Tissue engineering of a vascularized bone graft

Development of a Pre-vascularized 3D Composite Scaffold- Hydrogel System Using an Artery-Venous Loop for Tissue Engineering Applications; submitted to Biomaterials.. Experiment III: Appl

SUBHA ARAYA RATH

(MBBS, MMST) GRAFT

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR

OF PHILOSOPHY

DIVISIO OF BIOEGIEERIG

ATIOAL UIVERSITY OF SIGAPORE

2009

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

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

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

National University of Singapore

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

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

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

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

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

5.3.8 Immunohistochemistry 99

5.3.9 Real time PCR 101

5.4 Discussion 103

5.5 Conclusion 111

CHAPTER 6 COCLUSIOS AD RECOMMEDATIOS 113

6.1 Conclusions 113

6.2 Recommendations for future research 116

Reference: 118

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

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

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

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

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 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

artery-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

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 µm 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 µm, and (D) the

spacing between vessels in µm (D) in different specimens after eight weeks in vivo HA=

hyaluronan only; lowBMP = hyaluronan with low dose of BMP-2 at 500ng/ml;

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 200µm 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 µm 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 µm 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 µg 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), RUX-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 µg of BMP2

generates new bone matrix (unpublished data) 107

List of symbols

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

PCR Polymerase chain reaction

PEG Polyethylene glycol

PGA Polyglycolic acid

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

1.1 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

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

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

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 µm 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

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

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

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

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

OBJECTIVE: Osteoinductive protein such as BMP-2 or differentiated cells such as

osteoblasts can act on in-growing fibro-vascular tissue in vivo to form bone matrix,

which along with new angiogenesis formed by the A-V loop, can ultimately make a vascularized bone graft in composite scaffold-hyaluronan hydrogel system

• Hyaluronic acid (Extracel-HP) hydrogel containing osteogenic growth factor,

2 will be studied for 5 weeks in vitro in 1x PBS for the release of

BMP-2 by using enzyme linked immuno-sorbent assay (ELISA)

• Three-dimensional modified hyaluronan hydrogel will be used for rat osteoblast culture to assess their growth and biocompatibility

• Two different concentrations of BMP-2 will be incorporated in modified hyaluronan hydrogel matrix surrounding the PLDLA-TCP-PCL scaffold

• The pattern and architecture of new angiogenesis in vivo will be evaluated by

micro CT imaging after perfusing the rats after 3 months with Microfil contrast medium, while the blood vessels and fibro-vascular tissues will be measured from histological cross-sections

• All samples will be analyzed by immunohistochemistry for possible bone formation and vascular development

• RNA samples will be collected from all different specimens to analyze the genetic expression profile patterns

1.4 Organization of the Thesis

Chapter 1 provides the background to bone tissue engineering, a brief overview of current need of bone graft, and defines the aims and hypothesis of this research

Chapter 2 provides a detailed literature review of relevant topics, leading to the formulation and strategies adopted in this research

Chapter 3-5 documents individual experiments that formed parts of original published peer-reviewed papers or submitted manuscripts

Chapter 6 rounds up the thesis with the final conclusions and recommendations for future research in the topic

CHAPTER 2 Background to the Research

2.1 Angiogenesis and vascularization

Angiogenesis, the process by which new blood vessels develop from pre-existing ones, impacts significantly on many important disease states, including cancer, ischemic cardiovascular disease, wound healing, and inflammation [29] It needs to be distinguished from several related processes, namely, vasculogenesis, arteriogenesis and lymphangiogenesis In the process of angiogenesis, endothelial cells in the parent vessel penetrate the vascular basal lamina, migrate into the surrounding tissue in multi-directional fashion and subsequently divide with repeated sprouting and anastomosis forming ultimately a vascular network [32] Vasculogenesis was originally referred exclusively to the de novo formation of new blood vessels from primitive cells that occur early in embryonic development However, this distinction has recently become blurred by findings that under some circumstances endothelial precursor cells present in bone marrow and circulating endothelial cells in blood contribute to adult vasculogenesis [35] The term angiogenesis again refers to the formation of small blood vessels and therefore, needs to be distinguished from arteriogenesis, the formation of new arteries [9]; nonetheless, some agents (e.g VEGF-A) that induce angiogenesis also generate arterial blood vessels [36] For ease

of understanding, we used the terms angiogenesis and vasculogenesis interchangeably

in the present thesis as the main goal of both the approaches is to provide nutrition to the graft for survival

Vasodilatation Degradation of BM EC migration

& proliferation Capillary lumen formation & tube

Nascent endothelial tube

Pericytes & vascular

smooth muscle recruited

Primary vascular network formed

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

Angiogenesis is very vital for wound healing where the process is initiated by numerous growth factors and terminated by factors such as thrombospondin and angiostatin [37] The control of physiological angiogenesis is very remarkable without which the process continues in malignant growth associated with tumor growth and formation of metastases Surgical transfer of composite tissue requires adequate angiogenesis to occur before that tissue can survive independently of the vascular

pedicle, and it occurs randomly from the host vessels to bridge with the graft blood vessels [38]

Henceforth, the research trend is increasingly progressing towards making the graft supply with a well-developed vascular architecture inside it, especially for the central portions of the graft Keeping this view as the main target, a number of research approaches were directed to make a graft viable The cells and factors which are necessary for development of a normal vasculature during embryonic development are recapitulated during situations of neoangiogenesis in adults 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]

There are many models of angiogenesis to show the process, its quantitative measurement by vessel counting and its effect on survival of tissue Neovascularization is shown by models such as chick chorio-allantoic membrane, corneal micro-pocket and cutaneous wound In the chorio-allantoic membrane model, endothelial cells first grow by mitosis to expand the blood vessels before forming a mature micro-vascular network [39] In this study, the behavior of endothelial cells with respect to the maturation of blood vessels was analyzed In the corneal micro-pocket model, the vascular growth into a non-vascular tumor cell mass was studied, especially some angiogenesis-inducing tumor tissues [40] In the rat cutaneous model,

it was found that initial angiogenic growth was stimulated by growth factors, whereas the overall response is dependent on microenvironment and vessel origin rather than

growth factors [41] However, they did not study the angiogenesis process in an isolated model where the affecting factors can be precisely controlled The response

in the subcutaneous model might be from the surrounding subcutaneous tissue anatomy, and might not precisely from the growth factors A bridging angiogenesis model was there to show how skin survival is critically dependent upon the angiogenesis [37] Recently, the A-V loop model attempts to show the fabrication of new tissue in a chamber model with specific factors of interest in the chamber where

it acts like a bioreactor [42, 43]

2.2 Importance of vascularization in tissue-specific tissue engineering

Tissue engineering and reconstructive surgery are based upon the principle of replacing defective tissues with viable, functional alternatives The two main types of grafts currently in use are autografts and allografts An autograft or autogenous graft

is a section of tissue taken from the patient's own body, whereas an allograft is taken from a cadaver In spite of many available treatment modalities for tissue loss, currently, the gold standard is the autograft transplant; for bone, it is taken from iliac crest; for skin, it is taken from a healthy dermis The autografts are reliable, are immunologically compatible, provide excellent tissue integration, and incorporate well in defect sites Additionally, the vasculature inside them is fully intact for quick unification of host and graft vascular systems providing many of the above-mentioned benefits

2.2.1 Bone tissue engineering

In the United Nations alone, approximately 7.9 million fractures occur annually, among whom, 5-10% proceed to be delayed or impaired healing [9, 10] Again 10%

of the fractures require additional surgical procedures for complete healing In absolute terms, about 1.5 million patients need bone grafting procedures annually in United Nations [10] Simple bone fractures are treated by a plaster cast which immobilizes the joint above and below the fracture sites However, compound fractures need a suitable bone tissue transplant The standard treatment modality for bone loss cases is bone grafting of autologous cortical and cancellous bones harvested from iliac crest [12] From bone tissue deficiencies resulting from diseases to complications arising from an accident, bone transplants may provide an appropriate solution

When there is a necessity of bone transplantation, two options are there: bone from the patient himself may be utilized, which is called autograft or donor bone tissue may

be arranged in different forms Although autografts are still considered the best graft, there are limitations to its supply and associated morbidity to already moribund patients [11, 12] With advancement of medical technology and better understanding

of bone biology, tissue engineering of bone is actively pursued to meet the demand The basic concept of bone tissue engineering might combine the advantages of autologous bone transplants with a reduction of secondary harvesting operations

A functional tissue engineered bone can be constructed by applying different types of biomaterials, growth factors, and cell sources However, for successful bone healing, four components are fundamental as per the following: an osteoconductive matrix, osteoinductive factors, osteogenic cells which can respond to these factors and an intact blood supply [11] Skeletal tissue regeneration requires the interaction of three basic biological elements: cells; growth and differentiation factors; and extracellular matrix Hence, sophisticated designs have even tried taking these three main strategies, alone or in combination with one another, for the replacement bone defect

During the past decade, multiple researchers have observed a synergistic response when bioactive factors, scaffolds, and cells were used together for tissue engineering purposes [44] But the survival of the graft is dependent upon an intact vascular supply, without which the graft functionality is hampered [13] A bone loss site may

be treated by direct application of autografts or osteogenic growth factors for bone formation However, in many cases the local site is compromised by huge bone mass loss, scarring, or irradiation for cancer therapy, without having any healthy tissue If

we apply only a bone graft, whether autograft or allograft, it will be necrotic without getting enough nutrition These defects can be successfully treated with microsurgical transfer of vascularized bone Secondary corrective operations are required invariably due to limited donor site availability and difficulties in matching the shape of the defect site, and they are associated with significant morbidity [45] At this point, proper vascularization to the scaffold based bone grafts might be promising

2.2.2 Skin tissue engineering

Skin deficiency is the major cause of morbidity and mortality in burn patients Additionally, incidence of chronic wounds is very high due to diabetes and skin cancers Despite such heavy demands for skin grafts, a number of tissue-engineered skin products are now available in the market These products can be classified based

on their strategy of regeneration, namely acellular grafts, epidermal replacements, dermal replacements and bi-layered grafts [7] The clinical benefit of skin tissue engineered products has been poorer than expected because its use is associated with high infection risk and low regeneration rates The lack of vascularization is believed

to be the main reason for these problems because it impairs the delivery of competent cells, oxygen and nutrients Therefore, enhancement of vascular tissue regeneration in skin is one of the main goals in its current research [46]

immune-2.2.3 Genito-urinary tissue engineering

Genitourinary tissues can be engineered in-vitro and in-vivo for reconstruction using selective cell transplantation in combination with acellular matrices Tissues and organs in urology, such as the bladder, clitoris, corpus cavernous, kidney, testis, ureter and urethra have been created in the laboratory, with varying degrees of functionality [47] Cells have also been recently used in patients as bulking agents for the treatment

of vesico-ureteral reflux and urinary incontinence However, ischemic fibrosis during tissue maturation remains the main problem Even when the native vessels are retained, better functional results were obtained when ischemia was prevented by adding further vasculature to the construct with the omentum vasculature This influence of ischemia on bladder function is in line with studies on native human detrusor in which decreased bladder blood flow correlated strongly with decreased bladder compliance and with animal experiments where bladder ischemia increased transforming growth factor-β1 expression leading to fibrosis, smooth muscle atrophy, and noncompliance [14] Therefore, the importance of presence of an intact vasculature is understood recently in the success of genitourinary tissue engineering

2.2.4 Limitations of current status of tissue engineering

There are a lot of available procedures for variable tissue engineering applications However, many procedures are either in research stage or in small animal models making autograft as the graft of choice A number of current experimental and clinical

models necessitate autologous cells implanted in scaffolding matrices in vitro to generate tissue constructs, which can be subsequently implanted in vivo [43]

However, these constructs must necessarily be thin and two-dimensional as in cases superficial skin and heart valves, so that the cells can survive by only diffusion until

the construct is randomly vascularized from surrounding native tissue The process is called bridging angiogenesis, but even then the tissues survive only for initial days [43, 48] To generate truly three-dimensional tissues such as liver, bone, and thick skin a ready made vascular tree is required to prevent necrosis of cells especially towards the centre of the construct and subsequent graft failure [43]

Broadly, two approaches can be adopted to address this problem [43] In the first

approach, the vascular tree is developed in vitro in the scaffold before actual

implantation so that the host blood vessels can be linked with the vascular tree by microvascular anastomosis In the second approach, the intrinsic ability of the body to form angiogenesis inside the scaffold is relied upon in response to certain angiogenic stimuli In the first approach, the process is mostly non-adequate and non-uniform, while in the second approach, the effect is far from ideal

Hence, if the graft is pre-fabricated in a predetermined shape and also vascularized for viability, it may deliver sustained functions starting from its implantation If an intact blood supply is maintained, it can incorporate newly viable tissue into host tissue, independent of the surrounding recipient bed The predetermined shape of newly formed tissue may provide the most effective replacement of lost tissue, especially tissues such as bone and muscle The technique can provide a shorter healing time and

a graft which is resistance to infection and extrusion

This method is called pre-vascularization, which is a process of making neovascularization in tissues by implanting a vascular pedicle into them that can later

be transferred to defect site as a graft by micro vascular anastomosis [49] This method has long been applied to skin and other soft tissues in plastic and reconstructive surgery Numerous studies have been published in an attempt of

vascularization of tissues with pedicle graft with varying degrees of success [50-52]

At the same time studies are being carried out to test the pre-vascularization of implant before it can be actually used as a transplant in the defect site [53-55]

2.3 Methods of generating vascularized grafts

A free graft does not survive to its full extent, when transferred from one site to the defect site The cells near the surface may survive, but the cells deeper in the centre die Vascularized graft is defined as the graft taken complete with a supplying artery and a draining vein [56] Such vascularized grafts are usually procured from the fibula

or the iliac crest for bone; from latissimus dorsi for muscle or breast; from nearby healthy skin as vascular flaps for skin; because the related blood vessels are constant and reasonably suitable for micro surgical manipulations [57] The advantage of vascularized graft is that it can effectively resist infection, can repair the loss of large segmental defects even in a diseased bed, and it does not undergo graft failure [58] In tissue engineering field, many ways are manipulated to make the graft vascularized

2.3.1 Cell Source

The cell source for a successful tissue engineered graft is very critical, as it is the only viable source in all components The ideal cell source should be expandable to achieve enough numbers for graft, be non-immunogenic, and form microvascular network formation with proper stimulus

One strategy for creating such a network would be by promoting vasculogenesis in situ by seeding the basic cells within the biopolymer construct The cells are vascular endothelial progenitor cells (EPC), which are the fraction of CD133+/KDR+ subpopulation of CD34+ cells, isolated from peripheral blood, umbilical cord blood, bone marrow, or fetal liver [59] EPCs can participate directly in neoangiogenesis by

differentiating ex vivo into EPC derived endothelial cells (EC), but growing data suggest that they also affect tissue repair through paracrine mechanisms that are not well defined Currently, EPCs are pursued to utilize them as cellular therapy to potentially augment vascularization in ischemic areas or in tissue engineering

The EPC derived EC can be used to develop a microvascular network in tissue engineered grafts The logic behind the use of EC is that the engineered microvessels connect with host blood vessels as soon as the graft implanted accelerating the vascularization of the graft and its survival [59] This hypothesis was supported by other studies: pre-formed human microvascular networks in collagen/fibronectin gels were shown to form complex vascular structures perfused by the host circulation

within 31 days of implantation into immunodeficient mice [60] Nor et al reported

similar results with human microvascular EC seeded on biodegradable polymer matrices: functional microvessels were evident 7–10 days after implantation into mice [61] The use of cells from peripheral blood eliminates the need to sacrifice a blood vessel or tissue to obtain EC

However, the number of EPC in adult blood is much lower, even after cell purification and culture methods have improved a lot recently The isolation of enough EPC for a graft is cumbersome and their culture to grow them until suitable numbers is time consuming Using ECs as the only source for vascular development, many researchers have failed to demonstrate the complete permanent development of vascular tree [62] Additionally, the main defect site, where an implant is needed, is diseased and unhealthy to rely on the cells to actively form the required product The cells also maintain this endothelial phenotype when expanded and subsequently seeded on polyglycolic acid-poly-L-lactic acid (PGA-PLLA) scaffolds, but interestingly microvessels formation was not observed when applied alone, though,

when they are seeded with human smooth muscle cells, they form capillary-like structures throughout the scaffold [59]

Therefore, the development and sprouting of blood vessels from the preexisting vasculature in the vicinity, is recognized to be highly effective in tissue engineered grafts With bigger dimensions of the graft, the preexisting vasculature exists at a non-accessible distance from major portions of the graft making the process difficult and time-consuming The obstacle can be overcome by feeding parent vessels through the graft

2.3.2 Scaffold Material and design

To engineer a complex vascularized tissue, the requirements of scaffold material and architecture are manifold and extremely challenging In addition to their properties such as biocompatibility, non-inflammatory, non-immunogenic, they need special properties with respect to their design and composition for suitable vascularization into it Scaffolds with highly porous interconnected structures and large surface-to-volume ratios produce the highest rates of mass transfer of oxygen, nutrients, and metabolic byproducts [63] A typical porosity of 60-80% and a pore diameter of 200

µm to 900 µm are requisites for cell penetration and a proper vascularization of the ingrown tissue [64] [65], as pore sizes of 150 µm do not support neovascularization [66] Scaffold geometry, which restricts vascular in-growth, produces cartilage instead

of bone in bone tissue engineering applications [67] These findings underline the fundamental effect of scaffold pore size on vascular in-growth

Though scaffolds can be designed to promote vasculogenesis, they are not able to induce enough blood vessels to make the graft fully survive The design and the materials are only helpful for assisting the on-going vasculogenesis by other inducers

The process can be multifold increased when the scaffold is kept near a highly vascular bed source

2.3.3 Use of growth factors

Therapeutic angiogenesis can be possible with the use of local administration of growth factors (GF) There are three ways to deliver GFs to stimulate vascular in-growth: (i) systemic administration of growth factors, (ii) localized delivery by incorporating in a carrier matrix, and (iii) gene therapy Much of the evidence stimulating angiogenesis is by applying VEGF as a localized delivery VEGF expression precedes blood vessel formation in developing many tissues and its expression is tightly associated with cells involved in vessel formation Other studies have shown that Inhibition of VEGF using soluble VEGF-R1 in wounds dramatically reduces healing, as well as angiogenesis and tissue formation Among all growth factors, VEGF appears most promising, which is a heparin binding, endothelial cell-specific mitogen [68]

Many other cytokines, such as TGF-β and PDGF, play a key role in promoting angiogenesis in inducing molecules such as VEGF [20] There is also a protective and

potentiating effect of heparin on VEGF binding in vitro to lead some researchers to

analyze the effects of heparin with VEGF [69] In addition to VEGF, many other growth factors and cytokines are also able to generate new blood vessels [29] Prominent among them are fibroblast growth factors (FGF-1 and -2), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), angiopoietins (Ang)-1 and -2, transforming growth factor (TGF)-α and -β, IL-8, EG-VEGF, leptin, prostaglandins, various lipids, etc Some of those listed (e.g TGF-α, HGF, FGF-2) act

at least in part by regulating VEGF expression [70] Others have secondary roles in

vessel formation and differentiation [71] For example, PDGF secreted by endothelial cells serves to attract pericytes; TGF-β secreted by perivascular cells inhibits endothelial cell proliferation, induces vessel maturation and matrix deposition; and Ang-1 has a role in regulating vascular permeability.

Systematic administration of VEGF and FGF administered by repeated intramuscular injections or as an intra-arterial bolus has shown to augment collateral vessel development in ischemic parts [19, 72, 73] VEGF-A, -B, and -E play an essential role

in vascular angiogenesis, while VEGF-C and -D regulate lymphatic vessel growth [74, 75]

Because of its strong angio-stimulatory action, VEGF is incorporated into glycolic acid-poly-L-lactic acid (PGA-PLLA) matrices for in-growth of microvessels from the host vasculature [76] However, only sufficient functional human EC-derived microvessels were formed, when VEGF containing matrices were seeded with human dermal microvascular endothelial cells

poly-The short half-life and instability of the protein require the delivery of higher than physiological quantities and multiple dosages of the GF Additionally, for its proper action nearby blood vessels are mandatory Therefore, for a well-vascularized graft VEGF like GFs can add supporting role to cells or vessels, than the role provided by

on its own It is challenging to design therapies that deliver sufficient quantities of GFs over a time period for a noticeable vascular growth

2.3.4 Gene therapy for angiogenesis

Biologic therapies to promote vascular growth by using gene therapy for VEGF and other growth factors are being increasingly employed in research scenarios Gene therapy is the transfer of genetic information into host cells to achieve a therapeutic