University of Southern California Dissertations and Theses

Functionalization of scaffolds with anti-BMP-2 antibody: role in antibody mediated osseous regeneration (AMOR)

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Functionalization of scaffolds with anti-BMP-2 antibody: role in antibody mediated osseous regeneration (AMOR)

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FUNCTIONALIZATION
OF
SCAFFOLDS
WITH
ANTI-‐BMP-‐2
ANTIBODY:

ROLE
IN
ANTIBODY
MEDIATED
OSSEOUS
REGENERATION
(AMOR)

by

Sahar
Ansari

A
Dissertation
Presented
to
the

FACULTY
OF
THE
USC
GRADUATE
SCHOOL

UNIVERSITY
OF
SOUTHERN
CALIFORNIA

In
Partial
Fulfillment
of
the

Requirements
for
the
Degree

DOCTOR
OF
PHILOSOPHY

(CRANIO-‐FACIAL
BIOLOGY)

June
2014

Copyright
2014

Sahar
Ansari

ii

DEDICATION

This dissertation is dedicated to my love, Alireza, and my parents who have
provided me endlessly with their love, support, and motivation.

iii

ACKNOWLEDGMENTS

I would like to express sincere appreciation to my advisor, Dr. Homayoun H.
Zadeh, for the ubiquitous role he has had in all aspects of my academic program
and dissertation work, also for his tuition and his enthusiasm throughout this
project. I would like to extend my gratitude to my PhD program director, Dr.
Michael Paine. I am deeply grateful to him for all his support and kindness. I would
also like to thank Dr. Janet Moradian-Oldak, Dr. Sillas Duarte and Dr. Noah
Malmstadt, for their willingness to passionately participate in my committee and
also for their encouragement and challenge throughout my academic program.
Lastly, many thanks to my family for their unconditional love, support and
encouragement without which I would never have got this far.

iv
TABLE OF CONTENTS

Dedication ii
Acknowledgments iii
List of Figures vii
Abstract xi

Chapter 1: Introduction to Antibody Mediated Osseous regeneration (AMOR) 1
1.1 Bone Tissue Engineering 1
1.2 Antibody Mediated Osseous Regeneration (AMOR) 3
1.3 Biomaterials in Bone Tissue Engineering 6
1.4 Summary of the Project 12

Chapter 2: Evaluation of Different Scaffolds Functionalized with Murine
Anti-BMP-2 Antibody in AMOR

14
2.1 Material and Methods 14
2.1.1 Materials 14
2.1.2 Cell culture and Flow Cytometry 15
2.1.3 In vitro mAb binding and release kinetics study 15
2.1.4 Rat critical size calvarial defect 16
2.1.5 Histochemical Analysis 17
2.1.6 Micro-CT Analysis 17
2.1.7 Scanning Electron Microscopy 18
2.1.8 Confocal laser scanning microscopy (CLSM) 18
2.1.9 Statistical analysis of data 19
2.2 Results 20
2.2.1 In vitro cross-reactivity of anti-BMP2 mAb 20
2.2.2 In vitro binding and release characteristics 22
2.2.3 In vivo osteogenesis of anti-BMP2 mAb 24
2.2.4 SEM analysis of different scaffolds 27
2.2.5 CLSM analysis 27
2.3 Discussion 29
2.4 Conclusions 32

v
Chapter 3: Functionalization of Scaffolds with Chimeric Anti-BMP2
Monoclonal Antibodies for Osseous Regeneration

33
3.1 Material and Methods 33
3.1.1 Antibodies 33
3.1.2 Flowcytometry 35
3.1.3 Cell culture 36
3.1.4 In Vitro ostegenesis 36
3.1.5 Scaffold Biomaterials 37
3.1.6 In vivo calvarial defect model 38
3.1.7 Histological and histomorphometric analysis 38
3.1.8 Micro-CT Analysis 39
3.1.9 Confocal laser scanning microscopy (CLSM) 40
3.1.10 Statistical analysis of data 40
3.2 Results 41
3.2.1. Binding of anti-BMP2 mAb/BMP immune complexes cell 41
3.2.2. In vitro osteogenic response of C2C12 cells 43
3.2.3. In vivo AMOR in response to anti-BMP-2 mAb 45
3.2.4. In vivo persistence of anti-BMP-2 mAb 49
3.3 Discussion 51
3.4 Conclusions

55

Chapter 4: Biomechanical Analysis of Engineered Bone with Chimeric anti-
BMP-2 antibody Immobilized on Different Scaffolds

56
4.1 Material and Methods 56
4.1.1 Antibodies 56
4.1.2 Scaffold Biomaterials 56
4.1.3 In vivo calvarial defect model 57
4.1.4. Harvesting of tissue and biomechanical testing 58
4.1.5 Scanning Electron Microscopy 59
4.1.6 Statistical analysis of data 59
4.2 Results 60
4.2.1. Biomechanical evaluation of regenerated bone 60
4.2.2. SEM characterization results 63
4.3 Discussion 64
4.4 Conclusions

67

vi

Chapter 5: Effects of the Orientation of Immobolized Anti-BMP2 Monoclonal
Antibody on Scaffold in AMOR

68
5.1 Material and Methods 68
5.1.1 Antibodies and Protein G 68
5.1.2 Cell culture and Flowcytometry 69
5.1.3 In vivo study 70
5.1.4 Micro-CT Analysis 71
5.1.5 Histological and histomorphometric analysis 71
5.1.6 Immunofluorescence staining 72
5.1.7 Statistical analysis of data 72
5.2 Results 73
5.2.1. In vitro binding of the immune complex between ProteinG 73
5.2.2. Effects of orientation of binding of anti-BMP2 mAb 75
5.3 Discussion 80
5.4 Conclusions

83

References 86

Chapter 6: Conclusions and Future Work 84

vii
LIST OF FIGURES
Figure 1. AMOR: (a) anti-BMP-2 mAb is immobilized on a scaffold. (b) mAb
captures endogenous BMP-2 (and other homologous osteogenic BMPs) from
the microenvironment. (c) BMP-2 captured by specific mAb binds its cellular
receptor on osteoprogenitor cells, promoting their osteogenic differentiation.

5
Figure 2. Flow cytometric analysis of binding of immune complexes between
murine anti-BMP2 mAb and rhBMP2, rhBMP4 and rhBMP7 to BMP-
receptor-positive C2C12 cells. Immune complex between murine anti-BMP2
mAb and rhBMP2, rhBMP4 and rhBMP7 were formed by incubation for 30
min. The resultant immune complexes were incubated with C2C12 cells,
followed by their detection with phycoerythrin (PE)-conjugated secondary
anti-IgG Ab (matched with the isotype of primary Ab). Fluorochrome-labeled
cells were analyzed by flow cytometer and the mean fluorescence intensity
(MFI) of PE was calculated. Controls included cells alone (-) or substitution of
anti-BMP2 mAb with isotype-matched mAb (Iso). The mean fluorescence
intensity (MFI) of flowcytometric analysis showed significant binding
between anti-BMP2 mAb and BMP2, BMP4 and BMP7. Results confirmed
the presence of cross-reactivity between the murine anti BMP2 and BMP2,
BMP4 and BMP7. Asterisk symbols show the groups that are significantly
different (p<0.05).
21

Figure 3. Characterization of the in vitro binding and release profile of murine
anti-BMP2 mAb-loaded scaffolds. (a) CLSM analysis showing binding of
anti-BMP2 mAb on each scaffold detected by FITC-conjugated goat anti-
mouse secondary antibody. Day 1 represents detection of binding of anti-
BMP2 mAb immediately after immobilization of the mAb on the scaffolds
confirming that murine mAb is retained on all tested scaffolds for up to two
weeks in vitro. (b) Quantitative analysis of fluorescence intensity showing
initial binding (day 1) of anti-BMP-2 mAb to the 3 scaffolds and the in vitro
persistence of anti-BMP-2 at 7 and 14 days later (n=4). (c) The in vitro release
of anti-BMP2 mAb was calculated by measuring mAb concentrations in
solution at various time points. *p < 0.05.

23

Figure 4. (a) Micro-CT images of rat calvarial defects after 8 weeks of
implantation of different biomaterials preloaded either with anti-BMP2 mAb
or isotype mAb as the negative control. (b) Quantitative analysis via micro-CT
images showing the bone volume fraction (BV/TV) for each group.

24

Figure 5. (a) Histological analysis of rat calvarial bone defects implanted with
anti-BMP2 mAb immobilized on 3 different scaffolds. Results showed
presence of vital bone with osteocytes in lacunae around each of the scaffolds
with immobilized anti-BMP2 mAb. No evidence of bone formation was
observed in sited implanted with isotype-matched control Ab. Collagen

26
viii
exhibited the most compression, followed by alginate and titanium had the
best tissue volume maintenance. (b) Histomorphometric analysis of rat
calvarial bone defects implanted with anti-BMP2 mAb immobilized on 3
different scaffolds. Histomorphometric analysis was performed on Trichrome-
stained sections and percentage of new bone formation was quantified. No
significant difference was observed between the proportions of new bone
formation for each biomaterial. *p<0.05.

Figure 6. Representative SEM photomicrographs of scaffolds prior to
implantation (-) or with immobilized anti-BMP2 retrieved 24 hours after
implantation into rat calvarial defects. Significant cellular infiltration and
adhesion was observed on scaffolds functionalized with anti-BMP2 mAb.

27

Figure 7. (a) Implanted biomaterials showing positive results for BMP2
epitopes are stained red. Scaffolds immobilized with non-specific isotype mAb
failed to show any positive staining. (b) Quantitative analysis of fluorescence
intensity of scaffolds immobilized with murine anti-BMP2 mAb eight weeks
after implantation in rat calvarial defects. N=4 for each group. *p < 0.05, **p
< 0.01, and NS= Not Significant.

28
Figure 8. (a) Schematic representation of the flow cytometric assay to study
the binding ability of anti-BMP mAb + rhBMP-2 immune complexes to BMP-
receptor positive cells. Anti-BMP-2 mAb and rhBMPs were incubated to form
immune complexes, and the resultant complexes were followed by incubation
of immune complexes incubated with C2C12 cells. The cell-bound immune
complexes were detected with a fluorescently- conjugated secondary antibody
and flow cytometric analysis. (b) Flow cytometric data examining binding of
anti-BMP-2 mAb immune complexes with rhBMP-2, rhBMP-4 and rhBMP-7
to BMP-receptor-positive C2C12 cells. The mean fluorescent intensity (MFI)
of flow cytometric analysis showed significant binding between anti-BMP-2
mAb and BMP-2, BMP-4 and BMP-7. Results confirmed the presence of
binding interaction between the chimeric anti-BMP-2 and BMP-2, BMP-4 and
BMP-7. **, p<0.01.
42

Figure 9. (a) Alizarin red staining indicating mineralized nodule formation of
cultured C2C12 cells after treatment with either BMP-2 or chimeric anti-
BMP-2 mAb after four weeks. (b) Quantitative analysis of the amount of
alizarin staining. (c) Western blot analysis showing the effect of chimeric mAb
on the levels of expression of regulators of osteogenesis in C2C12 cells. (d)
Graphical summary of interactions between chimeric anti-BMP-2 mAb and
the receptors (BMPR-I and BMPR-II) that mediate AMOR through the BMP
signaling pathway. NS: not significant.

44

Figure 10. Dose response study of chimeric anti-BMP-2 mAb adsorbed on
ACS implanted in rat calvarial defects. Representative 3D reconstruction of

46
ix
micro-CT images (a) and histomicrographs (b) of rat calvarial defects 8 weeks
after implantation with different concentrations of chimeric anti-BMP-2 mAb
adsorbed on ACS (scale bar = 1 mm). (c) Quantitative analysis of micro-CT
data, expressed as % bone fill within calvarial defects. (d) Histomorphometric
analysis of Trichrome-stained sections in panel b, expressed as % osteoid
bone. Each concentration was compared to isotype mAb as the negative
control. Anti-BMP-2 mAb at 25 µg/mL and rhBMP-2 showed the largest
amounts of new bone formation. *, Asterisk symbols show the groups that are
significantly different (p<0.05).

Figure 11. (a) Representative 3D reconstruction of micro-CT images of rat
calvarial defects 8 weeks after implantation with chimeric anti-BMP-2 mAb
adsorbed on 4 different scaffold materials. (b) Quantitative analysis of micro-
CT data, expressed as % bone fill within calvarial defects. **, p< 0.01.

47

Figure 13. Confocal laser scanning microcopy analysis of scaffolds
functionalized with anti-BMP-2 mAb retrieved at different time points.
Representative CLSM images (a) and quantitative analysis (b, c) of
immunofluorescence of 4 different biomaterials with immobilized anti-BMP-2
mAb. Calvarial specimens were retrieved at 1, 14 and 56 days and
immunofluorescently labeled with FITC-conjugated goat anti-Human IgG Ab
and imaged by CLSM. To control for non-specific labeling, controls were
immunolabeled with donkey anti-goat IgG-FITC. (b) Quantitative analysis of
fluorescence intensity. (c) Reduction in the fluorescence intensity for each
scaffold from day 0 to day 56 after implantation. Three specimens for each
group were tested. *p<0.05.

50

Figure 14. Results of biomechanical evaluation of the regenerated bone using
different biomaterials immobilized with chimeric anti-BMP-2 mAb showing
Ti microbeads and ABBM scaffolds regenerated the strongest new bone in
comparison to ACS and alginate.*p<0.05, NS= not significant.

61
Figure 15. Biomechanical evaluation of the regenerated bone in comparison to
native bone. ABBM, Ti, alginate and ACS achieved 77%, 80%, 40% and 28%
of the biomechanical strength of native bone, respectively. rhBMP-2 with
ACS, used as positive control achieved 66% of the strength of native bone.
62

Figure 16. Representative SEM photomicrographs of the fracture site showing
specimens immobilized with chimeric anti-BMP2 mAb formed new bone with
organized collagen fibrils bridging the crack areas while the negative control
group did not promote any bone regeneration.

63

Figure 17. Schematic representation of the binding assay for binding of the
immune complex between protein G/anti-BMP-2 mAb/BMP’s to cellular
receptors. Initially, anti-BMP-2 mAb was incubated with protein G-conjugated
microbeads, followed by incubation with BMP-2,-4 or -7. The resultant

73
x
immune complexes were incubated with C2C12 cells and their presence on
these cells was detected with phycoerythrin- conjugated goat anti-human Ab.

Figure 18. Investigation of the effect of orientation of anti-mAb on the binding
of the immune complex of anti-BMP2/BMP2 to target cells. Flow cytometric
analysis of binding of the immune complex between anti-BMP-2 mAb
/Protein-G-conjugated microbeads/BMP-2, BMP-4 or BMP-7/BMP-2 cellular
receptor on C2C12 cells. Fluorochrome-labeled cells were analyzed by flow
cytometer and the mean fluorescence intensity (MFI) of PE was calculated.
Controls included cells alone (-) or substitution of chimeric anti-BMP2 mAb
with isotype-matched Ab (Iso mAb). The MFI of flowcytometric analysis
showed significant binding between PG/chimeric antibody complex and
BMP2. NS: not significant, **p<0.01, and ***p<0.001.

74

Figure 19. Repair of calvarial defects with anti-BMP2 mAb with and without
Protein G linkers immobilized on scaffold (A) Representative 3D
reconstruction of micro-CT images of bone volume within rat calvaria. Anti-
BMP-2 mAb immobilized on absorbable collagen sponge (ACS) with or
without Protein G linker implanted within rat calvarial defects. Isotype-
matched mAb immobilized on ACS served as the control. (B) Quantitative
measurement of new bone formation within calvarial defects. * p<0.05,
**p<0.01, and ***p<0.001.

76

Figure 20. Histological analysis of rat calvarial bone defects implanted
withanti-BMP-2 mAb immobilized on ACS with or without Protein G linker.
Animals were sacrificed at 8 weeks after surgery and calvarial bones were
processed for histologic and Histomorphometric analysis. (A)
Histomicrographs in low (4x) and high magnification (40x) of H&E stained
calvaria. (B) Histomorphometric analysis was performed on H&E stained
sections and percentage of new bone formation was quantified. The
percentage of osteoid bone coverage was measured within histomicrographs
by histomorphometric analysis. * p<0.05, **p<0.01, and ***p<0.001.

77

xi

ABSTRACT

The ultimate goal of bone tissue engineering is the regeneration of a construct that
matches the physical and biological properties of the natural bone tissue.

For the
reconstruction of pathologically damaged craniofacial bones in particular, an array of
surgical procedures is available. Repair and regeneration of craniofacial bone defects has
widely been achieved with bone grafting procedures. However, there are several
disadvantages associated with this treatment modality. Bone morphogenetic proteins
(BMPs) have been identified as major mediators in the regeneration of adult bones. BMP-
2 has been extensively studied for its ability to promote ectopic bone formation in vitro
and in vivo. Recombinant human (rh)BMP-2 has been approved by the FDA to facilitate
the reconstruction of craniofacial bones. However, administration of rhBMP-2 has a
number of biological and logistic drawbacks. Recently, we demonstrated that anti-BMP-2
monoclonal antibodies (mAbs) immobilized on a solid scaffold can be utilized to capture
endogenous BMP-2, as well as other homologous osteogenic BMP’s (BMP-4 and BMP-
7). Our data have further demonstrated that anti-BMP-2/BMP complexes induce the
osteogenic differentiation of mesenchymal stem cells, and accelerate bone regeneration.
We refer to this process as Antibody-Mediated Osseous Regeneration (AMOR). Hence,
the goal of this study was to evaluate the relative merits of alternative scaffolds with
varying chemical, physical and mechanical properties, including titanium and alginate.
The efficacy of three different biomaterials has been compared in the immobilization of
anti-BMP-2 mAbs for AMOR.

xii

In addition, the efficacy of a newly generated chimeric anti-BMP-2 mAb in mediating
AMOR was assessed, as well as the suitability of different biomaterials as scaffolds to
participate in AMOR was evaluated. The experiments outlined in the present thesis were
designed to investigate the feasibility of functionalization of different scaffolds with anti-
BMP-2 mAbs for application in bone tissue engineering via AMOR. Additionally, the
biomechanical properties of the regenerated bone through AMOR using different
scaffolds were evaluated. To that end, a chimeric anti-BMP-2 mAb was fabricated and
immobilized on four different types of scaffolds (Absorbable Collagen Sponge (ACS),
Alginate, Ti microbeads, and bioceramic material) to mediate de novo bone formation in
rat critical sized calvarial defect model via AMOR. These studies demonstrated the
presence of cross-reactivity between the newly generated chimeric anti BMP-2 mAb with
BMP-2, BMP-4 and BMP-7 in vitro and in vivo. Additionally, it was confirmed that
AMOR can be regulated through the BMP signaling pathway. Our data demonstrated the
ability of chimeric anti-BMP-2 mAb to functionalize different biomaterial with varying
characteristics to mediate osteogenesis. Additionally, Mechanical properties of
bioengineered bone varied depending on the scaffolds used. Furthermore, through
biomechanical analysis we demonstrated that the mechanical properties of bioengineered
bone varies depending on the scaffolds used, where, Ti microbeads and bioceramics
regenerated new bone significantly stronger than alginate or ACS. Finally, we showed for
the first time that application of antibody binding proteins such as: protein G, by binding
to the Fc region of the mAb, there would be more free antigen binding sites improving
the binding of endogenous BMP-2 to the mAb and enhancing antibody mediated bone

xiii

regeneration. Altogether, these series of experiments exhibited a novel and superior bone
regenerative modality of treatment based on chimeric anti-BMP-2 mAb immobilized on
different scaffolds leading to improved outcomes beneficial to clinicians and patients.

1

CHAPTER 1

INTRODUCTION TO ANTIBODY MEDIATED OSSEOUS
REGENRATION (AMOR)

1.1 Bone Tissue Engineering

Bone regeneration and repair is frequently necessary due to congenital anomalies,
infection, trauma, and skeletal diseases (Sachlos and Czernuszka, 2003). Autologous and
allogenic bone grafts currently comprise about 90% of grafts performed each year (Lin et
al., 2008; Porter et al., 2009). Synthetic bone graft materials are the other modality of
treatment and they comprise the remaining 10%. However, there are several
disadvantages associated with each of these treatment modalities. Autologous graft
harvesting has serious drawbacks including: donor site morbidity, such as infection,
hematoma, inflammation, pain, additional operating and recuperation time and high cost
(Chen et al., 2010; Monaco et al., 2011; Arrington et al., 1996). Moreover,
osteoconductive graft materials such as allografts, xenografts and alloplastic material
have limited ability to repair large defects, due to their inherent inability to initiate bone
formation.

2

Bone tissue engineering is one of the most promising approaches to develop biological
bone substitutes that restore, maintain or improve bone tissue function. In bone tissue
engineering concepts, it has been sought to combine the biomaterial scaffolds, cells, and
growth factors/signals to create synthetic matrices for bone tissue regeneration that match
the physical and biological properties of the native bone tissue (Porter et al., 2009; Betz
et al., 2008). There are several bone tissue engineering treatment modalities have been
reported in the literature such as: application of acellular scaffolds, gene therapy, stem
cell therapy, application of growth factors, or a combination of these strategies. Growth
factors such as platelet-derived growth factors (PDGFs), bone morphogenetic proteins
(BMPs), and insulin-like growth factors (IGFs) have been used for bone tissue
engineering with promising results (Chen et al., 2010; Chen et al., 2004).

Among the abovementioned growth factors, studies have shown that BMP signaling
signal transduction cascade activates the bone formation. These growth factors in this
signaling pathway have shown to be potent upregulators of gene and protein expression
osteogenic differentiation and bone formation (Shu et al., 2011; Yamaguchi et al., 1996;
Shum et al., 2003). Among these growth factors, BMP-2, BMP-4 and BMP-7 proteins
have been reported to have crucial role in bone development and repair (Tsuji et al.,
2008; Katagiri et al., 1998). In addition, other studies have shown that osteogenic BMPs
such as: BMP-2, BMP-4 and BMP-7 have the ability to stimulate osteoprogenitor
differentiation into mature osteoblasts. Both pre-clinical and clinical studies have

3
demonstrated that these BMPs have the osteoinductive potential leading to the FDA
approval of recombinant human (rh) BMP-2 and rhBMP-7 as biologic agents used in
regenerative medicine and dentistry (Wikesjo et al., 2005; Knippenberg et al., 2006; Chin
et al., 2005; Khan and Lane 2004). However, due to several disadvantages the clinical
applications of these osteogenic BMPs are limited. Several disadvantages have been
attributed to these recombinant growth factors including: the fact that super-physiologic
doses are needed for bone formation (milligrams versus pictograms), high cost, short in
vivo half-life and their lower biologic activity in comparison to their endogenous
counterparts (Oldham et al., 200; Ishikawa et al., 2007; Chen et al., 2004).

1.2 Antibody Mediated Osseous Regeneration (AMOR)

Therapeutic antibodies development has lead to a significant enhancement in the
treatment for patients with several disease indications. Originally, all therapeutic
antibodies were polyclonal, but discovery of hybridoma technology by (Zhang, 2012)
allowed large volumes of antibodies with a single specificity to be produced. In this
method antibody-producing cells are made with autonomic proliferation abilities by
fusing individual antibody- producing cells with myeloma cells. Simply, rodent antibody-
producing cells are fused with myelomas (immortal tumor cells) derived from the bone
marrow of mice to produce hybridomas. A hybridoma combines the cancer cell’s ability
to reproduce almost indefinitely with the immune cell’s ability to produce antibodies
(antibodies of a single type monoclonals) (Zhang, 2012; Ober et al., 2001).

4

Today, monoclonal antibodies (mAbs) have become one of the most vital categories of
therapeutic agents, with more than 24 mAbs currently approved for the treatment of a
broad range of diseases such as: autoimmune diseases, infectious diseases and cancers. In
the beginning, almost all the therapeutic monoclonal Abs were of murine origin and were
developed using hybridoma technology. Due to the presence of some differences between
the human and mouse immune systems, murine mAbs were normally ineffective as
therapeutic agents for treatment of disease such as: cancer because of the inability to
administer repeat infusions (Tonegawa 1983; Schroff et al., 1985).

Murine monoclonal antibodies are derived entirely from mice using hybridoma
technology. This methodology involves the fusion of immortalized myeloma cells with
B-cells from immunized mice. However, in human body, these developed murine
antibodies often had limited clinical application due to their short circulating half-lives,
their immunogenic nature and presence of difficulties such as: human immune effector
responses (Tonegawa, 1983). They also were able to trigger human anti-mouse antibody
(HAMA) or human anti-rat antibody (HARA) development, which are extremely
undesirable. It has been reported by Schroff et al., (1985) that, this anti-antibody response
(AAR), also known as the human anti- mouse antibody response (HAMA), can develop
shortly after initiation of treatment and prevents long-term treatment procedures.
According to Jaffers et al. (1986) there are two types of the HAMA response: either anti-
isotypic or anti-idiotypic (antibody response directed to the variable domains). The other

5
problems associated with the application of murine-derived monoclonal antibodies are
including: identifying better antigenic targets of therapeutic value with which to raise
mAbs and making useful fragments of mAbs that can be produced using microbial
expression systems.
The application of therapeutic antibodies for tissue engineering was first described in our
laboratory (Freire et al., 2011). In the seminal study, Freire et al., 2011 reported on the
application of anti-BMP-2 mAb immobilized on a solid scaffold as a strategy to capture
endogenous BMP2 and mediate de novo bone formation. This approach is termed
antibody-mediated osseous regeneration (AMOR) (Figure 1). Freire et al. immobilized
murine antibodies (mAbs) against BMP2 on absorbable collagen sponge (ACS) and
implanted them within rat calvarial defects (Freire et al., 2011; Freire et al., 2013). They
clearly showed the possibility of utilization of the murine anti-BMP2 mAb for bone
regeneration.

Figure 1. AMOR: (a) anti-BMP-2 mAb is immobilized on a scaffold. (b) mAb captures
endogenous BMP-2 (and other homologous osteogenic BMPs) from the
microenvironment. (c) BMP-2 captured by specific mAb binds its cellular receptor on
osteoprogenitor cells, promoting their osteogenic differentiation.

a
b
c

6
However, the mAbs in the previous experiment had been immobilized on ACS. This
scaffold is a convenient scaffold and is approved by FDA for the application of rhBMP2
(Ober et al., 2001). Nevertheless, ACS has some drawbacks such as very low mechanical
strength and rapid resorption. This shortage necessitates investigation of the feasibility of
application of other more versatile biomaterials such as alginate hydrogel, Titanium, and
bioceramics for antibody mediated bone regeneration.

1.3 Biomaterials in Bone Tissue Engineering

Several biomaterials have been used over the past few decades for application as scaffold
for bone repair. These materials can be: natural or synthetic, organic (polymers) or
inorganic (ceramics and metals). Starting with the synthetic polymers: these type of
materials present several advantages that make them desirable for bone tissue
engineering: the possibility of the rate of degradation of a scaffold (biodegradability).
The other key advantages of synthetic polymers are the ability to tailor their mechanical
properties for different applications. Also, due to the fact that they can be fabricated into
various shapes with desired pore size they can induce tissue in-growth. In addition,
polymers can be designed with chemical functional groups that can induce further tissue
growth. Examples of synthetic polymeric scaffold are: PLA (poly lactic acid), PLG (poly
glycolic acid), PLGA (poly lactic glycolic acid), polycaprolactone (PCL), and
polyanhydrides (Holy et al., 2003). As mentioned above, the first two are biodegradable
and are broken down in the body hydrolytically to produce lactic acid and glycolic acid,

7
respectively, leading to formation of a more acidic environment. The degradation and
resorption process of these types of materials are through hydrolysis via bulk erosion,
except polyesters and polyanhydrides that are through surface erosion. Generally, the
mass loss start when the molecular chains are reduced to a size, which allows them to
freely diffuse out of the polymer matrix. The mass loss is accompanied by a release
gradient of acidic by-products. Studies have shown that the release of these acidic by-
products may result in inflammatory reactions. If the host tissue is suffering from poor
vascularization or low metabolic activity, then these by-products might cause local
temporary disturbances (Salgado et al., 2004). It is necessary to mention the difference
between the bulk degradation and surface degradation of the abovementioned polymeric
scaffolds. In bulk degradation, the rate of water penetration exceeds the degradation and
solubilization rate of surface molecules resulting in a bulk material degradation and
consequently the loss of macroscopic mechanical properties of the scaffold. On the other
hand, in surface degradation, the surface molecules degrade and solubilize faster than the
water penetration rate resulting in surface erosion while bulk material maintains its
structural integrity. There are other classes of synthetic polymers too: non-biodegradable
synthetic polymeric scaffolds. Biomaterials such as: Polymethyl-methacrylate (PMMA)
and polytetrafluoroethylene (PTFE) fall into this group. The former has been used for
more than three decades in orthopedic surgery to as bone cement and the latter has been
used for augmentation and guided bone regeneration (Gunatillake and Adhikari, 2003).

8
The next class of polymeric scaffold is the natural biodegradable polymers that obtained
from natural sources, either from animal or plant source. The examples of these types of
materials are: collagen, alginate, hyaluronic acid and chitosan. It has been mentioned that
the main advantages of these materials are their low immunogenic properties, their
bioactivity and the capability of interacting with the host’s tissue, chemical tunability,
and unlimited source (O’Brien 2011). Collagen is the most common protein in the body
and provides strength and structural stability to tissues in the body including skin, blood
vessels, tendon, cartilage and bone. Some collagen advantages are: biocompatibility (low
immune response), biodegradability, optimal pore structure facilitating bone tissue
formation with excellent cell adhesion, infiltration and vascularization (Yannas et al.,
1989). Limitation of the collagen scaffold includes: batch-to-batch variations, very low
mechanical properties, undesirable degradation profiles and immunogenicity (O’Brien
2011).
Alginates are natural polysaccharides extracted from brown sea algae. Alginate belongs
to a family of linear block polyanionic copolymers composed of (1-4)-linked -D-
mannuronic acid (M units) and (1-4)-linked -L-guluronic acid (G units) residues (Figure
1) which vary in amount and sequential distribution along the polymer chain depending
on the source of the alginate. Alginate forms stable hydrogels in the presence of certain
divalent cations such as Ca
2+
and Ba
2+
at low concentrations via the ionic interaction
between the cation and the carboxyl functional group of G units located on the polymer
chain (Smidsrod and Skjakbraek, 1990). Alginate has long been recognized as an
excellent candidate material for implantation in biological systems because of its inherent

9
biocompatibility as well as its easily modifiable immunosuppression and degradation
properties. Furthermore, the gel properties of alginate allow for diffusion of biologically
active species both into and out of the matrix, ensuring cell viability and allowing the
secretion of pharmacological molecules as well as the proper excretion of waste
molecules. While allowing for fast diffusion, alginate gels maintain the proper
mechanical strength and stability to house cells. Alginate can be chemically modified to
enable degradation or enhance cell binding or deliver any other number of signaling
molecules to the encapsulated cells. In summary they have the following advantages:
biocompatibility, biodegradability (Degrades without changing local pH), gentle gelation,
hydrophilicity, low cost, easy handling (injectability), highly hydrated tissue-like
environment that encourage cell viability and prolonged shelf life. Due to unique
properties of alginates such as hydrophilicity, biocompatibility and low cost of
production, they are widely utilized in many biomedical applications, including bone
tissue engineering (Drury and Mooney, 2003). Studies have confirmed that when alginate
is used as a biomaterial for bone regeneration, it will increase the amount of
mineralization and cause up-regulation of the genes related to bone formation such as
OCN and RUNX2 (Drury et al., 2004). However, like other natural hydrogels, they have
very low mechanical properties that limit their application in bone tissue engineering to
small defects.

Hyaluronic acid is the other promising natural polymeric matrix material with
applications for bone tissue engineering. Hyaluronic acid is a linear glycosaminoglycan

10
(GAG). In vivo studies have conformed that Hyaluronic acid has the ability to induce
chondrogenesis and angiogenesis during remodeling. Specifically, for bone tissue
engineering, Hyaluronic acid has been coupled with polysaccharide polymer (ACP)
showing promising results in terms of bone f growth and biodegradation rate (Allison and
Grande-Allen, 2006).

The other class of biomaterials with applications in bone tissue engineering is Ceramics.
Bioceramics used for tissue engineering may be classified as non-resorbable (relatively
inert), bioactive or surface active (semi-inert), and biodegradable or resorbable (non-
inert). These materials can be either from natural origin such as coralline hydroxyapatite
or are synthetic such as synthetic HA or beta-tricalcium phosphate (β-TCP). They have
unique properties for bone tissue engineering: osteoconductivity and osteoinductivity.
Studies have confirmed that HA is biocompatible, and stimulates osseoconduction by
recruiting osteoprogenitor cells leading to differentiating into osteoblast-like bone-
forming cells (Di Silvio et al., 1998). This material can be resorbed and be replaced by
bone at a slow rate. On the other hand β-TCP materials have showed higher resorption
rates necessitating to have a combination of HA and TCP in in order to have predictable
degradation profile. Their advantages are: biocompatibility, non-toxic, resorbable,
excellent osteoconductive and osteinductive properties, similar structure to bone mineral
and ability to bond directly to bone structure. Due to these unique properties they have
been considered for bone tissue engineering applications. Drawbacks of ceramic
materials: very brittle and exhibit low mechanical strength and stability, which prevents

11
their use in the regeneration of large bone defects. It is difficult to predict their
degradation/dissolution profile for optimal results. Also, it is very difficult to fill the
surface of irregular bone defect using ceramic particles (Schnettler et al., 2003) (Di Silvio
et al., 1998). However, most of the in vivo studies with the ceramic materials have shown
excellent results regarding bone regeneration. It has to be mentioned that properties such
as sintering temperature, configuration and pore size can affect the bioactivity of the
ceramic materials for bone regeneration. In order to overcome the disadvantages of the
ceramic materials and still use them in the bone regeneration, third classes of composite
biomaterials have been developed. Usually ceramic materials such as tri-
calciumphosphate (TCP), hydroxyapatite (HA) or other basic salts are incorporated into a
polymer matrix. For instance, bovine collagen has been manufactured with HA
(Hutmacher, 2001). In this case, the ceramic particles can act as a filler that can modify
the degradation profile and mechanical properties if the polymer matrix. In addition, the
basic resorption products of HA or TCP would compensate for acidic by-products of
some the polymeric scaffolds such as PLA or PGA improving the outcome of the
implantation procedure.

One of the most popular class of ceramic materials is β-TCP. This material is a porous
form of calcium phosphate, with similar proportions of calcium and phosphate to
cancellous bone (Reynolds et al., 2010). However, the mechanical property of this
material is much less than the cortical bone. Studies have confirmed that TCP is a
promising material for bone tissue engineering. Haimi et al. 2009 reported the cell

12
attachment, proliferation and differentiation of osteoblasts and mesenchymal cells with
resulting bone growth with application of TCP. However, the resorption rate of TCP is
relatively unpredictable and to some extent short in comparison. This fact necessitates the
combination of TCP and HAp in order to have a material with higher mechanical
properties and enhanced degradation profile (MPCP: microporous biphasic calcium
phosphate where we have TCP/HA: 80/20).

Recently, porous Ti granule has been introduced as a promising, non-resorbable,
osteoconductive bone substitute by Alffram et al. (2007). This material was originally
aimed for hip stabilization prostheses. Studies by Sabetrasekh et al. (2011) confirmed that
porous titanium granules have superior microstructural properties (porosity,
interconnectivity, open pore size and surface area-to-volume ratio), cell viability and
proliferation. Titanium is used extensively in orthopedic (Brunette et al., 2001) and
dental implant (Meffert et al., 1994) therapies, where anti-BMP-2 mAb can potentially be
used for improving the efficacy of osseointegration.

1.4 Summary of the Project

The main objectives of the current project were threefold: first, to investigate the
possibility of application of different biomaterials as scaffolds for use in AMOR to
immobilize murine or chimeric anti-BMP-2 mAbs; second to investigate the feasibility of
utilizing chimeric anti-BMP-2 mAb for AMOR using both in vitro assay and an in vivo

13
animal model and evaluate the biomechanical properties of regenerated bone using
different biomaterials and chimeric mAb. The following biomaterials were tested in our
in vivo rat critical size calvarial model: alginate hydrogel, titanium microbeads,
macroporous biphasic calcium phosphate (MBCP) bioceramic and ACS.

Finally, we hypothesized that anti-BMP-2 mAb captures BMPs, which are then presented
to their cellular receptors, triggering their osteogenic differentiation. This will require
availability of the antigen-binding region of antibody to bind to BMPs in domain(s),
which do not interfere with interaction with their cellular receptors. To begin to further
test this hypothesis, it was sought to determine whether binding of anti-BMP-2 mAb to
the scaffold through its Fc region might be a more effective strategy, since this is likely to
leave antigen-binding sites available to binding BMP ligands. To that end, Protein G,
which is a bacterial cell wall protein with specific affinity for immunoglobulin (IgG)
(Erntell et al., 1988; Reis et al., 1986) was utilized. If confirmed, this information will
have utility in optimizing AMOR for translational application.

14

CHAPTER 2

EVALUATION OF DIFFERENT SCAFFOLDS FUNCTIONALIZED
WITH MURINE ANTI-BMP-2 ANTIBODY IN PROMOTING
ANTIBODY MEDIATED OSSEOUS REGENERATION (AMOR)

2.1 Materials and Methods
2.1.1 Materials
3G7 mAb (Abnova, Taipei, Taiwan) a murine monoclonal anti-BMP2 antibody was used
in this study. Isotype-matched mAb (Iso, anti rabbit IgG mAb, Biovision, Mountain
View, CA) with no specific affinity to BMP2 were used as the negative control. Anti-
BMP2 and isotype control mAbs were diluted with plain phosphate-buffered saline (PBS)
at 25 µg/mL and immobilized on each of the scaffolds according to the protocol
previously reported by Freire et al. Three different scaffold materials were used in this
study, including: grade IV Titanium microbeads with 125 µm diameter (Sybron Dental
Implants, Orange, CA), alginate hydrogel (NovaMatrix FMC Biopolymer, Norway), and
ACS (Helicote, Miltex, Plainsboro, NJ). The effect of alginate volume of the dilution of
the mAb was considered.

15
2.1.2 Cell culture and Flow Cytometry
Mouse myoblast cell line C2C12 was used in this study (American Type Culture
Collection, Manassas, VA). Briefly, the cells were cultured in Dulbecco’s modified
Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 100 units/mL penicillin,
100 mg/mL streptomycin (Sigma-Aldrich), and 15% fetal bovine serum (FBS,
Invitrogen) at 37°C in a humidified atmosphere supplied with 5% CO
2
. A high degree of
homology is present among osteogenic BMPs
18
; therefore, a flow cytometric analysis was
performed in order to study the possibility of binding of the immune complex formed
between 3G7 (anti-BMP2 mAb) and BMP2, BMP4 and BMP7 with BMP2 cellular
receptor. Briefly, rhBMP2, 4 and 7 (all 100 ng/mL, Medtronic, Minneapolis, MN) were
incubated with 3G7anti-BMP2 mAb (25mg/mL) for 30min at 4°C. The resultant immune
complexes were then incubated with C2C12 cells, which express BMP2 receptors.
Consequently, the formed immune complexes were immune labeled using phycoerythrin-
conjugated goat anti-mouse mAb (Santa Cruz Biotchnology, Dallas, TX). The intensity
of fluorescent labeling was determined by measuring mean fluorescent intensity by a
flow cytometer (FACS Calibur; Becton Dickinson, Laguna Hills, CA). Controls included
cells alone (-) and substitution of anti-BMP2 mAb with isotype-matched mAb with no
specificity (Iso).

2.1.3 In vitro mAb binding and release kinetics study
In order to evaluate the kinetics of murine anti-BMP-2 mAb release from each scaffold,
25 µg/mL of mAb was immobilized on each scaffold (Titanium microbeads, alginate

16
hydrogel, and ACS) according to the methods already described in literature (Ansari et
al. 2013). The mAb-loaded scaffolds were suspended in 5 mL of PBS (pH=7.4). At
various time points (1, 3, 7, and 14 days), the amount of released mAb was determined by
UV absorption spectroscopy (Beckman, Brea CA). In addition, the retained mAb was
detected with FITC-conjugated goat anti-mouse IgG antibody (Santa Cruz Biotechnology
Inc, CA) and measured using confocal laser scanning microscopy (CLSM). The
fluorescence intensity was quantified by Spot analysis software (SPOT Imaging
Solutions, Sterling Heights, MI).

2.1.4 Rat critical size calvarial defect
Thirty two month-old virgin female Sprague-Dawley rats (Harlan Laboratories,
Livermore, CA) were housed at 22°C under a 12-h light and 12-h dark cycle and fed ad
libitum (Purina Inc, Baldwin Park, CA). All animals were treated according to the
guidelines and regulations for the use and care of animals at the University of Southern
California. Full-thickness skin flaps were raised, exposing the parietal bones. Five mm
diameter defects in parietal bones were generated using a trephine under copious saline
irrigation. Each of the scaffold materials containing 25 µg/mL of mAbs were placed
inside each of the calvarial defects. At the end of the treatment period, 8 weeks post
implantation, animals were sacrificed in a CO
2
chamber and the skulls were harvested
and stored in buffered formalin until further analysis.

17
2.1.5 Histochemical analysis
For histochemical analysis, the retrieved specimens were fixed with 4% (v/v)
paraformaldehyde for 30 min at room temperature and then placed in PBS for 15 min
prior to dehydration. Serial dehydration was achieved by placing in a sequential series of
increasing ethanol concentrations to remove all the water. The ethanol was then
completely replaced with increasing xylene concentration solutions followed by a 100%
xylene step prior to incubation with paraffin saturated xylene at room temperature
overnight. The specimens were then serially sectioned (6 µm) and were adhered to glass
slides. Additionally, the paraffin was completely removed by immersion in xylene,
decreasing ethanol concentrations and then by washing with tap water. The sections were
stained with hematoxylin and eosin (H&E) and Masson’s Trichrome. Images were
captured using an Olympus DP50 digital camera (Olympus Optical Co, Japan) and
analyzed using Analysis imaging software (Soft Image System GmbH, Germany).

2.1.6 Micro-CT Analysis
Retrieved specimens from the animals were scanned using a high-resolution micro-CT
system (MicroCAT II, Siemens Medical Solutions Molecular Imaging, Knoxville, TN)
for evaluation of ectopic mineralization. The specimens were scanned at widths of every
10 µm at 60 kV and 110 µA at a spatial resolution of 18.7 µm (Voxel dimension). Bone
volume fraction (BV/TV) for each stem cell-alginate construct was calculated.

18
2.1.7 Scanning electron microscopy (SEM)
In order to characterize the morphology of the scaffold materials used in this study and
study the early interaction of the implanted scaffolds and cells, scanning electron
microscopy (SEM) (JEOL 5300, Peabody, MA) was used. The specimens were harvested
24 hrs after implantation from the animal bodies. They were then rinsed with 2 ml of PBS
and fixed with 1% glutaraldehyde overnight. Samples were dehydrated using graded
alcohol solution and sputter coated with gold.

2.1.8 Confocal laser scanning microscopy (CLSM)
The retrieved specimens, eight weeks after implantation, were fixed in 10% formalin
solution and dehydrated in an ascending series of ethanol and embedded in paraffin. Six-
micrometer sections were cut using a microtome and mounted on glass slides. For
immunofluorescence staining, de-paraffinized samples were treated with 3% H
2
O
2
,
followed by a blocking buffer (1% BSA and 0.25% Triton X-100 in PBS), and stained
with rabbit polyclonal anti-BMP2 antibody (Abcam, Cambridge, MA) at 4
o
C overnight
and detected using Alexa fluor conjugated secondary antibody (1:200 dilution;
Invitrogen) utilizing CLSM (Fluoview FV10i, Olympus Corp, Tokyo, Japan). The
fluorescence intensity was analyzed and quantified by Spot analysis software (SPOT
Imaging Solutions, Sterling Heights, MI) with the same fluorescence threshold.

19
2.1.7 Statistical analysis of data
Quantitative data were expressed as mean standard deviation (SD). One-way and two-
way analysis of variance (ANOVA), followed by Tukey`s test at significant level of
α = 0.05, was used for the comparison among multiple sample means.

20
2.2 Results

2.2.1 In vitro cross-reactivity of anti-BMP2 mAb with different BMPs
To determine whether the anti-BMP2 mAb tested here (3G7) cross-reacts with BMP4 and
BMP7 a flow cytometric assay was employed. Accordingly, the binding of the immune
complexes between each of the BMP antigens (rhBMP2, rhBMP4 or rhBMP7) and anti-
BMP2 mAb (3G7) to C2C12 cells with BMP cellular receptor was examined. Results of
flow cytometric analysis of binding of immune complexes between murine anti-BMP2
mAb and rhBMP2, rhBMP4 and rhBMP7 to BMP-receptor-positive C2C12 cells are
shown in Figure 2. Results demonstrated that the immune complexes between anti-
BMP2 mAb with each of rhBMP2, rhBMP4 and rhBMP7 bound specifically to C2C12
cells. These results confirmed the presence of cross-reactivity between the murine anti
BMP2 mAb and BMP2, BMP4, and BMP7.

21

Figure 2. Flow cytometric analysis of binding of immune complexes between murine
anti-BMP2 mAb and rhBMP2, rhBMP4 and rhBMP7 to BMP-receptor-positive C2C12
cells. Immune complex between murine anti-BMP2 mAb and rhBMP2, rhBMP4 and
rhBMP7 were formed by incubation for 30 min. The resultant immune complexes were
incubated with C2C12 cells, followed by their detection with phycoerythrin (PE)-
conjugated secondary anti-IgG Ab (matched with the isotype of primary Ab).
Fluorochrome-labeled cells were analyzed by flow cytometer and the mean fluorescence
intensity (MFI) of PE was calculated. Controls included cells alone (-) or substitution of
anti-BMP2 mAb with isotype-matched mAb (Iso). The mean fluorescence intensity
(MFI) of flowcytometric analysis showed significant binding between anti-BMP2 mAb
and BMP2, BMP4 and BMP7. Results confirmed the presence of cross-reactivity
between the murine anti BMP2 and BMP2, BMP4 and BMP7. Asterisk symbols show
the groups that are significantly different (p<0.05).

22
2.2.2 In vitro binding and release characteristics of anti-BMP-2 mAb
An in vitro binding and release kinetics study was performed to examine potential
differences in the binding and release profile of the murine mAb on the three different
scaffolds. Results demonstrated that immediately after immobilization of anti-BMP-2
mAb, the levels of the antibody detected on all 3 scaffolds was equivalent (Figure 3a, b).
Approximately 20% of mAb remained on the scaffolds after 2 weeks of in vitro
incubation. The release profile of the murine mAb from each of the scaffolds showed
sustained release for up to 14 days (Figure 3c). Moreover, alginate hydrogel showed a
significantly lower (p<0.05) initial release profile, however, no significant difference
(p>0.05) was observed in the amounts of release profile after day 3. Since ACS and
alginate are bioresorbable scaffolds, it is likely that the kinetics of mAb retention and
release will be different in vivo.

23

Figure 3. Characterization of the in vitro binding and release profile of murine anti-
BMP2 mAb-loaded scaffolds. (a) CLSM analysis showing binding of anti-BMP2 mAb on
each scaffold detected by FITC-conjugated goat anti-mouse secondary antibody. Day 1
represents detection of binding of anti-BMP2 mAb immediately after immobilization of
the mAb on the scaffolds confirming that murine mAb is retained on all tested scaffolds
for up to two weeks in vitro. (b) Quantitative analysis of fluorescence intensity showing
initial binding (day 1) of anti-BMP-2 mAb to the 3 scaffolds and the in vitro persistence
of anti-BMP-2 at 7 and 14 days later (n=4). (c) The in vitro release of anti-BMP2 mAb
was calculated by measuring mAb concentrations in solution at various time points. *p <
0.05.

24
2.2.3 In vivo osteogenesis of anti-BMP2 mAb functionalizing different scaffolds
Micro-CT analysis (Figure 4a) showed significant volume of bone formation within
calvarial defected implanted with each of the three scaffolds functionalized with anti-
BMP2. Control treatment with isotype-matched mAb did not mediate significant degrees
of calvarial bone repair after 8 weeks of implantation. Quantified micro-CT results
confirmed that sites with anti-BMP2 mAb on Ti microbeads exhibited the largest volume
of bone formation. No significant difference was observed between ACS and alginate
groups (P>0.05) (Figure 4b).

Figure 4. (a) Micro-CT images of rat calvarial defects after 8 weeks of implantation of
different biomaterials preloaded either with anti-BMP2 mAb or isotype mAb as the
negative control. (b) Quantitative analysis via micro-CT images showing the bone
volume fraction (BV/TV) for each group.

25
The histological analysis results of rat calvarial bone defects implanted with anti-BMP2
mAb immobilized on 3 different scaffolds are presented in Figure 5a. The histological
results demonstrated the presence of vital bone with osteocytes in lacunae within each of
the scaffolds with immobilized anti-BMP2 mAb. No evidence of bone formation was
observed in sites implanted with isotype-matched control mAb with no specificity. Anti-
BMP2 mAb immobilized on collagen scaffolds exhibited the most volumetric shrinkage,
followed by alginate. Anti-BMP2 mAb immobilized on titanium exhibited the largest
volume of bone within calvarial defects. Additionally, the histomorphometric analysis
(Figure 5b) exhibited no significant difference between the proportions of de novo bone
formation for each biomaterial. Ti microbeads showed the highest amount of bone
regeneration, followed by ACS. Alginate hydrogels samples showed the least values.
Isotype mAb group, with no specificity, demonstrated significantly lower amount of bone
regeneration (P>0.05).

26

Figure 5. (a) Histological analysis of rat calvarial bone defects implanted with anti-
BMP2 mAb immobilized on 3 different scaffolds. Results showed presence of vital bone
with osteocytes in lacunae around each of the scaffolds with immobilized anti-BMP2
mAb. No evidence of bone formation was observed in sited implanted with isotype-
matched control Ab. Collagen exhibited the most compression, followed by alginate and
titanium had the best tissue volume maintenance. (b) Histomorphometric analysis of rat
calvarial bone defects implanted with anti-BMP2 mAb immobilized on 3 different
scaffolds. Histomorphometric analysis was performed on Trichrome-stained sections
and percentage of new bone formation was quantified. No significant difference was
observed between the proportions of new bone formation for each biomaterial. *p<0.05.

27
2.2.4 SEM analysis of different scaffolds
The representative SEMs photomicrographs of scaffolds prior to implantation (-) or with
immobilized anti-BMP2 retrieved 24 hours after implantation into rat calvarial defects
are shown in Figure 6. Significant cellular infiltration and adhesion was observed on
scaffolds functionalized with anti-BMP2 monoclonal antibody.

2.2.5 CLSM analysis
In order to evaluate presence of immobilized murine anti-BMP2 mAb in vivo on three
tested scaffolds, mAb was immobilized on the 3 scaffolds and implanted in calvarial
defects. After 8 weeks of implantation animals were sacrificed. CLSM analysis
confirmed that scaffolds immobilized with murine anti-BMP2 mAb exhibited significant
Figure 6. Representative SEM photomicrographs of scaffolds prior to implantation (-) or
with immobilized anti-BMP2 retrieved 24 hours after implantation into rat calvarial
defects. Significant cellular infiltration and adhesion was observed on scaffolds
functionalized with anti-BMP2 mAb.

28
binding of BMP2 ligands following implantation (Figure 7a). As expected, the defects
implanted with non-specific isotype mAb failed to bind BMP2 ligand. After 8 weeks of
implantation, low levels of residual murine anti-BMP2 were observed in each retrieved
scaffold. Titanium specimens showed higher fluorescence intensity (p<0.05), while no
significant difference was observed between the fluorescence intensity levels of alginate
and ACS (p < 0.05) (Figure 7b).

Ti
Alginate ACS
Iso mAb
200 µm
0
10
20
30
40
50
Ti Alginate ACS
anti-BMP2 mAb
Iso mAb
**
**
**
NS
*
Fluorescence Intensity %
Murine anti-BMP2 mAb
b a
Figure 7. (a) Implanted biomaterials showing positive results for BMP2 epitopes are
stained red. Scaffolds immobilized with non-specific isotype mAb failed to show any
positive staining. (b) Quantitative analysis of fluorescence intensity of scaffolds
immobilized with murine anti-BMP2 mAb eight weeks after implantation in rat calvarial
defects. N=4 for each group. *p < 0.05, **p < 0.01, and NS= Not Significant.

29
2.3 Discussion

Application of autogenous, allogeneic, xenogenic, and synthetic biomaterials are the
current treatment modalities for bone regeneration in craniofacial reconstructive surgeries
and regenerative medicine. However, each of these treatment modalities have significant
limitations. In an effort to obviate the need for autologous tissue harvesting, while
utilizing material with high osteoinductive potential, recombinant BMPs have been
applied clinically (Parada et al., 2012; Li and Chen, 2013; Kruithof et al., 2012;
Moshaverinia et al., 2013; Guo et al., 2012; Guo et al., 2009; Semb, 2012). BMP2 is a
member of the TGF-β family, assembles into a biologically active homodimer, which
binds to heterodimeric type I and type II receptors for BMP-2 (Liu et al., 2013). Other
osteogenic BMP’s include BMP-4 and BMP-7 and currently rhBMP-2 and rhBMP-7
have been approved by US FDA for repair and regeneration of skeletal defects. However,
there are several drawbacks to the application of recombinant growth factors, including
their supra-physiologic dose requirement, some potentially serious side effects, as well as
high cost. Our laboratory has introduced AMOR, as an alternative strategy to the current
approaches of administering exogenous growth factors.

In tissue engineering, it has been proposed that the convergence of appropriate signaling
molecules on progenitor cells within a suitable scaffold can lead to tissue regeneration.
Our previously studies have established that when anti-BMP-2 mAbs are implanted in
vivo, they can capture endogenous BMPs that can provide the osteogenic signals to

30
progenitor cells to regenerate bone (Freire et al., 2011; Freire et al., 2013). Therefore, the
current study sought to investigate the role of various scaffolds in the pursuit of
optimizing this novel strategy. To that end, we examined the suitability of three different
biomaterials with different physical and chemical properties as scaffolds when
functionalized with anti-BMP2 Abs for AMOR. Results of the present study
demonstrated that though all three scaffolds when functionalized with the murine anti-
BMP-2 mAb mediated bone regeneration within calvarial defects, several differences in
the outcome were noted, which can affect their potential clinical application. Both
collagen sponge and alginate are biodegradable material and as such their application led
to significant volumetric shrinkage. In contrast titanium is a biologically stable material
and when functionalized with anti-BMP-2 mAb, it maintained its volume. Titanium is
used extensively in orthopedic and dental implants and as such its functionalization with
anti-BMP-2 mAb can potentially improve its capacity for osseointegration. Titanium
beads can also be considered as potential graft material for the repair of skeletal defects
(Brunette et al., 2001). However, in some applications, it may be desirable to have a
biodegradable scaffold, where the regenerated tissue does not contain remnants of the
scaffold material. In such situations alginate and collagen may be more appropriate
options. There are many strategies available to modulate the rate of degradation of
collagen by cross-linking and alginate by oxidation (Meffert et al., 1992; Alsberg et al.,
2001; Ansari et al., 2013). Currently, we are investigating physical properties of sites,
which have been regenerated with each of these scaffolds using AMOR to characterize
the physical strength of these repaired tissues. This information will further aid in the

31
selection of appropriate scaffold in each tissue engineering application.

Taking into account the high degree of homology of BMP2 with other osteogenic BMPs
such as BMP4 and BMP7, the binding capacity of murine anti-BMP2 mAb with BMP4
and BMP7 was additionally examined. The cross-reactivity of murine anti-BMP2 used
here with BMP4 and BMP7 can have significant implications on the potential mechanism
of action of AMOR. This may imply that the efficacy of AMOR may be in part
attributable to the capacity of the specific monoclonal Ab to capture multiple osteogenic
mediators. In view of the significant degree of homology (92.2%) between the human
and rat genes for BMP2
19
, the results of our calvarial defect model are likely to extend to
clinical and translational applications of our novel murine mAb for mediating bone
regeneration and repair. The feasibility of immobilizing this mAb on different types of
scaffolds with unique physical properties makes this novel treatment modality even more
versatile.

32
2.4 Conclusions
In the current study, we report the application of murine anti-BMP2 mAb in
functionalizing three different types of scaffolds and investigated their ability to mediate
AMOR in a rat calvarial defect model. Results demonstrated significant de novo bone
formation with all 3 scaffolds functionalized with murine anti-BMP-2 mAb. Osseous
defects regenerated with anti-BMP-2 mAb immobilized on collagen sponge and alginate
exhibited more volumetric shrinkage than titanium. The present data have demonstrated
the cross-reactivity between the anti BMP2 mAb and BMP4 and BMP7. These data have
potential implications on the mechanism of action of AMOR, suggesting that anti-BMP-2
may capture multiple endogenous osteogenic BMPs, which may in turn mediate de novo
bone formation. During the early healing, significant cellular infiltration and adhesion
was observed on scaffolds functionalized with murine anti-BMP2 mAb. The present
study demonstrated the possibility of utilizing multiple scaffolds with different physical
properties as scaffolds functionalized with anti-BMP-2 to participate in AMOR.

33

CHAPTER 3

FUNCTIONALIZATION OF SCAFFOLDS WITH CHIMERIC ANTI-
BMP-2 MONOCLONAL ANTIBODIES FOR OSSEOUS
REGENERATION

3.1 Materials and Methods

3.1.1 Antibodies

The hybridoma clone of a murine anti-BMP-2 mAb (3G7, Abnova Inc, Taiwan) was
expanded in non-selective hybridoma medium (Invitrogen, Carlsbad, CA), total RNA
was purified, and mRNA coding for the immunoglobulin genes were purified using the
Oligotex mRNA Kit (QIAGEN Inc., Chatsworth, CA). The mRNA was utilized to
synthesize total complementary DNA (cDNA), which was subsequently amplified using
PCR to yield light chain and heavy chain variable regions. After amplification, the PCR
products of the variable regions were cut with restriction endonucleases SalI and EcoRI
(New England Biolabs Inc, Ipswich, MA) for the heavy chain and SalI and BamHI (New

34
England Biolabs Inc) for the light chain. The cut variable regions were individually
ligated into pBluescript plasmids (SK
+
, Invitrogen), and the variable region genes were
amplified from the pBluescript vectors via PCR using oligonucleotide primers designed
to introduce appropriate restriction endonuclease sites and the Kozak translation initiation
sequence. Specifically, Hind3 and BsiWI (New England Biolabs Inc) restriction sites
were introduced for the light chain variable gene and XbaI and NheI restriction sites were
added for the heavy chain variable gene. The light chain variable regions were ligated
into the parent expression vector, into which the human kappa constant region had
already been cloned. The heavy chain variable region was ligated into the parent GS
expression vector, into which the human gamma 4 constant region had already been
cloned. The final expression vectors contained transcription cassettes for the chimeric
light and heavy chains, respectively.

The chimeric antibody was then expressed by NS0 cells (Invitrogen) using plasmid
technology, and high-expressing subclones of chimeric mAb were placed in liquid
suspension culture using selective medium containing 3% dialyzed fetal calf serum
(Invitrogen) and penicillin and streptomycin antibiotics (Invitrogen). The cells were
expanded to produce sufficient quantities of antibodies for subsequent testing. After 7
days of aeration (two weeks in culture), spent cultures were filtered through 0.2 µm filter
units (Sartorius TCC Company, CO) and purified by tandem protein A affinity
chromatography and ion exchange chromatography to yield antibody products with

35
greater than 98% purity. Antibody was collected in PBS and syringe-filtered (Millipore,
Billerica, MA) into sterile 5 ml glass vials for use in this study.

3.1.2. Flow cytometry

A flow cytometric assay was developed in order to study binding of the BMP-2 cellular
receptor with the immune complex formed between chimeric anti-BMP-2 mAb and
BMP-2, 4 and 7. Briefly, rhBMP-2, 4 and 7 (all 100 ng/mL, Medtronic, Minneapolis,
MN) were incubated with chimeric mAb (25 µg/mL) for 30 min at 4°C. The resultant
immune complexes were then incubated with C2C12 cells (American Type Culture
Collection, Manassas, VA), which express BMP-2 receptors. Subsequently, the immune
complexes were immunolabeled using phycoerythrin-conjugated goat anti-human Ab
(Santa Cruz Biotchnology, Dallas, TX). The intensity of fluorescent labeling was
determined by measuring mean fluorescent intensity (MFI) using a flow cytometer
(FACS Calibur; Becton Dickinson, Laguna Hills, CA). Controls included cells alone (-)
and substitution of anti-BMP-2 mAb with isotype-matched mAb with no specificity
(isotype mAb).

36
3.1.3. Cell culture

A mouse myoblast cell line (American Type Culture Collection) was used in this study.
C2C12 cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich)
supplemented with 100 units/mL penicillin, 100 mg/mL streptomycin (Sigma-Aldrich),
and 10% fetal bovine serum (Biocell Laboratories, Rancho Dominguez, CA) at 37°C in a
humidified atmosphere supplied with 5% CO
2
.

3.1.4. In vitro osteogensis

In order to study the effects of the chimeric anti-BMP-2 mAb/BMP-2 immune complex
on osteogenesis, an in vitro assay was utilized accordingly to a recently published method
[34]. Briefly, the C2C12 myoblast cell line was selected since it does not express BMP-2.
Accordingly, 25 µg/ml anti-BMP-2 mAb or isotype control mAb with no specificity
(isotype mAb, negative control) was incubated in 6-well plates for one hour at room
temperature, followed by three washes and blocking with PBS containing 0.5% BSA
(Invitrogen) to block unoccupied binding sites on the plastic surfaces. Plates were then
incubated with sub-osteogenic concentrations (100 ng/ml) of BMP-2 for 1 hour at room
temperature followed by six washes to remove any unbound BMP-2 from the solution.
Triplicate cultures of 1 × 10
5
C2C12 cells were grown in α-DMEM (Invitrogen) culture
media containing 10% FBS (Biocell Laboratories), 1% penicillin and streptomycin.
Positive control wells contained BMP-2 (100 ng/ml) in solution. Alizarin red S staining

37
was utilized to assess osteogenic differentiation of C2C12 cells. Briefly, cell cultures
were washed twice with PBS, fixed for 1 min in 60% isopropanol and rehydrated with
distilled water. Cultures were then stained with Alizarin red S solution for 5 min,
followed by two washes with deionized water. Positively stained cells were visualized
under a microscope and quantified using NIH ImageJ software (NIH, Bethesda, MD).

Western blot analysis was performed after two weeks of culturing in the abovementioned
conditions. Briefly, the cells were washed twice with PBS and lysed with protein
extraction buffer for 30 sec (BIO-RAD, Irvine, CA). After centrifugation, the supernatant
solution was collected, and protein concentrations were determined using a BCA assay
(Pierce, Rockford, IL). Equal amounts of protein extracts were fractionated in 10%
sodium dodecyl sulfate-polyacrylamide gels and electrophoretically transferred to a
nitrocellulose membrane (Bio-Rad). These nitrocellulose membranes were incubated
with mouse mAbs against p-Smad 1 (Santa Cruz Biotechnology), smad 6 (Abcam,
Cambridge, MA), Runx2 (Abcam) and the housekeeping gene beta-actin (Abcam).

3.1.5. Scaffold biomaterials

Alginate hydrogel (NovaMatrix FMC Biopolymer, Norway), macroporous biphasic
calcium phosphate (MBCP, Hydroxyapatite/β-Tricalcium phosphate 20/80, Biomatlante,
Vigneux de Bretagne, France), grade IV Titanium microbeads with 250 mm diameter
(Sybron Dental Implants, Orange, CA) and ACS (Helicote, Miltex, Plainsboro, NJ) were

38
used in this study. Chimeric anti-BMP-2 mAb (25 µg/mL) and isotype control mAb (25
µg/mL) were immobilized on each scaffold material, as previously described
(Moshaverinia et al., 2013).

3.1.6. In vivo calvarial defect model

Two-month-old virgin female Sprague-Dawley rats (n=32, Harlan Laboratories,
Livermore, CA) were housed at 22°C under a 12 h light and 12 h dark cycle and fed ad
libitum (Purina Inc, Baldwin Park, CA). All animals were treated according to the
guidelines and regulations for the use and care of animals at the University of Southern
California. Calvarial defects were created in 8-week-old rats under general anesthesia.
Full-thickness skin flaps were raised, exposing the left and right parietal bones. Defects
in the midline of the parietal bone, 7 mm in diameter, were generated using a trephine
under copious saline irrigation. Scaffold materials, each containing 25 µg/mL of mAb,
were placed inside the calvarial defects. At the end of the treatment period, the animals
were sacrificed in a CO
2
chamber and the skulls were harvested and stored in buffered
formalin while awaiting analysis.

3.1.7. Histological and histomorphometric analysis

For histological analysis, the retrieved specimens were fixed with 4% (v/v)
paraformaldehyde for 30 min at room temperature and placed in PBS for 15 min prior to

39
dehydration. Serial dehydration was achieved by placing specimens in a sequential series
of increasing ethanol concentrations to remove all of the water. The ethanol was then
completely replaced with a series of solutions containing increasing concentrations of
xylene, culminating in 100% xylene, prior to incubation with paraffin-saturated xylene at
room temperature overnight. The specimens were then serially sectioned (6 µm) and
affixed to glass slides. Additionally, the paraffin was completely removed by immersion
in xylene, followed by immersion in decreasing ethanol concentrations and then washing
with tap water. The specimens containing titanium microbeads were imbedded in resin
mounting solution and sectioned using Exakt grinder (Exakt 312, EXAKT, Germany).
The sections were stained with hematoxylin and eosin (H&E). Images were captured
using an Olympus DP50 digital camera (Olympus Optical Co, Japan) and analyzed using
Analysis imaging software (Soft Image System GmbH, Germany).

3.1.8. Micro-CT analysis

Specimens were scanned using a high-resolution micro-CT system (MicroCAT II,
Siemens Medical Solutions Molecular Imaging, Knoxville, TN) for evaluation of ectopic
mineralization, with scans of 10 µm width at 60 kV and 110 µA at a spatial resolution of
18.7 µm (Voxel dimension). Bone volume fraction (BV/TV) was calculated.

40
3.1.9. Confocal laser scanning microscopy (CLSM)

The concentration of the remaining immobilized chimeric mAb on each of the
previously-implanted scaffolds was analyzed using CLSM (Fluoview FV10i; Olympus
Optical Co). The samples were retrieved after 0, 1, 14 or 56 days of implantation, fixed
with 4% PFA, incubated for 30 min at room temperature, washed, blocked with BSA and
stained with secondary antibody (FITC-conjugated goat anti-Human IgG Ab), after
which CLSM and analysis was performed. Additionally, in order to confirm the
specificity of our chimeric anti-BMP-2 mAb, the specimens after one week of
implantation were immunolabaled with donkey anti-goat IgG-FITC antibody (Santa Cruz
Biotechnology) as negative control (-). The fluorescence intensity was quantified by Spot
analysis software (SPOT Imaging Solutions, Sterling Heights, MI) with the same
fluorescence threshold.

3.1.10. Statistical analysis of data

Quantitative data were expressed as mean ± standard deviation (SD). A two-tailed
Student’s t-test and one-way analysis of variance (ANOVA), followed by Tukey’s test at
significance level of α = 0.05, were used for the comparison of multiple sample means.

41
3.2 Results

3.2.1. Binding of anti-BMP-2 mAb/BMP immune complexes to cells

In view of the significant structural homology between BMP-2, -4 and -7, it was sought
to determine whether chimeric anti-BMP-2 mAb cross-reacts with BMP-4 and BMP-7.
Western blot analysis confirmed cross-reactivity between chimeric anti-BMP-2 mAb
cross-reacts with BMP-4 and BMP-7 (data not shown). Since antibodies can potentially
serve as antagonists and prevent interactions of ligands with their cellular receptor, it was
important to determine whether the immune complexes formed by BMP-2, BMP-4 or
BMP-7 and chimeric anti-BMP-2 mAb bind with the BMP-2 cellular receptor. To that
end, 100 ng/ml of BMP-2, BMP-4 or BMP-7 were incubated with chimeric anti-BMP-2
mAbs (25 µg/mL) (Figure 8a). The resultant immune complexes were incubated with
C2C12 cells, followed by immunofluorescent labeling with fluorochrome-conjugated
secondary Abs and flow cytometry to detect cells that bound to an immune complex. The
results demonstrated that anti-BMP-2 mAb immune complexes with BMP-2, BMP-4, or
BMP-7 were each able to bind to C2C12 cells at significantly higher level compared with
isotype control mAb incubated with BMP-2, -4 or -7 (Figure 8b).

42

Figure 8. (a) Schematic representation of the flow cytometric assay to study the binding
ability of anti-BMP mAb + rhBMP-2 immune complexes to BMP-receptor positive cells.
Anti-BMP-2 mAb and rhBMPs were incubated to form immune complexes, and the
resultant complexes were followed by incubation of immune complexes incubated with
C2C12 cells. The cell-bound immune complexes were detected with a fluorescently-
conjugated secondary antibody and flow cytometric analysis. (b) Flow cytometric data
examining binding of anti-BMP-2 mAb immune complexes with rhBMP-2, rhBMP-4
and rhBMP-7 to BMP-receptor-positive C2C12 cells. The mean fluorescent intensity
(MFI) of flow cytometric analysis showed significant binding between anti-BMP-2 mAb
and BMP-2, BMP-4 and BMP-7. Results confirmed the presence of binding interaction
between the chimeric anti-BMP-2 and BMP-2, BMP-4 and BMP-7. **, p<0.01.

43
3.2.2. In vitro osteogenic response of C2C12 cells to anti-BMP-2 mAb/BMP immune
complex

An in vitro assay was developed to evaluate the ability of immobilized anti-BMP-2
mAb/BMP-2 immune complexes to mediate osteogenic differentiation of C2C12 cells.
Cells were cultured under three different conditions: (1) rhBMP-2 added in culture media
(positive control), (2) immobilized chimeric anti-BMP-2 mAb + rhBMP-2 immune
complex and (3) immobilized isotype control mAb with no specificity substituted for
anti-BMP-2 mAb (negative control). Osteogenic differentiation of C2C12 cells after 2
weeks of culture was analyzed using Alizarin red staining. Positive control cultures, as
well as those with immobilized chimeric anti-BMP-2 mAb + BMP-2 immune complex
differentiated into Alizarin red-positive cells (Figure 9a). In contrast, negative control
cultures remained negative for Alizarin red (Figure 9b). No statistically significant
difference between positive control and experiment cultures were noted (Figure 9b).

To investigate the intracellular pathways of signaling involved following binding of
chimeric anti-BMP-2 mAb/BMP-2 immune complex to C2C12 cells, the expression of p-
Smad1, p-Smad6 and Runx2 was investigated. Results in Figure 9c demonstrated high
expression levels of osteoblast-specific molecules, including Runx2 and p-Smad1 in cells
cultured in the presence of the anti-BMP-2 mAb + BMP-2 immune complex for two
weeks. In contrast, a low expression level for a downregulator of osteogenesis (Smad 6)
was observed (Figure 9c).

44

Figure 9. (a) Alizarin red staining indicating mineralized nodule formation of cultured
C2C12 cells after treatment with either BMP-2 or chimeric anti-BMP-2 mAb after four
weeks. (b) Quantitative analysis of the amount of alizarin staining. (c) Western blot
analysis showing the effect of chimeric mAb on the levels of expression of regulators of
osteogenesis in C2C12 cells. (d) Graphical summary of interactions between chimeric
anti-BMP-2 mAb and the receptors (BMPR-I and BMPR-II) that mediate AMOR
through the BMP signaling pathway. NS: not significant.

45
3.2.3. In vivo AMOR in response to anti-BMP-2 mAb

To investigate the ability of chimeric anti-BMP-2 mAb to mediate AMOR in vivo, the
calvarial defect model in rats was utilized. A dose response experiment was performed in
order to determine the optimal dose of mAb for mediating bone formation. De novo bone
formation was detected in calvarial defects implanted with as little as 1 µg/mL of
chimeric anti-BMP-2 mAb (Figure 10a and 10b). A dose of 25 µg/mL of mAb was
sufficient for complete repair of calvarial defects. Based on this, as well as our previous
data on murine anti-BMP-2 mAbs [19], 25 µg/mL was selected as the dosage of chimeric
mAb for all subsequent experiments.

To investigate the ability of various biomaterials to serve as scaffolds for the mAb, 25
µg/mL of chimeric anti-BMP-2 mAb was immobilized on each of 4 different scaffold
materials and implanted in rat calvarial defects.

46

Figure 10. Dose response study of chimeric anti-BMP-2 mAb adsorbed on ACS
implanted in rat calvarial defects. Representative 3D reconstruction of micro-CT images
(a) and histomicrographs (b) of rat calvarial defects 8 weeks after implantation with
different concentrations of chimeric anti-BMP-2 mAb adsorbed on ACS (scale bar = 1
mm). (c) Quantitative analysis of micro-CT data, expressed as % bone fill within
calvarial defects. (d) Histomorphometric analysis of Trichrome-stained sections in panel
b, expressed as % osteoid bone. Each concentration was compared to isotype mAb as the
negative control. Anti-BMP-2 mAb at 25 µg/mL and rhBMP-2 showed the largest
amounts of new bone formation. *, Asterisk symbols show the groups that are
significantly different (p<0.05).

47
To evaluate de novo bone formation within the defects, micro-CT and histological
analyses were performed after 8 weeks. Micro-CT analysis (Figure 11a) demonstrated
significantly greater amounts of bone fill and repair in defects implanted with scaffolds
containing anti-BMP-2 mAb than in defects implanted with scaffolds containing isotype
matched mAb (P<0.05). The micro-CT data revealed that MBCP and Ti microbeads
generated the largest volume of new bone, while alginate hydrogel scaffold showed the
smallest amount of bone regeneration (Figure 11b).

Figure 11. (a) Representative 3D reconstruction of micro-CT images of rat calvarial
defects 8 weeks after implantation with chimeric anti-BMP-2 mAb adsorbed on 4
different scaffold materials. (b) Quantitative analysis of micro-CT data, expressed as %
bone fill within calvarial defects. **, p< 0.01.

48
Histological evidence supported the micro-CT observations. Staining with H&E
demonstrated a significant degree of bone fill within calvarial defects implanted with
scaffolds containing adsorbed chimeric anti-BMP-2 mAb (Figure 12a). The presence of
vital bone with osteocytes in lacunae was detected with each of the scaffolds (Figure
12a). The histomorphometric data (Figure 12b) were in good correlation with
quantitative micro-CT data, revealing that MBCP and Ti beads exhibited the greatest
amounts (p<0.05) of bone regeneration, while alginate hydrogel scaffold showed the
smallest
amount.

Figure 12. (a) Histological analysis of rat calvarial bone defects implanted with chimeric
anti-BMP-2 mAb immobilized on 4 different types of scaffolds (scale bar = 1 mm for low
magnification images and 50 µm for histomicrographs in high magnification). (b)
Quantitative histomorphometric analysis performed on H&E-stained sections showing
percentage of new bone formation. **, p< 0.01.

49
3.2.4. In vivo persistence of anti-BMP-2 mAb

To examine in vivo persistence of anti-BMP-2 mAb immobilized on various scaffold
materials, mAb was immobilized on the 4 scaffolds and implanted in calvarial defects.
Animals were sacrificed at 1, 14 or 56 days after implantation. Calvarial specimens were
examined with CLSM, following immunoflurescent labeling with FITC-conjugated goat
anti-human mAb, to visualize the chimeric anti-BMP-2 mAb. Results demonstrated that
the local concentrations of chimeric anti-BMP-2 mAb, as well as isotype control mAb
(data not shown) progressively decreased over time (Figures 13a and b). During the first
day following in vivo implantation, between 25 to 50% of the chimeric anti-BMP-2 was
lost, depending on the biomaterial used as the scaffold (Figure 13c). By Day 14,
approximately 25% of the initial concentrations of chimeric anti-BMP-2 persisted on all 4
biomaterial. At 8 weeks, low levels of chimeric anti-BMP-2 that was statistically higher
than background were detectable (p<0.05).

50

Figure 13. Confocal laser scanning microcopy analysis of scaffolds functionalized with
anti-BMP-2 mAb retrieved at different time points. Representative CLSM images (a)
and quantitative analysis (b, c) of immunofluorescence of 4 different biomaterials with
immobilized anti-BMP-2 mAb. Calvarial specimens were retrieved at 1, 14 and 56 days
and immunofluorescently labeled with FITC-conjugated goat anti-Human IgG Ab and
imaged by CLSM. To control for non-specific labeling, controls were immunolabeled
with donkey anti-goat IgG-FITC. (b) Quantitative analysis of fluorescence intensity. (c)
Reduction in the fluorescence intensity for each scaffold from day 0 to day 56 after
implantation. Three specimens for each group were tested. *p<0.05.

51
3.3 Discussion

BMPs have been identified as the major mediators in bone regeneration and formation. It
is well known that exogenous administration of rhBMP-2 can initiate a healing cascade
that induces bone regeneration through the TGF-β/BMP signaling pathway (Guo et al.,
2012; Guo et al., 2009). BMP-2 is a member of the TGF-β family, assembles into a
biologically active homodimer, which binds to heterodimeric type I and type II receptors
for BMP-2 (Liu et al., 2013). Our laboratory has introduced AMOR as a tissue
engineering strategy alternative to administration of exogenous growth factors. Since our
ultimate objective is translational application of AMOR, we generated a chimeric anti-
BMP-2 mAb to reduce the potential for xenogenic immune response to the mAb. One of
the objectives of the present study was to determine whether application of the chimeric
anti-BMP-2 mAb is associated with any adverse responses. Careful examination of
histologic slides of calvaria treated with chimeric anti-BMP-2 mAb or isotype control
mAb failed to find any evidence of significant inflammatory or pathologic changes.
Detailed necropsy of major organs, as well as examination of serum for acute phase
inflammatory markers also failed to find any significant aberrant systemic effects.
Previous studies with chimeric mAb’s have generally shown their clinical safety profile
(Sapir et al., 2011; Waldmann 2003). There are currently eight chimeric therapeutic
antibodies, which have been approved by US FDA and are being used clinically
(Waldmann 2003).

52
In view of the fact that the anti-BMP-2 mAb used in this study has been generated against
a human BMP-2 immunogen, one of the questions was whether it will be efficacious in
binding rat BMP-2 after in vivo implantation. The results of calvarial study clearly
demonstrated the efficacy of chimeric anti-BMP-2 mAb in mediating de novo bone
formation. This was not surprising, in light of the significant degree of homology (92.2%)
between the human and rat BMP-2 genes (Ober et al., 2001). Taking into account the
high degree of homology of BMP-2 with other osteogenic BMPs such as BMP-4 and
BMP-7, the binding capacity of chimeric anti-BMP-2 mAb with BMP-4 and BMP-7 was
also examined. The cross-reactivity of murine anti-BMP-2 parent clone of our chimeric
anti-BMP-2 mAb with BMP-4 and BMP-7 has previously been demonstrated. Moreover,
it has been reported that implantation of immobilized murine anti-BMP-2 mAb leads to
in vivo binding of BMP-2, BMP-4 and BMP-7. Our in vitro assay confirmed the cross-
reactivity of chimeric anti-BMP-2 mAb with BMP-4 and BMP-7. A potential
interpretation of these data with regards to the in vivo mechanism of anti-BMP-2 mAb
action is that this antibody can capture at least three osteogenic BMPs. These BMPs can
act in an additive or synergistic manner to induce de novo bone formation. Follow-up
studies are pending to determine what other immunologically related osteogenic
molecules are bound to the chimeric anti-BMP-2 mAb following in vivo implantation.
The present study has documented some of the downstream events following engagement
of the BMP receptor, including the expression of p-Smad 1 and Runx2. Moreover, the
expression level of downregulators of osteogenesis (such as Smad 6), which play a
critical role in the inhibition of the BMP signaling pathway, was apparently not affected.

53

In tissue engineering, biomaterials are increasingly utilized for sophisticated functions
such as scaffolds to guide the development of new tissues and organs (Engler et al.,
2006). To direct cell and tissue response, biomaterial design has focused on controlling
the material chemistry to promote highly specific binding interactions between the
materials and surrounding cells (Valamehr et al., 2008). Here we studied the feasibility of
using four different types of scaffolds with immobilized chimeric anti-BMP-2 mAb in
AMOR. This modality of treatment involves implanting a scaffold impregnated with anti-
BMP-2 mAb in a bone defect to capture endogenous BMP-2, as well as other related
osteogenic growth factors, which can induce bone regeneration. The ability of AMOR to
regenerate bone may be attributed to the capacity of anti-BMP-2 mAb to capture and
tether multiple endogenous osteogenic BMP’s and therefore increase their local
availability and persistence. Moreover, a robust cellular infiltration has been observed in
sites implanted with anti-BMP-2 mAb. The co-delivery of anti-BMP-2 mAb and
mesenchymal stem cells has also been shown to lead to greater degree of bone repair in
calvarial defects (Moshaverinia et al., 2013).

The current study confirmed the ability of chimeric anti-BMP-2 mAb immobilized on all
four of the biomaterials studied to mediate robust de novo bone formation in rat calvarial
defects, as confirmed by micro-CT and histological analyses. The bioceramic MBCP and
titanium microbeads showed the largest volume of bone regeneration, while alginate
hydrogel scaffold showed higher volumetric shrinkage. The differences between the

54
volumes of bone regeneration may be attributed to the ability of the prospective scaffold
to withstand compressive forces. In view of the fact that different skeletal defects present
with varying physical requirements, it was important to test the feasibility of
immobilizing chimeric anti-BMP-2 mAb on different types of scaffolds with different
physical properties. Each of the scaffolds utilized have unique properties, which make
them suitable for different applications. Titanium is used extensively in orthopedic and
dental implant therapies, where anti-BMP-2 mAb can potentially be used for improving
the efficacy of osseointegration (Brunette et al., 2001; Meffert et al., 1996). Alginate
hydrogel can be formulated as an injectable scaffold and has been used extensively in
tissue engineering (Alsberg et al., 2001; Moshaverinia et al., 2012). Therefore, alginate
or ACS may be suitable for intraosseous defects, where it is desirable to have a
biodegradable scaffold and compressive strength will not be required. MBCP is likely to
have application in repair of osseous defects, which require resistance to compression.
Current studies are in progress to investigate the physical mechanical properties of
regenerated bone using each of the scaffolds functionalized with chimeric anti-BMP-2.

55
3.4 Conclusions

Here we report the application of chimeric anti-BMP-2 mAb immobilized on different
types of scaffolds in the process of antibody mediated bone regeneration. The propose
system has the ability to mediate de novo bone formation efficiently, as confirmed by our
in vitro and in vivo studies. Furthermore, we report the cross-reactivity of the newly
generated chimeric anti BMP-2 mAb with BMP-4 and BMP-7 through specificity studies.
The anti-BMP-2 antibody is capable of binding to endogenous BMP-2, BMP-4 and
BMP-7, leading to enhanced antibody-mediated bone regeneration. Finally, AMOR can
be regulated through the BMP signaling pathway, as confirmed by the in vitro
experiments.

56

CHAPTER 4

BIOMECHANICAL ANALYSIS OF ENGINEERED BONE WITH
CHIMERIC ANTI-BMP-2 ANTIBODY IMMOBILIZED ON
DIFFERENT SCAFFOLDS

4.1 Materials and Methods

4.1.1 Antibodies

The hybridoma clone of a murine anti-BMP-2 mAb (3G7, Abnova Inc, Taiwan) was
expanded and used in order to fabricate chimeric anti-BMP-2 mAb according to Ansari et
al., 2013.

4.1.2. Scaffold biomaterials

Alginate hydrogel (NovaMatrix FMC Biopolymer, Norway), anorganic bovine bone
mineral (ABBM) (Nibec, South Korea), grade IV Titanium microbeads with 250 mm

57
diameter (Sybron Dental Implants, Orange, CA) and ACS (Helicote, Miltex, Plainsboro,
NJ) were used in this study. Chimeric anti-BMP-2 mAb (25 µg/mL) and isotype control
mAb (25 µg/mL) were immobilized on each scaffold material, as previously described
[18].

4.1.3. In vivo calvarial defect model

Two-month-old virgin female Sprague-Dawley rats (n=32, Harlan Laboratories,
Livermore, CA) were housed at 22°C under a 12 h light and 12 h dark cycle and fed ad
libitum (Purina Inc, Baldwin Park, CA). All animals were treated according to the
guidelines and regulations for the use and care of animals at the University of Southern
California. Calvarial defects were created in 8-week-old rats under general anesthesia.
Full-thickness skin flaps were raised, exposing the left and right parietal bones. Defects
in the midline of the parietal bone, 7 mm in diameter, were generated using a trephine
under copious saline irrigation. Scaffold materials, each containing 25 µg/mL of mAb,
were placed inside the calvarial defects. At the end of the treatment period, the animals
were sacrificed in a CO
2
chamber and the skulls were harvested and stored in buffered
formalin while awaiting analysis.

58
4.1.4. Harvesting of tissue and biomechanical testing

Eight weeks post implantation surgery the rats were sacrificed. The implanted
biomaterials with a layer of surrounding bone were removed using a trephine drill (inner
diameter = 7 mm) and the host bone surrounding the implants was trimmed with dental
burs, leaving a disc-shape specimen. These boney specimens were then rinsed in
physiological saline and stored in sterile plastic containers with saline at 4
o
C to avoid
damage to the fragile healed section. The bones were mechanically tested on the day of
sacrifice.

The mechanical response of the implants was evaluated using a universal mechanical
testing machine. The specimens were secured in position while the load was applied
vertically, by a steel rod with a diameter of 4 mm, to the center of the former defect
(compression rate = 0.5 mm min
-
1). For each group, five specimens were tested. During
compression, load-deformation values were recorded and stored with the computer
software supplied with the testing machine. The fracture strength (Fmax) was determined
as the maximum force applied during the biomechanical test, which is the force, applied
to cause fracture of the healed defect site (Spicer et al., 2012; Jones et al., 2007).

59
4.1.5. Scanning electron microscopy (SEM)

After mechanical testing, the specimens were in order to characterize the morphology of
the newly-formed bone and scaffold materials at the defect sites, scanning electron
microscopy (SEM) (JEOL 5300, Peabody, MA) was utilized. Briefly, the specimens were
washed with PBS at 37°C, submerged in fixative (1% formaldehyde in 0.15 M at
pH=7.3) overnight. Specimens were dehydrated using graded alcohol solution and sputter
coated with gold.

4.1.6. Statistical analysis of data

Quantitative data were expressed as mean ± standard deviation (SD). A two-tailed
Student’s t-test and one-way analysis of variance (ANOVA), followed by Tukey’s test at
significance level of α = 0.05, were used for the comparison of multiple sample means.

60
4.2 Results

4.2.1. Biomechanical evaluation of regenerated bone

The results of biomechanical evaluation (Figure 14) showed Ti microbeads and ABBM
scaffolds showed the greatest amount of force to fracture in comparison to ACS and
alginate. ABBM, Ti, alginate and ACS functionalized with chimeric anti-BMP-2 mAb
were able to achieve 77%, 80%, 40% and 28% of the biomechanical strength of native
bone, respectively. rhBMP-2 with ACS, used as positive control achieved 66% of the
strength of native bone. Alginate scaffold showed the lowest amounts of mechanical
properties (P<0.05) followed by ACS scaffold. The negative control group did not
promote any bone regeneration showing significantly lower mechanical properties
(P<0.05).

61

0
35
70
105
140
ACS
OCS-B
Ti
Alg
ACS+ rhBMP2
Bone
Anti BMP2 mAb
Iso mAb
F max (N)
*
*
*
*
NS
OCS-B + anti BMP2 mAb vs Alg + anti BMP2 mAb p<0.05
OCS-B + anti BMP2 mAb vs ACS + anti BMP2 mAb p<0.05
OCS-B + anti BMP2 mAb vs Ti + anti BMP2 mAb p>0.05
ACS + anti BMP2 mAb vs Alg + anti BMP2 mAb p>0.05
Figure 14. Results of biomechanical evaluation of the regenerated bone using different
biomaterials immobilized with chimeric anti-BMP-2 mAb showing Ti microbeads and
ABBM scaffolds regenerated the strongest new bone in comparison to ACS and
alginate.*p<0.05, NS= not significant.

62

Figure 15. Results of biomechanical evaluation of the regenerated bone in comparison to
native bone. ABBM, Ti, alginate and ACS achieved 77%, 80%, 40% and 28% of the
biomechanical strength of native bone, respectively. rhBMP-2 with ACS, used as positive
control achieved 66% of the strength of native bone.

63
4.2.2. SEM characterization results

The SEM images (Figure 16) clearly showed specimens immobilized with chimeric anti-
BMP2 mAb formed new bone with organized collagen fibrils bridging the crack areas. In
contrast, the negative control group did not promote any bone regeneration showing
connective tissue formation with unorganized collagen fibrils.

ACS
Alginate
anti-BMP2 mAb Iso mAb anti-BMP2 mAb Iso mAb
OCS-B
Titanium
ACS + rhBMP2
Bone
Figure 16. Representative SEM photomicrographs of the fracture site showing
specimens immobilized with chimeric anti-BMP2 mAb formed new bone with organized
collagen fibrils bridging the crack areas while the negative control group did not
promote any bone regeneration.

64
4.3 Discussion

Bone regeneration represent a large component of clinical practice aimed at repairing
defects due to numerous conditions including: congenital defects or trauma. There are
several treatment modalities available, however, several disadvantages are attributed to
the current treatment modalities (Spicer et al., 2012). Therefore, alternative bone
regenerative strategies are desired. Our recent studies have demonstrated the ability of
chimeric anti-BMP-2 monoclonal antibodies (mAb) immobilized on different types of
scaffold materials to mediate de novo bone formation (Ansari et al., 2013). However, to
our knowledge there is no single study available in literature investigating the
biomechanical properties of regenerated bone using different scaffolds functionalized
with chimeric anti-BMP-2.

Therefore, the objective of the current study was to determine whether the scaffold used
for tethering anti-BMP-2 mAb can affect the biomechanical properties of the regenerated
bone. To that end, chimeric anti-BMP-2 mAb was immobilized on four different
biomaterials: titanium microbeads (Ti), alginate hydrogel, anorganic bovine bone mineral
(ABBM) and absorbable collagen sponge (ACS) and the scaffolds immobilized with
chimeric anti-BMP-2 mAb were surgically implanted into rat critical-size calvarial
defects. After 8 weeks of surgery, the biomechanical properties of the new formed bone
was measured. Briefly, the maximum load to fracture was determined as the maximum

65
force applied during the biomechanical test, which is the force, applied to cause fracture
of the healed defect site.

The results of the current study showed that Ti microbeads and ABBM scaffolds showed
the greatest amount of force to fracture in comparison to ACS and alginate. ABBM, Ti,
alginate and ACS functionalized with chimeric anti-BMP-2 mAb were able to achieve
77%, 80%, 40% and 28% of the biomechanical strength of native bone, respectively.
rhBMP-2 with ACS, used as positive control achieved 66% of the strength of native bone.
Alginate scaffold showed the lowest amounts of mechanical properties (P<0.05) followed
by ACS scaffold. The negative control group did not promote any bone regeneration
showing significantly lower mechanical properties (P<0.05). The SEM images clearly
showed specimens immobilized with chimeric anti-BMP2 mAb formed new bone with
organized collagen fibrils bridging the crack areas. In contrast, the negative control group
did not promote any bone regeneration showing connective tissue formation with
unorganized collagen fibrils.

The possible explanation for the abovementioned findings might be attributed to the
biomechanical (compressive strength) properties of the biomaterials used. For instance,
alginate and ACS are biodegradable and inherently are weak biomaterials, while, OCS-B
(bioceramic) and Ti are not biodegradable (they are stable) and studies have confirmed
that the newly formed bone integrates with them and they become well fused with newly
created bone. Additionally, it has to be taken into account that Ti and OCS-B are

66
Based on the outcome of the current study, it can be concluded that each of the scaffolds
utilized can suitable for different clinical situations and applications. For example,
Titanium is used widely in implant dentistry and orthopedic, where anti-BMP-2 mAb can
potentially be used for improving the osseointegration of Ti to bone (Brunette et al.,
2001; Meffert et al., 1996). Due to their physical properties alginate or ACS may be
suitable for intraosseous defects, where it is desirable to have a biodegradable scaffold
and compressive strength will not be required. Bioceramics such as: ABBM is likely to
have application in repair of osseous defects, which require resistance to compression.

67
4.4 Conclusions

Here we report the application of chimeric anti-BMP-2 mAb immobilized on different
types of scaffolds in the process of antibody mediated bone regeneration. Additionally,
we showed that the scaffold used for tethering anti-BMP-2 mAb can affect the
biomechanical properties of the regenerated bone. The ability of ABBM and titanium
functionalized with chimeric anti-BMP-2 mAb to achieve 77%, and 80% of the
biomechanical strength of native bone demonstrated that antibody mediated osseous
regeneration (AMOR) is an effective tissue engineering approach.

68

CHAPTER 5

EFFECTS OF THE ORIENTATION OF IMMOBOLIZED ANTI-
BMP2 MONOCLONAL ANTIBODY ON SCAFFOLD IN AMOR

5.1 Materials and Methods

5.1.1 Antibodies and protein G

We generated and used a chimeric anti-BMP2 IgG2 monoclonal antibody (mAb)
according to the method previously reported (Ansari et al., 2013). An isotype-matched
mAb (Iso mAb- anti rabbit IgG mAb, Biovision, Mountain View, CA) with no specificity
for BMP2 was utilized as the negative control. The µMACS and MultiMACS Protein G
Kit (Miltenyi Biotec Inc., Auburn, CA) were utilized in this study.

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5.1.2 Cell culture and flow cytometry

The C2C12 mouse myoblast cell line (American Type Culture Collection) was used in
this study and cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich)
supplemented with 100 units/mL penicillin, 100 mg/mL streptomycin (Sigma-Aldrich),
and 10% fetal bovine serum (Biocell Laboratories, Rancho Dominguez, CA) at 37°C in a
humidified atmosphere supplied with 5% CO
2
.

A flow cytometric assay was developed in order to study binding of the BMP2 cellular
receptor with the immune complex formed between chimeric anti-BMP2 mAb and
Protein G with BMP-2, -4, and -7. Briefly, chimeric anti-BMP2 mAb (25 µg/mL) or
isotype matched control mAb (25 µg/mL) were incubated with protein G-coupled
microbeads (Dynabeads Protein G, 30 mg/ML, Life Technology AS, Oslo), washed with
PBS, and non-specific binding sites were blocked with bovine serum albumin (0.5
mg/mL BSA, Invitrogen). Then rhBMP-2 (100 ng/mL, Medtronic, Minneapolis, MN),
BMP-4 and BMP-7 (100 ng/mL, R&D Systems Minneapolis, MN) were incubated with
mAb/Protein G complex for 30 min at 4°C, and were washed thoroughly with PBS to
remove free rh-BMP2, -4, and -7 ligands (Fig. 17). The resultant immune complexes
were then incubated with C2C12 cells (American Type Culture Collection, Manassas,
VA), which express BMP-2 receptors. Subsequently, the immune complexes were
immunofluorescently labeled using phycoerythrin-conjugated goat anti-human Ab (Santa
Cruz Biotechnology, Dallas, TX). The intensity of fluorescent labeling was determined

70
by measuring mean fluorescent intensity (MFI) using a flow cytometer (FACS Calibur;
Becton Dickinson, Laguna Hills, CA). Controls included cells alone and substitution of
anti-BMP2 mAb with isotype-matched mAb with irrelevant specificity.

5.1.3 In vivo study

The USC Institutional Animal Care and Use Committee (IACUC) approved all
procedures involving vertebrate animals. Two-month-old virgin female Sprague-Dawley
rats (n=24, Harlan Laboratories, Livermore, CA) were housed at 22°C under a 12 h light
and 12 h dark cycle and fed ad libitum (Purina Inc, Baldwin Park, CA). Calvarial defects
were created in 8-week-old rats under general anesthesia. Full-thickness skin flaps were
raised, exposing the left and right parietal bones. Defects in the midline of the parietal
bone, 7 mm in diameter were generated using a trephine under copious saline irrigation.
Protein G-coupled microbeads were incubated with ACS scaffold for an hour, washed
three times with PBS and incubated with anti-BMP2 mAb (25 µg/ml) or isotype-matched
control mAb (25 µg/ml) to form mAb-Protein G complexes on ACS. Functionalized ACS
scaffolds were surgically implanted into rat calvarial defects. After 8 weeks, the animals
were sacrificed in a CO
2
chamber and the skulls were harvested and stored in buffered
formalin while awaiting analysis.

71
5.1.4 Micro-CT Analysis

Harvested calvarial specimens were examined using a high-resolution micro CT system
(MicroCAT II, Siemens Medical Solutions Molecular Imaging, Knoxville, TN) to
evaluate the healing of the defects. The specimens were scanned every 10 µm at 60 kV
and 110 µA at a spatial resolution of 18.7 µm (Voxel dimension) and three-dimensional
(3D) analysis was performed on the resulting images. Bone volume fraction divided by
total volume (BV/TV) of newly regenerated bone was calculated using Amira software
(Visage Imaging Inc. San Diego CA).

5.1.5 Histological and histomorphometric analysis

For histological analysis, the retrieved specimens were fixed with 4% (v/v)
paraformaldehyde for 30 min at room temperature and placed in PBS for 15 min prior to
dehydration. Serial dehydration was achieved by placing specimens in a sequential series
of increasing ethanol concentrations to remove all of the water. The ethanol was then
completely replaced with a series of solutions containing increasing concentrations of
xylene, culminating in 100% xylene, prior to incubation with paraffin-saturated xylene at
room temperature overnight. The specimens were then serially sectioned (6 µm) and
affixed to glass slides. Additionally, the paraffin was completely removed by immersion
in xylene, followed by immersion in decreasing ethanol concentrations and then washing
with tap water. The sections were stained with hematoxylin and eosin (H&E). Images

72
were captured using an Olympus DP50 digital camera (Olympus Optical Co, Japan) and
analyzed using Analysis imaging software (Soft Image System GmbH, Germany).

5.1.6 Immunofluorescence staining

Harvested specimens were prepared for immunefluorescence imaging according to
previous publications (Freire et al., 2011; Freire et al., 2013). Briefly, specimens were
treated with 3% H
2
O
2
, followed by a blocking buffer (1% BSA and 0.25% Triton X-100
in PBS). Specimens were then incubated with rat anti-rabbit anti-BMP2, BMP4, and
BMP7 (Abcam, 1:200 dilution) and detected using Alexa Fluor conjugated secondary
antibody (1:200 dilution; Invitrogen). The samples were then counterstained with DAPI.
The positively stained areas were determined from 3 independent samples for each
experimental group. Five areas were randomly selected from each sample, then the
positive area in the field was calculated with NIH Image-J software (NIH, Bethesda, MD)
and shown as a percentage of the area over total field area.

5.1.7 Statistical analysis of data

Quantitative data were expressed as mean ± standard deviation (SD). One-way and two-
way analyses of variance (ANOVA) followed by Tukey’s test at a significance level of α
= 0.05 were used for the comparison among multiple sample means.

73
5.2 Results

5.2.1 In vitro binding of the immune complex between Protein G/anti-BMP-2 mAb/BMP’s
to BMP cellular receptors

In order to determine whether the orientation of anti-BMP-2 mAb can affect its binding
to BMPs, Protein G-conjugated microbeads were incubated anti-BMP2 mAb or isotype-
matched mAb (Figure 17). The immune complex of anti-BMP2 mAb or isotype-
matched mAb bound to Protein G microbeads were incubated with BMP-2, BMP-4, or
BMP-7. The binding of Protein G/mAb/BMP complexes to C2C12 cells with BMP
cellular receptor was studied with flow cytometry.

Figure 17. Schematic representation of the binding assay used to assess the binding of
the immune complex between protein G/anti-BMP-2 mAb/BMP’s to cellular receptors.
Initially, anti-BMP-2 mAb was incubated with protein G-conjugated microbeads,
followed by incubation with BMP-2, BMP-4 or BMP-7. The resultant immune complexes
were incubated with C2C12 cells and their presence on these cells was detected with
phycoerythrin- conjugated goat anti-human Ab.

74
Results of flow cytometric analysis revealed significantly higher binding of immune
complexes between anti-BMP-2 mAb and BMP-2, BMP-4, or BMP-7 to C2C12 cells,
when the mAb was previously bound to protein G (Figure 18). These results suggested
that since mAb binds to protein G through its Fc region, antigen-bindings sites were
preferentially available to interact with BMP antigens. Binding of mAbs to microbeads
may be responsible for increased binding to C2C12 cells because it clusters mAb
molecules together on microbeads.

Figure 18. Investigation of the effect of orientation of anti-mAb on the binding of the
immune complex of anti-BMP2/BMP2 to target cells. Flow cytometric analysis of
binding of the immune complex between anti-BMP-2 mAb /Protein-G-conjugated
microbeads/BMP-2, BMP-4 or BMP-7/BMP-2 cellular receptor on C2C12 cells.
Fluorochrome-labeled cells were analyzed by flow cytometer and the mean fluorescence
intensity (MFI) of PE was calculated. Controls included cells alone (-) or substitution of
chimeric anti-BMP2 mAb with isotype-matched Ab (Iso mAb). The MFI of
flowcytometric analysis showed significant binding between PG/chimeric antibody
complex and BMP2. NS: not significant, **p<0.01, and ***p<0.001.

75
5.2.2 Effects of orientation of binding of anti-BMP2 mAb on scaffold on in vivo
osteogenic properties

To determine the effects of orientation of binding of anti-BMP-2 to scaffold, Protein G
was first immobilized ACS as a linker, followed by binding of anti-BMP-2 mAb or
isotype-matched control mAb. Protein G/anti-BMP-2 mAb/ACS, Protein G/isotype
mAb/ACS, anti-BMP-2 mAb/ACS or isotype mAb/ACS were each implanted in rat
calvarial defects. After 8 weeks, healing of calvarial defects was studied by micro-CT
and histology.

Micro-CT analysis (Figure 19a) showed increased volume of bone formation within
calvarial defects implanted with Protein G/anti-BMP2 mAb/ACS in comparison to the
defects implanted with anti–BMP2 mAb immobilized directly on ACS (p<0.05) or
isotype-matched control mAb with or without (data not shown) Protein G linker (p<0.05)
(Figure 19b).

76

Histological results demonstrated the presence of vital bone with osteocytes in lacunae
within defect sites implanted with ACS scaffolds immobilized with protein G-anti-BMP2
mAb complex, as well as ACS with anti-BMP-2 mAb (Figure 20a). No evidence of bone
formation was observed in sites implanted with isotype-matched control mAb with or
Figure 19. Repair of calvarial defects with anti-BMP2 mAb with and without
Protein G linkers immobilized on scaffold (A) Representative 3D reconstruction of
micro-CT images of bone volume within rat calvaria. Anti-BMP-2 mAb immobilized on
absorbable collagen sponge (ACS) with or without Protein G linker implanted within rat
calvarial defects. Isotype-matched mAb immobilized on ACS served as the control. (B)
Quantitative measurement of new bone formation within calvarial defects. * p<0.05,
**p<0.01, and ***p<0.001.

77
without Protein G. Histomorphometric analysis (Figure 20b) revealed increased volume
of bone formation within calvarial defects implanted with Protein G/anti–BMP2 mAb in
comparison to the defects implanted with anti–BMP2 mAb immobilized directly to ACS
(p<0.05) or isotype-matched control mAb (p<0.001). Careful examination of histologic
sections failed to demonstrate any evidence of adverse reactions within tissues, such as
excessive inflammatory infiltration.

Figure 20. Histological analysis of rat calvarial bone defects implanted withanti-BMP-2
mAb immobilized on ACS with or without Protein G linker. Animals were sacrificed at 8
weeks after surgery and calvarial bones were processed for histologic and
Histomorphometric analysis. (A) Histomicrographs in low (4x) and high magnification
(40x) of H&E stained calvaria. (B) Histomorphometric analysis was performed on H&E
stained sections and percentage of new bone formation was quantified. The percentage
of osteoid bone coverage was measured within histomicrographs by histomorphometric
analysis. * p<0.05, **p<0.01, and ***p<0.001.

78
To further characterize the phenotype of regenerated tissues in response to the application
of protein G as a linker for immobilizing anti-BMP-2 mAb on ASC scaffold,
immunofluorescence labeling was utilized. Representative sites implanted with protein
G/anti-BMP2 mAb complex or anti–BMP2 mAb immobilized directly on ASC exhibited
high BMP2, BMP4, and BMP7 protein expression throughout regenerated tissues
(Figure 21a). As expected, sites implanted with isotype control mAb exhibited no
expression of BMP2, -4, or -7 (Figure 21b). Semi-quantitative analysis revealed higher
expression of BMP-2, BMP-4 and BMP-7 proteins within sites implanted with protein
G/anti-BMP2 mAb complex than those with anti–BMP2 mAb immobilized directly on
ASC (p<0.05) or isotype-match control mAb (p<0.05) (Figure 21b).

79

Figure 21. Immunofluorescence labeling of tissue specimens harvested from rat calvarial
bone defects implanted with anti-BMP-2 mAb immobilized on ACS with or without
Protein G linker. Primary antibodies included those with specificity against BMP-2,
BMP-4, and BMP-7. (A) Representative immunofluorescent CLSM images
demonstrated positive labeling within sites implanted with anti-BMP-2 mAb
immobilized on ACS with Protein G linker, as well those sites with anti-BMP-2 mAb
immobilized directly on ACS. (B) Analysis of the percentage of positively stained area
for anti-BMP-2, BMP-4, and BMP-7 antibodies, showing that Protein G/anti-BMP2
mAb complex exhibited the highest expression of BMP2, 4, and 7 proteins in comparison
to those sites with anti-BMP-2 mAb immobilized directly on ACS, or isotype-matched
control mAb, *p<0.05 and **p<0.01, respectively.

80
5.3 Discussion

Recombinant human bone morphogenetic protein (rhBMP)-2 and -7 have been approved
by FDA for bone reconstruction of specific indications. However, the clinical
applications of these exogenous recombinant growth factors are fraught with potential
drawbacks. Therefore, as an alternative bone regenerative approach was introduced by
Freire et al., who demonstrated the proof-of-concept for AMOR(Freire et al., 2011;
Freire et al., 2013). More recently, Ansari et al. (2013) generated a chimeric anti-BMP-2
mAb to reduce the potential for xenogenic immune response to the mAb. In their study,
the ability of chimeric anti-BMP-2 mAb immobilized on different types of biomaterials
to mediate robust de novo bone formation in rat calvarial defects was confirmed (Ansari
et al., 2013). In an attempt to optimize AMOR, it was sought to determine whether the
orientation with which antibody is immobilized on scaffold could affect the safety and
efficacy of AMOR. It may be envisioned that optimization of AMOR will require
maximal availability of antigen-binding sites of anti-BMP-2 mAb molecules. Moreover,
it may be speculated that exposure of Ab Fc regions can potentially allow binding of
inflammatory cells. To that end, protein G was used as a linker to immobilize anti-BMP-2
mAb molecules onto scaffold. This will allow binding of the anti-BMP-2 mAb through
its Fc receptor on scaffold, exposing the antigen binding sites and limiting the availability
of Fc regions. Results of this study have indeed confirmed that, application of protein G
as a linker increased both in vitro binding of anti-BMP-2 mAb/BMP immune complexes
to cells, as well as in vivo repair of calvarial critical size defects.

81

Careful analysis of histological sections failed to demonstrate any significant
inflammatory immune response within sites implanted with anti-BMP-2 mAb/protein
G/ACS or anti-BMP-2 mAb/ ACS. These results are consistent with our previous
observations regarding lack of significant inflammatory immune response associated with
AMOR. Furthermore, immunofluorescence staining data confirmed that the application
of G Protein linker in immobilization of anti-BMP-2 to ACS scaffold lead to increased
expression of BMP-2, BMP-4, and BMP-7 ligands within reconstructed tissue. While,
specimens immobilized with anti-BMP2 mAb alone showed significantly less amounts of
positive staining.

The present study utilized protein G to control the orientation of binding of anti-BMP-2
mAb to scaffold. It is well known that this bacterial cell wall protein represents a more
general and versatile IgG-binding reagent as compared with protein A, the well-known
staphylococcal Fc-binding protein (Erntell et al., 1988; Reis et al., 1986). Compared with
protein A, the magnitude of the binding properties and the range of specificities of
protein G may prove to be of great value, not only when the use of protein A is limited
but also as an improvement in applications already developed for protein A (Akerstorm et
al., 1985; Grubb et al., 1985).
The present study provided experimental evidence for the significance of the orientation
of anti-BMP-2 tethering to the scaffold. Follow-up studies are under way to determine the

82
molecular mechanisms by which anti-BMP-2 modulates the healing response of bone
defects.

83
5.4 Conclusion

In this study, it was shown that when Protein G was used as a linker to immobilize anti-
BMP2 mAb through its Fc region to scaffold, the in vitro and in vivo capacity of anti-
BMP2 mAb to bind BMP-2, BMP-4 and BMP-7 and mediate their binding to cellular
receptors was enhanced. Moreover, the application of Protein G in binding anti-BMP2
mAb to scaffold was associated with increased bone regeneration in critical size calvarial
defects.

84

CHAPTER 6

CONCLUSIONS AND FUTURE WORK

In the series of experimental work presented here the presence of cross-reactivity
between the murine/ chimeric anti BMP-2 mAb - osteogenic BMPs (e.g. BMP-2, -4, and
-7) immune complexes with C2C12 cells was confirmed. Additionally, through
immunofluorescence staining it was shown that murine and chimeric anti-BMP-2 mAb
can capture and hold endogenous osteogenic BMPs in vivo. Furthermore, through the
critical calvarial defect model in rats we demonstrated that the immobilized murine/
chimeric anti-BMP-2 mAb on different types of scaffolds can efficiently mediated de
novo bone formation. Moreover, the biomechanical analysis of the regenerated bone
exhibited that the mechanical properties of bioengineered bone may vary depending on
the type of the biomaterial used. For instance, regenerated bone using Ti microbeads and
ABBM tolerated significantly higher loads to fracture in comparison to bioengineered
bone using alginate or ACS. Therefore, it can be concluded that based on the
physiochemical properties of each scaffold, they can be used in different biomedical
applications through AMOR. Finally, through the presented experimental work in the last
aim the important role of the orientation of anti-BMP2 mAb in AMOR was revealed.

85
Protein G, when used as a linker to tether the Fc region of the chimeric anti-BMP2 mAb
to scaffold, significantly increased the binding capacity of anti-BMP2 mAb- osteogenic
BMPs immune complexes to cellular receptors in vitro and in vivo.

In future studies, other mediators of osteogenesis through AMOR will be identified. In
addition, the possibility of anti-BMP2 mAbs in stem cell-mediated bone regeneration will
be evaluated through in vitro and in vivo studies. To further analyze the molecular
mechanism of the action of AMOR, the phenotypic characteristics of the cell infiltrate,
around implanted biomaterials functionalized with anti-BMP2 mAb, will be evaluated.
Furthermore, the mechanical properties of the regenerated bone will be further analyzed
using alternative methods such as: nano-indentation. Finally, more sophisticated
techniques (e.g. positron emission tomography (PET)) will be utilized to detect the mAbs
and BMPs in vivo.

86
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Abstract (if available)

Abstract The ultimate goal of bone tissue engineering is the regeneration of a construct that matches the physical and biological properties of the natural bone tissue. For the reconstruction of pathologically damaged craniofacial bones in particular, an array of surgical procedures is available. Repair and regeneration of craniofacial bone defects has widely been achieved with bone grafting procedures. However, there are several disadvantages associated with this treatment modality. Bone morphogenetic proteins (BMPs) have been identified as major mediators in the regeneration of adult bones. BMP‐2 has been extensively studied for its ability to promote ectopic bone formation in vitro and in vivo. Recombinant human (rh)BMP‐2 has been approved by the FDA to facilitate the reconstruction of craniofacial bones. However, administration of rhBMP‐2 has a number of biological and logistic drawbacks. Recently, we demonstrated that anti‐BMP‐2 monoclonal antibodies (mAbs) immobilized on a solid scaffold can be utilized to capture endogenous BMP‐2, as well as other homologous osteogenic BMP’s (BMP‐4 and BMP‐7). Our data have further demonstrated that anti‐BMP‐2/BMP complexes induce the osteogenic differentiation of mesenchymal stem cells, and accelerate bone regeneration. We refer to this process as Antibody‐Mediated Osseous Regeneration (AMOR). The experiments outlined in this study were designed to investigate the feasibility of functionalization of different scaffolds with anti‐BMP‐2 mAbs for application in bone tissue engineering via AMOR. Additionally, the biomechanical properties of the regenerated none through AMOR using different scaffolds were evaluated. To that end, a chimeric anti‐BMP‐2 mAb was fabricated and immobilized on four different types of scaffolds (Absorbable Collagen Sponge (ACS), Alginate, Ti microbeads, and bioceramic material) to mediate de novo bone formation in rat critical sized calvarial defect model via AMOR. These studies demonstrated the presence of cross‐reactivity between the newly generated chimeric anti BMP‐2 mAb with BMP‐2, BMP‐4 and BMP‐7 in vitro and in vivo. Additionally, it was confirmed that AMOR can be regulated through the BMP signaling pathway. Our data demonstrated the ability of chimeric anti‐BMP‐2 mAb to functionalize different biomaterial with varying characteristics to mediate osteogenesis. Additionally, mechanical properties of bioengineered bone varied depending on the scaffolds used. Furthermore, through biomechanical analysis we demonstrated that the mechanical properties of bioengineered bone varies depending on the scaffolds used, where, Ti microbeads and bioceramics regenerated new bone significantly stronger than alginate or ACS. Finally, we showed for the first time that application of antibody binding proteins such as: protein G, by binding to the Fc region of the mAb, there would be more free antigen binding sites improving the binding of endogenous BMP‐2 to the mAb and enhancing antibody mediated bone regeneration. Altogether, these series of experiments exhibited a novel and superior bone regenerative modality of treatment based on chimeric anti‐BMP‐2 mAb immobilized on different scaffolds leading to improved outcomes beneficial to clinicians and patients.

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Asset Metadata

Creator Ansari, Sahar (author)

Core Title Functionalization of scaffolds with anti-BMP-2 antibody: role in antibody mediated osseous regeneration (AMOR)

School School of Dentistry

Degree Doctor of Philosophy

Degree Program Craniofacial Biology

Publication Date 06/17/2014

Defense Date 04/02/2014

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

Tag antibody mediated bone regeneration,biomaterials,OAI-PMH Harvest,scaffolds,tissue engineering

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Zadeh, Homayoun H. (committee chair), Duarte, Sillas (committee member), Malmstadt, Noah (committee member), Moradian-Oldak, Janet (committee member), Paine, Michael L. (committee member)

Creator Email ansaari.ansari@gmail.com,sansari@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-420437

Unique identifier UC11285854

Identifier etd-AnsariSaha-2558.pdf (filename),usctheses-c3-420437 (legacy record id)

Legacy Identifier etd-AnsariSaha-2558.pdf

Dmrecord 420437

Document Type Dissertation

Format application/pdf (imt)

Rights Ansari, Sahar

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...

Repository Name University of Southern California Digital Library

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