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Local and systemic responses to craniofacial osteolytic defects in an animal model

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Local and systemic responses to craniofacial osteolytic defects in an animal model

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Local and systemic responses to craniofacial osteolytic
defects in an animal model

Susan Mahabady
University of Southern California, Graduate School
Ostrow School of Dentistry, Craniofacial Biology, M.S.
Thesis Committee:
Dr. P. Parish Sedghizadeh, Dr. Michael Paine, Dr. Homayoun Zadeh
Oral Exam: June 19, 2013

2

Table of Contents:
Title page 1
Table of contents 2
Summary 3
Introduction 3
Results 4
Materials and Methods 12
Discussion 17
References 21
Bibliography 23

3

Summary
Aggregatibacter actinomycetemcomitans (Aa) is a well-known disease pathogen
associated with oral infections. Although Aa readily forms biofilms in vitro and in vivo,
little is known about its pathogenesis or the role of host immune responses. In this study,
we used an Aa biofilm-inoculated titanium implant animal model and a calvarial lesion
animal model to study local and systemic responses to craniofacial osteolytic defects.
Analysis included evaluation of clinical inflammation, micro-computed tomography of
bone volume, polymerase chain reaction of Aa persistence, and Western blot analysis of
antibody production. Necropsies were performed for all study animals. Five out of 15 rats
treated with Aa biofilm-inoculated implants died during treatment. Following necropsy of
this subset of animals, cause of death was likely Aa sepsis leading to multi-system organ
dysfunction. These results may be important for future investigations in understanding
pathogenic mechanisms of local and systemic host-pathogen responses as they relate to
oral biofilms.

Introduction
Peri-implantitis is an inflammatory process of peri-implant tissues, involving the
alveolar bone as well as the mucosa (Berglundh et al., 2004). Little is known about the
pathogenesis of peri-implantitis or the role of host immune responses. Peri-implantitis
and periodontitis, an infection affecting the periodontium, both have similar clinical
characteristics including redness, edema, pocket formation, bleeding on probing,
inflammatory infiltrate, loss of supporting tissue, and alveolar bone loss (Heitz-Mayfield
and Lang, 2010; Offenbacher et al., 2008). Previous peri-implant studies have used
4

periodontitis animal models involving inoculation of specific pathogenic biofilm bacteria
by oral delivery or ligature placement to study the inflammatory process of affected
tissues (Carcuac et al., 2013; Lindhe et al., 1992; Trombone et al., 2009). These multi-
species biofilms present challenges in accurately characterizing and understanding the
contribution of different pathogens that elicit an immune response (Singh et al., 2002).
Furthermore, biofilms induced via ligatures are artificial in that they do not accurately
demonstrate the pathogenic processes occurring in vivo.
In the present study, we used an Aggregatibacter actinomycetemcomitans (Aa)
biofilm-inoculated titanium implant animal model previously developed in house (Freire
et al., 2011a). Aa is a well-known pathogen that readily forms biofilms in vitro and in
vivo, and is associated with oral infections including periodontitis and peri-implantitis
(De Boever and De Boever, 2006; Fine et al., 2006; Hultin et al., 2002). As a comparison
for local and systemic responses to iatrogenic craniofacial osteolytic defects, we used a
separate animal model also developed in house involving calvarial defects inoculated
with monoclonal antibodies (mABs) in the absence of Aa biofilm (Freire et al., 2011b).

Results
I. Aa biofilm-inoculated implants
Aa Biofilm Colonization In vitro
Aa biofilm formation on smooth and rough implants evaluated by Scanning
Electron Microscopy (SEM) is illustrated in Figure 1. After 1 day of cultivation, SEM
revealed agglomerates of coccoid-shaped Aa cells on the implant surfaces. These
agglomerates increased in both size and quantity by day 2, with a higher frequency of
5

coccoid and short-rod cell morphologies observed. On day 3 of cultivation, Aa biofilms
were well established with the majority of implant surfaces covered by multiple layers of
tubular and rod-shaped bacteria, each 2-6 µm in size. These findings indicate that Aa was
capable of forming well-established biofilms (Slots, 1986) on both smooth and rough
implants; however, there was significantly greater biofilm mass and growth on rough
compared to smooth implants (P <0.05) when evaluated by histomorphometry. Next, Aa
bacterial viability on rough surface implants was determined according to the Live/ Dead
fluorescent cell system as previously detailed (Freire et al., 2011a). Culture results from
smooth and rough implants indicated that rough implants on day 3 had the most viable
Aa biofilm bacteria (75%); consequently, these implants were selected for transfer to rats.

Figure
1.
SEM
images
of
titanium
mini-implants
pre
and
post
modification
and
inoculation.
Pre-
inoculated
smooth
titanium
implant
is
illustrated
in
image
A
(scale
bar
lower
right
=
500
µM).
High

power
magnification
of
pre-inoculated
smooth
(B)
and
rough
(C)
implants
demonstrate
the
effects
of

6

implant
modification
(scale
=
5
µM).
Increasing
magnification
of
day
3
Aa-inoculated
smooth
implants
is

shown
in
images
D
(scale
=
500
µM),
E
(scale
=
50
µM),
and
F
(scale
=
10
µM);
rough
implants,
G
(scale
=

500
µM),
H
(scale
=
50
µM),
and
I
(scale
=
10
µM).

Clinical Results of Aa Biofilm-inoculated Implants
Rats treated with biofilm-inoculated implants exhibited clinical signs of
inflammation, including erythema, edema, and bleeding, 2 days post-operatively and
continued to worsen. Rats treated with control implants exhibited minor post-operative
signs of mucosal inflammation also at day 2, but resolved by day 7. Osseointegration of
control implants was successful by the end of the treatment period. Clinical signs of peri-
implant mucositis progressed in rats treated with Aa biofilm-inoculated implants, which
failed to osseointegrate and exfoliated by day 14 leaving bony defects with granulation
tissue. Clinical bone volume measured by microcomputed tomography (microCT) at
week 6 indicated significant bone loss (P <0.05) in Aa biofilm-inoculated implants
compared to control implants, as shown in Figure 2.
7

Figure
2.
MicroCT bone volume quantification reveals statistically significant (P <0.05) bone loss surrounding
Aa biofilm implants compared to control implants by week 6.

Microbial Characteristics of Aa Biofilm-inoculated Implants
In vivo persistence of Aa biofilm on titanium implants was demonstrated by
culture and polymerase chain reaction (PCR) for the treatment period. Microbiological
and PCR data (Figure 3) for Aa biofilm on implants placed in vivo were positive for
implant surface samples and negative for control samples. Viable Aa was detected on
biofilm-inoculated implants post-operatively at 1 and 3 weeks; Aa was not detected on
control implants at any time point. Western blot evaluated over a period of 3 weeks
indicated antibody production against Aa in serum of rats treated with Aa biofilm-
inoculated implants (Figure 4). Rats treated with control implants did not have antibody
production against Aa at any time point.
8

Figure
3.
Persistence of Aa biofilm on implants over 3 weeks. A.) PCR data indicates the persistence of Aa
biofilm post-operatively on implant surfaces at 1 and 3 weeks; B.) Microbiological culture data confirms this
finding.
9

Figure
4.

Western
blot
of
antibody
production
in
rat
serum.
Aa
lysate
was
used
in
gel
lanes
1-3;
afrofilus

protein
control,
4-6.
Rats
treated
with
Aa-biofilm
implants
had
increased
levels
of
antibody
production

post-operatively
at
1
and
3
weeks.
Rats
treated
with
control
implants
did
not
have
antibody
production

at
any
time
point.

Necropsy
Five of the 15 rats implanted with Aa biofilm-inoculated implants died unexpectedly
2 to 14 days after implantation. Necropsy results for these rats indicated disseminated
foci of infection with detectable Aa purulence from the urethra. Among Aa biofilm
implant treated animals that survived, clinical signs of peri-implant mucosal
inflammation was observed. In the five rats that died during the experiment, the
integumentary system showed no clumping of fur and normal skin turgor, indicating that
animals had sufficient hydration and normal grooming. As determined by hematoxylin
and eosin (H&E) microscopic evaluation of the systems (Figure 5), the genitourinary
system revealed diffuse cortical coagulative necrosis of the kidney (Fig. 5A). The liver
10

showed acute passive congestion (Fig. 5B), while the remainder of the gastrointestinal
system was unremarkable. The cardiopulmonary system showed multiple thrombi in the
heart vasculature (Fig. 5C). The brain was normal showing no evidence of stroke or
masses.

Figure
5.
Histologic
sections
of
Aa
biofilm
implant
treated
animals.
A.)
Histopathology
of
the
kidney

(H&E,
20x
original
magnification)
indicates
diffuse
cortical
necrosis
with
“ghostly”
remnants
of

glomeruli
and
tubules
due
to
loss
of
cell
nuclei
and
cell
death,
while
overall
architecture
remains
intact;

this
is
consistent
with
coagulative
necrosis.
B.)
Histopathology
of
the
liver
(H&E,
40x
original

magnification)
indicates
mild
passive
congestion
with
erythrocyte
accumulation
and
stasis
surrounding

the
central
hepatic
vein
(middle).
The
lack
of
hemosiderin
deposition
in
the
sinusoids
is
indicative
of

acute
as
compared
to
chronic
passive
congestion.
C.)
Histopathology
of
the
heart
(H&E,
10x
original

magnification)
indicates
a
characteristic
thrombus
in
a
blood
vessel
adjacent
to
myocardiocytes
(left).

The
thrombus
contains
coagulated
erythrocytes,
cellular
and
inflammatory
debris,
and
hemosiderin

deposition
(dark
pigmentation).

Gross evaluation of the genitourinary system revealed a purulent discharge from the
urethra, which matched our experimental strain of Aa upon PCR analysis (Figure 6).
Necropsy and systemic evaluation of remaining animals (control and treatment groups) at
the end of the experimental period revealed normal findings grossly and histologically
compared to the aforementioned animals that experienced mortality.
11

Figure
6.
Clinical
image
of
experimental
rat
illustrates
purulent
exudate
appearing
as
a
white
discharge

from
the
urethra
(arrow).
Upon
PCR
analysis,
exudate
matched
Aa
strain
D7S-1
used
to
establish
biofilm

on
titanium
implants.

II. In vivo Calvarial Defect model
The rat calvarial defects implanted with anti-bone morphogenetic protein (BMP)
mABs via various scaffolds mediated significant bone regeneration as previously detailed
(Freire et al., 2011b). The effects of this osteolytic lesion on local and systemic responses
were evaluated as it relates to this study. Healing following calvarial defect surgery was
uneventful and complete within several days post-operatively. There were no signs of
chronic inflammation or delayed healing associated with the lesions in any study animals.
Necropsy
In all experimental animals in this portion of the study, the integumentary system
showed no clumping of fur and normal skin turgor, indicating animals had sufficient
hydration and normal grooming. As determined by microscopic evaluation of H&E
12

stained sections, rats with calvarial defects treated with various mABs showed normal
gross and microscopic findings in all treatment and control groups (Figure 7). The brain,
in addition to all other organs, was unremarkable showing no evidence of stroke or
masses.

Figure
7.
Histologic sections of calvarial defect treated animals. Microscopic evaluation (H&E, 20x original
magnification) indicates normal cell morphology in kidney (A), liver (B), heart (C), and lung (D).

Materials and Methods
I. Aa biofilm-inoculated implants

Microbial Culture: Aa strain D7S-1 was originally recovered from a patient with
aggressive periodontitis (Chen et al., 2010; Wang et al., 2002). Aa strain D7S-1 serotype
A was grown for 2 to 3 days on modified trypticase soy broth (mTSB, 3% trypticase soy
broth with 0.6% yeast extract), 5 to 10 colonies were transferred to 5 mL of liquid mTSB
13

then vortexed to disperse the bacteria. The suspension was transferred to 1 to 2 mL of
fresh mTSB at 1:20 dilution and incubated at 37°C with 5% CO
2
.
Biofilm Colonization on Treated Implant Surfaces: Prior to inoculation with Aa,
modified rough surfaces of machined (smooth) 1.2 x 4.5 mm titanium mini-implants (Ace
Surgical Supply, Brockton, MA) were produced by grit blasting with Al
2
O
3
(100 mm)
and HCl etching (pH 3, 20 minutes, 80°C). This modified implant roughness is
considered to promote bacterial adhesion and colonization when exposed to the oral
environment in vivo (Bollen et al., 1996). All implants (smooth and modified rough) were
submerged in Aa strain D7S-1 inoculated mTSB for 1 to 3 days in vitro with 5% CO
2
at
37°C.
Biofilm Viability: Aa biofilm cells were evaluated for viability 3 days after culture on
the implant surfaces by Live/ Dead staining (Boulos et al., 1999). Direct visualization and
analysis of the Aa biofilm was performed using a fluorescent cell system (BacLight
Molecular Probes, Invitrogen, Carlsbad, CA) composed of 2 nucleic acid binding stains:
SYTO 9, which penetrates all bacterial membranes (stains green), and propidium iodide,
which penetrates cells with damaged membranes (stains red). These stains together
produce yellow fluorescing cells. Optimal incubation conditions for visualization were
determined to be 20 minutes in the dark at room temperature, and total (red and green) as
well as viable (green) cells were visualized using confocal laser scanning microscopy.
Scanning Electron Microscopy: Aa biofilm-inoculated and control titanium implants
were visualized with SEM after 1 to 3 days in vitro. Implant samples were prepared with
Karnovsky fixative (6% glutaraldehyde and 4% paraformaldehyde in 0.2 M cacodylate
buffer) for 2 hours. Samples were then washed twice in 0.1 M cacodylate buffer for 15
14

minutes, post-fixed with 2% osmium tetroxide in 0.2 M cacodylate buffer for 2 hours at
4°C, and dehydrated with increasing ethanol concentration (30 to 70%). Samples were
placed in an increasing concentration gradient of hexamethyldisilazane, embedded in
100% hexamethyldisilazane, and air-dried at room temperature for 24 hours. Samples
were then gold coated and observed with a JSM-6610 (JEOL USA Inc., Peabody, MA)
SEM at 30 kV. Biofilm volume on smooth versus rough implants was quantified by
histomorphometry (MetaMorph, Molecular Devices LLC, USA), and statistical
significance was accepted at P <0.05.
Animals: Thirty 5-month old, virgin, female rats (Sprague-Dawley, Charles River
Laboratories, Hollister, CA) were housed in a laboratory at 22.2°C under a 12-hours light
and 12-hours dark cycle and fed ad libitum (Purina Laboratory Rodent Chow, Purina
Milk, Richmond, IN). Animal care was in accordance to the NIH Guide for the Care and
Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources,
National Research Council, all applicable government regulations, and university policies
governing the care and use of laboratory animals. All protocols and procedures were
approved by the Institutional Animal Care and Use Committee (IACUC) of the
University of Southern California, and were in accordance with the Panel on Euthanasia
of the American Veterinary Medical Association. The appropriate number of animals for
investigation was determined to be 30 by power analysis. The power calculation allowed
us to detect statistically significant effects between treatment and control groups. G
Power 3 software (Faul et al., 2007) determined sample size of 30 animals to be suitable
in detecting large effects, correlating to an effect size f of 0.40 (F tests- ANOVA:
repeated measures, within-between interaction). Rats were anesthetized with isoflurane,
15

and Aa biofilm-inoculated implants (15 rats) or control implants (15 rats) were
transmucosally placed into the palate adjacent to the maxillary alveolar ridge. Animals
were followed for 6 weeks post-operatively.
Detection of Aa Biofilm on Surgical Implants: Bacterial samples of Aa biofilm and
control were collected from the oral surfaces of implants after 1 to 3 weeks to confirm the
presence of Aa biofilms on the implants. Samples were obtained by touching a sterile
toothpick on the surgically placed implants for 10 seconds and vortexing in 500 mL of
phosphate-buffered saline. Aa was detected by PCR and culture identification methods.
PCR was based on amplification of 16S ribosomal RNA gene (16S RNA) signature genes
(primer sequence for species-identity PCR forward: 59 AAA CCC ATC TCT GAG TTC
TTC TTC 39 and reverse 59 ATG CCA ACT TGA CGT TAA AT 39) using standard
conditions for Taq DNA polymerase (Bollen et al., 1996). 16S rDNA was amplified from
Aa strain D7S-1 DNA template as a positive control, and without added template as a
negative control. Dilution of mTSB (1:10) was made for culture detection and plated in
triplicate on trypticase soy with serum, bacitracin, and vancomycin. Aa colonies on
trypticase soy with serum, bacitracin, and vancomycin were incubated with 5% CO
2
for 3
to 5 days at 37°C, then identified and counted under a stereomicroscope to determine Aa
density on implant surfaces.
Microcomputed Tomography: MicroCT radiologic evaluation with concomitant three-
dimensional reconstruction of peri-implant bone and tissues was performed 3 and 6
weeks post-implantation. Each rat was placed in a sample holder in the cranial-caudal
direction and scanned using a high-resolution microCT system (Micro CAT II, Siemens
Medical Solutions Molecular Imaging, Knoxville, TN) at a spatial resolution of 18.676
16

mm (voxel dimension) and 1536 x 1536 pixel matrices. Rats were maintained under
general anesthesia with isoflurane during the scanning procedure. The resulting two-
dimensional image data was stored in the Digital Imaging and Communications in
Medicine (DICOM) format, transferred to a computer, then three-dimensional
reconstruction and analysis was performed. To reduce the size of data for computation,
the craniofacial region was cropped and saved from obtained consecutive
microtomographic slice images as a volume of interest using software (Amira, Visage
Imaging, San Diego, CA). In this step, the original spatial resolution was maintained
because data was not re-sampled. Peri-implant bone volume was measured using
software (V-Works 4.0, Cybermed, Seoul, Korea), and bone tissue was segmented using
a global thresholding procedure. Threshold equaling -360 HU (Hounsfield Units) was
used to investigate peri-implant bone tissues. Peri-implant bone was measured by
applying a virtual cylindrical divider of 50 voxels in diameter and 105 voxels in length.
The proportion of bone volume occupying this virtual cylindrical space around implants
was measured and compared. A student t-test was used for pair-wise comparisons of bone
volume for biofilm-inoculated implants as compared to controls. Statistical significance
was accepted at P <0.05.
Necropsy: Rats were euthanized at the end of the treatment period; skulls were harvested
and stored in 10% buffered formalin for additional analysis. Systemic physical, gross
macroscopic, and microscopic examinations of H&E stained sections were evaluated for
various organs. Genitourinary exudate, present in some animals, was evaluated by PCR
for detection of Aa. To evaluate serum for presence of Aa, Western blot analysis for
antibody production was obtained using Aa lysate and afrofilus protein control.
17

II. Calvarial Defect Model

The experimental animal model used was previously described in detail (Freire et al.,
2011b). In brief, rat calvarial defects were created and inoculated with anti-BMP mABs
to evaluate the effects of local and systemic responses to the osteolytic lesion.
Animals: Thirty 5-month old, virgin, female rats (Sprague-Dawley Charles River
Laboratories, Hollister, CA) were selected and housed as previously described in detail
above.
In vivo Calvarial Defect Model: Calvarial defects were created in rats under general
anesthesia, and full thickness skin flaps were raised to expose left and right parietal
bones. Parietal bone defects of 7 mm diameter were created using a trephine with saline
irrigation. Anti-BMP-2 mABs or isotype control mABs (25 ug/ mL each) were incubated
with 4 different scaffolds (titanium microbeads, macroporous biphasic calcium
phosphate, alginate, and collagen sponge) and placed inside the calvarial defects. The rats
were euthanized at the end of the treatment period (45 days); skulls were harvested and
stored in 10% buffered formalin for further analysis.
Necropsy: Systemic physical, gross macroscopic, and microscopic examination of H&E
stained sections were evaluated for various organs to determine health status of all study
animals at time of euthanization.

Discussion
Oral biofilm-associated diseases may have local and even systemic effects by: 1)
spread of infection to adjacent tissues and spaces, 2) hematogenous dissemination of oral
biofilm bacteria, and 3) inflammatory mechanisms (Beikler and Flemmig, 2011). Oral
18

biofilms represent a complex collection of microorganisms that can disseminate into the
bloodstream and spread by transient bacteremia, as determined by oral biofilm types
isolated from infections distant to the oral cavity (Li et al., 2000). Oral biofilm bacteria
have been associated with shedding and hematogenous dissemination in forms of acute
bacterial myocarditis, infective endocarditis, brain abscess, liver abscess, lung abscess,
cavernous sinus thrombosis, and prosthetic joint infections (Beikler and Flemmig, 2011).
Oral biofilms may also impact systemic health by inflammatory mechanisms; and
increased levels of systemic inflammatory markers are reported in patients with
periodontitis (Craig et al., 2003).
Several bacterial agents of periodontitis are also associated with peri-implant
diseases, including Aa, P. gingivalis, T. denticola, and T. forsythia (Hultin et al., 2002).
An association is known between periodontitis and peri-implantitis; however, it is
reported that bacteria unrelated to periodontitis may also be associated with peri-implant
tissue inflammation (Koyanagi et al., 2013). Nevertheless, the full range of species
responsible for peri-implantitis remains unclear due to the complexity of microbiota in
the disease. To reduce this complexity and minimize confounders or effect modifiers, we
chose to focus on a single well-known pathogen in oral infectious disease. Thus, an Aa
monoculture was used in this study via an implant delivery system, rather than ligature-
based approaches previously utilized. Further rationale for selection of Aa as our model
pathogen includes the availability of significant in vitro data with immune responses to
this particular clinical pathogen and its deletional mutants (Nalbant et al., 2003; Zadeh et
al., 2001). In future studies, additional pathogens can be introduced singly and in
19

combination to systematically study pathogenetic mechanisms and contributions of
different species along with host responses.
The present study employed an Aa biofilm-induced oral osteolytic lesion animal
model to study its effects on local and systemic responses. As a comparison of local and
systemic effects, we also utilized a calvarial osteolytic lesion animal model without the
presence of Aa. In the Aa osteolytic model, chronic local inflammation was observed
when Aa was introduced via an implant delivery system. In non-Aa controls, however,
only transient acute inflammation was observed with subsequent resolution post-
operatively. Systemic complications occurred in a subset of animals that died
unexpectedly. Necropsies were performed to evaluate possible causes of death in this
subset of animals. Necropsy is of fundamental importance for the examination of
morphological changes associated with pathology or states induced by experimental
treatments. This evaluation of each animal allowed us to identify the cause of mortality
by a series of systematic operations: systemic physical, gross macroscopic, and
microscopic histopathology. As determined by our necropsy findings, the underlying or
primary cause of death in this subset of animals was likely Aa sepsis leading to multi-
system organ dysfunction. The immediate cause of death may have been acute renal
failure. We cannot rule out the possibility that other microorganisms may also have been
involved in the Aa-associated pathogenesis or bacteremia.
Local and systemic findings in the comparative calvarial defect animal model
confirmed that a craniofacial osteolytic lesion alone without introduction of Aa does not
result in local and systemic pathology or mortality. We recognize that our model system
has inherent limitations like any animal model or system, and findings are not directly
20

applicable to human disease. However, these studies and findings represent an important
step in the translation of this work to future clinical insight regarding host-pathogen
interactions. Future studies in this line of investigation will explore pathogenic
mechanisms and host immune responses to understand why some animals experience
systemic morbidity and mortality when exposed to oral Aa biofilms while others do not.

21

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Abstract (if available)

Abstract Aggregatibacter actinomycetemcomitans (Aa) is a well-known disease pathogen associated with oral infections. Although Aa readily forms biofilms in vitro and in vivo, little is known about its pathogenesis or the role of host immune responses. In this study, we used an Aa biofilm-inoculated titanium implant animal model and a calvarial lesion animal model to study local and systemic responses to craniofacial osteolytic defects. Analysis included evaluation of clinical inflammation, micro-computed tomography of bone volume, polymerase chain reaction of Aa persistence, and Western blot analysis of antibody production. Necropsies were performed for all study animals. Five out of 15 rats treated with Aa biofilm-inoculated implants died during treatment. Following necropsy of this subset of animals, cause of death was likely Aa sepsis leading to multi-system organ dysfunction. These results may be important for future investigations in understanding pathogenic mechanisms of local and systemic host-pathogen responses as they relate to oral biofilms.

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Asset Metadata

Creator Mahabady, Susan (author)

Core Title Local and systemic responses to craniofacial osteolytic defects in an animal model

School School of Dentistry

Degree Master of Science

Degree Program Craniofacial Biology

Publication Date 07/21/2013

Defense Date 06/19/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Aggregatibacter actinomycetemcomitans,biofilm,craniofacial,OAI-PMH Harvest,osteolytic,peri-implantitis

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Sedghizadeh, P. Parish (committee chair), Paine, Michael L. (committee member), Zadeh, Homayoun H. (committee member)

Creator Email mahabady@usc.edu,susan_mahabady@yahoo.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-292774

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Legacy Identifier etd-MahabadySu-1761-0.pdf

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Document Type Thesis

Rights Mahabady, Susan

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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