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Bone targeted antimicrobials for biofilm-mediated osteolytic infection treatment

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Bone targeted antimicrobials for biofilm-mediated osteolytic infection treatment

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Content
Bone Targeted Antimicrobials for Biofilm-Mediated Osteolytic Infection
Treatment
by
Raffie Garabedian, DMD

A Thesis Presented to the
FACULTY OF THE USC HERMAN OSTROW SCHOOL OF DENTISTRY
UNIVERSITY OF SOUTHERN CALIFRONIA
In Partial Fulfillment of the
Requirement for the Degree
MASTER OF SCIENCE
BIOMEDICAL IMPLANTS AND TISSUE ENGINEERING

August 2020

Copyright 2020 Raffie Garabedian

ii
ACKNOWLEDGEMENTS
This work was supported by grants (R41-DE025789-01 and R42-DE025789-02) from the
National Institutes of Health, National Institute of Dental and Craniofacial Research (NIDCR), to
PPS and FHE.

iii
TABLE OF CONTENTS

Acknowledgments .......................................................................................................................... ii
List of Tables .................................................................................................................................. iv
List Of Figures ................................................................................................................................ v
Abstract ........................................................................................................................................... vi
Introduction ..................................................................................................................................... 1
Materials and Methods .................................................................................................................... 4
Results ............................................................................................................................................. 8
Discussion ...................................................................................................................................... 10
Conclusion ..................................................................................................................................... 13
References ..................................................................................................................................... 14
Tables and Figures ......................................................................................................................... 18

iv
LIST OF TABLES

Table 1 Histomorphometric Analysis for Conjugated Graft Groups 18
Table 2 Histomorphometric Analysis for Control Graft Group 19
Table 3
Statistical Analysis of mean values of histomorphometric analysis of both
conjugated graft groups and control graft group
20

v
LIST OF FIGURES
Figure 1 Chemical structures of BCC and BCX conjugates.
21
Figure 2
Clinical photograph showing hemi-section of second, third, and fourth
premolars using surgical handpiece and diamond disc under copious
irrigation.
22
Figure 3
Clinical photograph showing endodontic treatment on the retained hemisected
root of each premolar after tooth sectioning.
23
Figure 4
Clinical photograph showing grafting of surgical defects (left) and placement
of collagen membrane and flap approximation using 4.0 synthetic resorbable
interrupted sutures.
24
Figure 5 CT scans show surgical defects in axial, coronal, and 3D reconstruction.
25
Figure 6 Histologic sections from trephine core biopsies of the surgical grafts.
26
Figure 7
Color coded histologic sections from trephine core biopsies of the surgical
graft sites for morphometric analyses.
27
Figure 8
Clustered columns show the statistical analysis differences between
conjugated grafts and control groups.
28

vi
ABSTRACT
Keywords: dental; bisphosphonate; antibiotic; fluoroquinolone; conjugate; bone graft; canine
model.

Objective: Bacterial biofilms are the major cause of debilitating bone diseases such as
osteomyelitis, osteonecrosis, prosthetic joint infections, peri-implantitis, and septic arthritis. In
dental settings, infections of bone grafts which are routinely placed after extractions for ridge
preservation or for dental implant therapy are also due to biofilm mediated infections. Current
treatment protocols for dental and orthopedic bone infections mainly involve surgery and/or
antimicrobial therapy. However, antibiotics suffer from poor bone resorption and
pharmacokinetics. Thus, the goal of this study was to design and test novel bone-targeted
antimicrobial drugs utilizing bone-binding bisphosphonates (BP) moieties conjugated to
fluoroquinolone antibodies in order to treat bone infections.

Materials and Methods: In this study we designed BP-Ciprofloxacin conjugates as a novel bone
graft formulation and tested them as a combination in animal model to assess initial safety and
bone grafting efficacy in vivo. Our study was designed as a canine (beagle dogs, n=3 based on
power analysis) split-mouth surgical extraction and anroganic bovine bone-grafting model to test
our conjugates against negative (collagen sham) and positive (bone graft without conjugate)
controls. Animal experiments conformed to the ARRIVE guidelines for reporting on animal
research to ensure the quality, reliability, validity, and reproducibility of results.

Results: Quantitative 3D volumetric CT and histologic morphometric analysis were performed to
examine the volume of de novo bone present in surgical defects as compared to residual graft
material and fibrous tissue. No adverse events were identified in any of this study animals
throughout the study period, and all sites when examined histologically revealed no evidence of
necrosis, infection, osteolysis or foreign body reaction. Bone volume or regeneration in the
surgical defects was greatest in the conjugate bone graft group as compared to the bone graft
substitute without conjugate group (p<0.0001, ANOVA) and also to the negative controls
(p<0.001, ANOVA). Our results indicate that BP-fluoroquinolone infused bone grafts did not
adversely impact graft survival and de novo mineralization was superior to that of bone graft
substitute alone, indicating that the antimicrobial and/or antiresorptive properties of conjugates
improve bone remodeling and healing outcomes.

Conclusion: Our dual-action conjugates employing bisphosphonates bound to releasable
antibiotics chemisorbed to bone graft material is a safe and promising therapeutic tool to enhance
bone grafting in the scope of dentistry.

1
INTRODUCTION
The predecessors to modern bisphosphonates (BP) were previously evaluated for their
activity on teeth and bones.
8,9
Human trials studied the use of BP compounds in various toothpaste
formulations demonstrated some anti-caries activities.
16
Also, anti-calculus effects seen in large
animal studies emerged promising results in treating periodontal disease secondary to oral biofilm
and calculus accumulation along with associated inflammation.
1
More recent long-term
longitudinal clinical studies showed no enhancement in maintaining alveolar bone levels in
subjects received oral BPs; however, the adjunctive use of oral BPs in conjunction with local
delivery medications for the management of periodontal disease may be efficacious.
11

BP coated dental implants showed increased bone density and significant resistance to pull-out
forces in animal models, and significantly increased implant stability and marginal bone stability
in humans.
24

Bacterial biofilm pathogens are a major cause of devastating infectious bone diseases such
as osteomyelitis, osteonecrosis, and prosthetic joint infections.
17,25
Similarly in dentistry, bacterial
biofilms are responsible for jaw-bone infections, and can present clinical challenges in terms of
diagnosis and management. Pathologic bone loss in oral cavity can be secondary to localized
infections, such as jawbone osteomyelitis, periodontitis, and peri-implantitis. The current approach
to manage these dento-alveolar infections includes surgical debridement, local and/or systemic
antimicrobial therapy, and bone grafting to reconstruct osseous defects. However, antibiotics
suffer from poor bone adsorption and pharmacokinetics.
15,10
In addition to poor penetration and
retention of antimicrobial agents into bone, systemic antibiotic therapy can be associated with
adverse events related to systemic toxicity. Furthermore, bone graft failure can occur and is usually
secondary to infection. According to Junka et al, bone-pathogens can invade hydroxyapatite and

2
even deeper within osseous tissues throughout the 3-dimensional canalicular network. This
invasion can directly destroy and resorb bone without eliciting host immunity or
osteoclastogenesis.
12

Antibiotic eluting bone graft cements are commonly used in orthopedic surgeries for
implant fixation and local antibiotic delivery following surgical debridement. However, balancing
the anti-infective properties with potential incidence of nephrotoxicity remains a major
challenge.
19
In contrast, bone cements are not commonly used in dentistry or dental implant
therapy, and dentists mainly utilize various bone graft substitutes with no inherent anti-infective
properties. Hence, prescribing systemic anti-microbials as well as blending antibiotics with bone
substitute materials are common hone among dental specialists, to anticipate nearby jawbone and
graft infections. Such methodologies still endure from poor pharmacokinetics, lack of bone
retention and reproducibility, and systemic exposure to adverse effects. To conquer the numerous
difficulties related with current conventions to treat bone contaminations, there is expanding
enthusiasm to develop drug delivery mechanisms using bone-targeting conjugates to achieve
higher and/or more sustained local therapeutic concentrations of antibiotic in bone while
minimizing systemic exposure.
20

To address the aforementioned clinical challenges and current therapeutic limitations in
dental bone graft settings, our team has developed novel anti-infective bone graft substitutes for
dental applications. We have designed bisphosphonate (BP) moieties that lack cellular
pharmacological or antiresorptive activity, but that retain hydroxyapatite-binding ability,
conjugated to fluoroquinolone antibiotics like ciprofloxacin and moxifloxacin.

We utilize
releasable carbamate linkers for conjugation to synthesize and test our BP-carbamate-
ciprofloxacin (BCC) and BP-carbamate-moxifloxacin (BCX) conjugates, chemisorbed to bone

3
graft material, in an animal model to assess initial safety and bone graft efficacy in vivo. Our
findings reveal that novel bone grafts with chemisorbed BP-fluoroquinolone conjugates represent
a safe and promising apparatus for applications to clinical bone grafting in dentistry.

4
MATERIALS AND METHODS
Chemistry
Chemical structures of the conjugate compounds are shown in Figure 1. BCC was prepared
as described by Sedghizadeh et al,
23
and BCX was prepared by a similar chemistry sequence with
modifications.
Binding affinity of antibiotics to hydroxyapatite
To test the affinity of the conjugates (BCC and BCX) and each parent antibiotic to
hydroxyapatite, which is relevant to bone targeting and graft retention, we performed HPLC
analysis as follows: 1µg/mL of each antibiotic compound (ciprofloxacin, moxifloxacin, BCC, and
BCX) was added to a solution containing 10µg/mL of hydroxyapatite spherules and incubated at
37°C/4 hours under magnetic stirring. Next, the mixture was allowed to sediment for 1h/4°C. After
this time the quantity of drug within the supernatant was assessed using HPLC (AGILENT 1220
Infinity LC system); gradient applied: phase A - deionized water stabilized to pH=7; phase B:
acetonitrile of HPLC grade. Column applied: Supelco ascentris express C18: 15cm x 4.6mm x
5µm. Gradient: 0-1min – 2% phase B and 1-20min – 100% of phase B. To evaluate the quantity
of conjugate bound to hydroxyapatite, we used methodology described in detail in our previous
publication.
23
Also, 1µg/mL of each compound served as a control sample for testing. Affinity of
compounds to hydroxyapatite was estimated as follows: [(100% - peak area of control) / peak area
of tested sample] *100%.
In vivo animal study
This animal study was approved by a university Institutional Animal Care and Use
Committee (USC protocol # 20474) and in accordance with the Animal Welfare Act and PHS
Policy on Humane Care and Use of Laboratory Animals. Radiation Safety Committee approval

5
(USC # RPAHS-18-00002) was obtained for live CT imaging of animals. Institutional Biosafety
Committee approval (USC # BUA-17-00053) was obtained for Biohazard Use Authorization of
chemicals and compounds used in the study. The study was designed as a canine (female beagle
dogs, n=3) split-mouth surgical extraction and bone-grafting model to test our conjugates against
controls. Experiments conformed to the ARRIVE guidelines for reporting on animal research to
ensure the quality, reliability, and reproducibility of results.
14
Sample size estimations are detailed
below under “statistical analysis.” Survival surgeries were performed in a vivarium sterile
operatory room. All procedures were performed under the direction of a veterinarian. Animal
weight was recorded pre-operatively and then regularly throughout the course of the study to
determine anesthesia dosages and monitor the health of animals post-operatively and during
healing. For anesthesia, animals were inducted with intramuscular injection of Acepromazine 0.05
mg/kg and Butorphanol 0.02 mg/kg. An intravenous catheter was inserted into the cephalic vein.
At the time of induction subcutaneous Buprenorphine sustained release (0.03 mg/kg) was given
and a first dose of Carprofen (4.4 mg/kg) was given subcutaneously, and then Carprofen (2 mg/kg)
was given orally for 48 hours post-op once a day. Vital signs (HR, BP, Resp, CO2, O2 saturation)
were constantly monitored throughout the procedure. Dogs were intubated with a size 7-8
endotracheal tube and attached to a Sevoflurane/oxygen machine, ventilated and maintained by
Sevoflurane (1-4%). Temperature was maintained by water blanket to prevent hypothermia.
During surgery, animals were monitored every five minutes by the veterinarian. The surgical site
was clipped and prepped aseptically with a minimum of three alternating rounds of alcohol and
Betadine. For pain management and to minimize discomfort and distress, local anesthetic
(Bupivicaine 0.5%, 1mg/kg) infiltration was given at surgical sites and included mandibular nerve
blocks and vestibular infiltrations.

6
In this split mouth design, bilateral (right and left) mandibular premolars (PM2, PM3 PM4)
were hemisected with a surgical handpiece and diamond disc under copious irrigation Figure 2,
and one-half of each tooth/root was atraumatically extracted using periotomes, elevators, and
forceps while the remaining tooth/root hemisection was endodontically treated. This approach was
utilized in order to provide a radiopaque marker (gutta percha) adjacent to extracted tooth sites and
allow for easy identification and improved analysis of grafting sites radiographically post-op and
after the healing phase of the study. Root canal treatment of the retained hemisected root of each
premolar involved pulp access with round carbide burs, cleaning and shaping of each canal with
hand files, irrigation with 2% chlorhexidine gluconate, and obturation with gutta percha and sealer
with heated lateral condensation Figure 3. At each adjacent extraction socket site, bone graft
material (anorganic bovine bone xenograft, 1g particle size of 1mm) chemisorbed with BP-
fluoroquinolone conjugates (treatment group; n=6 BCC, n=6 BCX) or without chemisorbed
conjugates (control group; n=6) was applied as 1mg conjugate/1g graft material at 37°C/4 hours
under magnetic stirring prior to delivery. Primary wound closure was achieved using a collagen
membrane and 4-0 vicryl synthetic resorbable interrupted sutures Figure 4. Surgeons were blinded
as to which sites were treatments versus controls, and sites were coded for future blinded analyses.
Immediately post-op, animals were taken to an imaging center for CT scanning (GE 16 slice
scanner, 59.7 mGy/exposure). For recovery, dogs were extubated and monitored clinically for
respiration, temperature, and eye color until 100% recovery. Surgical sites were allowed to heal
for three months and clinical evaluation was performed weekly until study end. After this three-
month healing period, animals were re-anesthetized as previously described and again CT scanning
was performed followed by surgical biopsy where standardized trephine core biopsies (4 mm
internal diameter) were obtained at the extraction sites for subsequent histomorphometric

7
assessments. All samples were fixed in 10% neutral buffered formalin, decalcified with 14%
EDTA, and repeatedly rinsed with 70% ethanol for one week until paraffin embedding and
sectioning for hematoxylin and eosin staining. Finally, animals were clinically evaluated for two
more weeks to ensure complete healing of biopsy sites and no adverse events, after which all
animals were successfully adopted.
Statistical analysis
Histomorphometric analysis utilizing Bioquant (Nashville, TN) image analysis software
with a Leica light microscope (20X, 0.50 PH 2 PL FLUOTAR 1X FW 239um, 756 Pixel,
0.316um/Pixel) was used for calculating referent data. Total surface area of the core biopsy, new
bone formation, residual graft, and fibrous tissue were calculated as mm
2
and represent 2D not 3D
data such as volume. Descriptors and measurements were in general accordance with
recommendations of the American Society for Bone and Mineral Research histomorphometry
nomenclature committee.
6
Statistical comparisons and tests were performed with IBM SPSS 22.0
(Armonk, NY, USA) and Microsoft Excel (Redmond, WA, USA). Power analysis was performed
to determine sample sizes for in vivo studies prior to experimentation using G Power 3 software.
7
Power calculations were predicated on the number of dogs per experimental group to detect
statistically significant effects between groups, with the sample size correlating to an effect size f
of 0.40 (F-tests ANOVA: repeated measures, within-between interaction). Quantitative data from
experimental results for each group were analyzed first with descriptive statistics to understand the
distribution of the data and to generate the mean, standard error, standard deviation, kurtosis and
skewness, and 95% confidence levels. Data was analyzed via ANOVA and then t-tests for
confirmation, and statistical significance was accepted at p<0.05 when comparing treatment to
control groups.

8
RESULTS
Chemistry
For an expedient synthesis of bisphosphonate-carbamate-moxifloxacin (BCX), 4-
hydroxyphenylethylidene BP was selected as a common pharmacologically inert starting
compound, allowing us to retain the bone-binding ability of the BP moiety while minimizing
toxicity and focusing on utilizing moxifloxacin as a more potent clinical fluoroquinolone as
compared to ciprofloxacin. Synthesis of the BP-carbamate-ciprofloxacin (BCC) conjugate has
been detailed in previous work of Sedghizadeh et al.
23
BCX was made via similar chemistry with
minor modifications.
Binding affinity of antibiotics to hydroxyapatite
HPLC results evaluating the binding affinity of tested compounds to hydroxyapatite were
as follows, from greatest to lowest binding affinity: BCX conjugate 92%, BCC conjugate 91%,
moxifloxacin 51%, ciprofloxacin 36%.
In vivo animal study
No adverse events were identified in any study animals throughout the study period, and
all surgical sites when examined clinically, radiographically, and histologically revealed no
evidence of infection, inflammation, necrosis, osteolysis, or foreign body reaction.
Radiographic examination allowed identification of grafted sites adjacent to radiopaque
markers, enabling reproducible anatomic identification of grafted sites (Figure 5) and accurate
biopsy cores to be taken for histomorphometric analyses.
Histologic features of the biopsy specimens from regenerated defects showing de novo
bone formation as compared to residual graft material and fibrous connective tissue are shown
in Figure 6. Histologic sections that were color coded for histomorphometric analysis shown in

9
Figure 7. The new bone appeared as woven bone with several large rounded osteocyte lacunae.
Bone remodeling is evidenced by prominent reversal lines. Direct connection observed between
the newly formed bone and the residual graft particles forming “bridges” of trabecular bone.
Table 1 and 2 report the results of the histomorphometric evaluation of conjugated graft
groups (BCX and BCC) and controlled graft groups respectively. Three specimens of the
conjugated graft group were excluded from the statistical analysis due to fractured facial wall and
only nine specimens analyzed. All specimens for control graft groups (total of 4) included in the
statistical analysis. New bone formation area (NBA) was 43.47% ± 8.91% vs 32.97% ± 2.13% for
the conjugated graft and control graft groups, respectively. The residual graft area (RGA) was
19.82% ± 11.16% for conjugated graft groups and 13.90% ± 11.06% for control graft group.
Statistical analysis (Table 3), revealed that area of de novo bone formation in the surgical
defects was greatest in the conjugate bone graft groups (BCX and BCC) as compared to the control
bone graft group without conjugate. This difference was statistically significant (p=0.04, p<0.05,
ANOVA), as shown in Figure 8A.
Between the two different conjugate groups (BCC vs. BCX), no significant differences in
the new bone area were observed (p=0.51, p<0.05, ANOVA).
There was no statistically significant difference in the residual bone graft area between the
conjugate treated group versus the control group (P=0.94, p<0.05, ANOVA), as shown in Figure
8B. The area of fibrous tissue in the defects was greater in the control group versus the conjugate
treated group (p=0.01, p<0.05, ANOVA), as shown in Figure 8C.

10
DISCUSSION
Infectious bone diseases, similar to most orthopedic infections, are mediated by bacterial
biofilm.
28
In most cases, the treatment of such infections are very challenging, although antibiotics
are the standard of care, they have limited bone bioavailability. Current antibiotic therapeutic
agents do not show a preferential affinity for bones but rather distribute throughout the body after
administration.
4
Among other classes, fluoroquinolone antibiotics are used in treatment of bone
infections because of their efficacy against some of the causative pathogens. On the other hand,
bisphosphonates (BP) class of drugs has high affinity for bone binding and retention in addition to
a long track-record of clinical use. BP drugs preferentially accumulate at sites of active bone
disease or biofilm infection, resorption, and remodeling.
3
BPs also penetrate the canalicular
network to osteocytes and osteocytes lacunae where no blood flow exists and where biofilm
organisms are known to embed.
5,22
More recently, concerns about safety and adverse effects such
as osteonecrosis of the jaw and atypical femoral fractures have impacted clinical use and BP
pharmacotherapy.
13,18,22
An ideal therapeutic drug for bone infections should act only on bone
tissue, with no pharmacological activity at other anatomical sites.
27
These adverse effects of BPs
class are only seen with the nitrogen-containing BP drugs and not the non-nitrogen variants.
Therefore, our team have been exploiting the bone targeting property of various BP-
fluoroquinolone conjugates and designed unique non-nitrogen BPs conjugated to fluoroquinolone
antibiotics for clinical applications. This extended bone targeting property allows for the bone
binding of the BP moiety without antiresorptive pharmacology or local or systemic adverse effects
and provides local antibiotic release at bone via a labile chemical linkage. Hydroxyapatite binding
affinity as tested by HPLC revealed much higher affinity of conjugates as compared to the parent
antibiotics alone.

11
It was previously reported on the antibiotic release kinetics of the parent antibiotic from its
conjugated form, and that this release varied depending on the type of chemical linkage used
(which also had a direct impact on antimicrobial efficacy).
19
Sedghizadeh et al, synthesized and
tested a non-cleavable amide-linked antibiotic conjugate, and when compared to a carbamate-
linked conjugate, they found it to be nearly inactive against osteomyelitis pathogens grown on
hydroxyapatite surfaces in vitro.
23

Research into bone-binding medicinal agents such as the work presented here is
progressively laying the foundation for next-generation 'magic bullets' that demonstrate targeted
and greater efficacy and retention at the disease site, minimizing systemic exposure or toxicity. In
previous study, Sedghizadeh et al, tested BP-conjugated fluoroquinolone in a small animal (rodent)
model, and in the current study we utilized a large animal (canine) model which is commonly used
in dental research as a gold standard model for grafting studies.
2,26
Larger animal models overcome
many of the limitations of small animal models as far as anatomical size and observable effects.
In the current study we created a novel bone graft formulation wherein BCC or BCX conjugates
were chemisorbed to bone graft material for testing in vivo. The split-mouth canine model allows
for well-controlled analyses in this context.
21
Although not directly comparable to our technology,
in the past antibiotic eluting beads and cements have been used for similar applications in
orthopedic settings. These materials suffer from several limitations. For example, many of these
materials are non-resorbable and thus a second surgery is required to remove them. They also tend
to release antibiotics in an initial bolus that quickly depletes the bulk of the antibiotic, followed by
a slow release at sub-therapeutic concentrations that may not control infection but may promote
development of resistance.
4

12
In dentistry, the addition of antibiotic agents to bone graft material is routinely performed
ad hoc in order to minimize the potential for infection. This is done for various bone grafting
indications including ridge preservation after tooth extraction or for dental implant therapy.
Further, such approaches are imprecise and there is currently no anti-infective bone graft approved
for use in the dental market. The use of our BP-antibiotic conjugation technology allows for
chemical perturbations and modifications that can fine tune parameters such as bone binding,
antibiotic release profiles and concentrations, and accordingly clinical and antimicrobial efficacy.

13
CONCLUSION
Bisphosphonate – fluoroquinolone conjugates chemisorbed to bone graft material
can function normally as a bone graft substitute for clinical application in dentistry as in this
extraction and ridge preservation model. Similarly, to control grafts, conjugated grafts were
deemed to be safe in vivo and not accompanied with any adverse events such as infection,
inflammation, graft failure, or osteonecrosis.
Our data showed that de novo graft area in conjugated groups was higher than that
of bone graft substitute alone and in vivo graft survival was not adversely impacted by
conjugate bone grafts. It can be hypothesized that healing and bone remodeling outcomes
were improved by the antimicrobial properties of conjugates in a non-sterile oral grafting
model. Future studies will explore the antimicrobial efficacy of our novel bone graft
substitutes using an oral peri-implant biofilm-mediated infection canine model.

14
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Sotereanos NG, Costerton JW, Demeo P. Orthopaedic biofilm infections. Curr Orthop
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29:592-602.
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28. Zimmerli W, Sendi P. Orthopaedic biofilm infections. APMIS 2017; 125:353-64.

18
TABLES AND FIGURES
Table 1: Histomorphometric Analysis for Conjugated Graft Groups.

Table-1
Histomorphometric Analysis for
Conjugated Graft Groups
Site ID NBA (%) RGA (%) FTA (%)
1LP3 49.9 21.33 28.75
2LP3 36.4 21.22 42.34
2RP2 44.2 23.98 31.81
3LP2 34.9 14.82 50.26
3RP2 42.5 8.71 48.81
1LP4 60.8 6.87 32.35
1RP2 48 11.9 40.07
2LP2 43.2 26.14 30.65
2RP4 31.3 43.39 25.33
Mean ± SD 43.47 ± 8.91 19.82 ± 11.16 36.71 ± 8.98
95% CI 36.62-50.32 11.24-28.40 29.80-43.61
NBA: new bone area. RGA: residual graft area. FTA: fibrous tissue area. SD: standard deviation.
CI: confidence interval.

19
Table 2: Histomorphometric Analysis for Control Graft Group.

NBA: new bone area. RGA: residual graft area. FTA: fibrous tissue area. SD: standard deviation.
CI: confidence interval.
Table-2
Histomorphometric Analysis for Control
Graft Groups
Site ID NBA (%) RGA (%) FTA (%)
1RP3 49.9 21.33 28.75
2LP4 36.4 21.22 42.34
3LP3 44.2 23.98 31.81
3RP3 34.9 14.82 50.26
Mean ± SD 32.97 ± 2.13
13.90 ±
11.06
53.14 ± 11.18
95% CI 29.57-36.35 -3.72-31.51 35.36-70.93

20
Table 3: Statistical Analysis of mean values of histomorphometric analysis of both conjugated
graft groups and control graft group.

Table-3
Statistical Analysis of mean values of histomorphometric analysis of both
conjugated graft groups and control graft group
Conjugated Grafts Control Graft P-value (p<0.05)
NBA 43.47% 32.97% 0.04
*
RGA 19.82% 13.90% 0.39
FTA 36.71% 53.14% 0.01
*
NBA: new (de novo) bone area. RGA: residual graft area. FTA: Fibrous tissue area. *statistically
significant. ANOVA: single factor (p<0.05).

21
Figure 1: Overall structure of the BP-ciprofloxacin (left) and BP-moxifloxacin (right) conjugates.

22
Figure 2: Clinical photograph showing hemi-section of second, third, and fourth premolars using
surgical handpiece and diamond disc under copious irrigation.

In this split mouth design, bilateral (right and left) mandibular premolars (PM2, PM3 PM4) were
hemisected with a surgical handpiece and diamond disc under copious irrigation.

23
Figure 3: Clinical photograph showing endodontic treatment on the retained hemisected root of
each premolar after tooth sectioning.

Endodontic treatment on the retained hemisected mesial root of each premolar after tooth
sectioning. Gutta percha used to fill the root canals after reshaping, disinfection with 2%
chlorhexidine gluconate, and dryness with paper-points.

24
Figure 4: Clinical photograph showing grafting of surgical defects (left) and placement of collagen
membrane and flap approximation using 4.0 synthetic resorbable interrupted sutures.

Left: bone graft placed into the surgical defects after thorough debridement. Right: grafted
surgical defects were covered with collagen membrane and flaps approximated using 4.0 0
synthetic resorbable interrupted sutures.

25
Figure 5: CT scans show surgical defects in axial, coronal, and 3D reconstruction.

CT scans show axial (left image) view of the mandible with radiopaque markers and each graft
site can be seen distal to each marker for anatomic reference; coronal (center image) view of the
mandibular grafted sites with measurements of the anatomic compartment; 3D reconstruction
(right image) of the jawbones demonstrating the surgical defects adjacent to each hemisected
premolar in the mandible.

26
Figure 6: Histologic sections from trephine core biopsies of the surgical grafts.

Histologic sections from trephine core biopsies of the surgical grafts show de novo lamellar bone
(purple) with prominent reversal lines and osteocytes in lacunae, with residual amorphous graft
material indicated by the black arrows and associated marrow or fibrovascular connective tissue
indicated by the white arrows.

27
Figure 7: Color coded histologic sections from trephine core biopsies of the surgical graft sites
for morphometric analyses.

Color coded histologic sections from trephine core biopsies of the surgical graft sites for
morphometric analyses. Red = de novo bone. Yellow = residual graft material. Blue = represents
fibrous connective tissue. Images from left to right represent examples of a control with graft
material but no conjugate, a graft with BCC conjugate, and a graft with BCX conjugate.

28
Figure 8: Clustered columns show the statistical analysis differences between conjugated grafts
and control groups.

A: De novo bone formation of conjugate grafts was significantly greater than control grafts without
conjugate (* p<0.05, ANOVA). B: No statistically significant difference in the residual bone graft
area between the conjugate treated group versus the control group. C: Statistically significant more
fibrous connective tissue in control graft group compared to conjugated graft groups (* p<0.05,
ANOVA).
0
5
10
15
20
25
% Residual Graft Area
Conjugated Graft Control Graft
B Residual Graft Area

Abstract (if available)

Abstract Objective: Bacterial biofilms are the major cause of debilitating bone diseases such as osteomyelitis, osteonecrosis, prosthetic joint infections, peri-implantitis, and septic arthritis. In dental settings, infections of bone grafts which are routinely placed after extractions for ridge preservation or for dental implant therapy are also due to biofilm mediated infections. Current treatment protocols for dental and orthopedic bone infections mainly involve surgery and/or antimicrobial therapy. However, antibiotics suffer from poor bone resorption and pharmacokinetics. Thus, the goal of this study was to design and test novel bone-targeted antimicrobial drugs utilizing bone-binding bisphosphonates (BP) moieties conjugated to fluoroquinolone antibodies in order to treat bone infections. ❧ Materials and Methods: In this study we designed BP-Ciprofloxacin conjugates as a novel bone graft formulation and tested them as a combination in animal model to assess initial safety and bone grafting efficacy in vivo. Our study was designed as a canine (beagle dogs, n=3 based on power analysis) split-mouth surgical extraction and anroganic bovine bone-grafting model to test our conjugates against negative (collagen sham) and positive (bone graft without conjugate) controls. Animal experiments conformed to the ARRIVE guidelines for reporting on animal research to ensure the quality, reliability, validity, and reproducibility of results. ❧ Results: Quantitative 3D volumetric CT and histologic morphometric analysis were performed to examine the volume of de novo bone present in surgical defects as compared to residual graft material and fibrous tissue. No adverse events were identified in any of this study animals throughout the study period, and all sites when examined histologically revealed no evidence of necrosis, infection, osteolysis or foreign body reaction. Bone volume or regeneration in the surgical defects was greatest in the conjugate bone graft group as compared to the bone graft substitute without conjugate group (p<0.0001, ANOVA) and also to the negative controls (p<0.001, ANOVA). Our results indicate that BP-fluoroquinolone infused bone grafts did not adversely impact graft survival and de novo mineralization was superior to that of bone graft substitute alone, indicating that the antimicrobial and/or antiresorptive properties of conjugates improve bone remodeling and healing outcomes. ❧ Conclusion: Our dual-action conjugates employing bisphosphonates bound to releasable antibiotics chemisorbed to bone graft material is a safe and promising therapeutic tool to enhance bone grafting in the scope of dentistry.

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Asset Metadata

Creator Garabedian, Raffie (author)

Core Title Bone targeted antimicrobials for biofilm-mediated osteolytic infection treatment

School School of Dentistry

Degree Master of Science

Degree Program Biomedical Implants and Tissue Engineering

Publication Date 07/21/2020

Defense Date 05/20/2020

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

Tag antibiotic,bisphosphonate,bone graft,canine model,conjugate,dental,fluoroquinolone,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Sedghizadeh, Parish P. (committee chair), Bakhshalian, Neema (committee member), Kar, Kian (committee member)

Creator Email rg.online@outlook.com,rgarabed@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-334309

Unique identifier UC11665817

Identifier etd-Garabedian-8730.pdf (filename),usctheses-c89-334309 (legacy record id)

Legacy Identifier etd-Garabedian-8730.pdf

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Document Type Thesis

Rights Garabedian, Raffie

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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