Tissue engineering of a vascularized bone graft

SUBHA ARAYA RATH
(MBBS, MMST)

TISSUE EGIEERIG OF A VASCULARIZED BOE
GRAFT

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR
OF PHILOSOPHY

DIVISIO OF BIOEGIEERIG
ATIOAL UIVERSITY OF SIGAPORE
2009


i
Acknowledgements
It is a pleasure to thank those who made this thesis possible and the list cannot be
completed with few names. I understood at the end of my PhD work that the journey
of few years was possible only with the help of the innumerable number of friends,
teachers and mentors whose unique help was absolutely important in maintaining
proper direction. Therefore, first of all, I would like to thank all those persons who
came across in my PhD journey and their contribution was very important, however

insignificant it may look.
I would like to express my special thanks to Prof. Michael Raghunath, who not only
taught us basic methods of research as a mentor and teacher but also helped me
beyond limits for my PhD completion. This thesis would not have been possible
without his supportive lessons throughout my PhD carrier and his special role for my
thesis completion.
I owe my deepest gratitude to Prof Dietmar W. Hutmacher, who constantly guided me
from the beginning until the very end of this PhD journey. I am very grateful to work
with such a scientific and fatherly figure. His supportive words through skype web
chatting and his magical emails made me feel his presence in each step of this journey
even without his physical presence. I would like to express my sincere and
overwhelming gratitude to Dr. med. Ulrich Kneser, and Dr. J. Thorsten Schantz for
their guidance and invaluable advice imparted throughout this project work. I remain
very much indebted to all my supervisors for their magnanimous and unflinching
inspiration instilled in me. The knowledge I gained from each of them in different
areas of my work will be helpful to dedicate my life to science.


ii
The support and guidance I received from the Lab of Tissue Engineering in NUS and
DSO NUSTEP, Singapore, were exemplary and made my research experience
memorable. I also take this opportunity to thank Lam Xu Fu Chirstopher, PhD student
in Lab. of Tissue Engineering, who guided and helped me in all scaffold related work
throughout my research work. I am indebted to him to support right until the end of
my thesis work. I express my sincere thanks to Barney for his microCT lessons;
Andrew for his works related to Extracel-HP. I am grateful to Clarice, Dr. Gajadhar
Bhakta, Evelyne, Anurag, David, Monique, Anand and Dr. Kee Woei for their timely
advice and knowledge in every aspect of my PhD work. I am indeed fortunate to have
worked alongside such committed and hardworking people.
I would like to show my special gratitude to Dr. Sambit Sahoo and Dr. Sampurna

Sattar for their moral support as well as scientific inputs in every new experiment.
The support for this work by my seniors, especially Dr. Sriram Vedula, Dr Dev
Chatterjee, and Dr. Karthik Harve are gratefully acknowledged.
I humbly express my sincere gratitude to Dr. Galyna Pryymachuk for her invaluable
guidance, and advice imparted during this investigation. I remain very much beholden
to her to teach me the microsurgical techniques for the orderly and successful
execution of the experiments for the completion of this thesis.
I would like to thank Dr Andreas Arkudas for his wise thoughts and kind words of
advice at every point in my endeavour without which it would not have been possible
to realize the thesis work. My special thanks go to Dr. Elias Polykandroitis for
contributing many ideas during the initial phase of my project. I would also like to
thank Dr. Oliver Bleizifer, Dr. Saskia Schnabl, and Dr. Justus Bier to be helpful with
friendly advices throughout my work.


iii

I am also grateful to many people who have helped and contributed, in one way or
another, to my research project. It has been a great pleasure to have worked with
Stefan Fleischer, Katja Schubert, and Dorothee Klummp, who have been
indispensable in the smooth completion of the project work.
I would like to thank all staff in division of bioengineering, NUS and department of
plastic surgery, University Hospital, Erlangen for their official and financial support.
Last, but not the least, I would also like to thank my family, especially my parents;
my wife, Bagmi; my brothers Bibhu and Prabhu for being very encouraging and
supportive throughout my PhD career.

July 2009 (Dr. Subha Narayan Rath)
National University of Singapore

iv
List of published work
• Leong DT, Abraham MC, Rath SN, Lim TC, Chew FT, Hutmacher DW. Investigating the
effects of preinduction on human adipose-derived precursor cells in an athymic rat model.
Differentiation. 2006 Dec; 74(9-10):519-29.
• Rath SN, Woodruff MA, Susanto E, Haupt LM, Hutmacher DW, Nurcombe V, Cool SM.
Sustained release and osteogenic potential of heparan sulfate-doped fibrin glue scaffolds
within a rat cranial model. J Mol Histol. 2007 Sep 12.
• Rath SN, Cohn D, Hutmacher DW. Comparison of Chondrogenesis in Static and Dynamic
Environment Using PCL-PEO Scaffold. Accepted in Journal of Virtual and Physical
Prototyping.
•
H
Fiegel HC
H
,
H
Pryymachuk G
H
,
H
Rath S
H
,
H
Bleiziffer O
H
,
H

Beier JP
H
,
H
Bruns H
H
,
H
Kluth D
H
,
H
Metzger R
H
,
H
Horch RE
H
,
H
Till H
H
,
H
Kneser U
H
. Fetal Hepatocyte Transplantation in a Vascularized AV-Loop
Transplantation Model in the Rat. Journal of cellular and molecular medicine. 2008 May 24.
• Rath SN, Arkudas A, Pryymachuk G, Polykandroitis E, Christopher LXF, Bier JP, Horch RE,
Hutmacher DW, Kneser U. Development of a Pre-vascularized 3D Composite Scaffold-

Hydrogel System Using an Artery-Venous Loop for Tissue Engineering Applications;
submitted to Biomaterials.
• Rath SN, Pryymachuk G, Bleiziffer OA, Lam CXF, Arkudas A, Ho STB, Bier JP, Horch RE,
Hutmacher DW, Kneser U. Hyaluronan-based heparin-incorporated hydrogels for generation
of axially vascularized bioartificial bone tissues: in vitro and in vivo evaluation in a PLDLLA-
TCP-PCL-composite system; submitted to Tissue Engineering.



v
Table of contents
Acknowledgements i
List of published work iv
Table of contents v
Summary x
List of tables xiii
List of figures xiv
List of symbols xvii
CHAPTER 1. Introduction 1
1.1. Background 1
1.2. Tissue engineering 2
1.2.1 Importance of vascularization in tissue engineering 2
1.2.2 Making a graft vascularized 4
1.3. Aims and hypotheses 5
1.3.1 Specific aim 1: To establish the scaffold architecture and optimize the
artery-venous (A-V) loop model in rat using the scaffold and to standardize
the explantation procedure. 6
1.3.2 Specific aim 2: To develop and evaluate pre-vascularized 3D
composite scaffold-hydrogel systems using an A-V loop for a possible graft.
7

1.3.3 Specific aim 3: Application of bone morphogenetic protein-2 (BMP-2)
or osteoblasts in the vascularized composite scaffold-modified hyaluronan
hydrogel system for development of a vascularized bone graft. 8
1.4. Organization of the Thesis 9
CHAPTER 2. Background to the Research 10
2.1. Angiogenesis and vascularization 10
2.2. Importance of vascularization in tissue-specific tissue engineering 13
2.2.1 Bone tissue engineering 13
2.2.2 Skin tissue engineering 15


vi
2.2.3 Genito-urinary tissue engineering 16
2.2.4 Limitations of current status of tissue engineering 16
2.3. Methods of generating vascularized grafts 18
2.3.1 Cell Source 18
2.3.2 Scaffold Material and design 20
2.3.3 Use of growth factors 21
2.3.4 Gene therapy for angiogenesis 22
2.3.5 Extracellular Matrix Components 23
2.4. Surgical approach with artery-venous loop 25
2.4.1 Cell free method: using body as bioreactor 30
CHAPTER 3. Experiment I: To establish the scaffold architecture and optimize
the artery-venous (A-V) loop model in rat using the scaffold and to standardize
the explantation procedure. 32
3.1. Introduction 32
3.2. Materials and methods 35
3.2.1 Scaffold fabrication 35
3.2.2 Porosity and pore interconnectivity calculation 37
3.2.3 Mechanical testing 38

3.2.4 A-V loop model 39
3.2.5 Explantation 40
3.2.6 Micro CT analysis 40
3.2.7 Histology 41
3.2.8 Statistical analysis 42
3.3. Results 43
3.3.1 Scaffold gross morphology 43
3.3.2 Porosity and interconnectivity 43
3.3.3 Mechanical testing 44
3.3.4 Explantation 44
3.3.5 Histology 46


vii
3.4. Discussion 46
3.5. Conclusion 51
CHAPTER 4. Experiment II: Development and characterization of a pre-
vascularized 3D composite scaffold-hydrogel system using an artery-venous loop
for tissue engineering applications. 53
4.1. Introduction 53
4.2. Materials & methods 56
4.2.1 Scaffold fabrication 56
4.2.2 Experimental design 57
4.2.3 Hydrogels 58
4.2.4 Surgical procedures 58
4.2.5 Explantation 59
4.2.6 Micro CT analysis 59
4.2.7 Histology 60
4.2.8 Histomorphometric analysis 60
4.2.9 Immunohistochemistry 61

4.2.10 Vascular corrosion cast preparation 61
4.2.11 Scanning electron microscopy 62
4.2.12 Statistical analysis 62
4.3. Results: 63
4.3.1 Surgery and animals: 63
4.3.2 Micro CT analysis: 63
4.3.3 Histology & Corrosion casting: 64
4.3.4 Histomorphometry 67
4.3.5 Immunohistochemistry 69
4.4. Discussion: 70
4.5. Conclusion: 74


viii

CHAPTER 5. Experiment III: Applying bone morphogenetic protein-2 (BMP-2)
or osteoblasts in the composite scaffold-modified hyaluronan hydrogel system
along with A-V loop for a vascularized bone graft formation. 76
5.1. Introduction 76
5.2. Materials and methods 78
5.2.1 Scaffold fabrication 78
5.2.2 Osteoblasts isolation and expansion 78
5.2.3 Osteoblast culture in modified hyaluronan hydrogel 81
5.2.4 AlamarBlue metabolic assay 82
5.2.5 PicoGreen DNA Quantification Assay 83
5.2.6 FDA/PI fluorescent staining of osteoblasts 83
5.2.7 Release kinetics of rhBMP2 from hyaluronan hydrogel (Extracel-HP)
84
5.2.8 Experimental design 85
5.2.9 Surgical procedures and explantation 86

5.2.10 RNA isolation and quantitative RT-PCR 86
5.2.11 Micro CT analysis 87
5.2.12 Histology 88
5.2.13 Histomorphometric analysis 88
5.2.14 Immunohistochemistry 88
5.2.15 Corrosion cast technique 88
5.2.16 Statistical analysis 88
5.3. Results 89
5.3.1 Osteoblasts in Hyaluronan hydrogel 89
5.3.2 Release kinetics 91
5.3.3 Surgery and animals 92
5.3.4 Micro CT analysis 92
5.3.5 Histology 95
5.3.6 Histomorphometry 97
5.3.7 Corrosion casting 98


ix
5.3.8 Immunohistochemistry 99
5.3.9 Real time PCR 101
5.4. Discussion 103
5.5. Conclusion 111
CHAPTER 6. COCLUSIOS AD RECOMMEDATIOS 113
6.1. Conclusions 113
6.2. Recommendations for future research 116
Reference: 118



x

Summary
The emergence of novel biomaterials and cell isolation techniques has revolutionized
the field of tissue engineering in fabricating tissue-like constructs in the laboratory.
The required mechanical and physical properties can be customized with available
biomaterials, whereas specific tissues might be fabricated with the application of
specific cell types and growth factors. However, if the size of the constructs is
increased, the applied number of cells must be proportionately more. Their survival in
vivo is very crucial in such constructs. The diffusion of micronutrients can be possible
for only few hundred micrometres from the capillary. The success of generating a
specific tissue-type is demonstrated only in relatively small-sized experimental
tissues. Additionally, in case of larger constructs, the specific differentiation is usually
demonstrated in only a fractional part of the whole construct. Moreover, the central
region gets frequently necrotic due to lack of nutrition.
Therefore, it is crucial to provide an extensive supplement of nutrition for the proper
function and survival of the graft, when its size is beyond few hundred micrometres.
Current attempts in achieving the goal rely on the application of specific growth
factors to induce vascular growth from nearby vessels or on the use of endothelial
precursor cells (EPC) for de novo vasculogenesis in the graft. In the former case, the
desired vasculogenesis is not usually achieved due to short half-life periods of growth
factors; while in the later case, the EPCs need nearby vessels to form a vascular tree.
In addition, the random formation of vascular tubes and their random connection to
parent blood vessels are unpredictably slow. The thesis reports a novel way of
fabricating a viable construct in a rat model by making an arterio-venous (A-V) loop
by microvascular anastomosis of the femoral artery and femoral vein with interposed


xi
contra-lateral femoral vein graft. This causes the formation of fibro-vascular tissue in
the construct relatively quickly. The biomaterial constructs are observed to be
successfully vascularized with mature blood vessels from the loop advancing into the

construct.
To keep specific cells or growth factors in a bioactive stable form, hydrogels are often
used as temporary vehicles for a successful graft. Though they provide hydrated
extracellular matrix-like environments, the properties differ from one another because
of their different degradation kinetics and tissue reactivity. To examine the different
rates of vascular growth in the A-V loop model, two hydrogels, fibrin glue and
hyaluronan, are tested in the loop supplied hard scaffold. The new vascular network is
successfully documented in each of the matrices, though the kinetic and magnitude
differ from each other. In fibrin glue, the vascular growth forms quickly with the
degradation of the hydrogel in few days. On the contrary, a uniformly increasing
vascular growth is observed in hyaluronan containing scaffolds along with a slowly
decreasing amount of remaining hydrogel in the constructs. The A-V loop permits the
angiogenesis pattern to grow centripetally from the loop invading the scaffold. Both
histological examination and micro CT scanning confirmed numerous sprouting blood
vessels from the A-V loop. However, at the end the number and pattern of blood
vessels are comparable between the two matrices. The finding is confirmed by
histomorphometric and micro CT analysis. This experimental study demonstrates that
hyaluronan-contained composite scaffold permits vascular in-growth slowly with its
slow degradation. This can be further explored for any growth-factor containing tissue
engineered graft.



xii
The utility of the vascularized scaffold-hyaluronan construct is further demonstrated
in vivo by attempting to fabricate a bone tissue-engineered product with an ectopic
bone-inducing growth factor, bone morphogenetic protein (BMP-2). To facilitate a
suitable amount of bone formation in such model, two different amounts of BMP-2
and the addition of osteoblasts are tested, expecting vascularization by A-V loop and
bone induction by BMP-2 or osteoblasts. Expression of bone-specific genes is

detected by real-time RT-PCR analysis, though no significant amount of bone is
detected histologically. The heterotopic isolation chamber setting in combination with
the absence mechanical stimulation might explain the insufficient bone formation.
However, the scaffold-matrix is vascularized to make a viable graft. Optimization of
the interplay of cells and growth factors in the scaffolds might eventually allow
generation of different axially vascularized grafts for application in reconstructive
surgery. This research project makes a promising approach for a vascularized graft for
further exploration.


xiii

List of tables
Table 3-1: The individual components of the composite scaffold and their properties. 35

Table 4-1: Groups and study design. In group FG animals, an arteriovenous (AV) loop was
constructed and put around the scaffold along with fibrin glue hydrogel; the loop was
placed in an isolation chamber for either 4 or 8 weeks. In group HA, the same procedure
was followed with a modified hyaluronan hydrogel as the matrix. Two out of the five
samples were analyzed by micro CT. 57

Table 5-1: Groups and study design. The PLDLLA-TCP-PCL scaffold along with an
arteriovenous (AV) loop in hyaluronan matrix was applied in all animals and kept for
eight weeks. Two out of the five samples were analyzed by micro CT. 86

Table 5-2: The primers of the genes analyzed by real time PCR. 87

Table 5-3: Summary of fold change of respective gene expressions compared to control group HA
normalized to internal β-actin expression as analyzed by qquantitative reverse
transcriptase polymerase chain reaction (qRT-PCR) for different groups: low-BMP,

high-BMP, and OB samples after 8 weeks. The fold changes of their expressions are
coded as per their expression fold changes: <0.1= ; 0.1-0.5= -; 0.5-2=±; 2-10= +; >10=
++. The asterisks indicate the results are significant at p=0.05 level (n=3). 103




xiv
List of figures
Figure 2-1: The sequential steps of angiogenesis as in literature. 11

Figure 2-2: The sequential steps of vasculogenesis as in literature. 11

Figure 2-3: The schematic diagram of five different types of A-V pedicle, possible to feed a graft.
(A) Arteriovenous (AV) shunt loop: 1. contralateral artery graft; 2. contralateral vein
graft*; 3. no graft*;(B) arteriovenous bundle: 4. ligation type; (C) 5.flow through type. *:
not shown, G: graft, A: artery, V: vein. 28

Figure 3-1: Schematic of the Screw Extrusion System developed in-house (robotic-arm not
shown). 37

Figure 3-2: (A) Scaffold pieces of 5×
××
×5×
××
×8 mm3 for characterization studies. (B) Bobbin shaped
scaffolds were fabricated with two cylindrical pieces. The groove was for the A-V loop
placement. 37

Figure 3-3: (A) The A-V loop was created microsurgically from femoral vessels with interposing

contralateral vein graft; (B) the loop was later placed surrounding the groove of the
scaffold in the middle. 39

Figure 3-4: The scaffolds were assessed for their pores and their interconnectivity by
reconstructing the virtual images by micro CT scanning. 43

Figure 3-5: (A) Stiffness of PLDLLA-TCP-PCL scaffolds in dry and wet states; (B) Yield stress of
PLDLLA-TCP-PCL scaffolds in dry and wet states. 44

Figure 3-6: After successful Microfil perfusion, the visceral micro capillaries visibly turn yellow
due to the compound as shown in (A) stomach and (B) intestines. (C) Before
explantation, the neck region of the loop, where the vessels enter into the chamber, was
inspected for two linear Microfil filled femoral vessels (arrows). 45

Figure 3-7: Hematoxylin and Eosin staining of loops to test their patency. (A) A non-patent loop
with a thrombosed vascular axis and (B) a patent loop with Microfil-filled vein and the
surrounding new capillaries can be delineated. 46

Figure 4-1: The schematic diagram of a bobbin-shaped scaffold used for in vivo experiments.
Two discs of 8 mm diameter were joined together in the middle with a piece of scaffold
making the construct. 56

Figure 4-2: (A) Schematic diagram of the bobbin shaped scaffold with A-V loop surrounding it.
The whole scaffold was placed in a chamber filled with the hydrogel. (B) An artery-
venous (A-V) loop as constructed from the left femoral vessels using a contralateral vein
graft. The loop was placed in a custom-made Teflon isolation chamber and was
embedded in 500 µL fibrin gel (C) or hyaluronic acid hydrogel (D). 59

Figure 4-3: Schematic representation of the scaffold for histomorphometric analysis to count the
number of blood vessels. All cross-sectional areas were divided into 10 equal sectors (6 at

the periphery and 4 at the central region) and the images were acquired under 100x
magnification. 60

Figure 4-4: Micro CT imaging of the constructs after Microfil perfusion. (A) umerous blood
vessels were observed after 4 weeks in fibrin glue constructs (group FG) which were still
visually comparable to those after 8 weeks (B). In contrast, the hyaluronan matrix
constructs (group HA) showed lower number of blood vessels (C) after 4 weeks


xv
compared to the increased number with numerous branching after 8 weeks (D). Scale
bars represent 2 mm. 64

Figure 4-5: Hematoxylin and eosin staining of 100X magnified specimen of (A,B) group FG
(fibrin glue) & (C,D) group HA (hyaluronic acid) after (A,C) 4 and (B,D) 8 weeks. S:
scaffold; HA: hyaluronic acid matrix; B: Microfil filled blood vessels; FVT: fibro
vascular tissue; V: vein of the loop. Scale bar showing 200 m in all the figures. 65

Figure 4-6: SEM images of the vascular corrosion cast of AV loop constructs of fibrin glue
scaffolds after 8 weeks demonstrating numerous new vessels originating from the AV
loop. The angiogenic sprouts, appearing as spikes (500x, asterisk) (A) or capillary loops
(arrows) (B), were observed. The variability in caliber of vessels within one field as signs
of vascular network maturity (250x) (C) and the pattern of nuclear imprints on
microvascular replica at higher magnification (1200x) (D) were also observed. Similar
observations were also made for hyaluronan scaffolds. 67

Figure 4-7: Histomophometric calculations of blood vessel formation in the histological slides of
the graft constructs from group FG (fibrin glue) and group HA (hyaluronan) specimens
after 4 and 8 weeks. (A) Mean number of blood vessels per cross section, (B) mean
percentage of fibro-vascular tissue, (C) mean number of blood vessels per mm2 area of

fibro-vascular tissue per cross section. (D) Mean percentage of remaining hyaluronan
hydrogel is shown. For all, n=4 and the values are in means±SEM. The significance of
values at p<0.05 levels between two time points is indicated by * and between two
matrices by **. 69

Figure 4-8: vWF (von Willebrand factor) immuno staining for fibrin glue (A) and hyaluronan (B)
specimens after 8 weeks of implantation. The dark colored Microfil filled vessels are
simultaneously positive for VWF immunostaining (stained brown, 200x). (C) ED1
immunostaining (dark brown color, arrow; inset at higher magnification) for foreign
body reaction in one of the fibrin glue specimens. However, this is rare in all specimens.
70

Figure 5-1: The microphotographs showing the osteoblasts in 2D culture in the culture plastic at
100x magnification; (A) the osteoblasts by day 7 and (B) at confluence by 18 days. 80

Figure 5-2: A disc shaped hydrogel with 5 mm thickness (Th) and 8 mm diameter was used for
release kinetic study (A); the disc-shaped hydrogel was kept inside 1x PBS to analyze the
released BMP2 (B). 85

Figure 5-3: Osteoblasts in hyaluronan hydrogel after 4 (A) and 8 (B) weeks. At 8 weeks, the same
microscopic field was observed for live cells (FDA-green) (C) and dead cells (PI-red) (D).
90

Figure 5-4: Alamar Blue assay of osteoblast-hyaluronan constructs for 8 weeks 91

Figure 5-5: PicoGreen assay of the osteoblast-hyaluronan hydrogels at four and eight weeks. The
value at eight weeks is significantly lower. 91

Figure 5-6: BMP-2 standard curve from known standards (A); Cumulative release of rhBMP-2
from hyaluronan hydrogel for a period of 35 days (B). 92

Figure 5-7: The micro CT reconstructed images of vascular pattern in different groups after
eight weeks; (A,B) hyaluronan hydrogel only, (C, D) with low dose of BMP-2 at 500
ng/ml, (E, F) with high dose of BMP-2 at 2500 ng/ml, (G, H) with 3 million osteoblasts
per construct. Scale bars represent 2 mm. 93

Figure 5-8: Micro CT analysis of specimens (n=2) showing (A) the volume of Microfil-filled blood
vessels, (B) the number of vessels per mm length, (C) vessel thickness in m, and (D) the
spacing between vessels in m (D) in different specimens after eight weeks in vivo. HA=
hyaluronan only; lowBMP = hyaluronan with low dose of BMP-2 at 500ng/ml;


xvi
highBMP= hyaluronan with high dose of BMP-2 at 2500ng/ml; OB = hyaluronan with 3
million osteoblasts per construct [16]. 94

Figure 5-9: Hematoxylin and eosin staining of 100X magnified specimens from arterial and
venous end: hyaluronan hydrogel only implanted loop (A, B); with low dose of BMP-2
(500 ng/ml) (C, D); with high dose of BMP-2 (2500 ng/ul) (E, F); and with 3 million
osteoblasts (G, H) after 8 weeks. S: scaffold; HA: remaining hyaluronic acid; BV:
Microfil-filled blood vessels; FVT: fibro-vascular tissue; V: vein of the loop. Scale bar
showing 200m in all the figures. 95

Figure 5-10: H & E staining of histological sections from peripheral regions after 8 weeks: (A) a
BMP2-added graft showing new vessels even in peripheral region, but visibly less
numerous and remaining hyaluronan (HA) matrix; (B) an osteoblast added specimen
showing numerous vasculoid rounded structures (V) with Microfil-filled (solid arrow) or
Microfil-unfilled vessels (dashed arrow); the later may be still in the process of getting
canalized. In these specimens, the hyaluronan has resolved completely. All scale bars
represent 200 m. 96

Figure 5-11: Histomorphometric calculations of blood vessel formation in the histological slides
of the graft constructs. (A) Mean percentage of fibro-vascular tissue and hyaluronan
matrix for comparison. (B) Mean number of blood vessels per mm2 area of fibro-
vascular tissue per cross section. (C) Mean number of blood vessels per cross section. (D)
Mean diameter of blood vessels in m. n=4 per experiment and the values are in
means±SEM. The * indicates the significance of the values at p<0.05 levels with the
values of numbered sample. (1) HA: hyaluronan, (2) lowBMP: 500 ng of BMP-2, (3)
highBMP: 2.5 g of BMP-2; (4) OB: 3 million osteoblasts per graft. 98

Figure 5-12: SEM images of the vascular corrosion cast of A-V loop construct after 8 weeks
demonstrating numerous blood vessels originating from the A-V loop. (A) With
osteoblast added loops, the initial stages of angiogenesis were grossly accelerated.
Irregular nascent capillaries combined with multiple neovascular sprouts (asterisks)
depicted the picture as an "angiogenetic hot spot" (SEM, x250). (B) Details from fig A
acquired with a 6 degree-tilt. A capillary loop forms both by an intussusceptive
angiogenesis (circle) and by means of interconnection (arrows) between two existent
neovascular conduits. (SEM, x 1200). 99

Figure 5-13: Immunohistochemistry with vWF antibody showing the new vascular architecture.
The samples are from constructs with hyaluronan matrix (A) without anything , (B) with
low dose of BMP2, (C) with osteoblasts. (D) egative control. 100

Figure 5-14: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis
of bone related gene expressions: Collagen-I (A), alkaline phosphatase (B), IBSP (C),
RUX-2 (D), osteocalcin (E), and osteonectin (F) in hyaluronan contained low BMP2,
high BMP2, and osteoblast samples after 8 weeks. Specific gene expression was
normalized to internal β-actin expression. Values represent the fold change compared
with controls (only hyaluronan). The error bar represents standard deviation and the
asterisk indicates the results are significant at p=0.05 level (n=3). 101

Figure 5-15: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis
of extracellular-matrix protein expressions: syndecan (A), biglycan (B) and growth
factors: VEGF (C), FGF (D), BMP2 (E) in hyaluronan contained low BMP2, high BMP2
and osteoblast samples after 8 weeks. Specific gene expression was normalized to
internal β-actin expression. Values represent the fold change compared with controls
(only hyaluronan implants). The error bar represents standard deviation and the
asterisk indicates the results are significant at p=0.05 level (n=3). 102

Figure 5-16: Subcutaneous implantation of hydroxyapatite-TCP (Baxter) with 2.5 g of BMP2
generates new bone matrix (unpublished data). 107




xvii
List of symbols
2D Two-dimensional
3D Three-dimensional
EC Endothelial cells
EPC Endothelial progenitor cells
BMP Bone morphogenetic protein
BMSCs Bone-marrow mesenchymal stem cells
DMEM Dulbecco’s modified Eagle medium
ECM Extracellular matrix
FBS Fetal bovine serum
FVT Fibro-vascular tissue
HA Hyaluronic acid or hyaluronan
HyA Hydroxyapatite
MSCs Mesenchymal stem cells

OPG Osteoprotegerin
PBS Phosphate buffered saline
PCL Polycaprolactone
PCR Polymerase chain reaction
PEG Polyethylene glycol
PGA Polyglycolic acid
PI Propidium iodide
PLDLLA-TCP-PCL Poly (L-lactide-co-D,L-lactide)- tricalcium phosphate- poly
caprolactone
PLGA Poly (lactic-co-glycolic acid)
PTH Para thyroid hormone
RANK Receptor activator of nuclear factor κβ
SEM Scanning electron microscope
VEGF Vascular endothelial growth factor
Chapter 1. Introduction

1
CHAPTER 1. Introduction
1.1. Background
Due to organ failure and tissue loss, millions of patients are admitted to the hospitals
in serious conditions. In the United States, estimated 8 million surgical procedures are
performed annually to treat millions of patients who experience organ failure or tissue
loss [1]. Although these approaches have saved many lives, they are imperfect for a
permanent cure. During 2006, approximately 29,000 donor organs were available for
more than 95,000 patients in need [2]. Although the number of organ donors is on the
rise recently, additions to the transplant waiting list have increased more rapidly, thus
increasing the burden and showing drastic shortage of donor organs. In fact, serious
patients are more likely to die while waiting for donor organ than in the first two years
after transplantation. Therefore, there is an unresolved issue of organ failure patients
even with current therapies such as organ transplantation, reconstructive surgery, or

by using mechanical devices such as kidney dialyzers or prosthetic hip joints.
To address this ongoing unmet need of organ shortage, the concept of tissue
engineering came in. About two decades ago, Langer and Vacanti first coined the
term tissue engineering as the application of principles and methods of engineering
and life sciences to restore, maintain, or improve tissue functions [3]. In the following
time, there were a lot of changes in research from the original views given by them to
fabricate tissue engineered substances. Although all tissues were probable candidates
of tissue engineering approach, few tissues such as skin, cartilage, and bone are in
advanced stage because of their potential need and relative ease of application.
Chapter 1. Introduction

2
1.2. Tissue engineering
The acute shortage of human tissues and organs for transplantation and the problems
associated with currently practiced treatment modalities made tissue engineering field
grow actively in the last few years, especially in research for its application purpose.
The main concept of this field lied in growing functional tissues and organs in a
laboratory set-up by applying biomaterials, cultured cells, and growth factors in a
variable array of combinations. Though some favorable results are seen, a number of
challenges must be successfully solved, in order to apply it for widespread clinical
practice [4].
Though the tissue engineering field is very broad, in brief, there are three principal
therapeutic strategies for treating diseased or injured tissues in patients: (i)
implantation of freshly isolated or cultured cells; (ii) implantation of tissues
assembled in vitro from cells and scaffolds; and (iii) in situ tissue regeneration [5] [3].
However, a number of tissue-specific growth factors and stimulants are used to
achieve the desirable results. For cell application, the specific cells or the stem cells
from the patient or a donor are either injected into the damaged tissue directly
or combined with a degradable scaffold in vitro and then implanted. For tissue
implantation, a complete 3D tissue is grown in vitro using cells and a scaffold, and

then is implanted after a maturation phase. For in situ regeneration, a scaffold
implanted directly into the injured tissue stimulates the body’s own cells to promote
local tissue repair.
1.2.1 Importance of vascularization in tissue engineering
Several tissues have shown encouraging results for tissue engineering, such as skin,
bone, cartilage, muscle, and bladder [6-8]. The demand for tissue engineered skin is
Chapter 1. Introduction

3
acute because of increased incidence of injuries, chronic ulcers, and burns causing
skin loss in patients. To date, the most advanced and biologically active products are
those that combine living cells with a supporting matrix. However, despite the many
clinical successes of current tissue engineered skin replacement products, the gold
standard in skin grafting is still the autograft harvested from the patients. Among the
major causes of shortcomings of tissue engineered products, the applied cells’
survival remains a question, especially in case of grafts of larger dimensions.
The need for bone substitutes is particularly important because bone loss and related
treatment complications account for a major burden in the healthcare system. In the
United Nations alone, it was estimated that 7.9 million fractures occur annually from
which 1.5 million patients need a bone grafting [9, 10]. Although autografts are still
considered the best graft, due to their limited supply and associated morbidity [11,
12], the tissue-engineering concepts came in order to meet the need. Different
combinations of biomaterials, growth factors, and cell sources are applied for a
successful bone healing. However, recently the importance of an intact blood supply
is understood for a viable functional graft [11]. Without a vascular supply, the graft
functionality is severely affected [13].
Reconstruction of genito-urinary tissues is important for bladder resected patients in
malignancy and in congenital genito-urinary abnormalities. Genito-urinary tissues can
be engineered using selective cell transplantation in acellular matrices. However, one
of the main problems is to limit the development of ischemic fibrosis during tissue

maturation [14]. An intact vascularization is needed to prevent such catastrophe.
Chapter 1. Introduction

4
1.2.2 Making a graft vascularized
Recently, it is understood that the most important factor for a successful tissue
engineered graft is the presence of a functional microvascular network within the
construct to provide the oxygen and nutrients to the cells. For the first few days the
cells within a graft without an intact microvascular network survive only by diffusion
of the nutrients which can be possible maximum 200-500 m of the vascular source
[15, 16]. Without a proper vascular supply, the major part of a graft becomes necrotic
and non-functional within days. Although the tissue engineering research in the
beginning time was concentrated more on developing biomaterials and cell sources,
the research on vascular supply gained importance recently.
A number of factors involved in neoangiogenesis can be used such as Vascular
Endothelial Growth Factor (VEGF) [15, 17, 18], basic Fibroblast Growth Factor
(bFGF) [15, 19], various members of the Transforming Growth factor beta (TGFβ)
family [20, 21], and Hypoxia-inducible transcription factor (HIF) [22] as well as a
number of progenitor cells helpful for development of blood vessels such as
endothelial cells [23, 24], endothelial progenitor cells [25, 26] and stem cells [27, 28].
The process of angiogenesis is recognized to play a key role in wound healing in
adults [29]. The overall rate of vascular in-growth can be stimulated more, if the
feeding parent vessels passed through the graft. Many researchers have accomplished
this by making an artery-venous (A-V) loop to supply blood to the graft [15, 30-33].
In most of the cases, a contralateral artery/vein graft is made anastomosis between an
artery and a vein for an A-V loop which can readily feed to any scaffold for a
vascularized graft. Though it was previously not reported to use a hard pre-formed
and pre-shaped scaffold in studying the vascularization process into it, this can be
Chapter 1. Introduction

5
feasible by applying the A-V loop into it. In this way, the scaffold can be customized
to the specifications of the required graft with a combination of biomaterials, matrix,
and growth factors.
1.3. Aims and hypotheses
In this thesis, a new strategy for a tissue engineered graft is proposed. Erol and Spira
first reported the importance of A-V shunt loop in inducing vascular networks for
survival of new flaps, and they even proposed A-V loops to improve the circulation of
implanted fingers [34]. Subsequently, many experiments demonstrate the pre-
vascularized flaps using an A-V shunt loop as a vascular carrier. The present thesis
attempts to apply the successful generation of a vascularized graft and then its
application in bone development by combining the potential benefit of A-V loop
along with bone inducing growth factors. An A-V loop is constructed by
microsurgical anastomosis between an artery and a vein using interposed contralateral
vein graft. The loop will be fed to a custom built bobbin shaped scaffold to make a
vascularized graft in a pre-defined shape. A suitable matrix is proposed, which can
permit the new angiogenesis from the A-V loop, carry the growth factors for tissue
development, as well as, support the in-growth of the extracellular matrix. A rich
matrix formation along the pores of the scaffold can take place spontaneously when
this is implanted subcutaneously in rat. The A-V loop and the generation of the rich
fibro-vascular matrix have been previously shown to be feasible and can support
viable tissue for a longer period of time in the fibrin glue matrix [15, 32]. To prevent
quick degradation of the matrix in vivo, a hydrogel matrix will be used and its
characteristics with respect to the in-growth of new blood vessels and holding of
growth factors will be examined. But the main supporting framework will still be
provided by a hard composite scaffold. The hydrogel is required for containment of
Chapter 1. Introduction

6
cells or growth factors at the beginning and for allowing the blood vessels and

extracellular matrix to grow into it.
With the potential vascularized tissue engineered graft in mind, a strategy is designed
with the hypothesis that:
“The use of highly porous and interconnected composite scaffold, in
combination with micro-surgically constructed artery-venous loop system will
allow to engineer a vascularized graft for the development of any specific viable
tissue engineered graft.”
To engineer a 3D vascularized construct in vivo, this approach of using a combination
of factors will enable tissues to grow into the scaffold along with vascular
ramification and generation of fibro-vascular tissue. The hard scaffold can provide the
main mechanical strength while the soft hydrogel can provide the support as the
extracellular matrix. To attain this proposed objective of testing it for a specific tissue
type in vivo, BMP-2 or osteoblasts are applied further to develop a vascularized bone
graft. Keeping this in mind, the following specific aims are designed:
1.3.1 Specific aim 1: To establish the scaffold architecture and
optimize the artery-venous (A-V) loop model in rat using the
scaffold and to standardize the explantation procedure.
OBJECTIVE: A-V loop, created micro-surgically by making an anastomosis of
femoral vessels with interposing contralateral vein graft, can be used to feed a
specially designed bobbin shaped PLDLLA-TCP-PCL scaffold.
• A special bobbin-shaped scaffold will be fabricated with PLDLLA-TCP-PCL
composite materials after blending and extruding with the help of rapid
Chapter 1. Introduction

7
prototyping method. It will be tested for its porosity and mechanical
properties.
• An A-V loop will be created micro-surgically by making an anastomosis of
femoral vessels with interposing contralateral vein graft.
• The possibility of development of new sprouted angiogenesis from the parent

A-V loop after four weeks will be tested and the process will be standardized
by injecting a vascular contrast medium into the vessels. The tissues will be
later analyzed by histology to test the patency of the loop.
1.3.2 Specific aim 2: To develop and evaluate pre-vascularized 3D
composite scaffold-hydrogel systems using an A-V loop for a
possible graft.
OBJECTIVE: In-growth of 3D fibro-vascular tissue, formed by A-V loop, will be
highly vascularized in presence of a hydrogel in composite scaffold-hydrogel systems.
Considering the different rates of degradation of hydrogels in vivo, the rates and
patterns of vascularization of the graft is expected to differ.
• In vivo formation of angiogenesis surrounding the PLDLLA-TCP-PCL
scaffold in two different hydrogels such as: a modified hyaluronic acid (HA)
and fibrin glue (FG) will be characterized.
• The pattern and architecture of new angiogenesis will be evaluated by micro
CT imaging after perfusing the rats with Microfil contrast medium.
• With histomorphometric method, the blood vessels and fibro-vascular tissues
of two hydrogel systems will be measured and compared for different time
points.