNA by
alternative splicing generates proteins with distinct and opposing actions on autophagy and cell
survival. Metabolic stress imposed by hypoxia or nutrient deprivation resulted in the synthesis of two
Bnip3 mRNA isoforms in post-natal ventricular myocytes in vitro and in vivo. Notably, one Bnip3
mRNA comprised of exons 1 through exon 6 encoded a protein of 26kDa, while a second mRNA
generated by the fusion of exon2 and exon4 encoded a truncated Bnip3 protein of 8.2kDa. Sequence
analysis revealed the truncated isoform encodes a conserved C-terminus domain that exclusively targets
Bnip3 to the endoplasmic reticulum and not mitochondrion. While the 26kDa Bnip3 induced
mitochondrial perturbations and autophagy, the spliced variant suppressed Bnip3- induced
mitochondrial defects and autophagy. Furthermore, genetic knock-down or mutations within the Cterminus of the spliced variant defective for ER targeting sensitized cardiac myocytes to mitochondrial
ROS production and death. To our knowledge our data provide the first direct evidence for a novel
survival mechanism whereby the metabolic status of the cell programs autophagy or apoptosis by
preferentially targeting Bnip3 isoforms to mitochondria or ER during metabolic stress.
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DOES RESEARCH ADD VALUE TO THE FACULTY OF DENTISTRY?
DR. CHRISTOPHER MCCULLOCH
The research enterprise has played a prominent role in the life of the Faculty of Dentistry for over 100
years but what is research doing for the Faculty now? How can we assess its value for students, patients
and faculty members? This presentation considers these questions and reviews some of the high water
marks of the Faculty’s previous research achievements. We will examine how this progress has affected
our approach to oral health care and what the future might hold in terms of the impact of the Faculty’s
current research on future dental health care. Importantly we will wonder about the value of research
for the long-standing progress of the dental profession and its associated personnel.
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Graduate Speaker
THE ENAMEL PROTEIN AMELOTIN PROMOTES HYDROXYAPATITE
MINERALIZATION IN VITRO
N ABBARIN*, B GANSS
Department of Biological and Diagnostic Sciences, Faculty of Dentistry, University of Toronto
Background: We have recently identified amelotin (AMTN), a novel protein that is specifically
expressed during the maturation stage of dental enamel formation. It is located at the interface between
enamel mineral and the apical surface of ameloblasts. Transgenic mouse models developed in our lab,
which overexpress or lack AMTN, have shown specific defects in the structure and organization of the
enamel hydroxyapatite (HA) microstructure. Objectives: Previous results suggest a possible
involvement of AMTN in regulating HA mineralization directly. The first goal of this study was to
determine the binding affinity of AMTN molecules to hydroxyapatite. The second goal was to study the
effect of recombinant AMTN protein on the formation and growth of calcium phosphate minerals in
vitro. Methods: Recombinant human AMTN protein was expressed in Escherichia coli cells and
affinity purified to near-homogeneity. Protein adsorption experiments were carried out on synthetic
hydroxyapatite powder using “Langmuir isotherm equation”. About 90g/ml AMTN was added to
SBF solution (molar Ca/P=1.67 and physiological pH) and incubated at 37C for 48hours. Calcium
phosphate deposits were then analysed using scanning and transmission electron microscopy
techniques. Phosphorylated osteopontin from bovine milk and myoglobin from equine heart were used
as positive and negative controls, respectively. Results: Adsorption isotherms of the proteins used in
this study on HA fit Langmuir model (R2 ≥0.97). The adsorption affinity of AMTN for HA binding
sites was lower than that of phosphorylated osteopontin, but higher than that of myoglobin. The
maximum adsorption sites on HA for binding to AMTN molecules were the highest. Our in vitro
crystallization results indicate that AMTN induced hydroxyapatite formation significantly. Control
experiments did not show any mineral precipitation from the buffer after 48 hours. Conclusion: These
findings suggest a direct interaction between AMTN and hydroxyapatite at the maturation stage of
amelogenesis.
Supported by: NSERC discovery grant
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Graduate Speaker
CULTURE ON SOFT SUBSTRATES SUPPRESSES THE FIBROTIC FATE OF
MESENCHYMAL STEM CELLS
N. TALELE*1, J. FRADETTE2, J. DAVIES3, B. HINZ1
1
Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group, Faculty of Dentistry, University of Toronto,
Toronto, Canada, 2Laboratoire d'Organogénèse Expérimentale, Centre de recherche FRSQ du CHA Universitaire de
Québec, Université Laval, Québec, Canada, 3Institute for Biomaterials and Biomedical Engineering, University of Toronto,
Toronto, Canada
Background: Mesenchymal stem cells (MSCs) have great potential to regenerate damaged tissues.
However, MSCs used for the treatment of fibrocontractive diseases, e.g., fibrosis and cancer, bear the
risk of differentiating into fibrotic myofibroblasts (MFs) that worsen the disease. One main stimulating
factor for MF differentiation is the stiffness of the fibrotic tissue. We hypothesize that mechanically
triggered MF differentiation abrogates the regenerative potential of MSCs. Objective: To demonstrate
whether the stiffness of fibrotic tissues drives MSCs into fibrotic cells with reduced regenerative
capacity. Method: Human MSCs were plated on normal-soft and fibrosis-stiff elastomeric substrates.
First, to identify a suitable MSC source, fibrogenesis was compared between MSCs from adipose tissue
(hAMSCs), bone marrow (hBMSCs) and umbilical cord (hUMSCs). Second, rat MSCs were directly
isolated on soft versus stiff substrates and then sub-cultured on a range of differently stiff substrates for
assessing their profibrogenic potential. Third, we tested whether MF differentiation affects the
multipotency of MSCs. Pure MF-MSC and non-MF-MSC populations produced from hBMSCs by flow
sorting were subjected to stiff and soft substrates. We then compared both populations for the presence
of MSC surface markers by immunofluorescence microscopy, flow cytometry and their potential to
undergo induced tri-lineage differentiation. Results: Growth of hAMSCs and hBMSCs on soft
substrates resulted in decreased expression of the MF marker α-SMA, in contrast to high levels of
fibrotic marker on stiff substrates. Direct isolation of rBMSCs on soft substrates suppressed fibrotic
development that was stimulated by sub-culture on stiff but not on soft substrates. Similarly, purified
hBMSC-MF populations lost fibrotic characteristics upon soft substrate culture. Non-MF-MSCs and
MF-MSCs both expressed MSC surface markers. However, MF-MSCs exhibited different tri-lineage
potential compared with non-MF-MSCs. Conclusions: Our approach of MSC isolation and subculture
on tissue-compliant substrates eliminates the culture plastic effect of inducing spontaneous MF
differentiation. Hence, soft culture systems potentially increase the success of regenerative medicine by
suppressing fibrogenesis in expansion culture.
Support: CIHR & EU-FP7 Training Network T3Net.
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UNDERGRAD STUDENTS CATEGORY
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1. The Role of MDP/VBATDT Ratio on Chemical Bonding to Zirconia
A AGGARWAL*, GMD DE SOUZA
Ceramic Processing Laboratory, Faculty of Dentistry, University of Toronto, Toronto.
Background: Adequate adhesion of a fixed prosthesis to the substrate is important for its success. Although
zirconia is lately attracting significant interest in prosthodontics due to its outstanding mechanical properties,
achieving adhesion with natural or synthetic substrate is a significant challenge with zirconia based prosthesis.
Our previous studies evidenced that primers for metal containing MDP (10-methacryloyloxydecyl dihydrogen
phosphate) and/or VBATDT (6-(4-vinylbenzyl-n-propyl) amino-1,3,5-triazine-2,4 dithiol) would enhance the
bonding to zirconia. We hypothesized that an appropriate combination of both would lead to enhanced bonding.
Objective: To evaluate the effect of the ratio of MPD/VBATDT monomers on micro-tensile bond strength
between zirconia and resin cement. Methods: Sintered zirconia (Lava, 3M ESPE) slices, 4.0mm thick, were cut
and roughened up to 600grit carbide silicon paper. Composite resin (3M ESPE) block, 6.0mm thick, were
fabricated and hydrated for 30 days. Seven combinations of MDP and VBATDT primers were developed.
Zirconia surface was coated with two layers of primer, air dried and cemented to composite resin substrates
using dual cure resin cement (Clearfil Esthetic Cement EX, Kuraray) under standard pressure. Bonded specimens
were stored in water for 48 hrs. Micro-tensile slabs were obtained by cutting the blocks vertically in two different
axes and were micro-tensile tested in an Instron machine (crosshead speed of 0.5mm/min). Mode of failure was
evaluated under microscope. Representative samples were studied under scanning electron microscope. Results:
One-way ANOVA and Tukey HSD (α=0.05) evidenced that Group 3 (0.5% MDP/0.5% VBATDT) presented the
highest bond strength (15.1±5.7MPa). The overall results evidenced that none of the molecule work by itself and
that a minimum of 0.5% MDP is necessary to achieve reasonable bond strength. Conclusion: Bond strength to
zirconia can be achieved by means of chemical bonding employing MDP and VBATDT in an appropriate ratio.
Supported by: University of Toronto, Start-up Funding.
2. Role of TLR4 in Enhancing Host Susceptibility To the Lyme Disease Pathogen Borrelia
Burgdorferi In Response To Diet-Induced Obesity
M Ahmed*, J Gananam, M Parikh, A Bansal, YR Kim, M Santana-Sosa, TJ Moriarty
Matrix Dynamics Group, Faculty of Dentistry, University of Toronto
Background: Lyme disease (LD) is caused by the bacterium Borrelia burgdorferi (Bb). We recently found that
high fat diet-induced obesity enhances susceptibility of mice to disseminated Bb infection, suggesting that
increasing obesity rates may contribute to the rising incidence of LD worldwide. Diet-induced obesity is
dependent on inflammation mediated by toll-like receptor 4 (TLR4), which also contributes to insulin-resistant
diabetes in response to high fat diet. Under normal diet, TLR4 is not required for the immune response to Bb
infection, as wild-type and TLR4 loss of function mice are equally susceptible to Lyme disease. Objective:
Determine if TLR4 is required for enhanced host susceptibility to disseminated LD in response to diet-induced
obesity. Methods: TLR4 wild-type and TLR4-deficient C3H mice were pre-conditioned with normal and highfat chow for 10 weeks before infection with Bb. Body weight and fed blood glucose were monitored on a weekly
basis before and during infection. Four weeks after infection, tissues were collected for qPCR measurement of
Bb burden and histological analysis of joint and cardiac inflammation. Results: Mean body weight was higher in
mice fed high fat diet compared to regular diet (P<0.001). Fed blood glucose levels exhibited a small (11%) but
significant (P<0.01) increase in TLR4 wild-type mice fed high fat versus regular diet, but not in TLR4-deficient
mice. High fat diet significantly elevated Bb infection in joints (2.6-6.1-fold) and liver (27.9-36.9-fold) of both
wild-type and TLR4-deficient mice (P<0.05). No significant difference in bacterial burden was observed in the
wild-type and TLR4-deficient mice. N=10 mice per group. Conclusions: TLR4-mediated inflammation is not
required for enhanced Bb infection in response to high fat diet. Elevated Bb burden in liver of obese mice
suggests that obese LD patients might be at a greater risk of developing hepatitis than non-obese patients.
Funding: NRFTD, CIHR, Faculty Enrichment Endowment Fund. Scholarships: Faculty of Dentistry (M Ahmed),
BBDC (J Gananam), CIHR (M Parikh), Ontario Ministry of Training (YR Kim).
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3. Risk of Developing Dental Caries among Preterm Children: A Systematic Review
N ALZAMANI *, H MOHAMED YAKUB, S ABUKLAM, A SAWANI, S JAMIL
Department of Community (Dental Public Health), Faculty of Dentistry,
Background: Dental caries is a multi-factorial disease in any age group but preterm and low birth weight
children maybe at greater risk. Adverse events in utero may impair immune development, elevating the risk
of infection, or extending the window of infectivity for acquisition of streptococci mutans and the
development of caries. Objectives: The goal was to assimilate the literature of the past decade to understand
this relationship in a better way. Methods: databases (Cochrane Library, Ovid MEDLINE(R), Ovid
MEDLINE(R) In-Process & Other Non-Indexed Citations, Pubmed, Scopus and Web of Knowledge) were
systematically searched. Criteria were set to include studies assessing caries risk/prevalence, published
between July 2000-April 2012 with cohort or case-control study designs and a study population from birth
to 6 years old who were born prematurely or with low birth weight. Studies were excluded if they were invitro, had no control group or when no English translation was available. Results: All four studies showed
no significant relationship between Pre-term Low Birth Weight (PTLBW) and caries development.
Conclusions: Further stratification of birth weight into Low Birth Weight (LBW) and Very Low Birth
Weight (VLBW) has led to some interesting findings confirming the dose-response relationship. While
more research is needed, it is very difficult to carry out studies that control all the confounding factors;
however, the scope of study needs to be broadened keeping in mind the dental components along with the
medical and social implications of low birth weight children.
4. Effect of A Novel Photosensitizer on the Antibiofilm Efficacy using Photodynamic
Therapy in Infected Dentin Tissue Management
R BAWA*, A SHRESTA, D O’SHEA, A KISHEN
Department of Endodontics, Faculty of Dentistry, University of Toronto
Department of Chemistry, University College Dublin, Ireland
Background: Apical periodontitis is a host immune response to microbial biofilm present within the root canal
system. Persistence of biofilms that are resistant to conventional antibacterial treatment has been a major
challenge. Photodynamic therapy (PDT) has shown promising results as an adjunct to conventional treatment.
However, the antibiofilm efficacy of PDT is influenced by the absorption spectrum of the photosensitizer and
ability of light to penetrate dentin. Objective: This study aims to test the antibiofilm efficacy of a novel
brominated tetraarylazadipyrromethene photosensitizer (BF2) on infected dentin. BF2 has a broad (spans
infrared region) and low absorption spectrum which suggests potential for greater penetration and greater
antibiofilm efficacy. Methods: Absorption spectrum of the photosensitizer was determined using UV-VIS plate
reader. The singlet oxygen release potential was determined by DPBF assay. Cellular uptake of the
photosensitizer was determined in planktonic Enterococcus faecalis using UV-VIS plate reader. PDT mediated
cytotoxicity and dark toxicity was assessed using MTT assay. The antibiofilm efficacy of photosensitizer was
assessed using microbiological culture to analyze cell viability in one week and three weeks old biofilms grown
on dentin sections. A phenothiazine photosensitizer methylene blue (MB) was used as control. Results: BF2 has
lower and broad absorption peak from 500-700nm. BF2 has slow and steady whereas MB has a rapid singlet
oxygen release. There was significantly higher concentration of uptake of BF2 than MB, p<0.05. There was no
significant difference in cytotoxicity observed. Greater antibiofilm efficacy was observed with BF2 in one week
and three weeks old biofilms, p<0.05. Conclusion: BF2 is a novel photosensitizer with low and broad absorption
spectrum which resulted in enhanced antibiofilm efficacy in infected dentin model. It can be explored as an
adjunct for the management of infected dentin in root canal treatment or dental caries. Supported by: Summer
Research program, Dental Research Institute.
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5. Effectiveness of a Novel Delivery System on Salivary Flow Rate, Quality of Life, and
Inhibition of Caries Associated Microbiota in Sjogren’s Syndrome Patients
J CHEUNG*, LP LAING
Department of Prosthodontics, Faculty of Dentistry, University of Toronto
Background: Sjögren's Syndrome (SS) is a systemic autoimmune disease characterized by dry mouth (xerostomia)
symptoms due to salivary gland destruction by lymphocyte infiltration. An extract from licorice root (Glycyrrhiza
uralensis) has been shown to exhibit growth inhibitory effects on plaque formation by S. mutans as well as bacterial
glycosyl-transferase activity. Meanwhile, a natural sugar alcohol called xylitol also demonstrates anti-cariogenic
properties by reducing the amount of bacterial acid production and to further promote tooth remineralization by
increasing the flow of saliva, which is a common effect observed in sweeteners. Objectives: 1) To increase salivary
flow rate (SFR), 2) To reduce growth of S. mutans and Lactobacilli spp., and 3) improve the overall quality of life
(QoL) for SS. Methods: SS patients were recruited and provided a 10-day regimen of lollipop consumption (2
lollipops per day). The lollipops contain only one of licorice root extract, xylitol, or an artificial sweetener, and
patients were randomly assigned to one of these treatments. Before and after the treatment period, saliva samples
(resting and mechanically stimulated) were collected to determine SFR in ml/min and microbial analysis was
performed to measure amounts of S. mutans and Lactobacilli spp. Self-administered questionnaires helped evaluate
subjective experiences: Oral Health Inventory Profile (OHIP-14), Xerostomia Inventory (XI), Dry Mouth
Questionnaire, and QoL. All statistical tests were performed at P<0.05. Results: 1) Amounts of Lactobacilli spp.
were reduced in all treatment groups, while only some S. mutans levels were reduced. 2). Two particular lollipop
treatments effectively increased saliva flow (resting and stimulated). 3) One treatment allowed SS patients to feel a
statistically significant improvement in their QoL after 10 days. 4) Sixty percent of SS patients in the study reported
that the lollipops showed effectiveness in dry mouth relief or that mouths felt more pleasant. Conclusions: SS patients
who used the lollipops twice daily for only 10 days exhibited an increase in saliva production (beyond the duration of
lollipop consumption) and demonstrated a decrease in oral bacterial load, especially Lactobacilli spp. A majority of
patients also found the lollipops to be a positive experience of xerostomia relief. Supported By: This project was
funded by the University of Toronto (Faculty of Dentistry) Rosenstadt Endowment Fund and Dr. John’s Candies.
6. Characterization of Esophageal Defects in the Fgfr2 W290R Crouzon Mouse Model
S DAB*,1 R SOKHI ,1, J-C LEE 1,3, B J SESSLE1,3, J E AUBIN4, S G GONG1,2#
1
Dental Research Institute, 2 Department of Orthodontics, 3 Department of Oral Physiology, Faculty of Dentistry, University
of Toronto, 124 Edward Street, Toronto, Canada, 4Department of Molecular Genetics, University of Toronto, Canada.
Background: Fibroblastic Growth Factor Receptor (FGFR) mutations have been associated with human
craniosynostotic birth defects such as Crouzon syndrome (CS). Several case reports and anecdotes have indicated the
higher frequency of gastrointestinal tract (GIT) disorders in these patients. However, the effects of FGFR mutations on
GIT development in craniosynostotic, specifically CS, patients have not been well documented in the literature. Our
laboratory has characterized a mouse model of human CS with a W→R mutation in codon 290 of FGFR2 W290R.
Objective: To characterize the esophageal defects in the FGFR2W290R mutant mice. Method: Dissected esophagi of
wild type (WT) and FGFR2W290R heterozygotes (HET) postnatal mice were analyzed for morphological, histological,
molecular and functional differences. Results: The esophagi of HET were noticeably shorter, with wider lumen and
thinner epithelia and the presence of strictures,. In HET, there was a difference in the thickness and orientation of the
esophageal muscle layers with bundles that were less organized compared to WT littermates. HET esophagi also
showed reduced amount of collagen as assayed by Masson’s Trichrome staining and and hydroxyproline (HP) assay.
There was reduced expression of Ki67 in the basal layer of HET esophageal epithelia concomitant with a decrease in
the expression of compared to WT. Functional analysis using strain gauge suggested reduced contractile properties in
HET. Conclusion: The reduced epithelial thickness may lead to decrease in mucosal resistance. This might further
make it susceptible to damage induced by recurrent esophageal reflux. Alterations in muscles may lead to
uncoordinated contractions and then likely lead to improper peristalsis and thereby to further esophageal motility
disorders. Collectively, our results provide evidence towards the importance of Fgf signaling in the growth and
patterning of the esophagus. Furthermore, our studies can also provide a sound rationale for any changes in the
diagnosis and clinical management of GIT problems in patients with craniosynostotic defects.
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7. Studying Host-immune Response Using C. elegans Interaction with Biofilm as a Model
A DOSANJH*, A KISHEN, B GUPTA
Background: Enterococcus faecalis is a hardy gram-positive facultative anaerobe with high antibiotic resistance.
Teeth with persistent post-treatment endodontic infection commonly contain E. faecalis in 24% to 77% of cases.
Additionally, as a biofilm, E. faecalis becomes more resilient, shown from its ability to survive in the root canals of
endodontically treated teeth where other bacteria may fail to thrive. Objective: The aim of this study was to examine
the host immune response of E. faecalis using a nematode, Caenorhabditis elegans, as a model organism. C. elegans
represents a simple host organism in which mechanisms of host defense can be dichotomized to probe host-pathogen
interface. Methods: N2 C. elegans in the early adult stage was allowed to interact with planktonic (planktonic group)
and 4 week old biofilm (biofilm group) of E. faecalis, and treatments of M9 and OP50 (control groups). The effect of
pathogen-nematode interaction was assessed by recording the movement, reaction to touch, progeny, and body length
of C. elegans post interaction. One-way ANOVA post-hoc Tukey test was used for statistical analysis. Results: There
was a significant difference in the body length between the M9 group (mean=16.5, SD=2.1) compared to the
planktonic bacteria group (mean=29.66, SD=4.6). However, it was found that C. elegans after interaction with biofilm
bacteria had on average about 20% (SD=3.4%) shorter body length compared to after planktonic bacterial interaction.
The biofilm interaction also produced 33% (SD=24.5%) fewer progeny than in the planktonic group. Response to
touching was similar in all treatment groups. Conclusion: After interaction with E. faecalis, C. elegans showed a
shorter body length and fewer progeny, with E. faecalis biofilm resulting in greater effect. C. elegans is a convenient
model to study bacteria and host-pathogen response; however observation maybe difficult owing to its small size and
fast movements, and further research on behavioural changes should be conducted. Supported By: Kishen Lab
8. Development of a Flow Chamber Model System For Investigating the BBK32-Dependent
B. Burgdorferi Vascular Adhesion Mechanism
R EBADY*, A KATZ, TJ MORIARTY
Background: A key step in blood-borne dissemination of the Lyme disease pathogen Borrelia burgdorferi is vascular
adhesion, a shear force-regulated process initiated by tethering and dragging bacterial interactions with endothelial
surfaces. Intravital microscopy studies in mice indicate that tethering and dragging are dependent on host fibronectin
(Fn), glycosaminoglycans (GAGs) and the Fn-, GAG-, and fibrinogen (Fg)-binding B. burgdorferi adhesin BBK32.
BBK32 mediates both the tethering and dragging steps of vascular adhesion by direct interactions with Fn and GAGs
respectively. Intravital microscopy studies in mice are of unknown relevance for understanding vascular adhesion
mechanisms in humans, and can be challenging due to differences in intravascular clearance of bacterial variants and
local variations in blood flow speed, vessel architecture and optical clarity. Objective: Develop a physiologicallyrelevant flow chamber model system for high resolution, biophysical, biochemical and genetic studies of the BBK32
vascular adhesion mechanism using primary human endothelial cells. Methods and Results: We have established a
powerful, highly quantitative model system for studying BBK32-dependent adhesion of B. burgdorferi to postconfluent monolayers of primary human endothelial cells under physiological shear force conditions. B. burgdorferi
tether and drag on human endothelia at rates similar to or greater than observed in the mouse microvasculature.
Interactions are significantly but not fully dependent on BBK32, host adaptation of bacteria, and perfusion with
medium containing a serum concentration equivalent to blood serum concentration. Adhesion to human endothelia
under a range of shear forces exhibits a slip bond mechanism, with distinct rates of decay for tethering and dragging.
The ability to perform high temporal and optical resolution imaging using enzymatically and genetically modified
endothelia, bacteria cultivated in media depleted of specific host factors, and in perfusion medium supplemented with
fractionated serum and purified serum factors provides a powerful repertoire of biophysical, biochemical and genetic
approaches for investigating the BBK32 vascular adhesion mechanism. We are using this system and a panel of
BBK32 variants to investigate the mechanistic contributions of BBK32-Fn, -GAG and -Fg interactions to human
endothelial interactions under shear force. Support: University of Toronto Graduate Fellowship (AK), Banting
Research Foundation, CIHR, NSERC, CFI/ORF, Faculty Enrichment & Bertha Rosenstadt Endowment Funds.
10
9. The Use of Orthodontic Splinting in the Management of Severe Compound Dental
Trauma
F EBRAHIM*, G KULKARNI
Department of Paediatric Dentistry, University of Toronto
Background: Dental trauma is a common occurrence that affects one out of every two children.
Trauma to the mixed dentition is complicated by the varying stages of tooth eruption and immaturity of
the root development. Objective: This report describes a case of complex trauma to the early mixed
dentition in which tooth avulsion, intrusion, extrusion and lateral luxation were managed effectively
using a fixed,-non-rigid orthodontic splint where previous attempts with a traditional wire-composite
splint had failed. Methods and Materials: In this case report, the orthodontic splinting procedure
utilized orthodontic brackets and a flexible nickel-titanium wire to treat the compound dental trauma.
Results: This technique provided several benefits over the traditional wire-composite splint – such as
ease of fabrication, the ability to splint severely malpositioned teeth, and the gradual re-establishment
of arch form allowing for future ease of prosthodontic rehabilitation. Conclusion: Orthodontic
splinting should be re-evaluated as a viable treatment alternative in managing complex dental trauma.
10. The Assessment of Meta-Analyses of Randomized Controlled Trials in Dentistry
M EL-RABBANY*, S LI, S BUI, M BHANDARI, A AZARPAZHOOH
Department of Dental Public Health, Faculty of Dentistry, University of Toronto,
Department of Clinical Epidemiology and Biostatistics, McMaster University
Background: As the volume of publications in dentistry is increasing, clinicians are becoming
increasingly reliant on systematic reviews and meta-analyses as their primary source of evidence. With
an increase in the dependence on dental meta-analyses, it is important to ensure that they are being
conducted with as little bias as possible. Objectives: The objective of this systematic review is to
assess the quality of therapeutic meta-analyses of randomized controlled trials (RCTs) on dental-related
topics that were not published in Cochrane Database of Systematic Reviews, and to analyze how it has
changed over time. Methods: All relevant studies were searched for through Medline, EMBASE,
PsychINFO, and the Cochrane Library. Title, abstract, and full text review, as well as data extraction
and quality assessment, was all conducted in duplicate. All reviewers used a pilot-tested extraction
form that included a modified form of the validated AMSTAR meta-analysis quality checklist. A logit
link function ordinal regression was conducted to evaluate quality improvement trends over time.
Results: Of the 3120 studies identified, 143 studies were selected for review. Of these, 13% provided
an a priori design, 50% screened and extracted data in duplicate, 29% included grey literature, 51%
assessed the quality of included studies, and 29% assessed publication bias. As was indicated by the
ordinal regression, the quality of meta-analyses, as per the AMSTAR criteria, has increased
significantly with time (p<0.001). Conclusions: This investigation illustrates that while the quality of
meta-analyses of RCTs have been increasing since 2000, there is still room for improvement within all
aspects of systematic review reporting and methodology. An emphasis is placed on the need for the
provision of an a priori design, the inclusion of grey literature, and the evaluation of publication bias
within the included studies. Supported by: University of Toronto, Faculty of Dentistry Summer
Research Program Grant.
11
11. Investigation of Mechanisms Coordinating Adhesion and Motility in Lyme disease
Bacteria
N GUPTA*, TC TANG, L LAMONACA-BADA, TJ MORIARTY
Background: Dissemination of the Lyme disease pathogen, Borrelia burgdorferi (Bb) through the host is a
critical step in Lyme disease progression, and requires the ability to simultaneously adhere to and crawl along
blood vessel surfaces. The mechanism of coordination of these two seemingly contradictory actions is currently
unknown for Bb and other spirochete bacteria. Objective: To develop quantitative methods to analyze
mechanisms of coordination of adhesion and motility in Bb under fluid shear force conditions. Hypothesis:
Rapid lateral diffusion of adhesion proteins in the fluid outer membrane of Bb facilitates coordinated adhesion
and motility under shear force. Methods and Results: Approximately 10 hours of existing intravital time-lapse
microscopy footage of Bb interactions with the vasculature of living mice were converted from VHS format to
DVD using the Toshiba HiFi VCR and DVD Recorder with 1080p* Upconversion. The DVD footage is being
converted to TIFF file time series using Adobe Premiere Pro, for import and automated tracking in Metamorph
image analysis software. Kinetic data for Bb interacting with vessels for at least two successive frames will be
analyzed. We will identify/measure the length of each Bb interacting with the vessel, the quadrant/s where the
interaction first occurs, total time of interaction, overall Bb velocity and velocity through each quadrant.
Conclusions and Significance: Developing quantitative methods for analyzing adhesion and motility kinetics of
Bb visualized interacting with blood vessels in vivo will enable us to determine whether Bb coordinate adhesion
and motility via rapid lateral diffusion of adhesion sites along bacterial surfaces. With new visualization
techniques established, the analysis of mechanistic features of Bb dissemination in vivo can lead to further
understanding of the mechanisms coordinating adhesion and motility of the spirochete phylum. Supported by:
NSERC Discovery Grant (TJM), CIHR Undergraduate Studentship in Musculoskeletal Health and Arthritis
(NG), NSERC USRA (TCT), University of Toronto Work Study Program (TCT).
12. Changes in Facial Mechanosensitivity Following Maxillary Molar Teeth Extraction in A/J,
C57BL/6J Mice and Five of Their Recombinant Lines
G LANDZBERG*, D NGUY, Z SELTZER, B SESSLE, L AVIVI-ARBER
Departments of 1Oral Physiology and 2Prosthodontics, Faculty of Dentistry, University of Toronto, Toronto,
Ontario
Background: Humans undergoing teeth extractions experience postoperative pain that is sometimes
accompanied by altered orofacial mechanosensitivity that is highly variable across subjects. This suggests that
this trait is under genetic control, but the genes involved are unknown. Since inbred A/J (‘A’) and C57BL/6J
(‘B’) mice contrast on levels of post-injury sensitivity, A and B mice and their 23 genetically-mapped AXBBXA recombinant inbred (RI) lines can be used to map candidate genes for orofacial sensitivity after tooth
extraction. Objectives: This pilot study aimed (i) to model post-extraction perioral sensitivity in mice; (ii) to
demonstrate that facial mechanosensitivity levels are under genetic control by comparing pre-and post-extraction
sensitivity in A, B and 5 AXB-BXA RI lines. Methods: Female mice of A (n=5), B (n=9) and AXB1 (n=8),
AXB4 (n=11), AXB24 (n=6), BXA14 (n=13) and BXA24 (n=11) lines had either extraction or sham operation
of all right maxillary molar teeth. Mechanical sensitivity was assessed by measuring withdrawal thresholds to
bilateral mechanical stimulation of the facial vibrissal pads at 1 day pre- and 4, 7, 14 and 21 days post-extraction.
Statistics: ANOVA, post-hoc Dunn’s test (p<0.05). Results: All strains and lines displayed bilaterally increased
mean withdrawal thresholds from baseline, with a high degree of intra- and inter-strain/–line variability. On
postoperative days 4 and 7, BXA14 had significantly higher thresholds as compared with AXB1, BXA24 and A
mice. No significant differences in mean withdrawal thresholds were observed between ipsi- and contra-lateral
vibrissal pads or between extraction and sham groups. Conclusions: The study successfully modeled postextraction altered perioral sensitivity to mechanical stimulation that is strain/line specific, thereby indicating that
this trait is under genetic control. Future studies will examine the full panel of 23 RI lines of male and female
mice and test gender and genetic effects contributing to alterations in postoperative mechanical sensitivity.
12
13. Susceptibility to Gingival Attachment Loss in AMTN-/- Mice
R LU*, B GANSS
Background: Amelotin (AMTN) is a recently discovered enamel protein that is predominantly
expressed in ameloblasts during the late stage of amelogenesis and in the cells of the junctional
epithelium (JE) after tooth eruption (Somogyi-Ganss E et al, Cells Tissues Organs 2012;195:535–549;
Nishio C et al, Bull Group Int Rech Sci Stomatol Odontol 2010; 49:111-112). AMTN -/- mice show
overt signs of enamel hypomineralization but their JE seems undisrupted. This may be attributed to the
fact that experimental mice live in a specific-pathogen-free (SPF) environment where the JE is not
challenged. Challenging the JE will help elucidate amelotin’s role there. Objective: To challenge the
JE with a clinically-relevant insult and to compare the symptoms between AMTN -/- and wild type
mice. Methods: Periodontitis was induced around the maxillary left second molar by tying a silk
ligature with the contralateral side as a control. Mice were euthanized at time points from one day to up
to two weeks after the procedure. Tissues were fixed, demineralized, embedded in paraffin, sectioned,
and stained with H&E. Results: Preliminary results show that gingival pockets in the AMTN -/- mice
were noticeably deeper compared with the wild type mice. In addition, signs of gingival detachment
also appear sooner in the knock-outs. Conclusions: AMTN -/- mice show more susceptibility to
periodontal insult and attachment loss compared with wild type in our ligature-induced periodontitis
model. Supported by: University of Toronto, Faculty of Dentistry Summer Research Program
14. Analyses of Matrix Adhesions in Diabetic Cardiomyopathy
J.B. MEYERSON*, V.I. PINTO, L STAUDINGER, I TALIOR, C.A. McCULLOCH
Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario
Background: Prolonged hyperglycemia in diabetics leads to glycation of extracellular matrix proteins.
Glycated matrix proteins mediate the conversion of cardiac fibroblasts to pro-fibrotic myofibroblasts,
specialized cells that produce the scarring of the cardiac interstitium seen in diabetic cardiomyopathy1.
The formation of myofibroblasts is driven in part by glycated matrix proteins that signal through matrix
adhesions. In diabetic cardiomyopathy, the critical molecules of matrix adhesions that regulate
myofibroblast differentiation are not defined. Objective: Define the molecular composition of matrix
adhesions in human cardiac fibroblasts that attach to glycated collagen. Methods: We adapted a new
method2 for isolating intact matrix adhesions from human cardiac ventricular fibroblasts. Cells were
plated on native or methylglyoxal-treated type I rat-tail collagen for 3 hours, swollen with a hypotonic
triethanolamine solution, and detached from collagen substrates by shear force. Isolated adhesion
proteins were examined by immunofluorescence and immunoblotting for the focal adhesion proteins
vinculin and talin. Results: Phase contrast microscopy confirmed complete removal of cell bodies with
an optimized shear force pressure of 3.549 Pa. Under these conditions, immunofluorescence imaging
showed retention of intact adhesions that exhibited vinculin and talin staining with characteristic
lamellipodia, filopodia and focal streaks. Conclusions: We optimized a hypotonic/shear method to
reproducibly isolate intact adhesions from human cardiac fibroblasts; utilizing this protocol permits
more precise molecular analyses of adhesions forming on glycated matrix proteins. Supported by:
Ontario Heart and Stroke Foundation grant to Dr. C.A. McCulloch
13
15. Public Preferences for Government Spending in Canada
S RAMJI*, C QUINOÑEZ
Community Dental Health Services Research Unit, Faculty of Dentistry,
Objective: This study considers three questions: 1. What are the Canadian public’s prioritization
preferences for new government spending on a range of public health-related goods outside the scope
of the country’s national system of health insurance? 2. How homogenous or heterogeneous is the
Canadian public in terms of these preferences? 3. What factors are predictive of the Canadian public’s
preferences for new government spending? Methods: Data were collected in 2008 from a national
random sample of Canadian adults through a telephone interview survey (n =1,005). Respondents were
asked to rank five spending priorities in terms of their preference for new government spending.
Bivariate and multivariable logistic regression analyses were conducted. Results: As a first priority,
Canadian adults prefer spending on child care (26.2%), followed by pharmacare (23.1%), dental care
(20.8%), home care (17.2%), and vision care (12.7%). Sociodemographic characteristics predict
spending preferences, based on the social position and needs of respondents. Conclusion: Policy
leaders need to give fair consideration to public preferences in priority setting approaches in order to
ensure that public health-related goods are distributed in a manner that best suits population needs.
Grants: This research was funded by the Applied Health Research Network Initiative of the Ministry
of Health and Long-Term Care, Government of Ontario.
16. Filamin A Mechanoprotects Cells by Recruiting Talin to Cell Adhesions
V SENINI*, V PINTO, C McCULLOCH.
Background: In mechanically loaded tissues such as periodontal ligament and myocardium, pathophysiological
forces disrupt cell-matrix contacts. These disruptions can induce cell death, which in turn leads to tissue and
organ dysfunction. Protection against cell death is mediated by filamin A, an actin-binding protein that regulates
integrin-mediated cell adhesion by recruiting talin. Notably, filamin A and talin compete for β1 integrin tail
binding, which may impact integrin-dependent cell adhesion to collagen and fibronectin. Objectives: Assess the
impact of tensile forces and filamin A expression on talin recruitment to vinculin-stained focal adhesions and
apoptotic cell death. Materials and Methods: 3T3 fibroblasts that expressed or were null for filamin A were
plated on fibronectin (0.1-100 μg/ml of coating solution); collagen-coated magnetite beads were attached to their
dorsal surfaces. Magnetically-generated tensile forces were applied to beads for 1 or 12 hr. The recruitment of
talin to vinculin-containing focal adhesions was examined by total internal reflection fluorescence microscopy of
immunostained cells. Data were analyzed by one-way analysis of variance; comparisons between groups were
assessed by Tukey’s test. TUNEL staining was used to measure apoptotic cell death. Results: Vinculin-stained
focal adhesions were larger and longer in cells plated on higher concentrations of fibronectin (p<0.05).
Application of tensile force induced significant time-dependent reduction of focal adhesion numbers, area and
length (p<0.05). Immunoblotting showed that these reductions were not associated with altered expression of
talin. Compared to cells that were null for filamin A, cells expressing filamin A exhibited: 1) more and longer
vinculin-stained focal adhesions (p<0.05); 2) time-dependent enrichment for talin in focal adhesions (p<0.05);
and 3) much lower proportions of TUNEL-stained apoptotic cells. Conclusions: Filamin A mediates talin
recruitment to focal adhesions and protects against apoptosis in cells subjected to tensile forces. Supported by:
Canadian Institutes of Health Research grant MOP-11106.
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17. Development of Oral Microbe-Induced Periodontitis Mouse Model and Fluorescent
Treponema Denticola
SONG, P.1, KIM, G.1, VISSER, M.2, GLOGAUER, M.2, AND MORIARTY, T1.
Moriarty Lab, Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto;
2
Glogauer lab, Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto.
1
Background: Periodontal disease (PD) is a major burden on the dental and healthcare systems. It is the result of
periodontal inflammation in response to oral microbes such as spirochetes, but the mechanisms of oral microbeinduced periodontitis are still not well understood. Two oral microbes associated with PD are the anaerobes
Treponema denticola (Td) and Porphyromonas gingivalis (Pg). A powerful approach to studying the role of oral
microbes in the etiology of PD is to directly visualize the behaviour of bacteria in living hosts using intravital
imaging. The aim of this project is to establish a mouse model of oral microbe-induced periodontitis and
fluorescent oral anaerobes which can be used to study PD progression using intravital microscopy. Methods: Td
alone, Pg alone, Td + Pg or vehicle were administered by oral gavage to 48 antibiotic-pretreated mice, followed
by PCR monitoring of microbe colonization over a 42 day disease induction period. Periodontal disease will be
assessed by measuring alveolar bone loss in maxillary molars, cytokine expression in gingival tissues, and
histological evidence of inflammation in mandibles. We are building targeted homologous recombination
constructs for antibiotic selection-independent expression of Flag-tagged anaerobic fluorophores Bs1, Bs2 and
Pp1 from the chromosomal tde0911 site of Td. Results: Animals have been sacrificed and are currently
undergoing alveolar bone loss measurements to determine whether periodontitis was induced. The fluorophore
expression constructs are awaiting transformation into T. denticola. Conclusions: In the next few weeks, we will
determine whether we have established an animal model of oral microbe-induced PD, and whether Td expresses
anaerobic fluorophores in the absence of antibiotic selection.
18. The Effectiveness of Green Tea Chewing Gum on Salivary Flow Rate, Quality of Life and
Inhibition of Caries Associated Bacteria in Sjögren's Syndrome Patients
L TAN*, L LAING
Faculty of Dentistry, University of Toronto
Background: Sjögren's Syndrome (SS) is an autoimmune disease that causes damage to the salivary glands. As
a result, SS patients experience xerostomia and an increased caries risk. Green tea (GT) polyphenols, such as (-)epigallocatechingallate (EGCG) have been shown to have potent antioxidant and anti-inflammatory and anticaries activities in SS patients. Drinking four cups of GT per day has been shown to have a significant effect on
salivary flow rate, viscosity and Quality of Life (QoL) on patients with SS. Objectives: To assess the effects of
a GT gum on salivary flow rate (SFR), QoL and caries-associated microbial load. Methods: 9 SS patients were
selected to participate in this study. Baseline data was collected in the form of questionnaires: Oral Health
Inventory Profile (OHIP-14), Modified Xerostomia Inventory (XI), Visual Analogue Scale Dry Mouth-Related
Symptoms (VAS) and QoL. In addition, saliva samples were collected to determine resting and stimulated
salivary flow rate as well as to determine the bacterial loads. Subjects were assigned to either green tea gum or
non green tea gum and were instructed chewed one piece three times a day for 14 days. There was a one-week
washout period followed by a cross-over to the other intervention and data collection. Results: SS patients had a
lower average count for caries associated bacteria after both gum interventions. However, GT gum was more
effective at reducing bacterial loads, especially lactobacilli. 2. There was no significant change on saliva
stimulation after both gum interventions. Mechanical stimulation by chewing was able to increase salivary flow
rate, regardless of the flavour: GT, NGT or flavourless. 3. There were no significant improvements in QoL
scores. Conclusions: In SS patients, where caries are rampant and occur at atypical sites, chewing three pieces of
GT gum a day may be beneficial in improving both oral health and what is otherwise a devastating QoL.
Supported by the Sjögren's Society of Canada
15
19. Bond Strength of Repaired Experimental Resins Incorporated with Chlorhexidine or
Catechin
Y VANDERSLUIS*, A PRAKKI
Department of Clinical Sciences – Restorative, Faculty of Dentistry, University of Toronto, Toronto
Background: Chlorhexidine (CHX) and a catechin (CAT), which inhibit matrix metalloproteinases and are
antibacterial, have been incorporated into restorative dental resins. At some ratios these compounds can impair
polymerization conversion by mopping up free radicals from the monomers via protonation. The availability of
unreacted methacrylate groups on the resin surface is however necessary to allow chemical bonding with a new
layer of resin and improve the repair strength. Objective: To evaluate the repair bond strength of CHX or CAT
incorporated comonomers, at different ratios, after a simulated in-service ageing process. Methods: BisGMA/TEGDMA (70/30 mol %) cylinders (r and h = 4 mm) were fabricated (n=4), aged in boiling water, and
assigned to the following groups: incorporation of CHX or CAT at 0, 0.2, 1.0 or 2.0 wt% each. The work surface
of each block was treated with 37% H3PO4 and adhesive resin. Each cylinder was restored with a 4 mm resin
layer incorporated with the same drug at different ratios totalling 32 groups. Specimens were sectioned in x and y
axes with a diamond disk into 8mm x 1mm x 1mm beams. Each beam was tested for microtensile bond strength
(cross-head speed = 0.5 mm/min). Data were analysed by two-way ANOVA and Tukey’s test (α=0.05). Results:
All CHX aged groups had significant decrease in bond strength when repaired with 2%-CHX comonomer
(p<0.01). Repaired drug ratio did not alter bond strength of catechin control aged groups (p=0.12). Conclusions:
Repair strength of examined groups was, in general, significantly affected by higher amounts of incorporated
drugs. Supported by: Dental Research Institute, University of Toronto, Faculty of Dentistry.
20. Temporomandibular Joint Cone Beam CT: Comparative Dosimetry for Various Patient
Technique Factors
J WONG*, T LUKAT, E LAM
Discipline of Oral and Maxillofacial Radiology, Faculty of Dentistry, University of Toronto, Toronto,
Canada
Background: There is universal agreement that both computed tomography (CT) and cone beam CT (cbCT)
offer excellent visualization of the osseous structures of the temporomandibular joint (TMJ). The use of limited
field-of-view cbCT to image the TMJ reduces the size of the irradiated field, the amount of scatter radiation, and
ultimately, the imparted dose. Dosimetric analysis for a limited field-of-view TMJ imaging technique was
previously evaluated at our institution using a Kodak 9000 3D cbCT system (Rochester, NY) operating at the
standard adult settings of 70 kVp and 10 mA. Separate, successive bilateral TMJ acquisitions rendered an
effective radiation dose of 20.51.3 Sv. Objective: The purpose of this follow-up study is to evaluate the
effective radiation doses of bilateral TMJ cbCT acquisitions, varying kVp and mA using the pre-set patient sizebased techniques available on the Kodak 9000 3D unit. Methods: Twenty-five optically stimulated
luminescence dosimeters placed in various locations in an anthropomorphic RANDO® Man phantom were used
to measure absorbed radiation doses for tissues of the head and neck using the child (68 kVp/6.3 mA),
youth/small adult (70 kVp/8 mA), and large adult (74 kVp/10 mA) technique settings. Dosimetric measurements
were performed in triplicate for each technique. Equivalent radiation doses were calculated for the three
techniques using the 2007 International Commission on Radiological Protection tissue weighting factor
recommendations. Results: Effective doses and standard deviations using the child, youth, and large adult
settings were 9.7±0.1, 13.5±0.5, and 19.7±0.6 μSv, respectively. ANOVA and Tukey HSD post-hoc analysis
demonstrated a significant difference in effective radiation dose between all groups (p<0.001), except for the
standard adult and large adult settings (p=0.474). Conclusions: This study compares the dose rendered during
TMJ imaging with limited field-of-view cbCT under various patient technique factors and emphasizes the
prudence of selecting the appropriate settings for patients of different size.
16
21. FLRT2 Interacts with Neurocan during Mid-face Development
CS YANG*, SG GONG
Department of Orthodontics, Faculty of Dentistry, University of Toronto
Background: Fibronectin Leucin Rich Transmembrane Protein 2 (FLRT2) is highly expressed in the migrating
cranial neural crest cells (CNCC) during mid-face development and plays a role in regulating cell-cell
interactions. A recent mass spectrometric study by our lab identified protein candidates that may interact with
FLRT2 in the developing mid-facial tissues. One of the identified proteins was neurocan, a secreted proteoglycan
found to inhibit N-cadherin and integrin-mediated cell adhesion. Objective: To verify the interactions of FLRT2
with neurocan in the developing mid-facial tissues. Methods: Protein complexes containing FLRT2 were
immunoprecipitated from the E11.5 mouse mid-facial tissue lysates with antibodies against the extracellular
domain of FLRT2 and separated using SDS-PAGE. Western blots with antibodies against neurocan were
subsequently conducted to test for the presence of neurocan in the FLRT2-containing protein complexes.
Results: In mouse embryonic mid-facial tissues, the antibody against FLRT2 recognized a ~85-90 kDa band,
and a ~75 kDa doublet band most likely representing the full length and the extracellular portion of FLRT2
respectively. The antibody against neurocan recognized two bands of sizes ~250 kDa and ~130 kDa, which most
likely represent the full length and the N-terminal fragment of neurocan respectively. Both the full length and the
N-terminal fragment of neurocan were co-immunoprecipitated with antibodies against the extracellular domain
of FLRT2, (n=2). Conclusions: There is evidence showing the interactions of FLRT2 with the extracellular
matrix. The interaction of FLRT2 with the N-terminal fragment of neurocan, as suggested in our study, may
explain the mechanism by which FLRT2 mediates cell movement and adhesion. Our findings therefore will help
us better understand the mechanisms through which FLRT2 regulates CNCC migration during mid-face
development. This work is supported by NSERC and CFI awards. Clair S. Yang is supported by the University
of Toronto, Faculty of Dentistry, Summer Student Research Program Fellowship.
22. Obesity and Hyperglycemia Promote Borrelia burgdorferi Dissemination
N ZLOTNIKOV*, A ARYA, M AHMED, A BANSAL, M PARIKH, J GANANAM, YR KIM, F
MATAR, M SANTANA SOSA, T MORIARTY
Matrix Dynamics, Department of Laboratory Medicine and Pathobiology, Faculty of Dentistry,
Background: Incidence of Lyme Disease, metabolic syndrome and obesity are rising in industrialized nations,
with high prevalence in middle-aged adults. A critical stage of Lyme disease is dissemination via the
cardiovascular system, which is frequently affected in metabolic syndrome, obesity and middle-age. Objective:
Evaluate the possible correlation between diet-induced obesity and accompanying hyperglycemia, and
dissemination of the Lyme Disease etiologic agent Borrelia burgdorferi in male mice infected with four
clinically relevant strains of B. burgdorferi (GCB726, BL214, N40, 297), and in female and male mice infected
with one strain (GCB726). Materials and Methods: Five-week old mice were preconditioned with high-fat diet
(HFD) or normal-diet (ND) for 12 weeks, and inoculated with 1x104 B. burgdorferi. Blood glucose and weight
were recorded weekly or bi-weekly. Four weeks post infection tissues were collected for qPCR analysis of
bacterial burden (blood, brain, liver, bladder, ear, patella, liver, lung, heart, skin), and histological analysis of
Lyme arthritis and carditis in tibiotarsal joints and hearts. DNA from tissues and blood was extracted using the
Qiagen DNeasy kit. Statistical analysis was performed using GraphPad Prism. Results: All animals exhibited a
statistically significant increase in weight on HFD compared to ND. Blood glucose levels increased significantly
over time in HFD males and females, but not in ND. qPCR and histological analyses are currently underway for
completion in early 2013. I predict the following hierarchy of infectivity and disease severity GCB726 > BL214
> N40 > 297. I believe that disease presentation will not differ significantly between male and female mice.
Supported by: National Research Fund for Tick-borne Diseases, CIHR; Scholarships: CIHR undergrad (M
Parikh), NSERC USRA (F Matar), Banting & Best Diabetes Centre (J Gananam), Faculty of Dentistry summer
research scholarship (M Ahmed), Ontario Ministry of Training (YR Kim).
17
23. Validation of an Artificial Aging Method for Dental Zirconia
A ZYKUS*, RG RAHBARI, GMD DE SOUZA
Ceramic Processing Laboratory, Faculty of Dentistry, University of Toronto, Toronto.
Background: Orthopedic Y-TZP (Yttria-tetragonal zirconia polycrystal) presents tetragonal to
monoclinic phase transformation. This phase transformation compromises the mechanical properties of
the prostheses and reduces its longevity under body temperature. Although Y-TZP is also employed as
a dental material in CAD-CAM blocks, an artificial aging method has not yet been investigated and
validated for dental Y-TZP. Objective: The aim of this study was to validate an artificial aging method
for dental zirconia by comparing the aging characteristics of both, dental and orthopedic Y-TZP.
Materials & Methods: Medical (“Z5” - C5, Medical Werks, US) and dental (“ZC” - IPS E-max
Zircad, Ivoclar Vivadent, Germany) zirconia blocks were used. Pre-sintered blocks were cut, polished
up to 1µm diamond paste and fully sintered. Specimens were aged in an autoclave unit (2 bars pressure,
134°C) for 0, 30, 60, and 90 minutes. Bi-axial flexural strength of samples (n=10) was evaluated
(piston-on-three balls device) and X-ray diffraction analysis (XRD) was performed in one sample from
each group to detect the presence of the monoclinic phase. Results were compared by a two-way
ANOVA and Tukey HSD (α=.05). Results: There was no effect of aging time on flexural strength for
both materials. Material significantly affected (P=0.001) flexural strength (Z5= 966.95 MPa; ZC=
847.82 MPa). Z5/60min presented the highest flexural strength (1,027.9 MPa) and Z5/90min presented
the lowest value (791.0 MPa). There was no significant difference among the other groups. XRD
evidenced a continuous increase in the amount of monoclinic phase related to the aging time for both
materials. Conclusions: The validation of an artificial aging for dental zirconia requires more intense
aging procedures in a way that flexural strength would reflect an increase in monoclinic phase ratio.
Supported by: University of Toronto Summer Research Institute Scholarship; University of Toronto
Start-Up Fund.
18
GRADUATE AND POSTGRADUATE
STUDENTS
Basic Science Category
19
24. Decreased Excitability of Face Primary Motor Cortex (Face-M1) Induced by Mustard Oil
Application to Rat Molar Tooth Pulp is Dependent on the Functional Integrity of Face-M1
Astrocytes
L AWAMLEH*, H PUN, L AVIVI-ARBER, J-C LEE, BJ SESSLE
Department of Oral Physiology, Faculty of Dentistry,
Background: Dental pain is a common clinical occurrence that is often associated with limited orofacial motor
functions. The underlying mechanisms are unclear but may involve face-M1 that plays a crucial role in the initiation
and control of orofacial movements. Growing evidence suggests that brainstem non-neural astrocytes are involved in
dental pain modulation but it is unclear whether face-M1 astrocytes also play a role in modulating pain-related faceM1 excitability. Objectives: To determine whether: 1) application of the inflammatory irritant mustard oil (MO) to a
rat tooth pulp affects face-M1 excitability manifested by an altered electrical current required to stimulate the face-M1
in order to evoke threshold electromyographic activity in the anterior-digastric jaw-opening muscles; 2) face-M1
application of the astrocytic inhibitor Methionine Sulfoximine (MSO) can influence MO effect. Methods: Under
Ketamine general anaesthesia, electromyographic electrodes were implanted into the anterior-digastric muscles of
Sprague-Dawley male rats; a craniotomy exposed the left hemisphere; a fine electrode was positioned within a faceM1 site from which electrical stimulation (35ms train, 12x0.2ms pulses, 333Hz) could evoke anterior-digastric
electromyographic activity at a low-intensity threshold (≤30µA). This baseline stimulation threshold was monitored
every 5min for 30min; then MO (N=6) or saline (N=5) was applied to a previously exposed right maxillary molar
tooth pulp and stimulation thresholds were monitored every 5min for 15min. MSO (N=5) or saline (N=5) was then
applied to the left face-M1 and stimulation thresholds monitored every 10min for 1hr. Results: Within 15min of MO
(but not saline) pulp application, stimulation thresholds increased significantly as compared to baseline (53%;
repeated-measures ANOVA, post-hoc Bonferroni, p<0.001). The decreased face M1 excitability was normalised to
baseline levels following MSO application to face-M1. Conclusion: These findings suggest that diminished face-M1
excitability associated with acute dental pain may be dependent on the functional integrity of face-M1 astrocytes.
Support: CIHR MOP4918, Rosenstadt funding
25. Elucidation of The Role of K+ Transporters in Streptococcus Mutans
GURSONIKA BINEPAL, MARTHA CORDOVA, DILANI B. SENADHEERA, DENNIS G.
CVITKOVITCH
Dental Research Institute, University of Toronto, Toronto, ON, Canada
Streptococcus mutans is a well known cariogenic pathogen that contributes to the biofilm formation, acidification of
the biofilm micro-environment and by itself tolerates acidic conditions. In the present study, we have identified the
role of potassium (K+) and the four specific K+ transport systems in S. mutans that include, Trk1 and Trk2 encoded
by trk/trkB/pacL and trkA/trkH, respectively, that are energy dependent, moderate affinity and high specificity
transporters, KcsA that is a putative K+ channel, encoded by kcsA, and GlnQHMP that is a glutamate/ K+ cotransporter encoded by the glnQHMP locus. Here, we report that the Trk2 system is important for the biofilm
formation in S. mutans and the knocking out of both the Trk1 and Trk2 system leads to complete disruption of
biofilms. We also show that the biofilms of S. mutans are sensitive to presence of K+ in the medium as the growth is
impaired at concentrations <25mM of K+. Further, to characterize each K+ transport system individually we have
used Escherichia coli K+ transport deficient strain TK2420 to complement with either of S. mutans K+ transport
systems i.e. Trk1, Trk2, KcsA and GlnQHMP. The Trk2 system was able to complement the deficiency due to K+ in
E.coli TK2420 as observed in our growth curves and complementation assays done on K+ limiting conditions. We
also observed that all the S. mutans K+ transport systems cloned and transformed in E. coli TK2420 were able to
improve the growth relative to wild type E.coli TK2420. Overall, our study establishes the importance of K+ and its
transport systems in S. mutans for its growth and propagation. We also show that these K+ transport systems can be
cloned and are functional in other bacteria such as E. coli TK2420 , which can help us to better understand these
systems. Our results considerably add to our knowledge of streptococcal K+ homeostasis and its likely contribution to
bacterial virulence.
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26. Cariogenic Bacteria Degrades Dental Resin Composites and Adhesives
M BOURBIA*, D MA, D CVITKOVITCH, J SANTERRE, Y FINER
Institute of Biomaterials and Biomedical Engineering, University of Toronto
Background: A major cause for dental resin composite restoration replacement is secondary caries.
Streptococcus mutans is a primary species associated with the formation and progression of dental caries.
Hypothesis: In addition to acid production, cariogenic bacteria are hypothesized to contain esterase activities
that degrade dental resin composites and adhesives. Objectives: To measure esterase activities from S. mutans,
and to measure the hydrolytic-mediated degradation of cured dental resin composites and adhesives in the
presence of S. mutans. Methods: Esterase activities of S. mutans UA159 were measured using pnitrophenylbutyrate, o-nitrophenylbutyrate, p-nitrophenylacetate, and butyrylthiocholine-iodide (BTC)
substrates. Standardized specimens of resin composite (Z250), total-etch (Scotchbond, SB), and self-etch
(Easybond, EB) adhesives were incubated with bacteria or bacterial media for up to 30 days. Quantification of
biodegradation-byproduct derived from BisGMA, bishydroxy-propoxy-phenyl-propane (BisHPPP), was
performed using high performance liquid chromatography in combination with UV-spectroscopy and massspectrometry. Surface analysis of the specimens was performed by scanning-electron-microscopy (SEM).
Results: S. mutans had activity towards the nitrophenyl-esters, but not towards BTC, in levels comparable to
human saliva. A trend of increasing BisHPPP release throughout the incubation period was observed for all
materials. The amount of BisHPPP was elevated in the presence of bacteria vs. control for EB and Z250 but not
SB (p < 0.05). The amount of BisHPPP released from EB after 30 days of incubation with S. mutans
(143.15±3.28 μgs/cm2) was 39 and 82 times higher than that released from Z250 (3.71±0.24 μgs/cm2) and SB
(1.74±0.31 μgs/cm2), respectively (p < 0.05). SEM analysis confirmed the increased degradation of all materials
with S. mutans vs. control. Conclusion: S. mutans has esterase activities at levels that degrade dental resin
composites and adhesives. These findings confirm that EB, a self-etching adhesive, is significantly inferior in
terms of biostability when compared to a traditional resin adhesive material. Grant: NIH # R01DE021385-0.
27. Acai Berry (Euterpe oleracea) Extract Inhibits Osteoclastogenesis: an in vitro Study
C BRITO*, A STAVROULLKIS, P HARRISON, G NOGUEIRA-FILHO
Department of Preventive Dentistry, Faculty of Dentistry, University of Toronto
Background: Acai berry, a South American "super fruit” which belongs to the Arecaceae family, has
been proven to have anti-oxidant and anti-inflammatory properties, deriving primarily from the many
flavonoids in its composition. There have been many claims that this fruit possesses an even greater
range of health benefits. During inflammatory responses, the number of large osteoclasts in the
inflamed area increases, a mechanism triggered by pro-inflammatory cytokines. The hypothesis was
that the acai-berry extract can regulate osteoclast size and differentiation. Objective: To investigate the
potential anti-osteoclastogenesis effects of the acai-berry in vitro. Methods: RAW 264.7 cells were
cultured and treated with various concentrations of acai extract (25, 50 and 100 µg/mL). Cell viability
assays were used to evaluate cytotoxicity. Tartrate-resistant acid phosphatase (TRAP) staining was
used to estimate osteoclast number and size after treatments and the extract effects on
osteoclastogenesis. Results: Statistical analysis showed that in comparison to control, an overall
decrease in total osteoclast number was observed (p < 0.05). Interestingly it was observed at 50 µg/mL,
the number of small and large osteoclasts was significantly reduced. Conclusion: Within the limits of
this study, it was concluded that the acai-berry extract inhibits the growth of osteoclasts in both size and
number. The extract had no deleterious effects on cell growth. Future studies are needed to fractionate
the acai-berry extract in order to verify its active compounds that may regulate osteoclastogenesis.
21
28. Interaction Between Lung Macrophages And Myofibroblast At Different Levels Of
Activation
E CAMBRIDGE*, B HINZ
Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group,
Faculty of Dentistry, University of Toronto, Toronto, Canada
Background: Pulmonary fibrosis is associated with chronic inflammation and micro-scarring in the alveolar sacs.
Previous studies indicate that the immune system exacerbates the fibrotic process by promoting fibroblast activation to
pro-fibrotic myofibroblasts through soluble factors. In explants of rodent lung, we regularly observe cell-cell adhesion
between (myo)fibroblasts and macrophages via yet uncharacterized mechanisms. This direct cell-cell communication
varies with the stiffness of cell culture substrates, a major determinant of myofibroblast activation. We hypothesize
that the activation state of fibroblasts to myofibroblasts will influence the efficiency of macrophage attachment and
thus the progression of fibrosis. Objective: To characterize the adhesion mechanism and kinetics between
macrophages and myofibroblasts. Methods: Mouse lung fibroblasts were treated with either transforming growth
factor (TGF)ß1 to promote myofibroblast activation or TGFß receptor 1 inhibitor to reduce fibroblast to myofibroblast
activation. Fibroblast activation was evaluated by quantification of the myofibroblast marker α-SMA. Direct cocultures were used to study the physical interaction between fibroblastic cells and macrophages. Macrophages were
identified on basis of F4/80 antigenicity. To test the rate of adhesion, macrophages were fluorescently labeled and cocultured with treated fibroblasts. Adhesion was quantified by measuring the relative light intensity.
Immunofluorescence of ß-catenin (adherens junctions), vinculin (integrins) and I-CAM were used to identify the
molecular mechanisms and components of attachment. Results: Macrophage adhesion to myofibroblasts is initially
slower compared to fibroblasts. However, adhesion increases and reaches saturation faster than macrophages to a nonfibrotic population. These results support the hypothesis of different adhesion properties of (myo)fibroblasts which
impacts the rate and strength of macrophage adhesion. Immunofluorescence of adhesion proteins were not specific
enough to exclude any candidates. Future aims: TGFß levels and TGFß receptor localization on fibroblasts, with or
without macrophages, will indicate if a preferential microenvironment exists encouraging both populations to persist
beyond normal resolution.
29. Trigeminal Neuropathic Pain and Central Sensitization are Attenuated by Pregabalin but
not Acetaminophen in a Mouse Model
P S CHERKAS*, V VARATHAN, L AVIVI-ARBER, S FRIEDMAN, B J SESSLE
Faculties of Dentistry and Medicine, University of Toronto
Background: Chronic post-endodontic pain, reported in 3-12% of patients, may be induced by trigeminal nerve
injury. Since central sensitization may underlie the development of chronic pain, the objectives of the study were to
examine (1) whether trigeminal nerve injury induces nociceptive behaviour and central sensitization in functionally
identified medullary dorsal horn (MDH) nociceptive neurons, and (2) whether these phenomena can be reduced by
analgesic drugs pregabalin and acetaminophen. Material and Methods: Facial mechanical nociceptive withdrawal
thresholds (MWT) were tested with von Frey filaments applied to the ipsilateral facial skin, pre-operatively and postoperatively up to day 56 following left infraorbital nerve transection (IONX) in adult A/J male mice. On postoperative days 7, 21 and 49, MWT was assessed before and at different time intervals after injection of pregabalin (75
mg/kg i.p., n=9), acetaminophen (100 mg/kg i.p., n=9) or isotonic saline (vehicle control, n=5). MDH nociceptive
neurons were also recorded at these post-operative days, and their decreased activation threshold and increased
responses to pinch/pressure were assessed as measures of central sensitization. Results: The MWT values were
significantly reduced (p<0.05, 2-way ANOVA, n=9) from post-operative days 1-56. At day 7, pregabalin and
acetaminophen but not saline reversed the reduced MWT. At days 21 and 49, only pregabalin was effective.
Pregabalin also significantly attenuated central sensitization at days 7, 21 and 49, while acetaminophen was effective
only at day 7. Conclusion: This study demonstrates that pregabalin, but not acetaminophen, attenuates IONX-induced
mechanical allodynia and the associated MDH central sensitization, and indicates that pregabalin may be useful for
treating orofacial neuropathic pain conditions. Grants: AAE Foundation, Canadian Academy of Endodontics
Endowment Fund, NIH, CIHR, Pfizer
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30. Influence of Superhydrophobicity on Initial Salivary Pellicle and Biofilm Formation on
Titanium in vitro
E CHUNG*, M LEGNER, C LEVESQUE, D SENADHEERA, H TENENBAUM, E FADEEVA, G
NOGUEIRA
Department of Periodontics, Faculty of Dentistry, University of Toronto
Background: Dental abutment surfaces are colonized by oral microorganisms that can promote infectious diseases such as
peri-implantitis. Studies have suggested that superhydrophobic surfaces may have properties that prevent bacterial
colonization and subsequent biofilm formation which would therefore prevent infection. Purpose: To evaluate in vitro the
influence of superhydrophobicity on initial salivary pellicle and biofilm formation on titanium surfaces. Methods: Half of
each titanium discs (n=10) and titanium abutments (n=5) were treated by laser ablation to develop superhydrophobic selforganized structures. The other half consisted of a polished titanium surface that serves as the control. Each sample was
placed in saliva to allow for salivary pellicle formation and then moved into a mixture of medium, saliva and a five species
mixed suspension of bacteria (Actinomyces naeslundii, Streptococcus oralis, Streptococcus sobrinus, Veillonella dispar and
Prevotella intermedia) and incubated at 37°C for 24 and 72 h. Salivary pellicle and biofilm was stained and observed by
confocal laser scanning microscopy and scanning electron microscopy. Confocal microscopy images were analyzed by
COMSTAT 2 to determine biofilm thickness and biomass. Results: When analyzing confocal data with COMSTAT 2,
biomass and average biofilm thickness of stained live bacteria was determined to be greater in the superhydrophobic treated
surface when compared to the polished titanium surface. On the other hand, when viewing stained dead bacteria, the
superhydrophobic treated surface had lesser amounts of biomass and average biofilm thickness. Conclusions: The results of
this study indicate that superhydrophobic titanium surfaces created by laser ablation do not prevent biofilm formation in an
in vitro model. However, the superhydrophobic surfaces appear to have less dead bacteria included in the biofilm which
may indicate that live bacteria use some (unknown) mechanism to overcome the hydrophobicity barrier. Further studies are
required to confirm this. Supported by: University of Toronto, Dental Research Institute - DRI.
31. Elucidating the Molecular Mechanisms involved in Targeting V-ATPases to the Plasma
Membrane: Discovering Potential Targets for Novel Anti-resorptive Therapeutics
S ESMAIL* R REITMEIER, M MANOLSON
Dental Research Institute, Faculty of Dentistry, University of Toronto
Background: V-ATPases are ATP driven proton pumps that when targeted to the plasma membrane of osteoclasts, are
responsible for acidifying the surface of bone, essential for bone resorption and bone remodeling. Evidence suggests that
the 100 kDa V-ATPase ‘a’ subunit is responsible for targeting the multi-subunit complex to the plasma membrane. The
protein sequence of the ‘a’ subunit contains one putative glycosylation site. Hypothesis: We hypothesize that the VATPase ‘a’ subunit is glycosylated in vivo and further hypothesize that glycosylation of the ‘a’ subunit is important to
targeting the rest of the V-ATPase complex to the plasma membrane. Overall Objective: Elucidate the molecular
mechanisms involved in targeting V-ATPases to the plasma membrane with the ultimate goal of preventing this process to
inhibit bone loss in osteolytic diseases. Methods: Mammalian cells were transfected with C-terminal FLAG tagged version
of the V-ATPase ‘a’ subunit with or without mutations that eliminate the putative glycosylation site (N499D and N499Q).
Expression and plasma membrane targeting were assessed by immunofluorescence and immunoblotting. Glycosylation
was assessed by immunoblotting whole cell extracts with or without treatment with endoglycosidases PNGase and Endo H
enzymes. Results: Immunoblotting and immunofluorescence evidence demonstrates that the C-terminal FLAG tagged
version of the V-ATPase ‘a’ subunit with or without the glycosylation mutations N499D and N499Q express in mammalian
cells. Whole cell extracts were immunoblotted with anti-FLAG resulting in a fuzzy band of about 96 kDa in the wild type
suggesting that the ‘a’ subunit has complex glycosylation. Whole cell extracts from ‘a’ subunits bearing the N499D and
N499Q mutations both resulted in a sharp band of about 93 kDa with reduced intensity relative to the wild type. These
results suggest that N499 is the site of glycosylation and that glycosylation is important for proper and efficient protein
folding. Immunoblots revealed that the wild type ‘a’ subunit is Endo-H insensitive but PNGaseF sensitive resulting in a 93
kDa sharp band again providing evidence that the ‘a’ subunit has complex glycosylation. Conclusions: Our preliminary
results suggest that the human V-ATPase ‘a’ subunit is complex-type glycoprotein with N-linked glycosylation at position
N499 and this glycosylation is crucial for the proper folding. Further investigations are required to determine whether the
glycolyslation is important to plasma membrane targeting of the V-ATPases. Supported by: CIHR and Cell Signal PhD
training award
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32. Role of Rac in Macrophage Polarization
H GOLDBERG*, M LIMA, M GLOGAUER
Objective: To determine if Rac1 and Rac2 are necessary for the polarization of macrophages into M1 and
M2. Methods: Bone marrow from the tibia and femur is taken from WT, Rac1 null and Rac2 null mice.
Cells are left over night and the non-adherent cells are taken, counted and plated (5 million) in a 6 well plate
where MCSF (20ng/ml) is added. After 96 hours, the cells are stimulated with IFN, IL4, or LPS and MCSF
is added again. Cells are stained for flow and run on a flow cytometer to see if they have M1 (CD36 and
CD80) or M2 markers (CD206). This experiment is also done with the use of WT, Rac1 null, and Rac2 null
RAW cells. Results: In cells treated with IFN, Rac2 has the greatest number of M2 macrophages when
compared to WT and Rac1. There is no significant difference in the number of M2 macrophages when
treated with IL4. There is also an increase in the number of M2 macrophages in the Rac2 null RAW cells
when compared to the WT and Rac1 RAW cells. There is no significant difference between the treatments
in the cell line, suggesting that these cells no longer respond to stimuli and they are already differentiated.
Conclusions: Rac2 is required for macrophage polarization into the M1 phenotype. Rac2 null cells produce
more M2 macrophages suggesting that they have a deficiency in producing M1 macrophages. However,
further investigation is required to discover the mechanisms that cause this.
33. Development of a Novel Cell Contraction Test for High-Throughput Screening
Applications
S HUME*, B HINZ
Background: Cell contraction plays a vital role in promoting crucial body functions. The regulation of
contraction is crucial to maintain tensional homeostasis of healthy tissue; dysregulation fosters disease
states such as fibrosis and chronic hypertension. Pharmaceutical companies are in need for a functional in
vitro test to evaluate compounds that target cell contraction. None of the currently available techniques are
efficient and quantitative on large cell populations. High-resolution traction force microscopy is limited to
single cells and incompatible with high-throughput applications. Objective: To engineer a novel
quantitative assay to test cell contractility in a high-throughput format. Methods: We build upon the
principle to quantify cell contraction by visualizing “wrinkles” in silicone substrates. As improvement over
conventional silicone oil substrates, we implemented an elastomer that is fully polymerized and is stiffnesstuned to accommodate differently contractile cell types. We functionalized the polymer surface to enhance
the visibility of cell-generated wrinkles for automatic image analysis. The assay was translated from a single
dish to a multi-well assay. Proof-of-principle experiments were performed with contractile primary rat lung
fibroblasts. Results: To produce optically impeccable silicone substrates, we optimized polymer degassing
from air inclusions and minimized polymer thickness to achieve an ideal focal range for microscopy.
Second, we modulated the bulk polymer stiffness and determined the maximum stiffness at which
contractile cells created wrinkles. With these refinements, cell-generated wrinkles can be preserved for up to
one week. High-throughput compatible 96-well plates were produced with a detachable bottom that was
coated with the polymer before assembly with the well cast. We established an automatic image analysis
routine to quantify the proportion of wrinkling cells per well.Conclusion: This novel system will allow to
measure cell contractility with applications in both fundamental research and for time-effective high
throughput processing used in industrial drug testing. Support: CIHR/NSERC (CHRP), CIHR.
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34. Microtensile Bond Strength Of Self-Adhesive Resin Cements To E-Max Crowns
M KANDIL*, G DE SOUZA, H ALKUMRU, O EL-MOWAFY
Department of Restorative Dentistry, Faculty of Dentistry, University of Toronto
Objectives: To determine microtensile bond strength (μTBS) of two CAD-CAM crown materials when bonded to
dentin or composite-resin substrate using two self-adhesive, an etch-and-rinse and a self-etch resin system; and to
compare the effect of two surface treatments of the ceramic crown material on μTBS. Methods: Lithium disilicate
ceramic (IPS e.max CAD, Ivoclar-Vivadent/EMX), composite-resin (Paradigm MZ-100, 3M-ESPE/PZ) blocks, and
custom-made composite-resin (Z100 restorative, 3M-ESPE) blocks were cross-sectioned into rods 1mm2 ± 0.1 mm2.
Similar dentin specimens were obtained from coronal portion of freshly-extracted molars. EMX rods were either gritetched (50μm Al2O3) or etched with HF acid and silanated. PZ rods were only grit-etched and silanated. EMX and PZ
specimens were then bonded to either dentin or composite rods using four cements (RelyX-ARC and RelyX-Unicem,
3M-ESPE; Multilink Automix, Ivoclar-Vivadent; Clearfil SA, Kuraray) using a custom-made device (n=10). A static
load of 146 g was maintained until initial setting of cement. Specimens were stored in water (37°C) for 24 h,
thermocycled (500 cycles, 5- 55°C), then subjected to μTBS test. Means and SDs were calculated in MPa and data
statistically-analyzed by factorial ANOVA test (α=0.05). Results: ANOVA revealed significant differences among
the groups (P<.05). For composite substrate, highest mean μTBS (32.7(±2) was obtained when RelyX-ARC was used
with PZ, while lowest mean μTBS (11.6(±1.3) when Rely-X Unicem was used with EMX. For dentin, highest mean
μTBS (28.8(±2.3) was obtained when RelyX-ARC was used with PZ, while lowest mean μTBS (8.61(±1.3) was
obtained when Clearfil-SA was used with EMX. Conclusions: In overall RelyX-ARC and Multilink-Automix resulted
in significantly higher mean μTBS irrespective of the substrate material. Whereas RelyX-Unicem and Clearfil-SA
(self-adhesive cements) resulted in significantly lower mean μTBS irrespective of the substrate. For EMX HFtreatment resulted in significantly-higher mean μTBS as compared to grit-etching. Acknowledgements: 3M/ESPE,
Ivoclar/Vivadent, Kuraray.
35. Mechanism of BBK32-Dependent Vascular Adhesion
1
WC KAO*1, D FARRINGTON2, R HOF2, C CAMERON2, T MORIARTY1
Matrix Dynamics Group, University of Toronto; 2Department of Biochemistry & Microbiology,
University of Victoria
Background: Lyme borreliosis is a multi-system, tick-transmitted infection caused by the spirochaete Borrelia
burgdorferi and several other closely related species. Systemic dissemination of microbial pathogens permits microbes
to spread from the initial site of infection to secondary target tissues and is responsible for most mortality due to
bacterial infections. Dissemination is a critical stage of disease progression by B. burgdorferi. However, many
mechanistic features of the process are not yet understood. A key step is adhesion of circulating microbes to vascular
surfaces in the face of the shear forces present in flowing blood. Vascular adhesion is dependent on host fibronectin
(Fn) and glycosaminoglycans (GAGs).The B. burgdorferi protein BBK32 is the first and only bacterial adhesin known
to mediate vascular adhesion in vivo, and is conserved in other disseminating extracellular bacterial pathogens.
Objectives: To determine if BBK32 Fn- and GAG- binding under static/shear force conditions are independent,
additive, competitive or allosteric, and to determine if BBK32 homologues from other disseminating bacteria can
mediate vascular adhesion in vitro and in vivo. Methods and Anticipated Results: We are using a flow chamber
system and monolayers of primary human endothelial cells to determine the properties of the BBK32/Fn/GAG
interaction mechanism under physiological fluid shear force. We are also building constructs to introduce the
Staphylococcus aureus and Streptococcus pyogenes BBK32 homologues FnBPA and Sfb1, as well as a number of
Treponema pallidum adhesins, into a GFP-expressing non-infectious B. burgdorferi strain we used previously to
establish a role for BBK32 in vascular interactions in gain-of-function experiments. We will investigate the ability of
heterologous adhesins to restore interaction under shear force to this strain. Conclusions: These studies will elucidate
a novel shear force-regulated vascular adhesion mechanism which may be shared by other pathogens, and will identify
adhesion mechanisms and molecules with potential for development as dissemination-blocking clinical drug targets.
Funding: CIHR, CIHR Bhagirath Singh Award, NIH, Bertha Rosenstadt Endowment Fund, Faculty Enrichment
Endowment Fund, CFI/ORF.
25
36. Mechanisms of Fibronectin-dependent B. Burgdorferi Vascular Adhesion
KATZ, A., EBADY, R., BANSAL, A., AND MORIARTY T.J.
A critical step in bloodborne dissemination of the Lyme disease spirochete Borrelia burgdorferi is bacterial adhesion to
lumenal vascular surfaces and subsequent transmigration into tissues, a specialized fluid shear force-regulated interaction
mediated by bacterial cell surface adhesins. The critical first step of B. burgdorferi vascular adhesion in vivo is mediated by
fibronectin (Fn) and Fn-binding sequences of the BBK32 adhesin which are known to induce formation of superfibronectin
(sFn) in vitro. Objective: Define the mechanistic contributions of Fn to the BBK32-dependent B. burgdorferi vascular
adhesion mechanism. Methods and Results: We have established a powerful, highly quantitative flow chamber-based in
vitro model system for studying the mechanistic contributions of Fn to adhesion of BBK32-expressing fluorescent B.
burgdorferi to post-confluent monolayers of primary human endothelial cells under controlled physiological shear force
conditions; this model system replicates key features of the initiating steps of B. burgdorferi vascular interaction in vivo.
Treatment with anti-plasma Fn (pFn) antibody or cultivation of bacteria in pFn-depleted serum nearly abolishes interactions,
indicating that B. burgdorferi-endothelial interactions under shear force are dependent on recruitment of soluble pFn, and
not on insoluble cellular Fn deposited in endothelial extracellular matrices. Interactions are significantly reduced but not
eliminated in bacteria in which BBK32 expression is genetically disrupted, and treatment with anti-pFn antibody eliminates
all residual BBK32-independent interactions, indicating that B. burgdorferi-endothelial interactions under shear force can be
mediated by adhesins in addition to BBK32, and by serum components other than pFn. Using fluorescently labelled pFn and
panels of BBK32 variants and monoclonal antibodies to specific pFn domains and sequences, we are currently investigating
the roles of pFn fibrillogenesis and sFn formation in BBK32/pFn-regulated endothelial interactions under shear force.
Conclusions: We expect that this system will permit us to precisely define the mechanistic contributions of pFn to B.
burgdorferi adhesion to endothelial cells under physiological shear force conditions. Supported by: University of Toronto
Graduate Fellowship (AK), Banting Research Foundation, CIHR, NSERC, CFI/ORF, Bertha Rosenstadt Endowment Fund.
37. Organized is More Efficient: TGF-β1 Activation from the Extracellular Matrix
F KLINGBERG*1, M CHOW1, A KOEHLER1, L BUSCEMI2, B HINZ1
1
University of Toronto, 2Laboratory of Cell Biophysics, Ecole Polytechnique Fédérale de Lausanne,
Switzerland
Background: Fibroblast-to-myofibroblast differentiation contributes to the detrimental tissue contractures
characteristic of fibrosis and depends on the presence of active transforming growth factor-β1 (TGF-β1) and
mechanically stress. Latent TGF-β1 is stored in the extracellular matrix (ECM), forming a complex with the latent
TGF-β1 binding protein-1 (LTBP-1). We previously demonstrated ‘pulling’ via integrins as the canonical mechanism
of TGF-β1 activation by myofibroblast. We hypothesize that organized LTBP-1 in the ECM will enhance the
availability of TGF-β1 for activation by pre-stressing the latent complex, analogous to the loading of a nanomechanical spring. Objective: Test whether organization of LTBP-1 into pre-stressed ECM improves activation of
TGF-β1 by myofibroblast contraction. Methods: To study the organization of LTBP-1 into ECM fibrils using live
microscopy, we expressed LTBP-1-EGFP in HEK293 cells. HEK293-derived unorganized ECM was then used to
follow incorporation, remodeling and fibrillogenesis of latent TGF-β1. In an interdisciplinary approach, we engineered
compliant substrates simulating healthy and fibrotic conditions to determine the effects of mechanical stress on latent
TGF-β1 assembly and release of active TGF-β1. Results: Our results from protein expression analysis and
immunohistochemistry demonstrated significantly increased organization of LTBP-1 during myofibroblast
differentiation of human dermal fibroblasts. Myofibroblasts seeded onto unorganized ECM produced by LTBP-1EGFP transfected HEK293 cells generated fibrillar LTBP-1 structures. Co-localization of EGFP and LTBP-1
demonstrated that fibrils were assembled exclusively from the HEK293-derived ECM. When ECM assembly was
disrupted in focal adhesion kinase knock-out fibroblasts, myofibroblasts released less TGF-β1 then from fibrillar
organized ECM. Conclusion: Myofibroblasts remodel pre-existing into fibrillar structures. The organization level of
latent TGF- determines the amount of active TGF-β1 released from the ECM. Translation of our findings to in vivo
treatment of fibrosis possibly involves pharmaceutical targeting of the integrins that are responsible for latent TGF-β1
complex pre-stressing. Support: CIHR and the EU FP7 International Training Network T3Net.
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38. Coordination of Adhesion and Motility of The Lyme Disease Pathogen
LAMONACA-BADA L1, GUPTA N1, TANG T1, PITZER J2, SULTAN S2, MOTALEB M.D.2,
MORIARTY T1
1
Matrix Dynamics Group, University of Toronto; 2East Carolina University Brody School of Medicine,
Department of Microbiology & Immunology
Background: Spirochetes are highly motile, invasive gram-negative bacteria that cause serious blood-disseminated
human diseases including syphilis, leptospirosis and Lyme disease (LD). LD is a rapidly emerging tick-borne
infection caused by Borrelia burgdorferi (Bb). Key events in Bb dissemination are bacterial adhesion to lumenal
vascular surfaces (initiated by the outer membrane lipoprotein BBK32), and crawling/dragging to endothelial cell
junctions where extravasation occurs. Spirochetes’ ability to disseminate requires the ability to simultaneously adhere
to and move along vessel surfaces despite fluid shear forces arising from blood flow. The mechanisms permitting
dynamic coordination of adhesion and motility are unknown. Objective: Define the mechanisms by which Bb
dynamically coordinates vascular adhesion and motility under fluid shear force. Hypothesis: Rapid lateral diffusion
of outer membrane adhesion complexes would allow bacteria to crawl/drag along vessel surfaces in the presence of
blood flow without disrupting bacterial adhesion. Methods: 1) Indirectly visualize diffusion of BBK32 and P66
transmembrane control protein along Bb surfaces using antibody-coated beads. 2) Directly visualize lateral diffusion
rates of fluorescent BBK32 and P66 fusion proteins in photobleached Bb adhering to immobilized host ligands. 3) Use
kymograph profiling to analyze Bb motility and adhesion under conditions where outer membrane fluidity is reduced
(low temperatures). Results: I have built constructs for: 1) disrupting chemotaxis and motility genes in Bb (cheX,
cheY3 and fliG1); and 2) expression of fluorescent GFP, mCherry and tdTomato-BBK32 and –P66 fusion proteins in
GFP-expressing Bb and motility mutants. I am developing methods for studying mobility of BBK32 and P66 bound to
antibody-coated latex beads. Conclusions: These studies will provide the first insight into the mechanisms
coordinating spirochete motility and adhesion during dissemination, a critical step of spirochete pathogenesis which is
a candidate for development of novel antibiotic-complementing therapeutic approaches. Funding: NSERC, CIHR,
NIH, CFI/ORF, Faculty Enrichment Endowment Fund, Bertha Rosenstadt Endowment Fund. Salary support: CIHR
undergrad scholarship (NG), UofT Work Study Program (TT).
39. Irrigation Dynamics Associated With Positive Pressure, Apical Negative Pressure And
Passive Ultrasonic Irrigations: A Computational Fluid Dynamics Analysis
G LAYTON*, J CHEN, B NURBAKHSH, M BUSSMANN, A KISHEN
Department of Endodontics, Faculty of Dentistry
Background: Complexities in root canal anatomy and surface adherent biofilm structures remain challenges in
endodontic disinfection. The ability of an irrigant to penetrate into the apical region of a canal, along with its
interaction with the root canal walls, will aid in endodontic disinfection. Objective: The aim of this study was
to qualitatively examine the irrigation dynamics of syringe irrigation with different needle tip designs (openended and closed–ended), apical negative pressure irrigation with the EndoVac system, and passive ultrasonicassisted irrigation, using a computational fluid dynamics model. Materials and Methods: The SST k-ω
turbulence model was enabled to study the four modes of irrigation and the flow fields for the four types of setup
were calculated and compared in terms of velocity, wall shear stress, and apical pressure with the commercial
CFD code ANSYS FLUENT 12.1.4 for all computational analysis. Results: Syringe-based irrigation with a
side-vented needle showed a higher wall shear stress than the open-ended but was localized to a small region of
the canal wall. The apical negative pressure mode of irrigation was found safest for use at the working length,
while the passive-ultrasonic irrigation group showed the highest wall shear stress along with the greatest
magnitude of velocity. Conclusion: The application of CFD models provides information on the flow and
exchange of irrigant within the root canal system for a particular mode of irrigation. The findings from this
analysis provide a fundamental understanding of the fluid dynamics associated with different irrigation methods.
Supported by: Natural Sciences and Engineering Research Council of Canada (NSERC) Undergraduate Student
Research Awards Program (USRA) and Faculty of Dentistry, University of Toronto Startup funds.
27
40. Characterization of the Streptococcus mutans LexA-Like Regulator and its Role in DNA
Damage Response
V LEUNG*, S KOYANAGI, C M LÉVESQUE
Department of Oral Microbiology, Faculty of Dentistry, University of Toronto
Background: Bacterial cells can suffer DNA damage by various agents, such as antibiotics, acid shock, and oxidative
stress. In many bacteria, DNA damage induces the activation of the SOS response, as a means to cope and repair
damaged DNA. Upon DNA damage, RecA becomes activated and promotes the self-cleavage of the LexA repressor
for the activation of the SOS-regulon for DNA damage tolerance and repair. Many bacteria including
Streptococcus mutans and Streptococcus pneumoniae lack a classically defined LexA homologue and SOS response.
Pneumococci activate their competence pathway in response to DNA damage instead. Previous results from our lab
have shown that activation of the CSP-ComDE competence pathway in S. mutans increases tolerance towards
antibiotics including fluoroquinolones targeting DNA gyrase through the formation of dormant persister cells. We
hypothesized that DNA damage may activate either or both the competence pathway and a separate DNA damage
tolerance mechanism similar to the SOS response. Objective: To characterize the LexA-like homologue (named
LexA-Sm) in S. mutans and determine its role in a general stress response. Methods: Mutant knockouts and an
overexpressing mutated lexA-Sm strain to prevent LexA-Sm autocleavage were constructed. Transcriptional analysis
was performed by QPCR. Persistence assay were performed to determine tolerance of strains towards DNA damage.
Results: Initial BLASTP searches revealed the presence of a LexA-like orthologue in S. mutans. This LexA-like
regulator exhibit characteristics similar to LexA, where it auto-regulates its own expression under various stresses,
undergoes RecA-dependent autocleavage, and the proteolytic degradation of its N-terminal domain is Clp-dependent.
The autocleavage of LexA is important in regulating genes involved in DNA damage tolerance. Conclusions:
S. mutans encodes a LexA-like repressor that activate a tolerance mechanism towards DNA damage. Further analysis
and identification of potential genes regulated by LexA-Sm will need to be determined to elucidate the pathway for
this “SOS”-like response.
41. Epigenetic Imprinting Effects of the Mechanical Environment on the Fibrogenesis of
Mesenchymal Stem Cells
C LI*, B HINZ
Background: When routine repair mechanisms of the body fail to regenerate large area skin burn wounds,
mesenchymal stem cell (MSC) therapy is considered. MSCs can give rise to fibroblastic cells that facilitate wound
healing. However, engrafted MSCs are prone to become myofibroblasts when exposed to high mechanical tension and
pro-fibrotic cytokines in the wound microenvironment. Myofibroblast activity increases wound stiffness and activate
healthy into contractile precursor cells, resulting in pathological tissue remodelling and development of hypertrophic
scars. Objective: To test if the mechanical conditions of cell culture prime MSCs towards or protect MSCs against the
myofibroblast fate by inducing epigenetic changes through fibrosis-associated microRNAs. Methods: Rat bone
marrow-derived MSCs were isolated and sub-cultured on skin-soft (elastic modulus of 5 kPa) or fibrosis-stiff silicone
substrates (100 kPa) for three passages before transferring to their respective other substrates for two additional
passages. Myofibroblast character was evaluated before and after substrate switching to verify if MSCs acquire a
‘memory’ through mechanical priming. The expression of fibrosis-associated microRNAs was assessed using qRTPCR. Results: After three passages, expression levels of the myofibroblast marker smooth muscle actin (SMA) in
MSCSs are higher on stiff than on soft substrates. High SMA levels are maintained even after switching MSCs from
stiff to soft substrates for another two passages. Conversely, switching from soft to stiff maintains low SMA levels.
Consistently, MSCs preserve their level of ED-A fibronectin production after substrate switching. MiR-21 is
responsive to mechanical stimuli as stiff substrates upregulate and maintain miR-21 expression. Conclusion:
Continuous culture on soft substrates imprints a mechanical memory that protects MSCs from activation by elevated
mechanical stress, such as engraftment into stiff dermal scars. By demonstrating that culture on skin-soft substrates
protects MSCs against myofibroblast activation, we are providing novel methods to expand ‘high-quality’ MSCs prior
to body engraftment. Support: Ontario graduate scholarship. CIHR training program in regenerative medicine.
28
42. Role of Type 2 Toxin-Antitoxin Modules in Survival of Streptococcus mutans
A MANKOVSKAIA*, K MOTAVAZE, C M LÉVESQUE
Background: Streptococcus mutans is highly adapted to life in the mouth. It has evolved various means of
coping with the deleterious effects of the environmental stressors and avoiding the host immune system. It has
been suggested that toxin-antitoxin (TA) modules are stress-response elements that help bacteria to cope with
stress. Type 2 TA modules are composed of a small toxin and its cognate unstable antitoxin that sequesters the
toxin through protein complex. Recent work done in our lab has shown that S. mutans uses a quorum-sensing
peptide (CSP) as a stress-inducible alarmone to synchronize the gene expression of a specific group of cells to
successfully survive competition in their ecological niche. Objectives: To study the role of S. mutans MazEF TA
in cell survival under specific stresses and characterize the activity of putative type 2 TAs induced by the CSP
alarmone. Methods: The phenotypic effects of MazEF on tolerance to acid shock, oxidative stress, and
inhibition of translation and DNA replication were determined by monitoring cell lysis turbidimetrically.
Transcriptional studies were conducted by constructing promoter transcriptional fusion. To investigate the role of
CSP-inducible toxins, expression vectors were constructed for the induction of the toxin in E. coli. Results: The
viability of a mutant deficient in MazEF was significantly higher than that observed for the wild-type strain when
challenged with the protein synthesis-inhibiting antibiotic spectinomycin. Under normal growth conditions, the
promoter activity of mazEF remained nearly unchanged until mid-log phase and then started to decline. Under
spectinomycin stress, cells displayed 2.5-fold reduction in mazEF gene expression. Overproduction of S. mutans
CSP-inducible toxins had a toxic effect on E. coli. Mild-ectopic expression of a novel TA increases the levels of
antibiotic-tolerant persisters in S. mutans. Conclusion: Type 2 TAs play an important role in S. mutans
physiology, including stress survival and persister formation.
43. Remote Mechanosensing by Cells on Thin Floating Collagen Matrices
H MOHAMMADI*, P ARORA, P JANMEY, C MCCULLOCH
Institute for Medicine and Engineering, University of Pennsylvania,
Background: The mechanical properties of the extracellular matrix impact many cellular functions but the
contribution of matrix deformations to cellular mechanosensing, especially at sites beyond the immediate cellmatrix interface, is not defined. Objectives: To assess remote, in-plane mechanosensing by cells on constrained
floating matrices. Methods: We examined remote mechanosensing with a novel cell culture model that employs
collagen gels supported circumferentially by nylon mesh grids floating on culture medium. This model obviates
mechanical interference from the rigid underlying foundation of tissue culture plastic and enables assessment of
remote, in-plane mechanosensing. Results: We found that 3T3 cells rapidly (<3h) formed cellular extensions
whose lengths and number per cell depended on the grid opening size. When the opening sizes were 200 µm or
500 µm wide, the mean number of extensions per cell and the sum of cell extension lengths was not significantly
different (p>0.6). In contrast, in larger grids (1700 µm width), the mean number of extensions per cell, the mean
extension length and the sum of cell extension lengths decreased (40-60%; p<0.0001). In grids of 200 µm and
500 µm widths, cell-generated deformation fields extended to, and were resisted by, the grid boundaries.
However, in grids of 1700 µm width, the deformation field did not extend to the grid boundaries. Generation of
cellular extensions required the ß1 integrin, focal adhesion kinase, filamin A and non-muscle myosin II activity.
Conclusions: The new model demonstrates that in the absence of a rigid underlying foundation, cells sense
remotely the presence of physical boundaries through creation of deformation fields in thin matrices, which
affects the formation of cell extensions. Grants: HM gratefully acknowledges financial support from a CIHR
Strategic Training Fellow, STP-53877. The research was supported by CIHR operating grant to CAM (MOP36332), who is also supported by a Canada Research Chair (Tier 1).
29
44. TSP-1 Synthesized by Stromal Cells is Involved in Invasion of Oral Squamous Cell
Carcinoma
S K PAL*, K SAKAMOTO, J W LEE, A YAMAGUCHI
Section of Oral Pathology, Department of Oral Restitution, Division of Oral Health Sciences, Graduate
School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in
Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo, Japan.
Background: Thrombospondin-1 (TSP1) is a secretory glycoprotein that binds to many proteins and mediates
diverse cellular functions. Its significance in oral carcinogenesis has been suggested, but is yet poorly understood.
Materials and methods: 30 formalin fixed, paraffin embedded specimens of surgically excised Oral Squamous
Cell Carcinoma (OSCC) were used for immunohistochemical analysis. For cell culture experiments human oral
cancer cell lines (HSC3, Ca9-22 and Ho-1N1) and a mouse stromal cell line (ST-2) were used to investigate the
cell proliferation, migration and invasion. Results: In normal oral mucosa, TSP1 expression was observed
preferentially in the capillary endothelial cells. In OSCC, TSP1 expression was significantly increased in the
cancer-associated stroma, showing TSP1 expression in the capillary endothelial cells, activated fibroblasts and its
accumulation in the extracellular matrix. Cell culture experiments revealed that production of TSP-1 mRNA was
increased in ST-2 cells by co-culture with OSCC cells. TSP-1 has no significant effect in cancer cell proliferation
but it stimulated the migration and invasion of OSCC cells (HSC-3 and Ho1-N1) by Wound healing assay and
Matrigel invasion assay respectively. These migration and invasion activities were inhibited by the supplement of
TSP-1 neutralizing antibody. All of OSCC cells used in this study synthesized substantial levels of TGF-β1 by
ELISA analysis. TGF-β1increased the expression of TSP-1 in ST-2 cells at mRNA and protein levels.
Conclusion: These findings suggested that TGF-β1 synthesized by OSCC cells may participated the induction of
TSP-1 expression in stromal cells, and the TSP-1 produced by stromal cells promote migration and invasion of
OSCC.
45. Bioactive Nanoparticles and Photodynamic Therapy Inhibit Collagen Degradation in vitro
A PERSADMEHR*, CD TORNECK, DG CVITKOVITCH, CAG MCCULLOCH, A KISHEN
Background: Collagen is the major protein constituent of human dentine but collagen degradation can
compromise the structural integrity and resistance to fracture of dentine. Objective: We evaluated the ability of
photodynamic therapy (PDT), chitosan nanoparticles (CSnp), or PDT in combination with CSnp, to inhibit
bacterial collagenase-mediated degradation of collagen. Material and Methods: Rat type 1 fibrillar collagen
matrices were treated as follows: Groups-A & B: untreated (control); Group-C: 2.5% glutaraldehyde; Group-D:
2.5% GA followed by 1% CSnp; Group- E: 1% CSnp; Group-F: PDT (Rose Bengal activated with 540 nm light
at 40 J/cm2); and Group-G: 1% CSnp followed by PDT. Samples (except Group A controls) were exposed to
Clostridium histolyticum collagenase for 24 h. Collagen degradation was assessed by hydroxyproline release.
Residual collagen remaining after enzyme treatment was quantified by picrosirius red staining. We analyzed
collagen cross-links with Fourier transform infra-red (FTIR) spectroscopy. The binding of collagenase to CSnp
was determined by immunoblotting. Results: We observed that collagen treated with CSnp, PDT, or a
combination of CSnp and PDT, exhibited less degradation than controls. GA-treated collagen was the most
resistant to collagenolytic. The abundance of post-treatment residual collagen correlated with the extent of
degradation. FTIR analysis showed that PDT and GA treatments enhanced collagen cross-linking.
Immunoblotting analysis showed that CSnp and collagenase did not bind. Conclusion: Application of PDT,
CSnp, or a combination of PDT and CSnp to root dentine may inhibit collagenolytic degradation of exposed
dentine collagen. Grants: Supported by American Association of Endodontists Foundation, Canadian
Association of Endodontics Endowment Fund, and Alpha Omega Foundation of Canada.
30
46. Tooth Pulp Noxious Stimulation Depresses Excitability of Rat Face Primary Motor Cortex
(Face-M1) that is Dependent on the Functional Integrity of Medullary Astrocytes
H PUN*, L AWAMLEH, L AVIVI-ARBER, B J SESSLE
Background: Dental pain is associated with neuronal and astrocytic processes in the medullary dorsal horn
(MDH) and neuroplastic changes in face-M1, and may be reflected clinically in disruption of orofacial
sensorimotor functions. Application of the inflammatory irritant mustard oil (MO) to the dental pulp and the
astrocytic inhibitor methionine sulfoximine (MSO) to the MDH in rats has been used to study brainstem
mechanisms associated with pulpal inflammation and pain. To examine if MDH astrocytes are also involved in
modulating face-M1 neuronal activity, our Objectives were to test whether: 1) MO application to the rat’s tooth
pulp affects face-M1 excitability defined by intracortical microstimulation (ICMS) thresholds capable of evoking
electromyographic activity in the jaw-opening anterior-digastric muscles, and 2) subsequent MDH application of
MSO influences the MO effect. Methods: Under Ketamine general anaesthesia, Sprague–Dawley male rats were
implanted with electromyographic electrodes into the anterior-digastric muscles. Following craniotomy, a
microelectrode was positioned within the left face-M1 at a site from which ICMS (35ms train, 12x0.2ms pulses,
333Hz) could evoke baseline anterior-digastric electromyographic activity at a low-threshold stimulation
intensity (≤30µA). Baseline ICMS threshold was monitored for 30min, then MO (N=6)(or saline, N=5) was
applied to the previously exposed right maxillary molar tooth pulp and ICMS thresholds monitored for 60min. In
other rats, 15min following MO pulp application, 0.1mM MSO (N=5)(or saline, N=5) was applied to MDH and
anterior-digastric ICMS thresholds monitored for 60min. Results: Within 15min of MO pulp application,
anterior-digastric ICMS thresholds significantly increased (39%; repeated-measures ANOVA, post-hoc
Bonferroni: p=0.014) as compared with baseline or saline controls. The decreased face-M1 excitability could be
normalized to baseline levels following MDH application of MSO. Conclusions: These findings suggest that
dental pain-related motor disturbances may involve reduced face-M1 excitability that is dependent on the
functional integrity of MDH astrocytes. Support: CIHR MOP4918, Rosenstadt-funding
47. Triethylene Glycol Up-regulates Virulence Genes in Cariogenic Oral Bacteria
L SADEGHINEJAD*, D G CVITKOVITCH, J P SANTERRE, Y FINER
Secondary caries is a major cause of dental composite restoration failure. Streptococcus mutans is a primary species
associated with the initiation and progression of caries. The breakdown of the resin-dentin interface by salivary and
bacterial esterases results in the generation of biodegradation by-products including triethyleneglycol (TEG). It was
previously reported that several S. mutans virulence genes including; Glucosyltransferases B(gtfB) and C(gtfC),
Glucan binding protein-B(gbpB), comCDE were significantly up-regulated in the presence of TEG at concentrations
of 0.01 and 0.1 mM (P<0.05) at pH 5.5, more significantly in biofilms versus planktonic cells. Objective: To further
investigate the effect of TEG on the same genes in S. mutans cultures under neutral pH. Methods: Gene expression
was measured by quantitative real-time PCR for both planktonic and biofilm cultures of S. mutans UA159 in the
presence of 0.00, 0.001, 0. 01 and 0.1mM of TEG at pH 7.0. Results: At pH 7.0 the tested genes were not affected by
TEG during biofilm growth of S. mutans UA159, however these genes were up-regulated at a concentration of
0.01mM TEG in planktonic S. mutans cells. These findings indicate that there is substantial dependence of gene
expression on pH. One possible explanation for the difference is that low pH triggers changes in the bacterial cell
membrane fatty acid composition that affect membrane permeability. This may result in easier penetration of
environmental stressors like TEG, a small molecule, and activation of stress-response genes. The close proximity of
cells in the biofilm cultures may facilitate sensing of environmental stresses when compared to the planktonic state.
Conclusion: These findings indicate that the TEG induced effect on S. mutans pathogenicity is not only concentrationand growth mode- dependent as reported previously but also pH-dependent, thereby providing another parameter that
may influence the progression of secondary caries and longevity of dental composite restorations. Supported by: NIH
# R01DE021385-0 and University of Toronto, Faculty of Dentistry Harron Scholarship.
31
48. Functional Characterization of the Dual CRISPR Systems within Streptococcus mutans
M A SERBANESCU1*, M CORDOVA1, R FLICK2, N BELOGLAZOVA2,
A F YAKUNIN2, D B SENADHEERA1, D G CVIKOVITCH1
1
Dental Research Institute, Faculty of Dentistry, University of Toronto, Toronto, Canada.
2
Department of Chemical Engineering and Applied Chemistry, Banting and Best Department of
Medical Research, University of Toronto, Toronto, Canada.
Clustered regularly interspaced short palindromic repeats (CRISPRs) together with cas genes (CRISPRassociated) provide adaptive immunity against phages and invasive genetic elements.
We investigated the involvement of CRISPRs in the physiology of the cariogenic organism Streptococcus mutans. Since
promoter regions of both CRISPRs revealed putative binding sites for the VicR response regulator, we used transcriptional
analysis to demonstrate that VicR/K differentially modulated gene expression of both operons. Further, two S. mutans Cas
proteins, Smu.1760c (Csd2) and Smu.1763c (Cas5d), were recombinantly expressed in Escherichia coli and analyzed for
nuclease activity. Both proteins cleaved single stranded (ss) RNA, whereas only Csd2 cleaved ssDNA. Since Cas5d cleaved
synthetic ssRNA we sought to identify potential substrates specifically targeted and cleaved by this protein. S. mutans
UA159 RNAs incubated in vitro with purified protein were used for cDNA synthesis and microarrays. Although the
expression of five genes was significantly downregulated by the addition of Cas5d protein (p<0.05), our “in vitro”
transcription analysis showed no apparent substrate specificity for candidate genes. Considering the significant cleavage
observed it was likely that interaction of Cas5d with other proteins was required for specific recognition and degradation.
Since Cas5d and Csd2 had RNase/DNase activity we sought to determine whether CRISPR/Cas systems protected the cell
from foreign DNA taken up by S. mutans during genetic transformation. Sequence similarity searches for seven spacers
within CRISPRs revealed 100% identity with nucleotide sequences of plaque residents, Aggregatibacter
actinomycetemcomitans (17 bp), Fusobacterium nucleatum (21bp), and Treponema succinifaciens (18 bp). Hence, we argue
that CRISPR systems in S. mutans may preferentially defend against DNA taken up by transformation as a counterattack
system and studies are underway to test the role of CRISPRs in horizontal gene transfer, a process linked with the control of
other virulent attributes of S. mutans. Grants: NIH R01DEO13230-03; CIHR MT-15431, Cell Signalls fellowship.
49. Targeted Antibacterial Properties of Functionalized Nanoparticles
A SHRESTHA*, A KISHEN
Discipline of Endodontics/ Dental Research Institute
Background: Biocompatibility and effectiveness in the presence of tissue fluids are two of the crucial factors for
antimicrobial nanoparticles to be applied in-vivo. This study hypothesized that a bioactive polymeric nanoparticle
functionalized with a photosensitizer would combine properties of chitosan i.e., polycationic and antibacterial; and that of a
photosensitizer i.e., to generate singlet-oxygen when photoactivated. This functionalized bioactive nanoparticle (chitosan
conjugated rose bengal-CSRBnp) may show targeted antibacterial activity and biocompatibility towards fibroblasts.
Objective: The study aimed to assess the interaction and uptake of CSRBnp on bacterial and fibroblast cells and the
resultant effect on their viability. Methods: CSRBnp was synthesized using previously set protocol. The interaction and
uptake of CSRBnp was evaluated on bacteria (Enterococcus faecalis ATCC-29212) and mouse fibroblast cells (NIH-3T3).
The effect of bovine serum (BS) on the toxicity of CSRBnp was also evaluated. The effect on bacterial cells was assessed
using culture methods, absorbance and fluorescence microscopy (viability and uptake); scanning-electron (SEM) and
atomic-force-microscopy (AFM) (surface morphology). Fibroblasts were assessed using mitochondrial assay (viability); and
bright-field microscopy and SEM (uptake and surface morphology). Results: The CSRBnp showed significant antibacterial
properties and uptake into the bacterial cells within 15min of interaction. In case of fibroblasts, the CSRBnp were taken up
into the cell cytoplasm after 15min of treatment. These CSRBnp did not show any cytotoxicity as compared to the strong
antibacterial activity. CSRBnp with/without photoactivation retained the antibacterial efficacy even in the presence of BSA.
SEM and AFM revealed CSRBnp adhered to the bacterial surface resulting in altered cell morphology. CSRBnp was
effluxed by the fibroblasts with longer interaction time without any toxicity. Conclusion: The CSRBnp interacted in a
distinctly different manner with bacteria and fibroblasts, resulting in toxicity towards bacteria while maintaining the
viability of fibroblasts. This highlighted the targeted antibacterial property of the newly developed CSRBnp.
Supported by: University of Toronto start-up fund (#928133), CIHR Training Fellowship, TGF-53877.
32
50. Temporal Controlled Drug Delivery System using Polycationic Chitosan Nanoparticles for
the Differentiation of Stem Cells from Apical Papilla
S SHRESTHA1*, A DIOGENES2, A KISHEN1
Discipline of Endodontics/Dental Research Institute, Faculty of Dentistry, University of Toronto, Canada
Endodontics, University of Texas Health Science Center San Antonio, San Antonio, TX, USA
Background: Delivery of a drug or a bioactive molecule is a major challenge in tissue engineering, mainly because of the
low solubility, rapid release and short lifetime of the bioactive molecules in vivo. Objective: To evaluate temporal
dexamethasone (Dex) release from polycationic chitosan nanoparticles (CSnps) for odontogenic differentiation of stem cells
from apical papilla (SCAP). Methods: CSnps with an average diameter 100 nm were prepared by ionic gelation of
polycationic chitosan with polyanionic tripolyphosphate. Dex loaded CSnps (Dex-CSnps) was prepared by physical
-potential of synthesized nanoparticles were measured using a Zetasizer
via dynamic light scattering method. The release of Dex in phosphate buffered saline was determined by quantifying the
free Dex absorption spectrum at 242 nm. The association of Dex to Csnp was characterized using Fourier Transform Infrared (FTIR) spectroscopy. The temporal controlled release of Dex and their potential for odontogenic differentiation was
validated by biomineralization assay. Results: FTIR analysis showed minor change in the band position of O-H and N-H
stretching from Dex and chitosan, respectively. This indicated intermolecular interaction between the lipophylic Dex and
polymeric chitosan. Lack of other characteristic absorption bands depicted that that there was no obvious chemical reaction
between the Dex and chitosan. Adsorption analyses using UV spectrophotometer determined the actual loading of Dex to be
0.25%. The release of Dex was biphasic in nature. There was an initial rapid release phase, followed by a slow and linear
release phase for 2 weeks. Both blank CSnps and Dex-CSnps were nontoxic to SCAP. Alizarin red staining after 2 weeks of
odontogenic induction showed that biomineralization occurred in the wells. The light microscopy revealed that staining was
positive for Dex-Csnp treated group as compared to positive control. The mineralized structures produced in SCAP cultures
were positive for markers of odontogenic differentiation. Conclusions: This study showed that the developed drug loaded
chitosan nanoparticles delivered Dex in a time-controlled manner for the odontogenic differentiation of SCAP. To our
knowledge, this is the first report showing the efficacy of biopolymeric polycationic CSnps to deliver Dex in a temporal
controlled manner for differentiation of SCAP into odontoblasts.
Supported by: University of Toronto Start up fund (#928133).
51. Understanding The Role Of CopYAZ Operon In Copper Homeostasis In Streptococcus
mutans
K SINGH*, D B SENADHEERA, C M LEVESQUE, D G CVITKOVITCH
Dental Research Institution, University of Toronto, Toronto, ON, Canada
Background: Copper acts as a crucial cofactor for certain enzymes, while its homeostasis and detoxification are important
for cell viability. In various bacterial systems, copper influences biofilm formation and dissemination. Copper can also be
potentially toxic and its homeostasis must then be carefully regulated. In Streptococcus mutans, a leading etiological agent
of dental caries, copper cations are involved in the dimerization of the ComE response regulator of the CSP-ComDE
quorum-sensing system involved in the regulation of biofilm development. The S. mutans copYAZ operon codes for a
copper transport and resistance system. Work previously done suggested that CopY is a negative regulator repressing cop
promoter activity, while copA and copZ code for a P-type ATPase and an anti-repressor, respectively. We recently identified
a putative binding site for GcrR in the promoter region of cop operon. GcrR is a negative transcriptional regulator of
surface-associated glucan binding proteins that contribute to the cohesive properties of dental plaque biofilms. Objective:
To examine the role of the cop operon on biofilm formation, genetic transformation, and tolerance to copper in S. mutans.
Methods: A copYAZ non-polar knockout mutant strain (ΔcopYAZ) was constructed and utilized for growth kinetics,
biofilm formation and transformation frequency assays. Quantittive Real time PCR was conducted using RNA isolated from
wild type and mutant strains in gcrR or copYAZ. Results: Using a CopYAZ deficient mutant, we showed significance
difference in susceptibility to copper cations as compared with the wild-type strain. Gene expression analysis revealed
increased expression of the cop operon in a GcrR deficient mutant. We also demonstrated that the cop operon is induced by
copper in vitro. Interestingly, a CopYAZ deficient mutant was unable to develop biofilm in the presence of copper and was
impaired in its ability to acquire DNA from the environment relative to the wild type. Conclusion: Altogether, our results
suggest that copper homeostasis is important for S. mutans growth, biofilm formation and natural transformation.
Acknowledgements: CIHR grant MT-15431, NIH-R01DE013230-03
33
52. The Regulatory Role of ComRS in Cell Viability and Biofilm Formation of Streptococcus
mutans
IB WENDERSKA*, M CORDOVA, DG CVITKOVITCH, DB SENADHEERA
Background: Competence contributes to genetic heterogeneity, allowing for faster adaptation to
changing environmental conditions. In Streptococcus mutans, the ComRS signaling system, comprising
of the ComR regulatory protein and the ComS precursor to the sigX-inducing peptide (XIP), has
recently been identified as the proximal regulatory system to ComX, an alternate sigma factor essential
for competence development. ComRS is regulated by an upstream signal transduction system,
ComCDE. ComCDE also regulates the transcription of bacteriocin genes implicated in cell lysis, and
glucosyltransferases (gtfs), important for biofilm formation. Objective: Due to the crucial role of
ComRS in ComCDE-mediated competence development, here we investigate whether ComRS also
plays a role in regulating ComCDE-mediated cell lysis and biofilm development. Results: Our
evaluation of cell viability in the presence of 10μM XIP resulted in the killing of nearly 82% of the
population. The killing activity was shown to be dependent on the presence of comR/S and comX. We
further observe abolished biofilm formation on polystyrene surface in the presence of XIP in wild-type
UA159. In the absence of comR, XIP had no effect on biofilm development. Since glucosyltransferases
modulate biofilm formation via polymer production, the role of ComR on the expression of gtfs was
investigated. Using gene expression analyses, we demonstrate a significant increase in the expression
of gtfB/C/D in the absence of comR, suggesting that ComR negatively regulates gtfB, gtfC and gtfD
expression. We further observe a downregulation of their expression in the presence of exogenous XIP.
Conclusions: Taken together, this is the first report that describes roles for ComRS in modulating cell
killing and biofilm formation of S. mutans. Our finding that XIP can act as a potent biological molecule
for targeted killing of S. mutans may have clinical implications in targeting S. mutans in an effort to
control dental caries. Supported by: NIH-R01DE013230-03 and CIHR-MT15431.
34
STUDENTS
Clinical Science Category
35
53. The Effect Of The Voxel Size On The Identification Of Vertical And Horizontal Root
Fractures By Cone Beam Computed Tomography: An In-Vitro Study.
AMINTAVAKOLI N*, PHAROAH MJ, BASRANI B, LAM EW.
Department of oral radiology, Faculty of Dentistry, University of Toronto
Background: Tooth root fractures represent cleavage planes in tooth structure, and can be subclassified as either
vertical or horizontal based on the orientation of the fracture plane. In general, the prognosis of tooth root
fractures is usually poor, so early, definitive identification is important to reduce damage to the adjacent tissues.
Although clinical and radiographic features may not definitively identify a fractured root, clinicians often base
their diagnoses on these investigations. Recently, cone beam computed tomography has been used to investigate
tooth root fractures. The aim of this study is to identify the effects of the voxel size (resolution) on the
identification of vertical and horizontal root fracture by cone beam CT scan in an in vitro study. Method and
Material:Forty-five intact extracted human teeth were used in this study. Vertical (15) and horizontal (15) root
fractures were induced in a total of 30 teeth, and 15 teeth were left intact. The teeth were randomly grouped into
9 tooth blocks, each consisting of one incisor, two premolars and two molars as they might appear in a segmental
dental arch. Each block was scanned with the Kodak 9000 3D (Carestream, Rochester, NY) operating at 65 kV
and 2.5 mA at the native voxel resolution of 76μm. The raw images were then downsampled to create images
with voxel sizes of 100, 200, and 300μm. Four observers then evaluated the images in a randomly fashion with a
one-week interval between each observation. Result:The interclass correlation coefficient ( ) for inter-observer
agreement for CBCT voxel sizes 76μm, 100μm, 200μm, and 300μm were 0.52, 0.61, 0.61 and 0.46, and the
mean areas under curve (AZ) for voxel sizes 76μm, 100μm, 200μm, and 300μm were 0.65, 0.65, 0.62 and 0.6,
respectively. These differences were not statistically significantly different. Conclusion:Voxel resolution of
cone beam CT images do not affect the interpretation of vertical or horizontal root fractures, in vitro.
54. The Role of Light Emitting Diode Phototherapy (LED) in Orthodontic Tooth Movement
(OTM).
S CHUNG*, S GONG, B TOMPSON.
Objective: An increase in orthodontic treatment efficiency has been reported with laser application.
However, lasers require a controlled clinical environment for safe and effective delivery. This study
will investigate if light emitting diode (LED) phototherapy is a viable alternative to lasers. Results from
our study will offer clues to the potential use of LED to accelerate the rate of OTM in the clinical
setting. Methods: Eleven patients undergoing orthodontic treatment at the Faculty of Dentistry, UT,
were selected. Inclusion criteria included bilaterally symmetric extraction of premolars and full banding
and bonding of appliances. During space closure of the extraction site, LED phototherapy was applied
to one side of the dental arch for 20 minutes daily for 4-12 weeks. LED phototherapy was recorded by
the LED unit as well as by the patient. To permit measurements of space closure on dental casts, dental
impressions were taken at 3 time points using a chromatic alginate with long dimensional
stability (Kromopan) immediately prior to (T0), during (T1) and after space closure (T2). The rate of
space closure of the control and LED treated sides were compiled and compared with each other.
Results: All eleven patients were compliant with LED application. On average the duration of usage
was 78% at T1 and 82% at T2. Preliminary results suggest that no significant differences in the rate of
tooth movement resulted from the application of LED phototherapy. Conclusions: The results suggest
that extra-orally delivered LED phototherapy does not significantly alter the rate of OTM. This is to
contrary previous findings with laser phototherapy mediated modulation of OTM and could be related
to the duration or method of LED delivery. Further investigations are needed to determine whether
LED phototherapy application can influence the rate of OTM.
Supported by: University of Toronto, Faculty of Dentistry Dental Research Institute.
36
55. Ectopic Eruption of the First Permanent Molar:
A Retrospective Assessment of Prevalence and Prognostic Factors in a Private Pediatric Dental
Practice
and
Survey of Specialists' Attitudes Regarding Intervention
B DABBAGH*, M SIGAL, K TITLEY, B TOMPSON, P ANDREWS
Department of Pediatric Dentistry, Faculty of Denistry, University of Toronto
Objectives: The purpose of this study was to assess retrospectively the incidence and predictive factors for
self-correction and occurrence of adverse events associated with ectopic eruption of maxillary first
permanent molars, in a sample population where no corrective intervention was initiated. The survey
portion’s objective was to assess the prevailing attitudes amongst pediatric dentists and orthodontists
regarding indications and timing of intervention of ectopically erupting first permanent molars. Materials
and Methods: Charts of patients diagnosed with ectopic eruption of the first permanent molars were
assessed and radiographs were analyzed for predictive factors. An online survey presenting three cases of
ectopic eruption was sent to active members of the AAPD, CAPD and AAO. Results: Sixty-five ectopically
erupting first permanent molars were included in the study. Forty-six (71%) self-corrected, of which one
third occurred after age nine. Space loss, with an average of approximately 3 mm, occurred in 18 cases
(28%). Increased impaction value and severe lock were positively correlated with irreversibility. Muliple
regression analysis was significant for males, bilateral cases, severe lock and no partial eruption as
predictors of irreversibility. Nine hundred and fifty pediatric dentists and 642 orthodontists responded to the
survey. Orthodontists initiated treatment more frequently than pediatric dentists in a bilateral case of ectopic
eruption and estimated the rate of spontaneous self-correction to be much lower. Conclusion: Significant
factors in determining the type of ectopic eruption are discussed. Spontaneous self-correction may occur
after 9 years of age and delaying treatment is a treatment option when uncertain of diagnosis.
56. Nitric Oxide Levels in Gingival Crevicular Fluid during Orthodontic Tooth Movement
H FORD*, D NILFOROUSHAN, S SURI, SG GONG
Background: Nitric oxide (NO) is a signaling molecule involved in bone remodeling induced by
mechanical loading. It has also been shown to enhance the rate of orthodontic tooth movement (OTM) in rat
models. In humans, however, the role of NO in OTM remains less clear. Our hypothesis is that NO levels
are increased in the gingival crevicular fluid (GCF) of teeth undergoing OTM in human subjects. Objective:
To measure the levels of NO in the GCF of specific teeth before and after the initiation of OTM. Methods:
Thirteen male participants (ages 11-18, mean 14y 2m) undergoing non-extraction treatment at the Graduate
Orthodontics Clinic, UT, were recruited based on the following inclusion criteria: 1) full non-extraction
treatment of maxillary permanent teeth, 2) absence of systemic and periodontal conditions, and 3) good oral
hygiene. Samples of GCF were collected immediately before, 1 hour after, and 3-4 days after bonding of the
maxillary right to left first molars and insertion of light Nitinol archwires. The maxillary second molars
served as controls. GCF samples were collected with PerioPaper points (Oraflow Inc.) on maxillary bilateral
central incisors and first and second molars. All GCF samples were analyzed by the Griess Reagent system.
Results: Preliminary data so far indicated that the levels of NO in GCF did not increase to a significant
degree when compared to baseline levels and to the second molar controls. Conclusions: The result of this
study shows that the levels of NO in GCF samples did not change to a significant degree following the
initiation of OTM with light continuous forces. It is speculated that, since the patients had minimal
crowding and the application of very light orthodontic forces, the orthodontic forces applied to the teeth
were too light to demonstrate a significant effect on NO levels present in the GCF.
37
57. Quantification of Ramus Bone for Harvest using Cone Beam CT and Orthopantomogram
Radiography
R GALLARDI*, C CLOKIE, E LAM, M WILEY, J HASSO, F EBRAHIM
Department of Oral and Maxillofacial Surgery, Faculty of Dentistry
Objective: The purpose of this study is to compare the accuracy of Cone Beam CT imaging with digital
Orthopantomograms in determination of bone quantity for harvest in the ramus region. Methods: Twenty-nine
cadaveric mandibles marked bilaterally with three separate fiducial markers in the posterior mandible were
imaged using both Cone Beam CT (CBCT) and digital Orthopantomogram radiography. The mandibles were
sectioned at each marker and four cross sectional measurements were made. The same measures were preformed
on the CBCT images and two corresponding linear measurements were made on Orthopantomograms. Six
separate observers preformed all measures. Statistical analysis was used to determine intra and interobserver
variability and to compare accuracy of CBCT and Orthopantomogram measures with the anatomic gold
standards. Results: The majority of mandibular canals were located 10 to 15 mm from the alveolar crest. Buccal
bone thickness was on average 3.2 mm in the area of the ascending ramus, 5.2 mm in the area of the second
molar and 5.5 mm in area of the first molar. There was no intra or interobserver variability for any of the
measures (p<0.05). CBCT measures were found to differ from anatomic gold standards by 24.9 percent or on
average 2.9 mm (P< 0.05). The linear measures from the Orthopantomograms varied from the anatomic gold
standards by 15.9 percent. Conclusion: Evaluation of the cadaveric mandibles revealed measures that were
consistent with past anatomic studies, identifying the greatest thickness of buccal bone in area of first molar.
CBCT and orthopantomogram measures were significantly (p< 0.05) different from those identified on the
anatomic specimens, the inaccuracies being explained by deficiencies in the radiographic technology or lack of
precision in landmark identification.
58. Craniosynostosis, the Fibroblastic Growth Fact Receptor and Gastrointestinal
Malformations – A Possible Link
C HIBBERD*, S BOWDIN, B TOMPSON, C FORREST, S GONG
Craniosynostotic birth defects are most commonly associated with mutations in Fibroblast Growth Factor
Receptors (FGFR)-2 and 3. Recent clinical and animal reports have suggested a link between the presence of
defects in the gastrointestinal tract (GIT) with FGFR-associated craniosynostotic syndromes. Objective: To
determine whether clinical symptoms associated with GIT disorders are more prevalent in children with FGFRassociated craniosynostosis. Methods: A retrospective chart review of craniosynostotic patients treated from
1980-2012 was conducted at the Hospital for Sick Children. Treatment records of craniosynostotic patients were
obtained from the SHIRE, Craniofacial and Molecular Genetic Laboratory databases and analyzed for any
history of visceral organ abnormalities. Inclusion criteria included: i) a diagnosis of craniosynostosis, ii)
molecularly confirmed mutation of FGFR gene, iii) availability of health records at HSC, iv) at time of treatment
patient was under 18 years of age. GIT abnormalities were classified using the European Surveillance
Classification of Congenital Anomalies (EUROCAT) system. Results: Fifty two charts have been reviewed for
symptoms of GIT problems. Some of the most common symptoms were gastroesophageal reflux, pyloric
stenosis, choanal atresia, umbilical hernias and bifid collecting systems. Conclusions: Preliminary results
suggest an association FGFR-associated craniosynostosis and GIT malformations. This is not unexpected in light
of the findings in animal studies that showed a direct effect of FGF signaling on proper formation of the GIT. An
awareness of the association between organ malformations, craniosynostosis and an FGFR mutation is extremely
important in the diagnosis and care of these afflicted individuals and potentially can change their management.
Supported by: University of Toronto, Faculty of Dentistry, Research Ethics Board at the Hospital for Sick
Children.
38
59. Root Canal Instrumentation Performed Through Conservative Endodontic Access: A
Micro-CT Assessment In Incisors, Premolars and Molars
R KRISHAN*, F PAQUE, T DAO, A KISHEN, S FRIEDMAN
Background: Conventional endodontic access is designed to facilitate canal instrumentation, but dentin removal while
gaining straight-line access may render root-filled teeth susceptible to fracture. Conservative endodontic access (CEA) may
reduce fracture risk, but also compromise canal instrumentation due to restricted access. Objective: This study aimed to
characterize canal instrumentation performed through CEA, with regards to dentin removal and efficacy as suggested by
canal-wall contact. Materials and Methods: Sixty intact extracted human maxillary incisors, mandibular premolars and
molars (N=20/type) were imaged with micro-CT (Scanco µCT 40; 70 kV, 114 µA) with 20µm resolution. Canals were
accessed either through CEA plotted on the micro-CT images so as to minimize removal of dentin (experimental group;
n=10/type) or conventional access (control group; n=10/type), cleaned and shaped with Wave-One instruments (DentsplyMaillefer) with 1.25% NaOCl irrigation, and imaged again with micro-CT as before. The volume of dentin removed (VDR;
mm3) and instrumented canal-wall surface (ICS; % of total canal-wall surface) were determined by 3-dimensional
reconstruction and repositioning of micro-CT images before and after instrumentation. Data was analyzed by repeatedmeasures ANOVA (p=0.05). Results: One-hundred-twenty micro-CT scans were completed, revealing significantly lower
(p≤0.001) VDR values in the experimental group (16.09±4.66 mm3 incisors, 8.24±1.64 mm3 premolars, 33.37±6.41 mm3
molars) compared to controls (23.24±3.38 mm3 incisors, 14.59±4.85 mm3 premolars, 67.71±14.12 mm3 molars). Analysis of
ICS data continues for the apical, middle and coronal canal levels. Conclusions: Canal instrumentation performed through
CEA required less dentin removal than a conventional access. Further data analysis will help understand whether CEA
impacts on efficacy of canal instrumentation and fracture. Supported by: Canadian Academy of Endodontics Endowment
Fund and by Endo/Tech.
60. Oral neutrophils display a site-specific phenotype characterized by expression of T-cell
receptors
F. LAKSCHEVITZ*, G. ABOODI, M. GLOGAUER
Background: Neutrophils, key cells of the innate immune system, are mistakenly described as terminally differentiated cells with
a highly condensed nucleus, incapable of altering their gene expression following differentiation and maturation in the bone
marrow. Only recently it has been shown that neutrophils carry out rapid and complex changes in gene expression during
inflammatory responses. Previous work from our lab has demonstrated differences in reactive oxygen species (ROS) production
between oral and peripheral blood neutrophils isolated from periodontitis patients, suggesting that oral neutrophils present with a
distinctive oral phenotype when compared with blood neutrophils. Understanding differences in the neutrophil transcriptome
following transit from circulation into the site of inflammation will give us new insights into how these innate immune cells
function during inflammation. Methods: Venous blood and oral rinse samples were obtained from healthy subjects. Blood
neutrophils were isolated using a standard gradient method. Oral neutrophils were isolated through nylon mesh filters of different
pore sizes. After isolation of neutrophils from both samples and further RNA purification, gene expression analysis was assessed
by microarray technology and confirmed by qRT-PCR. Additionally, immuno-fluorescence microscopy and Cytokine Array were
performed. Results and discussion: We were able to optimize oral neutrophil isolation which we show is critical when analyzing
gene expression as isolation through 40µM alone does not purify the sample, and results in epithelial cells contamination. We also
show that oral neutrophils present with a significant increase in T cell receptor expression compared to circulating neutrophils,
suggesting a role for oral neutrophils in cross-talk between innate and adaptive immune system in the mouth. Conclusions: Our
goal was to use a modified method to isolate highly pure and viable neutrophils from oral rinse samples in order to characterize
OPMN using a transcriptome approach. With this method we compared the oral neutrophil phenotype to the circulating neutrophil
phenotype using microarray analysis and qRT-PCR. Using a bioinformatics approach for clustering neutrophil gene expression, we
found that oral neutrophils present with a unique transcriptome compared to circulating neutrophils. In addition we show for the
first time that oral neutrophils express T-Cell receptors indicating that these cells may play a key role in linking the innate and
adaptive arms of the oral immune system. Grants: This work was funded by The Canadian Institutes of Health Research (CIHR,
Ottawa, ON) and the Alpha Omega Foundation of Canada (Toronto, ON). FL is supported by CIHR Training Fellowship, TGF53877 and the Harron scholarship (Faculty of Dentistry, University of Toronto) GA is also supported by Harron scholarship.
39
61. Mineral Trioxide Aggregate Pulpotomy for Vital Primary Incisors: A Randomized
Controlled Trial
T NGUYEN*, P JUDD, M CASAS, M SIGAL
The Hospital for Sick Children, Department of Paediatric Dentistry, Faculty of Dentistry, University of
Toronto
Objective: To compare clinical and radiographic outcomes of mineral trioxide aggregate (MTA)
pulpotomies and root canal therapy (RCT) in carious vital primary maxillary incisors. Methods:
Asymptomatic carious vital primary incisors with pulp exposure in healthy children aged 19 to 46
months were randomly allocated to receive MTA pulpotomy or RCT under general anesthesia. Incisors
were assessed clinically and radiographically at 6-month intervals up to 24 months post-treatment.
Two calibrated, disinterested raters classified each incisor into one of the following radiographic
outcomes: N=normal incisor without pathologic change; Po=pathologic change present, follow-up
recommended; Px=pathologic change present, extract. Results: One hundred sixty eight primary
incisors in 70 subjects were enrolled in the study. One year data demonstrated no statistical clinical
difference between MTA pulpotomy and RCT (P=.86, chi-square test). Incisors rated N or Po
comprised 97% of MTA and 97% of RCT incisors. Radiographic outcomes were not statistically
different between MTA and RCT incisors for Px outcomes (P=.86, chi-square test). The level of
agreement between raters was excellent for incisors with outcome Px (K = 1, Cohen’s kappa).
Conclusions: There were no statistically significant differences between mineral trioxide aggregate
pulpotomy and root canal therapy outcomes in primary vital incisors 1 year post treatment.
62. Catechin and Chlorhexidine Release from the Bis-GMA/TEGDMA Copolymer: Stability
of Drugs by 1H NMR
V PILLY*, H HRYNASH, T BURROW, A PRAKKI
Dental Public Health, Faculty of Dentistry, University of Toronto
Department of Restorative Dentistry, Faculty of Dentistry, University of Toronto
Objective: To evaluate the 30 days stability of catechin (CAT), chlorhexidine diacetate (CHX), and the
combination of both drugs after being released from the Bis-GMA/TEGDMA dental copolymer.
Methods: Resin discs (5mm x 3mm) were prepared from Bis-GMA (70 mol%) and TEGDMA (30
mol%) comonomer containing: i) no drug, ii) CAT (epigallocatechin-gallate; EGCg), iii) CHX, and iv)
CAT + CHX. Two concentrations of each drug (0.5 MIC and 1 MIC) were incorporated into the
resin discs. The minimum inhibitory concentrations (MICs) of CAT and CHX were determined
according to the microdilution method. Comonomers were activated for visible light polymerization by
the addition of camphorquinone and dimethylaminoethylmethacrylate (0.2 weight% each). Single
resonance 1H NMR spectra of CAT and CHX alone, and extracts from unloaded and drug containing
copolymers were collected and tested at baseline (after 48 hours immersion in distilled water), 7 days,
and 30 days periods. The spectrophotometer (Varian unity 500) operated at 499.67 MHz at a
temperature of 25 ᵒC. Samples were dissolved in deuterium oxide at 10% prior testing. Results:
Spectral analyses for CAT, CHX and the extracts revealed that the chemical structures of the drugs
remained unaffected for 30 days after comonomer manipulation, polymerization and storage.
Conclusions: Both CAT and CHX are likely to retain their therapeutic activities when incorporated
into the dental resin matrix. Supported by: Dental Research Institute (U of T Faculty of Dentistry) and
MI PoP MaRS.
40
63. Evaluation of the Accuracy of NaviDent, a Novel Dynamic Computer-Guided Navigation
System for Placing Dental Implants
E SOMOGYI-GANSS*, H I HOLMES, E W N LAM, A JOKSTAD
Background: To replace edentulous spaces, implant-retained prostheses have evolved from a questionable to
evidence-based treatment modality with a predictable outcome in the last 30 years. A significant contributor is
the evolution of radiographic diagnostic and therapy planning tools. New computed technologies enable threedimensional image reconstructions and interactive therapy planning softwares; the latter leading to fabrication of
computed tomography-derived surgical guides and computer guided surgery. Objectives: To implement,
evaluate and compare an experimental surgical navigation system (ESNS) in implant placement accuracy to four
commercially available static planning and transfer systems. Material and Methods: Partially edentulous,
surgical (master) typodonts were used to simulate prosthetically-driven implant osteotomies in a clinical setting.
After cbCT acquisition the DICOM files were used to reverse plan and fabricate four surgical guides for both
maxillas and mandibles. The five modalities were manual placement, three static guiding systems and the ESNS.
Eight osteotomies per jaw were transferred to 10 slave typodonts in five series, resulting in 400 osteotomies by 3
operators, each modality. The lateral, vertical, total and angular deviations were measured and statistically
compared. Results: The new ESNS was reasonably accurate for implant transfer and placement as compared to
the other four planning and transfer systems. We established that computer-assisted systems provide superior
accuracy related to manual implant placement, except vertically; placement in the maxilla was less accurate in
lateral deviations; there was a significant difference in the accuracy of implants placed in free-end positions as
opposed to anterior sites and all operators exhibited an initial learning curve with the different systems.
Conclusions: Based on our results, the null hypothesis, that the accuracy of the ESNS is not better than
commonly used freehand methods for the positioning the placement of dental implants, could be rejected.
Supported by: Claron Technology Inc., Toronto, Canada and Nobel Biocare Research Fellowship,
Prosthodontics
64. The Potential Role of NSAIDs in Impaired Bone Healing in Implant Prosthodontics
BPLC WINNETT*, B GANSS, HC TENENBAUM, A JOKSTAD
Objective: The phenomenon of implant failure occurring in a small subset of patients without systemic risk
factors remains poorly understood. One potential factor previously unaccounted for in the dental literature is the
inhibitory role of non-steroidal anti-inflammatory drugs (NSAIDs) on normal bone metabolism and healing.
This retrospective study aims to elucidate whether adverse events following oral implant placement may be
associated with peri-operative use of NSAIDS. Methods: During the period of 1979 to 2012, 5829 implants
have been placed in the Faculty of Dentistry Implant Prosthodontic Unit. Of these, 168 patients have suffered
292 implant failures. These patients were contacted to solicit updated medical and analgesic histories, and the
relevant surgical/restorative protocols employed and related complications, the clinical course and radiographic
appearance of any bone loss, and the presence/absence of other implants and their status were recorded. Results:
The study was approved by the University of Toronto research ethics office, #27644. 112 patients suffering from
implant loss consented to partake in the study. In these patients, 205 of a total of 468 implants in these were lost
(43.8%). Eighty-four of the 205 these implants (40.9%) failed either for reasons well-defined in the literature or
for no known reason without concurrent NSAIDs usage. An additional 19 implants in these patients had >50%
radiographic bone-loss. The remaining 121 implants lost could not be attributed with a particular etiological
factor. All of these implants were placed in the presence of high-dose and/or long-term NSAID analgesics. In
these patients, an additional 57 remaining implants demonstrated >50% radiographic bone loss. Conclusions:
The findings of the current study suggest that a substantial number of the otherwise-unexplained implant failures
may be directly related to the inhibitory effect of NSAIDs on bone healing. Further prospective research is
warranted to clarify this potential causative relationship in humans.
41
65. Work in Progress: Ultrasound Analysis of Tongue Function During Normal Chewing and
After Initial Bonding of Braces and Orthodontic Arch-wire Placement
M YARASCAVITCH*, S GONG*, B TOMPSON*, T BRESSMANNǂ
*Department of Orthodontics, Faculty of Dentistry, University of Toronto
ǂ
Department of Speech Language Pathology, Faculty of Medicine, University of Toronto
Background: Tongue function is central to the oral environment. Although changes to eating and
speaking following orthodontic procedures have been described, the tongue’s capacity to adapt to
bonded braces and arch-wires during chewing is not known. No description exists for normal coronal
tongue movements during chewing, or the nature or timeline of possible adaption to orthodontic
appliances. Objectives: 1) To record and analyze coronal and saggital tongue movements during
normal chewing using a novel application of 4D ultrasound; 2) To examine tongue movements during
chewing before and after initial bonding of braces and placement of arch-wires. Material and
Methods: Phase I: 12 subjects not receiving orthodontic treatment will undergo 4D ultrasound, videorecording of masticatory movements, and VAS questionnaires of qualitative aspects of chewing for
exploratory investigation. Phase II: 12 non-surgical non-extraction subjects starting orthodontic
treatment at the Graduate Orthodontic Clinic at the University of Toronto Faculty of Dentistry will
receive 2D coronal plane ultrasound pre-bonding, post-bonding, and one month following bonding,
with video-recording of masticatory movements, and VAS questionnaires at each time point.
Normative data in Phase I will be used to set parameters of analysis in Phase II. Data acquired from
ultrasound will be extracted with computer-based tracing of video frames. Video-recording of
masticatory movements will be matched to ultrasound data to inform the analysis. The number of
tongue movement events during chewing will be counted. ANOVA will be used to examine for
chewing differences at different time points between groups. Conclusion: Results from Phase I will
improve the understanding of chewing-related tongue movements, which may have application for
patients with tongue dysfunction. Results from Phase II will improve understanding of changes that
take place in tongue function at the initiation of orthodontic treatment, and may lead to improved
informed consent, patient cooperation, and possibly more satisfying patient experience.
42
STUDENTS
Education, Behavioural, &
Health Services Research
Category
43
66. Hospital-Based Visits and Admissions for Maxillofacial Injuries in Ontario, Canada
M AL-DAJANI*, C QUIÑONEZ*, A MACPHERSON, C CLOKIE*, A AZARPAZHOOH*
*Faculty of Dentistry, University of Toronto;  Faculty of Health, York University
Objectives: (1) To calculate rates for maxillofacial (MF) injury-related visits in emergency department
and hospitals in Ontario, Canada, which in turn will allow the identification of the trends in utilization
of health care services among Ontarians who are affected by MF injuries. (2) To identify the most
common types of MF injuries. (3) To investigate the variation and trends in epidemiologic distribution
of MF injuries according to sex, age and socioeconomic status. (4) To identify and rank common
causes for MF injuries. (5) To describe the geographic distribution of MF injuries. Methods: An 8-year
retrospective descriptive epidemiologic study design is underway to investigate the characteristics of
hospital-based visits and admissions for maxillofacial injuries in the Province of Ontario, Canada. Two
datasets were used: the Discharge Abstract Database and National Ambulatory Care Reporting System.
Patients with MF injury visits were identified by physician discharge diagnosis codes assigned based on
the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision,
Canada (ICD-10-CA). This analysis included calculating rates of MF injury-related emergency
department visits and hospitalizations. Comparisons are being performed with Chi-square tests or
Fisher’s exact tests, as indicated. All data will be analyzed using SPSS for Mac, Release Version 21.0.
Results: The expected results will provide a clearer image of MF injury among one-third of the
Canada’s population. The patient and visit characteristics will be described based on a number of
factors (i.e., frequency and type of injury, age group and sex, date and time of injury, location of injury,
postal codes, and concomitant injuries). Moreover, linking these inferred data to an area-based measure
of income quintile depending on census tract data is expected to provide vital socio-economic
information. Conclusion: This study will offer health policymakers with estimates that describe the
epidemiologic aspects of MF injuries across Ontario.
67. Knowledge, Practices and Opinions of Ontario Dentists When Treating Patients Receiving
Bisphosphonates
A ALHUSSAIN*, S PEEL, C CLOKIE, L DEMPSTER, A AZARPAZHOOH
Department of Oral & Maxillofacial Surgery, Faculty of Dentistry, University of Toronto
Background: Bisphosphonate related /associated/induced osteonecrosis of the jaws (BRONJ) is a
severe but extremely rare complication of prolonged treatment with bisphosphonates. Improper
treatment or misdiagnosis of BRONJ can have serious repercussions. In some cases, the treatment of
BRONJ can require jaw resection, prolonged use of antibiotics and long hospitalizations. Objective:
The primary objective is to measure the awareness of Ontario dentists about BRONJ and to identify any
gaps in their knowledge of the condition and its treatment. In particular, the study aims to answer
questions about the dentists’ knowledge of the current guidelines and their opinions and practices
related to performing oral surgery on patients taking bisphosphonate. Material and Methods: The
study involved sending a web-based questionnaire to oral surgeons, periodontists, prosthodontists,
endodontists and a random sample of general dentists in Ontario, Canada. Information about their
awareness of BPs, their experiences treating patients presenting with osteonecrosis, their experiences
with different surgery procedures (e.g., tooth extraction, dental implant placement, periodontal, and/or
endodontic surgery) in patients taking intravenous (IV) or oral bisphosphonates, and their awareness of
the BRONJ guidelines suggested by the AAOMS was collected. Results: In progress. Conclusion: In
progress. Supported by: Ron Warren Award, COMS Foundation
44
68. The Effect of Test Enhanced Learning on Diagnostic Accuracy
M BAGHDADY*, EWN LAM, H CARNAHAN, N WOODS
Department of Oral Radiology, Faculty of Dentistry, University of Toronto
Wilson Centre for Research in Education
University Health Network
In health professions education, tests have been used to assess the skills and knowledge of learners. More
recently, research in psychology and education has shown that tests can also be used to enhance student memory;
a phenomenon called the “testing-effect”. Much of the research in this domain has focused on rote memory of
simple facts, and not on the deeper comprehension and application of complex theoretical knowledge necessary
to diagnose and manage patients. The purpose of this study was to examine the effects of testing on students’
comprehension of the basic science mechanisms and diagnostic accuracy. Methods: Undergraduate students
were taught the radiographic features and pathophysiology underlying 4 intrabony abnormalities. Participants
were divided into two testing groups: test-enhanced (TE) and study (ST) groups. Following the learning phase
the TE group completed an interventional test that tested the basic science mechanisms, and the (ST) group were
given additional training passages without being tested. Participants in both groups then completed a diagnostic
test and a memory test immediately after the learning phase and 1 week later. Results: Participants in (TE) group
outperformed those in the (ST) group on immediate and delayed testing. Participants in the TE group obtained a
mean score of 0.74 , and those in the ST group had a mean score of 0.67 on the immediate test. On the delayed
test, the TE group had a mean score of 0.72, and the ST group a mean score of 0.68. A significant effect of
testing condition was found. Unlike the diagnostic test the memory test showed no difference between the
groups. Conclusion: The inclusion of the basic science test not only improve the students understanding of the
underlying disease mechanisms learned, but it also improved their ability to make more accurate radiographic
diagnoses.
69. Environmental Scan of Youth Engagement with General and Oral Health Promotion and
Access to Care
R. FIGUEIREDO*, H. STEWART, C. QUINONEZ
Discipline of Dental Public Health, Faculty of Dentistry, University of Toronto and Toronto Public
Health, City of Toronto
Objective: The aim of this environmental scan is to develop better understanding of behavior of Toronto
adolescents towards access to health information within their existing community.
Methods: This
environmental scan utilized a convenience sample of 198 participants, where the participation was on a voluntary
basis. Data were collected from nine different sites of the city of Toronto and was focused on adolescents
between the ages of 12 and 18. A self-administered feedback form, consisting of 13 multiple choice questions,
was used as the survey instrument. The questions focused on the self-perception of the youth regarding their
general and dental health and sources of health information they prefer and utilize. Results: Overall, the
participants classified their general health status better than their oral health status; 65.2% reported that their last
visit to the dentist was during the previous year and 15.7 % had not visited the dentist during the two previous
years; and over 24% reported “tooth pain” as their main reason to visit a dentist. Even though a range of dental
services are covered by the public sector to Toronto residents under the age of 18 years, 36.9% of this
convenience sample of youth indicated “cost of dental treatment” as a barrier to access dental care. As for the
dental information, the most preferred sources were dental clinics (70.2%) and family doctor (31.3%). Regarding
the health advertisements that draw their attention, “Internet” and “Poster in schools” represented 42.4% of their
preference, followed by “Pamphlets in health clinics” (34.3%) and “Poster in the TTC” (33.8%). Conclusion:
This study only describes the information obtained from a small subset of the Toronto youth population.
However, the findings of this scan present some discussion about the opportunities for improving ongoing
processes of health communication and advertisement among youth in Toronto.
45
70. No Association Between Periodontitis and Obesity in a Nationally Representative Sample
of Canadian Adults
S HERSHENFIELD*, S SUTHERLAND, H TENENBAUM, C QUIÑONEZ
Discipline of Periodontology, Faculty of Dentistry, University of Toronto
Background: Research has supported a plausible association between periodontitis and obesity, with
the suggested mechanism relating to the underlying inflammatory nature of both conditions. Current
literature consists primarily of cross-sectional studies with extensive variation in the epidemiological
definition of periodontitis. Objective: To study the relationship between obesity and periodontitis in
Canadian adults using the recently updated 2012 CDC-AAP case definitions of periodontitis. Material
and Methods: Data were obtained from the 2007/09 Canadian Health Measures Survey (CHMS). The
CHMS conducted both personal interviews and clinical assessments at 15 different sites across Canada.
The inclusion criteria of the present study were dentate individuals aged 20-79 who participated in both
the CHMS anthropometric examination, including body mass index (BMI) and waist circumference
(WC), and the periodontal assessment, which recorded the worst probing depth and attachment loss for
a total of six sites per mouth (n=2795). The definition of periodontitis was derived from the 2012 CDCAAP case definitions. BMI and WC cut-offs followed those set by Health Canada, adapted from the
World Health Organization. Statistical tests included univariate, bivariate, logistic regression and
correlation analyses. Results: 15% of the weighted sample had periodontitis, 35% were overweight and
20% were obese according to BMI, while 33% had above normal WC. There were more cases of
periodontitis among obese participants, as classified by BMI and WC (17.3% and 17.0% vs. 14.8% and
14.3%); however, this was not a statistically significant difference. According to the logistic regression
analysis: sex, age, smoking, cultural/racial origin and having no dental insurance were associated with
periodontitis. Conclusion: This study found no statistically significant association between
periodontitis and obesity.
71. Straight, White Teeth as a Social Prerogative: An Analysis
A KHALID*, C QUIÑONEZ
Discipline of Dental Public Health, Faculty of Dentistry,
One of the distinguishing features of North American society is its preoccupation with self-image, as
seen in the rigorous and ritualistic nature of body practices (hygiene, diet, exercise etc.) aimed at
constantly improving it. Nowhere is this more apparent than in the prevailing fixation with straight,
white teeth. While there is an ever-expanding literature on the sociology and anthropology of the body,
very little has been written on teeth in this context, and teeth arguably represent a rich site of analysis.
Using literature from various disciplines including dentistry, biology, socio-biology, anthropology,
sociology and social psychology, this study attempts to answer three questions: 1) Why have straight,
white teeth become a beauty ideal in North American society? 2) What is the basis behind this
preference for a perfect smile? 3) How is this ideal reproduced among the general population? This
study demonstrates the ways in which our dental aesthetic tendencies are biologically, culturally and
socially patterned. Concepts borrowed from the works of French sociologist Pierre Bourdieu and
French social theorist Michel Foucault are used to illustrate how straight, white teeth contribute
towards reinforcing class differences and how society exercises a disciplinary power on individuals
through the ideal of straight, white teeth.
46
72. Dentists’ Views and Practice of Sedation and General Anaesthesia in Ontario
S PATODIA*, D HAAS, V LEBLANC, L DEMPSTER
Department of Dental Anaesthesia, Faculty of Dentistry
Data support patients’ demand for sedation during dental treatment; however, little is known about the
availability and provision of sedation and general anesthesia (GA) by dentists. Objectives: (1) to investigate the
perceptions Ontario dentists have towards the use of sedation and GA for patients; (2) to investigate specific
patterns of sedation and GA use in Ontario dental practices; and (3) to compare dentist and patient responses
(obtained from a previous study) regarding patient preference for sedation/GA and if dentists are able to identify
fear. Methods: From the provincial regulatory body’s databases, 3001 practicing dentists were invited to
complete a self-administered questionnaire. A mixed mode format offered mailed and web survey options. Data
analysis included descriptive statistics, means, standard deviation, and frequency. The relationship between
dentist responses and demographic variables was assessed with mixed ANOVA followed by independent
samples t-tests or one-way ANOVAs. Results: Study participants (n=1076; 37.9% response rate) were 69.7%
male, 83.0% general practitioners, practicing 0.5-42 years (mean 20.6 years), with 40.6% from cities >500,000.
60.2% of respondents provided sedation. Dentists estimated patients’ disinterest in sedation/GA adjuncts to be
higher (66.8%) than patient stated preference (43.9%). Patients’ preference for sedation/GA by service was also
underestimated for specific procedures (p<0.001) except for extractions. Barriers to care were cost (72.2%) for
providers; lack of training (38.2%) and patient demand (25.3%) for non-providers. From the specified dental
procedures, dentists reported use of sedation highest for extractions with nitrous oxide being most commonly
used (5.7% frequency). Dentists’ (D) also overestimated patients’ (P) level of fear, reporting them to be
somewhat afraid 19.95% (D) vs. 9.8% (P); very afraid 10.6% (D) vs. 2.0% (P); and terrified 6.0% (D) vs. 3.5%
(P). Conclusion: This study confirms differences between dentists’ use and estimation of patient demand for
sedation/GA.
73. Undergraduate Dental Students’ Experience and Perceptions in the Provision of Dental
Treatment to Persons With Disabilities
D PERUSINI*, M SIGAL, V LEBLANC, L DEMPSTER
Discipline of Pediatric Dentistry, Faculty of Dentistry, University of Toronto
Purpose: To determine if experience in a hospital-based dental clinic (D-Clinic) influences undergraduate dental
students’ comfort and specific concerns regarding treating persons with disabilities (PWDs). Method: Senior
dental students at the University of Toronto (n=94) were surveyed before and after clinical rotations in D-Clinic.
Questions asked students about their comfort, concerns and experience providing dental care to PWDs; scored 1
(strongly disagree/not at all) – 5 (strongly agree/very much). Results: Pre- survey (n=45/48% response; 66%
female; mean age 28.7 years) and post-survey participants (n=42/45% response; 59% female; mean age 29.2
years) were representative of their class. Prior non-clinical experience with PWDs was a significant predictor of
comfort (r=0.549;P<.001); however, no significant difference (NSD) in comfort level was reported pre- (x=2.9)
and post-rotation (x=3.0). Respondents (55%) felt more experience would increase their comfort. Students with
higher comfort scores were less concerned about time constraints and poor patient cooperation, ascribed higher
capacity for consent in individuals with lower IQs, and felt basic dental care for PWDs was best provided by
general dentists (P<.0001). Students’ specific concerns treating PWDs were poor patient cooperation (highest
ranked), inadequate treatment, time constraints, poor communication, injury-to-patient and injury-to-self (lowest
ranked) with NSD reported in mean scores pre- (range=3.1-4.0) and post- (range=2.7-3.8) rotation. The majority
of students reported D-Clinic to be a valuable experience (71%). Conclusions: Experience in D-Clinic did not
change students’ comfort and specific concerns about treating PWDs; however, students considered the rotations
valuable with a desire for more experience. Level of comfort treating PWDs appeared dependent on prior nonclinical experience.
47
74. Validation of Screening Questions for Limited Oral Health Literacy
D. SABBAHI, HP LAWRENCE, H.LIMEBACK
Background: A set of brief and easy-to-use screening questions to identify patients with inadequate
oral health literacy (OHL) will be a useful tool in busy dental practices compared to the timeconsuming OHL instruments. Objective: to evaluate the performance of 4 screening questions for
identifying patients with limited OHL. Methods: In this study, 177 participants were recruited from the
pool of new patients attending the Faculty of Dentistry Clinics at University of Toronto (convenience
sample). Four OHL screening questions, the Rapid Estimate of Adult Literacy in Dentistry (REALD30) and Oral Health Literacy Instrument (OHLI) were administrated to the patients. The areas under the
receiver operating characteristic (AUROC) curve and their 95% confidence intervals (CI) were used to
compare the overall performance of the screening questions and their combinations against REALD-30
and OHLI as reference standards. An ideal question is one that achieves an area of 1 under the ROC,
and an area of 0.5 indicates a screening test that provides no useful information. Results: The mean age
of the participants was 46.72±14.12 years and about 54% of them were male. About 31% of the
participants had marginal or inadequate level of OHL, measured using OHLI, while 63% of the
participants had marginal or low level of OHL, measured using REALD-30. AUROC for the individual
screening questions, against OHLI, ranged between 0.608 and 0.689 with all questions significantly
higher than the null value (0.5) as detected by the 95%CI. The AUROCs for the screening questions,
against REALD-30, ranged between 0.586 and 0.657 with only 2 questions significantly higher than the
null value (0.5) as detected by the 95%CI. Combining the screening questions did not improve their
performance significantly. Conclusion: All four screening questions may be useful for identifying
patients with limited OHL skills. Acknowledgment: University of Toronto, Faculty of Dentistry
Research Committee.
75. The Impact of Dental Treatment on Employment Outcomes: A Systematic Review
S SINGHAL*, C R QUIÑONEZ
Community Dental Health Services Research Unit, Faculty of Dentistry, University of Toronto,
Ontario, Canada
Abstract: Background: Policy advocates in North America argue that access to dental care for low
income and unemployed populations can help improve the chances of acquiring a job or attaining a
better job, thus having positive economic and social benefits. Objective: Our objective is to review the
evidence in support of the policy hypothesis that timely access to dental care can improve employment
outcomes. Methods: A systematic review was conducted by searching various scientific databases and
search engines. Key words included Dental Care, Dental Intervention, Social Welfare, Unemployment,
Employment, and Job. Results: Seven articles were considered eligible for this review. They varied in
study design, target population and intervention studied. Overall, they presented low levels of evidence
due to small sample sizes, lack of control groups, combined interventions or being based on anecdotal
reports. Conclusion: There is a limited amount of evidence concerning the assumption that dental care
can improve employment outcomes. The scarcity of well-conducted studies and the poor quality of
evidence makes it difficult to judge the effect of dental care on employment outcomes. More studies
need to be conducted in order to confirm or dismiss this generalized assumption.
Grants: Population Health Improvement Research Network, Applied Health Research Network
Initiative, Government of Ontario.
48
76. Managing Simulated Patient Crises: Cognitive Appraisal as a Predictor of Stress and
Performance
C YARASCAVITCH*†, D HAAS, VR LEBLANC†
Discipline of Dental Anaesthesia, Faculty of Dentistry, University of Toronto
†The Wilson Centre, Faculty of Medicine, University of Toronto
Background: Acute stress has been shown to degrade performance. Perceptions of resources relative to
the demands of a situation (cognitive appraisal) may influence stress responses. As such, cognitive
appraisals may impact performance. Objective: To describe the relationship between cognitive
appraisal and stress responses, patient management, and teamwork in the context of dental teams
managing simulated patient medical emergencies. Materials and Methods: Each team (N=22) of one
general practice dentist and one assistant participated in four different medical emergency scenarios of
equal difficulty. Participants completed demographics and a knowledge pre-test. Scenarios were video
recorded and independently scored by four trained raters: patient management was assessed by
checklists (Ck) previously developed through a Delphi method and a global rating scale (pGRS);
teamwork was assessed using the Global Assessment of Obstetric Team Performance modified to the
dental context (GATP) and a global rating scale (tGRS). Pre and post-scenario stress was measured by
cognitive appraisal (ratio of perceived demands to resources; ≤1 = challenge appraisal; >1 = threat
appraisal,), self-reported anxiety (STAI-Y1) and salivary cortisol. Relationships between cognitive
appraisal, stress and performance, knowledge and demographics were evaluated using stepwise logistic
regression. Results: All scales demonstrated good inter-rater reliability (ICC Ck=0.85; pGRS=0.76;
GATP=0.759; tGRS=0.79). Cognitive appraisal emerged as a consistent predictor of both stress
response and performance: threat was associated with higher stress response (cortisol r=.30, p=.00;
anxiety r=.50, p=.01) but lower performance (Ck, r=-.23, p=.03; pGRS, r=-.22, p=.04; GATP, r=-.24,
p=.03; tGRS, r=-.25, p=.02). Knowledge and clinical experience were not consistent predictors of stress
response or performance. Conclusions: The pattern of findings suggests that cognitive appraisals of
threat by dental team members are related to increased stress and decreased performance. Dental
professionals may benefit from simulated training experiences which address cognitive appraisal to
reduce stress and enhance performance during medical emergencies.
77. Analysis of Provisions in Ottawa’s Government Funded Dental Program
E YOON*, C QUIÑONEZ, H TENENBAUM, L DEMPSTER, M SIGAL
Department of Paediatric Dentistry, Faculty of Dentistry, University of Toronto
Objective: To describe the provisions provided by general dentists and paediatric dentists in public
health dental clinics in Ottawa, Canada. Methods: Data was obtained from the Ottawa Public Health
through the Children in Need of Treatment (CINOT) program, which manages public dental clinics in
Ottawa. Data collected included patient demographic information, procedures and date provided, fees
and teeth restored or extracted. Data was tabulated in Microsoft Excel and analyzed using Statistical
Package for the Social Sciences. Results: Data obtained from 3 public health dental clinics ranged from
1987 to 2008. The total number of procedures provided was 235 816 which averaged to 11 229
procedures per year. The total expenditure of the CINOT program in Ottawa’s public health clinics
was $9 525 292. Further analysis of data is pending.
49
STAFF, POST-DOCS
and
RESEARCH ASSOCIATES
CATEGORY
50
78. TGF-ß1 is Activated by Integrin-mediated Contraction of Human Cardiac Fibroblasts
V SARRAZY, A KOEHLER*, E ZIMINA, M CHOW, C LI, B HINZ
Background: Contraction and stiffening of the heart tissue by myofibroblasts is the principle cause of cardiac
fibrosis progression, ultimately leading to organ failure. Myofibroblast differentiation from cardiac fibroblasts is
stimulated by transforming growth factor (TGF)-β1. TGF-β1 is secreted as an inactive large latent complex that
is integrated into the extracellular matrix. Myofibroblast traction forces mediated via transmembrane integrins
have been shown to induce a conformational change in the latent complex, resulting in TGF-β1 activation.
However, the molecular identity of the cardiac fibroblast integrins involved is unknown. In cardiac fibrosis, the
latent TGF-β1 binding integrins αvβ3 and αvβ5 have been shown to be upregulated. We hypothesize that cardiac
myofibroblast traction transmitted by αvβ3 and αvβ5 will activate TGF-β1. Objective: To identify the integrins
contributing to TGF-β1 activation in cardiac fibroblasts. Materials and Methods: Human cardiac fibroblasts
were grown on tissue culture plastic and soft culture substrates that mimic healthy heart tissue. Integrin
expression and myofibroblast differentiation were assessed by immunofluorescence and Western blotting.
Activation of TGF-β1 by cardiac myofibroblasts was quantified using a luciferase reporter assay. Function of
candidate integrins was either blocked by peptides, antibodies and shRNA or enhanced by overexpression.
Results: Expression of integrins αvβ3 and αvβ5, as well as the myofibroblast marker α-SMA, was up-regulated
on tissue culture plastic as compared to heart-soft substrates. Increasing integrin traction by Mn2+ increased
active TGF-β1, whereas blocking of integrin αvβ5 or the integrin binding site in the latent complex resulted in
significantly reduced TGF-β1 levels and decreased α-SMA expression. Conclusion: Blocking of integrin αvβ5
expression or interaction with latent TGFβ1 emerges as an attractive therapeutic strategy to selectively blocking
TGF-β1 activation by fibrotic fibroblasts. Supported by: Heart and Stroke Foundation Grant #NA7086 and
CIHR grant #210820 to BH.
79. Characterization of a Streptococcus mutans Intergenic Region Containing a Small Toxic
Protein and its Cis-Encoded Antisense Small RNA Antitoxin
S KOYANAGI*, C M LÉVESQUE
Background: Toxin-antitoxin (TA) modules are found widely across prokaryotic genomes. TAs involve a stable toxin
protein, and are categorized depending on their labile antitoxin: RNA (type I, III) or protein (type II, IV, V). Under
normal growth conditions, antitoxin levels are sufficient to repress toxin activities. During events such as
environmental stress, the labile antitoxin is degraded, and the toxin is free to perturb essential cellular processes.
Recently, we identified an unannotated ORF encoding a putative type I toxin (Fst-Sm) and its cis-encoded antisense
small RNA antitoxin (srSm) in the Streptococcus mutans genome. Objective: To characterize the first functional
chromosomal type I TA module in Streptococcus. Methods: Transcriptional analysis was performed by RT-PCR,
Northern blot, and RNA half-life determination. Toxicity assays were assessed using bacterial expression systems. A
two-plasmid gene reporter system was designed to investigate toxin repression at the transcriptional level. Persistence
assays were performed using cell wall targeting antibiotics. Results: Transcripts of fst-Sm mRNA (~200 nt) and srSm
RNA (~70 nt) were detected by Northern blot throughout S. mutans growth, with half-lives of ~30min and ~90min
respectively. Overproduction of Fst-Sm had a toxic effect on E. coli and S. mutans cells which can be neutralized by
coexpression of srSm RNA. Inhibition of fst-Sm expression occurred by the binding of srSm RNA to the 5’ fst-Sm
mRNA coding region. Mild-overproduction of Fst-Sm/srSm TA dramatically decreased the level of persister cells
after treatment with bacterial cell wall synthesis inhibitors. Conclusion: We described the first functional type I TA in
Streptococcus. Most prokaryotic chromosomes contain a number of toxic genes that can induce a reversible dormancy
state to enhance fitness and competitiveness of the microbial population. Consequently, the deciphering of the
mechanism of action of these toxic peptides represents an innovative and promising avenue for the development of
novel antimicrobial strategies.
51
80. The effects of Incisor trimming on Rat’s Mastication
J-C LEE*, L AVIVI-ARBER , A HUNTER, BJ SESSLE
Department of Oral Physiology, Faculty of Dentistry, University of Toronto
Objective: To test the hypothesis that the rat masticatory motor behaviour could be reversibly modified
in response to trimming of the mandibular incisors out of occlusal contacts and subsequent re-eruption
into occlusion. Methods: Two male Sprague-Dawley rats were trained in a feeding motor behaviour
that required the rat to stay in an observation cage and keep eating corn flakes (~0.5x0.8cm) for 10
minutes. Under isoflurane anesthesia, electromyographic (EMG) wires were implanted into target
muscles and a magnet was implanted subcutaneously under the chin for subsequent daily monitoring of
jaw-opening, jaw-closing and tongue protrusion kinematic and associated EMG activities in awake
freely moving rats while chewing corn flakes; an acrylic headcap collected all EMG wires to a socket
that could be connected to a computerized data acquisition system. Following 1 week of postoperative
healing, 30min of daily video, EMG and kinematic recordings resumed to establish baseline data.
Thereafter the rat received trimming of the mandibular incisors and subsequent daily monitoring for 1
week during which the incisor let to re-erupt back into occlusion. Customized design program was used
to analyze the cycle-by-cycle mastication to evaluate the effects incisor trimming. Results: Our
analysis program provided an easy way to identify and measure the grasp, biting and chewing phases.
Mandibular incisor trimming resulted in a significantly increased duration of biting phase that
subsequently decreased as the incisors re-erupt back into occlusion; a significantly increased chewing
phase frequency that last 3 days postoperatively but had no significant effect on the duration of the
chewing phase. Conclusions: Our novel mastication analysis system in a freely moving rat could
identified mastication behaviour on a cycle-by-cycle basis and demonstrated that the rat adapts its
masticatory motor behaviour to the loss of incisor occlusal contacts by spending longer time on biting
phase and chewing faster. Support: CIHR-MOP-4918
81. Acai Berry (Euterpe oleracea) Extract Modulates Osteoclast Activity: an in vitro Study
A STAVROULLAKIS*, C BRITO, K LI, P HARRISON, G NOGUEIRA-FILHO
Department of Preventive Dentistry, Faculty of Dentistry, University of Toronto
Background: The Acai berry is indigenous to the Amazon River flood plains. It has been investigated
for its many potential health benefits, most notably as an anti-oxidant. It is still unknown which active
compounds provide anti-oxidizing effects, but studies have shown that flavonoids which are contained
in the acai-berry may have anti-inflammatory properties. Osteoclast activity is dependent on specific
signalling pathways. Objective: To investigate the Acai berry extract’s effects on bone resorption and
how the signalling pathways of HIF and NFκB can be affected. Methods: RAW 264.7 cells were
cultured and treated with various concentrations of Acai extract (25, 50 and 100 µg/mL). Von Kossa
staining was performed to evaluate osteoclast activity in osteo plates. Western Blotting was utilized to
test expression of HIF and NFκB with β-actin as a control group. Results: Statistical analysis showed
that the Acai berry extract reduced bone resorption in all concentrations tested; at 100 µg/mL there was
little to no bone resorption present (p < 0.05). The HIF and NFκB pathways were down-regulated when
Acai berry extract was present. Conclusion: Within the limits of this study, the Acai berry extract may
modulate osteoclast activity via the HIF/ NFκB pathways. Further studies are needed to detect which
pathways it affects as well as use of in vivo bone resorption models.
52
82. Kindlin-2 Regulates Mechanical Activation of the Cardiac Myofibroblasts
E ZIMNA1, S CLEMENT3, L BRUCKNER-TUDERMAN2, C HAS2, B HINZ1
1Laboratory of Tissue Repair and Regeneration Matrix Dynamics Group, Faculty of Dentistry,
University of Toronto, Toronto, Canada, 2Department of Dermatology, University Medical Center
Freiburg, Freiburg, Germany, 3Department of Pathology and Immunology, Faculty of Medicine, CMU
University of Geneva, Switzerland.
Background and Objective: Excessive activation of cardiac myofibroblasts from local fibroblasts
results in the pathological heart stiffening characteristic for cardiac fibrosis. Mechanical stress is a
central factor in inducing myofibroblast differentiation. Myofibroblasts are highly contractile cells that
promote tissue repair after myocardial infarction. Integrin stransduce mechanical signals from the
microenvironment, such as extracellular matrix (ECM) stiffness, over the cell membrane. Cytoskeletal
integrin adaptor proteins are crucial for an intracellular transmission of extracellular cues. Kindlin-2 is a
recently characterized adaptor that mediates intracellular binding of integrins to the contractile actin
cytoskeleton. We test the hypothesis that kindlin-2 controls cardiac fibroblast differentiation by
mediating mechanosensing and -transduction. Methods and Results: Immunohistochemistry and
Western blotting demonstrated that kindlin-2 is upregulated in cardiac fibroblasts upon myofibroblast
activation in vivo and in vitro. Kindlin-2 accumulated in cell-ECM focal adhesions of primary human
cardiac myofibroblasts. Cardiac fibroblasts were subjected to different mechanical conditions to model
the long-term and short-term mechanical challenges of cardiac fibroblasts during cardiac repair and
fibrosis. Together with the myofibroblast marker α-SMA, kindlin-2 levels were increased in cardiac
fibroblasts cultured on fibrosis-stiff silicone substrates compared with fibroblasts cultured on soft
substrates. Short term mechanical stimulation of cardiac fibroblastswas performed by applying
magnetic fields to fibronectin-coated ferromagnetic microbeads and by stretching cells on deformable
silicone membranes. Local force application resulted in a portion of kindlin-2 translocating from focal
adhesions to the nucleus. Downregulating kindlin-2 in primary cardiac fibroblasts using siRNA resulted
in decreased α-SMA expresssion, indicative of reduced myofibroblast differentiation. Conclusion:
Mechanical stress controls the expression and localization of kindlin-2 in cardiac fibroblasts. Kindlin-2
is novel mechanosensor within the myocardium that contributes to the myofibroblast differentiation.
Support: CIHR Post-Doctoral Fellowship to EZ and CIHR and HSFO funding to BH.
53
INTERNATIONAL
SUBMISSIONS*
*Non-judging category
54
83. Roles of Lysine-Specific Gingipain in Osteoclast Differentiation Induced By Inflammatory
Cytokines
TOMOHITO AKIYAMA1,2*, YOICHI MIYAMOTO2, ATSUSHI YAMADA2, MASAMICHI
TAKAMI2, KENTARO YOSHIMURA2, MARIE HOSHINO1, TAKAHISA IMAMURA3, RYUTARO
KAMIJO2, KAZUYOSHI BABA1
Departments of 1Biochemistry, and 2Prosthodontics, Showa University School of Dentistry;
3
Department of Molecular Pathology, Faculty of Life Sciences, Kumamoto University
Background: Porphyromonas gingivalis is one of the potent etiological agents of periodontitis. P.
gingivalis secretes cysteine proteases called gingipains, which are classified by their substrate cleavage
sites, i.e., arginine gingipains (Rgps) and lysine gingipain (Kgp). We found that Kgp degrades
osteprotegerin (OPG) and accelerates osteoclast differentiation induced by various Toll-like receptor
ligands (Biochem J 419:159-166, 2009). Objective: We investigated the effects of Kgp on
inflammatory osteoclastogenesis in vivo as well as in vitro. Methods: Osteoclastogenesis was
evaluated in vivo by activity staining of tartrate-resistant acid phosphatase (TRAP) in mouse calvaria
after subperiosteal inoculation of wild-type, Kgp-deficient, and Rgp-deficient P. gingivalis. Osteoclast
differentiation in vitro induced by TNF-17A in the presence or absence of Kgp was
evaluated by TRAP staining. Degradation of these cytokines by Kgp was quantitatively analyzed by
western blotting. N-Terminal amino acid sequence of OPG fragments generated after incubation with
Kgp was determined. Results: The extent of osteoclastogenesis in calvaria in Kgp-deficient P.
gingivalis-inoculated mice was lower than those in mice inoculated with wild-type and Rgp-deficient
bacteria. Kgp augmented the osteoclast differentiation induced by TNFKgp suppressed the IL-17A-induced osteoclast differentiation. TNFsusceptible, whereas IL-17A was more susceptible to degradation by Kgp than OPG. N-Terminal
amino acid sequences of OPG fragments revealed that Kgp cleaved the death-domain-resembling
region of OPG. Conclusions: The enhancing and suppressing effects of Kgp on inflammatory
cytokine-induced osteoclastogenesis seemed to be dependent on the difference in degradation
efficiencies between each cytokine and OPG. Degradation of OPG in its death-domain-resembling
region possibly prevents dimer formation of OPG required for inhibition of RANKL. These results
suggest that degradation of OPG by Kgp is crucial in development of bone loss in periodontitis.
55
84. Porphyromonas gingivalis Aggravate Experimental Autoimmune Myocarditis in Mice
N ASHIGAKI*, J SUZUKI, M OGAWA, R WATANABE, N AOYAMA, N KOBAYASHI, T
HANATANI, A SEKINISHI, Y IZUMI, M ISOBE
Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and
Dental University
Global Center of Excellence (GCOE) Program, “International Research Center for Molecular Science
in Tooth and Bone Diseases”, Graduate School of Medical and Dental Sciences, Tokyo Medical and
Dental University
Objective: Periodontitis is a very common human infection. Recently, some published reports assert
that periodontitis is associated with the increased risk of cardiovascular disease. Although viral,
bacterial infections and autoimmune diseases may be the cause of myocarditis, the pathogenesis of it
remains unclear. The aim of this study was to investigate the influence of the periodontal pathogen on
experimental autoimmune myocarditis (EAM). Methods: Porphyromonas gingivalis (P.g.), PBS as a
control, were injected into the mice from the chamber. We performed induction of EAM. 0.2mL
emulsion (Alpha MHC peptide was emulsified with an equal volume of adjuvant) was injected.
Histopathological and immunohistochemical analyses were performed. We examined heart mRNA
levels using quantitative RT-PCR and serum level of cytokines. Results: The anti-P.g. IgG antibody
level in plasma samples of the P.g.-injected group significantly increased compared to the PBS injected
group. Histopathological analysis detected that the myocarditis affected areas and the fibrotic area in
the P.g.-injected EAM group significantly increased compared to the PBS injected EAM group.
Immunohistochemical analysis detected that more CD11b and F4/80-positive cells were shown in the
heart of the P.g.-injected EAM group compared to the PBS EAM injected group (P < 0.05). The
Hearts from the P.g.-injected EAM group showed significantly increased expression of MCP-1, IFN-γ
and MMP-9 mRNA compared to the hearts from the PBS injected EAM group (P < 0.05). On a day 7,
serum levels of IL-6 were significantly enhanced in the P.g. injected EAM group compared to the PBS
injected EAM group (P < 0.05). Conclusion: These results showed that P.g. injection could deteriorate
EAM in mice through CD11b-positive cells, cytokines and MMP-9 expression.
Supported by: Global Center of Excellence (GCOE) Program, “International Research Center for
Molecular Science in Tooth and Bone Diseases”, Graduate School of Medical and Dental Sciences,
Tokyo Medical and Dental University
56
85. HDAC Inhibitor Induces Cell Cycle Arrest and Apoptosis in Myeloma Cells
S FUJII*1,2, T OKINAGA1, W ARIYOSHI1, K TOMINAGA2, T NISHIHARA1
1. Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental
University
2. Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial
Surgery, Kyushu Dental University
Objectives: Multiple myeloma (MM) is hematological disorder accompanying the unregulated
terminally differentiation of mature B-lymphoid cells. Histone deacetylase (HDAC) inhibitors are
known to be used in molecular target theraphy for several malignant cancer. In the present study, we
investigated the effect of Ky-2, a novel class I HDAC inhibitor, on signaling pathway in the induction
of cell cycle arrest and apoptosis in myeloma cells. Methods: Myeloma cells, HS-72, P3U1, and Bcl-2
overexpressed HS-72 cells were used in this study. Effect of HDAC inhibitors to cell viability was
determined by WST-1 assay and trypan blue assay. Cell cycle was analyzed by flow cytometry. The
expression of cell cycle associated proteins (p21, Rb and CDK4, 6) and the apoptosis associated
proteins (caspase-9 and caspase-3) were examined by Western blot analysis. Hoechst’s staining was
used to detect apoptotic cells. Results: The hyperacetylation of histone H3 and a decrease in the level
of HDAC1 and HDAC2 were observed in Ky-2-treated myeloma cells. The cell proliferation of Ky-2treated myeloma cells were significantly inhibited. Flow cytometric analysis revealed that Ky-2
treatment induced a G1 phase cell-cycle arrest and accumulation of a sub G1 phase in myeloma cells.
Western blot analysis revealed that the expression of p21 and hypophosphorylation forms of Rb were
upregulated in Ky-2-treated myeloma cells. In addition, we confirmed that Ky-2 enhanced caspase-9
and caspase-3 cleavage and activations in western blot analysis. On the other hand, Ky-2 treatment had
no effect on the induction of apoptosis in Bcl-2 overexpressed HS-72 cells. Interestingly, Ky-2 had no
effect on the cytotoxicity of B cells and T cells isolated from mouse spleen. Conclusion: Our results
showed that Ky-2 induced G1 cell cycle arrest through the regulation of cell cycle associated proteins,
and apoptosis by caspase-dependent pathway in myeloma cells.
57
86. Low-Intensity Pulsed Ultrasound (LIPUS) Suppresses Adipogenesis and Promotes
Osteogenesis of Mesenchymal Stem Cells through ROCK-Cot/Tpl2-MEK-ERK Signaling
Pathway
J KUSUYAMA*, K BANDOW, K KAKIMOTO, T OHNISHI, T MATSUGUCHI
Department of Biochemistry and Molecular Dentistry, Field of Developmental Medicine,
Kagoshima University Graduate School of Medical and Dental Sciences
Background: Mesenchymal stem cells (MSCs) are pluripotent cells that can differentiate into
multilineage cell types, including adipocytes and osteoblasts. Mechanical stimulus is one of crucial
factors in regulating MSCs differentiation. However, it remains unknown how mechanical stimulus
affects the reciprocal relationship between adipogenesis and osteogenesis. Low-intensity pulsed
ultrasound (LIPUS) therapy is clinical application of mechanical stimulus and facilitates bone fracture
healing. Here, we applied LIPUS to MSCs for analyzing the effects of mechanical stimuli in cell
differentiations. Material and Methods: ST2, a mouse MSC line, was stimulated by LIPUS for 20min.
The activations of MAPKs family molecules were examined by western blotting. ST2 cells were
cultured in adipogenic or osteogenic differentiation media. They were stimulated by LIPUS for 20min
every day. After 10days, cell differentiations were evaluated by the cell staining and the gene
expressions of adipogenic or osteogenic markers. Results: LIPUS stimulations suppressed adipogenic
differentiation, represented by lipid droplet appearance and gene expressions of peroxisome proliferator
activated receptor gamma2 (PPARgamma2) and fatty acid binding protein 4, and promoted osteogenic
differentiation, characterized by cell calcification and inductions of Runx2 and osteocalcin mRNAs.
Cancer Osaka thyroid oncogene/tumor progression locus 2 (Cot/Tpl2) kinase was activated to
phosphorylate mitogen activated protein kinase 1 (MEK1) and p44/p42 extracellular signal-regulated
kinases (ERKs) by LIPUS treatments. Suppression of adipogenesis and promotion of osteogenesis were
also prevented by Cot/Tpl2 specific inhibitor. Furthermore, LIPUS-induced phosphorylations of
Cot/Tpl2 and effects in MSCs differentiations were attenuated by the inhibition of Rho-associated
kinase (ROCK). Interestingly, LIPUS stimulation induced not only the down-regulation of
PPARgamma2 gene expression but also the phosphorylation of PPARgamma protein, which leads the
decrease of transcriptional activation. Conclusion: These results indicate that LIPUS treatment
suppress adipogenesis and promote osteogenesis of MSCs. This differentiation control is mediated by
ROCK-Cot/Tpl2-MEK-ERK signaling pathway and the modulation of PPARgamma activity.
58
87. Panoramic-Based Mandibular Indices and Bone Mineral Density of Femoral Neck and
Lumbar Vertebrae in Women
S MARANDI*, M IMANIMOGHADDAM , M HATEF, A BAGHERPOUR
Department of Oral Radiology, School of Dentistry, Mashhad University of Medical Sciences
Objective: The aim of this cross-sectional analytic study was to evaluate the diagnostic efficacy of
panoramic-based indices of the mandible (Mental Index-MI, Mandibular Cortical Index-MCI and
Panoramic Mandibular Index-PMI) and to determine their correlation with bone mineral density
(BMD) of the femoral neck and lumbar vertebrae (L2-L4) in order to assess the possibility of using
these parameters as indicators of osteoporosis. Materials and Methods: The mandibular indices of 67
women over 35 years old were measured from panoramic radiographs, and bone densitometry was
performed in the femoral neck and lumbar vertebrae (L2-L4), using DXA (Dual Energy X-ray
Absorptiometry) technique. The patients were divided into three categories of normal, osteopenic and
osteoporotic in each skeletal region. One-way ANOVA and ROC curve analyses were applied. Results:
The mean BMD in the femoral neck in women between C1 and C3 subgroups of MCI were statistically
significant (P<0.05). Also the MI value between normal and osteopenic subgroups in the femoral neck
was statistically significant (P<0.05). However, there was no statistically significant difference in PMI
value in the three skeletal subgroups (P>0.05). Conclusion: Using radiomorphometric indices of the
mandible (MCI-MI) may be useful in determining the skeletal status of the patients, but is not sufficient
for precise evaluation.
59
LATE SUBMISSIONS*
*Non-judging category
60
88. Characterising Apolipoprotein A1 And Apolipoprotein B In Early Aortic Valve Disease In
Mice
VACHHANI, K.1, BLASER, M.C.1, SIMMONS, C.A 1,2,3
1
Cellular Mechanobiology Lab, Institute of Biomaterials and Biomedical Engineering, University of
Toronto; 2 Faculty of Dentistry, University of Toronto; 3 Department of Mechanical and Industrial
Engineering, University of Toronto
Aortic valve disease (AVD) is a cell-mediated disease of the aortic heart valve, associated with valvular
matrix remodelling and calcification. AVD is implicated in a significant proportion of cardiovascular
morbidity in North America, and its initiation shares several similarities with that of atherosclerosis.
Apolipoprotein A1 (ApoA1) and Apolipoprotein B (ApoB) are the primary protein components of
high- (HDL) and low-density lipoprotein (LDL), respectively. In atherosclerotic lesions, ApoA1
mediates cholesterol efflux while ApoB levels positively correlate to lesion cholesterol content. Due to
the shared pathogenesis between AVD and atherosclerosis, ApoA1 and ApoB may also play a role in
the development of early stage valve disease.
Male wild-type C57Bl/6J mice were fed a normal or a high-fat (HF) diet known to induce early
pathological valvular changes for two or four months (n = 4-6 per group). Immunohistochemical
staining of heart sections for ApoA1 and ApoB was performed to examine the presence of valvular
apolipoprotein content.
Valvular ApoA1 was found in mice on the HF diet for four months but not in those on normal diet
(1.13 ± 0.36 vs. 0.14 ± 0.08 % valve leaflet stained, p < 0.05). Mice on both diets for two months
displayed no significant ApoA1 staining. ApoB was absent in valves of mice on either diet for two or
four months. Thus, this study provides the first evidence of an increase in valvular HDL without LDL
accumulation during early AVD pathogenesis.
61
89. Mapping Quantitative Trait Loci in the Mouse Genome for Heat Pain Sensitivity and the
Effect of Stress
M MASHREGI*, D. FROIMOVITCH, D. TICHAUER, E. SOLEIMANNEJAD, B.J. SESSLE, Z SELTZER
Department of Pain/Neurosciences, Faculty of Dentistry,
Background: Naïve male and female mice of the A/J (‘A’) strain are known to be significantly less sensitive to
noxious heat than C57BL/6J (‘B’) mice, justifying using the AXB-BXA recombinant inbred (RI) panel of 23
mouse strains (derived by crossing A and B strains) for genetic mapping of quantitative trait loci (QTLs)
controlling heat nociception. The behavioural response of mammals to repetitive noxious stimulation, within or
between test sessions, may show sensitization or adaptation reflecting stress-induced hyper- or hypoalgesia,
respectively. However, neither the mechanisms involved nor the genetic underpinnings and/or sex effects have
been studied as of yet. Objective: To map the mouse genome for QTLs controlling for (i) heat nociception, and
(ii) modification of pain responses by repetitive noxious heat stimulation. Methods: Naïve male and female mice
(5-10 mice/strain/sex, n=1300; age=8-12 weeks) of the AXB-BXA RI panel (25 strains) were tested for their
behavioural response to noxious laser heat and tactile stimulation. There were two test groups of mice: Group#1
received laser heat stimulation on test days 1 and 2, Group#2 received tactile stimulation on days 1 and 3 and
laser heat on days 2 and 4. Laser heat stimulation: Three noxious laser heat pulses were directed at each ear
and tail (dorsal and ventral) and quantified on a scale of 0-4 (0=no response, 4= maximal response). Stimulation
of the hind-paws was measured with a stopwatch and quantified as the response duration (sec). Tactile
stimulation (Group #2 mice only): 7 applications of a noxious von Frey monofilament (0.2 gr bending force)
was applied to the ears, hind-paws and ventral tail and graded from 0-2 (0=no response, 2=max response).
Results: Repeated noxious heat stimulation did not cause sensitisation and/or adaptation within and between test
days. Exposure to noxious tactile stimulation 1 day prior to noxious heat resulted in an increase in the
behavioural response to noxious heat in some strains, but decreased it in others. The greatest increase in the
behavioural response was recorded at the ears, and more so in female mice. A significant QTL was mapped on
chromosome 10, in males and females, for the behavioural response to noxious heat stimulation at the ears.
Several candidate genes of interest were found, e.g. Tac2. Conclusions: The behavioural response to noxious
heat and the effects of stress on this trait depend on the tested body site, sex, and genetic background. Our results
implicate Tac2 in chr10 as a candidate gene.
Supported by: NIH RO1DEO18575 and the CIHR.
62
CLINICAL TABLES
63
PARTICIPATING DEPARTMENTS:
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The Faculty of Dentistry Library
64
Index
Acknowledgements
Program
Auditorium Lay-out
Guest Speakers
Dr. Lorrie Kirshenbaum
Dr. Christopher McCulloch
Graduate Speaker
Nastaran Abbarin
Graduate Speaker
Nilesh Talele
Undergrad Students’ Category
Anuj Aggarwal
1
Mijhgan Ahmed
2
Noor Alzamani
3
Roli Bawa
4
Johnson Cheung
5
Sandeep Dab
6
Amandeep Dosanjh
7
Rhodaba Ebady
8
Fouad-Hassan Ebrahim
9
10 Mohamed El-Rabbany
11 Nupur Gupta
12 Gillian Landzberg
13 Roger Yi Xin Lu
14 Joel Meyerson
15 Sabrina Ramji
16 Vincent Senini
17 Peng Song
18 Laura Tan
19 Yona Vandersluis
20 Jason Wong
21 Clair S. Yang
22 Nataliya Zlotnikov
23 Angelica Zykus
Graduate and Post-Graduate Students’ Category – Basic Science
24 Laith Awamleh
25 Gursonika Binepal
Page
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ii
iii
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2
3
4
4
5
5
6
7
8
8
9
9
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10
11
11
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12
13
13
14
14
15
15
16
16
17
17
18
18
19
20
20
65
26 Maher Bourbia
27 Carlos Henrique Lacerda Brito Junior
28 Elizabeth Cambridge
29 Pavel Cherkas
30 Eugene Chung
31 Sally Esmail
32 Hailey Goldberg
33 Stephanie Hume
34 Mohamed Kandil
35 Wei-Chien (Andrew) Kao
36 Alexandra Katz
37 Franco Klingberg
38 Lauren Lamonaca-Bada
39 Gillian Layton
40 Vincent Leung
41 Chen Li
42 Alexandra Mankovskaia
43 Hamid Mohammadi
44 Samir Kumar Pal
45 Anousheh Persadmehr
46 Henry Pun
47 Lida Sadeghinejad
48 Mihaela Anca Serbanescu
49 Annie Shrestha
50 Suja Shrestha
51 Kamna Singh
52 Iwona Wenderska
Graduate and Post-Graduate Students’ Category – Clinical Science
53 Niloufar Amintavakoli
54 Sean Chung
55 Basma Dabbagh
56 Heather Ford
57 Robin Gallardi
58 Christine Hibberd
59 Raj Krishan
60 Flavia Lakschevitz
61 Trang Nguyen
62 Vinay Pilly
63 Eszter Somogyi-Ganss
64 Brent Winnett
65 Marc Yarascavitch
21
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25
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41
41
42
66
Graduate and Post-Graduate Students’ Category – Education, Behavioural, &
Health Services Research Category:
66 Mahmoud Al-Dajani
67 Ahmed Alhussain
68 Mariam Baghdady
69 Rafael Luis Fiori de Figueiredo
70 Sari Hershenfield
71 Abeer Khalid
72 Sangeeta Patodia
73 Darsi Perusini
74 Dania Sabbahi
75 Sonica Singhal
76 Carilynne Yarascavitch
77 Edward Yoon
Staff, Post-docs and Research Associate Category
78 Anne Koehler
79 Stephanie Koyanagi
80 Jye-Chang Lee
81 Alexander Stavroullakis
82 Elena Zimina
International submissions
83 Tomohito Akiyama
84 Norihiko Ashigaki
85 Seiko Fujii
86 Joji Kusuyama
87 Sara Marandi
Late Submissions
88 Kathak Vachhani
89 Mariam Mashregi
Clinical Tables
The Faculty of Dentistry Library
Index
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46
46
47
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48
48
49
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to open the Abstract Book for Research Day 2013

Transcription

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