Parrag Ian C 201006 PhD Thesis.pdf

7/24/2019 Parrag Ian C 201006 PhD Thesis.pdf

1/238

THE DEVELOPMENT OF ELASTOMERIC BIODEGRADABLE POLYURETHANE

SCAFFOLDS FOR CARDIAC TISSUE ENGINEERING

by

Ian C. Parrag

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Graduate Department of Chemical Engineering and Applied Chemistry &

The Institute of Biomaterials and Biomedical Engineering

University of Toronto

Copyright by Ian C. Parrag (2010)


2/238

ii

THE DEVELOPMENT OF ELASTOMERIC BIODEGRADABLE POLYURETHANE

SCAFFOLDS FOR CARDIAC TISSUE ENGINEERING

Ian C. Parrag

Doctor of Philosophy, 2010

Department of Chemical Engineering and Applied Chemistry & Institute of Biomaterials and

Biomedical Engineering, University of Toronto

Abstract

In this work, a new polyurethane (PU) chain extender was developed to incorporate a

Glycine-Leucine (Gly-Leu) dipeptide, the cleavage site of several matrix metalloproteinases.

PUs were synthesized with either the Gly-Leu-based chain extender (Gly-Leu PU) or a

phenylalanine-based chain extender (Phe PU). Both PUs had high molecular weight averages

(Mw > 125,000 g/mol) and were phase segregated, semi-crystalline polymers (Tm ~ 42C) with

a low soft segment glass transition temperature (Tg < -50C). Uniaxial tensile testing of PU

films revealed that the polymers could withstand high ultimate tensile strengths (~ 8-13 MPa)

and were flexible with breaking strains of ~ 870-910% but the two PUs exhibited a significant

difference in mechanical properties.

The Phe and Gly-Leu PUs were electrospun into porous scaffolds for degradation and

cell-based studies. Fibrous Phe and Gly-Leu PU scaffolds were formed with randomly organizedfibers and an average fiber diameter of approximately 3.6 m. In addition, the Phe PU was

electrospun into scaffolds of varying architecture to investigate how fiber alignment affects the

orientation response of cardiac cells. To achieve this, the Phe PU was electrospun into aligned

and unaligned scaffolds and the physical, thermal, and mechanical properties of the scaffolds

were investigated.

The degradation of the Phe and Gly-Leu PU scaffolds was investigated in the presence of

active MMP-1, active MMP-9, and a buffer solution over 28 days to test MMP-mediated and

passive hydrolysis of the PUs. Mass loss and structural assessment suggested that neither PU

experienced significant hydrolysis to observe degradation over the course of the experiment.

In cell-based studies, Phe and Gly-Leu PU scaffolds successfully supported a high

density of viable and adherent mouse embryonic fibroblasts (MEFs) out to at least 28 days.

Culturing murine embryonic stem cell-derived cardiomyocytes (mESCDCs) alone and with


3/238

iii

MEFs on aligned and unaligned Phe PU scaffolds revealed both architectures supported adherent

and functionally contractile cells. Importantly, fiber alignment and coculture with MEFs

improved the organization and differentiation of mESCDCs suggesting these two parameters are

important for developing engineered myocardial constructs using mESCDCs and PU scaffolds.


4/238

iv

Acknowledgments

There are numerous people who have contributed to my scientific and personal

experiences during the last several years that have made this work possible. It is my hope that I

have thanked the people who have helped me along the way because I would not have made it

far enough to be writing this now without their help. I would first like to thank my supervisor

Dr. Kimberly Woodhouse for her guidance and support that have been invaluable for my career

development. I am very appreciative of the flexibility she has given me in pursuing my research

and personal interests. Without the opportunity to work with her, I would not have been able to

do and accomplish many things that are important to me. I would also like to thank my

committee members, Dr. Paul Santerre and Dr. Peter Zandstra, for their time and guidance with

this research project along with access to their lab equipment and resources. The members of the

Santerre, Zandstra, and Edwards labs have been very helpful in the training and use of lab

equipment and technical advice. Celine Bauwens, Sylvia Niebruegge, Ting Yin, Kuihua Cai,

and Cheryl Washer have been particularly accommodating in this regard. I would also like to

acknowledge Eric Altman, Frank Gibbs, Dionne White, Gary Skarja, and Tim Burrows for

technical consultations and sample analysis. Funding from the Department of Chemical

Engineering and Applied Chemistry and OGSST was much appreciated. It has been a realpleasure to work with all of the members of the Woodhouse group both in and out of the lab. I

would especially like to thank Joanna Fromstein, Patrick Blit, Cecilia Alperin-Dalley, Robin

Farmer, Lauren Flynn, Dave Laughren, and Elizabeth Srokowski for all their help with the work

in this thesis. Lastly, I would like to thank my family and friends for all their support and

encouragement that has gotten me through the difficult and enjoyable times that have come along

with this research project. Things never seem that bad when youve got good people in your life

and I am very appreciative of every single one of them.


5/238

v

Table of Contents

Chapter 1: Introduction...1

1.0. Clinical Problem......1

1.1. Hypothesis....2

1.2. Research Objectives.3

1.3 References3

Chapter 2: Literature Review..5

2.0. Introduction......5

2.1 Heart Tissue....5

2.1.1 Myocardial Cells.6

2.1.2. Extracellular Matrix Organization and Function.8

2.1.3. Reparative Response of the Heart to Myocardial Infarctions..9

2.2. Matrix Metalloproteinases and their Role in Heart Remodeling and Disease...10

2.2.1. MMP Expression Following Myocardial Infarctions and in Heart Failure...11

2.2.2. Cleavage Sites of ECM Proteins, Peptides and Biomaterials by MMPs...12

2.3. Regenerative Approaches to Repair the Heart...142.3.1. Inducing Endogenous Mechanisms in Heart Repair..14

2.3.2. Cellular Cardiomyoplasty..15

2.3.2.1. Fetal and Neonatal Cardiomyocytes....17

2.3.2.2. Embryonic Stem Cell-Derived Cardiomyocytes.....18

2.3.2.2.1. Differentiation of Murine Embryonic Stem Cells into Cardiomyocytes...19

2.3.2.2.2. Large-Scale Production of a Pure Population of Embryonic Stem Cell-Derived

Cardiomyocytes.21

2.3.2.2.3. Transplantation of Human and Murine ESC-Derived Cells into the Heart...23

2.3.3. Cardiac Tissue Engineering...25

2.3.3.1. Myocardial Tissue Engineering Using Biomaterials with Undefined Structures....25

2.3.3.2. In SituCardiac Tissue Engineering......27

2.3.3.3. Myocardial Cell Sheets....29


6/238

vi

2.4. Cardiac Tissue Engineering Using Pre-formed Three-Dimensional Scaffolds.31

2.4.1. Biomaterials for Cardiac Tissue Engineering31

2.4.1.1. Natural Biomaterials....33

2.4.1.2. Synthetic Biomaterials.....33

2.4.1.2.1. Traditional Polymers for Tissue Engineering...33

2.4.1.2.2. Elastomeric Biomaterials..34

2.4.2. Scaffold Fabrication Techniques...36

2.4.3. Cells for Cardiac Tissue Engineering38

2.4.4. Seeding and Cultivation Parameters for Cardiac Tissue Engineering...39

2.5. Biodegradable Segmented Polyurethanes for Tissue Engineering41

2.5.1. Chemistry and Properties of Degradable Polyurethanes...41

2.5.1.1. Segmented Polyurethane Synthesis.43

2.5.1.2. Reactant Chemistry for Biodegradable Polyurethanes44

2.5.2. Polyurethane Degradation.48

2.5.3. Enzyme-Degradable Polyurethanes..51

2.6. Electrospinning for Tissue Engineering Scaffold Formation54

2.6.1. Principles and Parameters..54

2.6.2. Electrospun Scaffolds for Cardiac Tissue Engineering.56

2.7. References..58

Chapter 3: Synthesis and Characterization of Phe and Gly-Leu-containing Segmented

Polyurethanes...84

3.0. Abstract..84

3.1. Introduction85

3.2. Materials and Methods...86

3.2.1. Dipeptide-based Chain Extender Synthesis...86

3.2.2. Gly-Leu-based Chain Extender Purification..88

3.2.3. Chain Extender Characterization...89

3.2.4. Polyurethane Synthesis and Film Casting..90

3.2.5. Polyurethane Characterization...91

3.3. Results and Discussion..92


7/238

vii

3.3.1. Chain Extender Synthesis and Purification92

3.3.1.1. Reaction Systems for Chain Extender Synthesis.93

3.3.1.2. Synthesis of Chain Extenders using Gly-Ile or Gly-Leu Dipeptides...96

3.3.1.3. Purification Strategies for the Gly-Leu-based Chain Extender...99

3.3.2. Polyurethane Characterization ...106

3.3.2.1. Molecular Weight Averages .106

3.3.2.2. Thermal Transitions and Phase Segregation..107

3.3.2.3. Chemical Composition...107

3.3.2.4. Mechanical Properties109

3.3.2.5. Effect of Amino Acid and Dipeptide-based Chain Extenders on Polyurethane

Properties...110

3.4. Conclusions..112

3.5. References113

Chapter 4: Electrospinning Phe and Gly-Leu Polyurethanes..116

4.0. Abstract116

4.1 Introduction..117

4.2. Materials and Methods.118

4.2.1. Electrospinning Phe and Gly-Leu Polyurethane Scaffolds..118

4.2.2. Scaffold Characterization.120

4.3. Results and Discussion121

4.3.1. Electrospinning Polyurethane Scaffolds..121

4.3.1.1. Effect of PU Concentration on Scaffold Morphology...123

4.3.1.2. Molecular Weight Averages and Thermal Properties129

4.3.1.3. Fiber Size in Electrospun PU Scaffolds for Soft Tissue Engineering...129

4.3.2. Aligned and Unaligned Phe PU Scaffolds...130

4.3.2.1. Scaffold Morphology.131

4.3.2.2. Molecular Weight Averages and Thermal Properties134

4.3.2.3. Mechanical Properties135

4.3.2.4. Electrospun PU Scaffolds for Cardiac Tissue Engineering...138



8/238

viii

4.5. References142

Chapter 5: Polyurethane Degradation by Matrix Metalloproteinases146

5.0. Abstract146

5.1. Introduction..146


5.2.1. Activation and Activity of MMPs147

5.2.2. Degradation of Polyurethanes by MMPs.149


5.3.1. Activation of MMPs150

5.3.2. Activity of MMPs after Incubation with Polyurethanes..153

5.3.3. Degradation of Polyurethanes by MMPs.155


5.5. References166

Chapter 6: Cell Response to Electrospun Polyurethane Scaffolds...170

6.0. Abstract170

6.1. Introduction..171


6.2.1. Mouse Embryonic Fibroblast Culture and Seeding onto Polyurethane Scaffolds...172

6.2.2. Characterization of MEFs on Phe and Gly-Leu-containing Polyurethanes.172

6.2.3. Culture and Differentiation of Murine Embryonic Stem Cells174

6.2.4. Monitoring the Differentiation of Cardiomyocytes from mESCs...175

6.2.5. Scaffold Preparation and Cell Seeding176

6.2.6. Characterization of mESCDCs and MEFs on Aligned and Unaligned Polyurethane

Scaffolds..176


6.3.1. Viability of MEFs on Phe and Gly-Leu-containing Polyurethanes.178

6.3.2. Differentiation of mESCs into Cardiomyocytes in Spinner Flasks.181

6.3.3. Effect of Fiber Alignment and Coculture with MEFs on Response of

mESC-derived Cardiomyocytes..187


9/238

ix

6.3.4. Aligned and Unaligned PU Scaffolds for Cardiac Tissue Engineering...200


6.5. References203

Chapter 7: Conclusions209

7.0. Conclusions.209

7.1. Significant Contributions to Literature214

7.2. Future Work.214

7.2.1. Polyurethane Design and Synthesis.214

7.2.2. PU Scaffold Formation and Characterization..214

7.2.3. PU Degradation215

7.2.4. Cell-based Testing of PU Scaffolds.215

7.3. References217

Appendix A: Supplementary Information for Dipeptide-based Chain Extender

Characterization220

A.1. C13

NMR Spectra of Reactants, Theoretical Predictions, and Raw Products.220


10/238

x

List of Figures

Figure 2.1. The structure of the myocardium.........7

Figure 2.2. The cardiac extracellular matrix..8

Figure 2.3. Alterations in MMP and TIMP levels in human heart disease..............12

Figure 2.4. Illustration of microphase separation in segmented polyurethanes...42

Figure 2.5. Standard two-step segmented polyurethane reaction.....44

Figure 2.6. Diisocyanates used to synthesize biodegradable PUs46

Figure 2.7. Polyols often used in biodegradable PU synthesis47

Figure 2.8. Model for environmental biodegradation of PUs..49

Figure 2.9. Schematic of electrospinning apparatus.55

Figure 3.1. Chain extender reaction system setups..87

Figure 3.2. Synthesis scheme for Gly-Leu-based diester, diamine chain extender..............88

Figure 3.3. Synthesis scheme for Gly-Leu PU.....91

Figure 3.4. Mass spectrum of raw Gly-Ile-CDM-PTSA product.....94

Figure 3.5. Mass spectra of raw Gly-Ile-based chain extender products synthesized indifferent solvent systems....95

Figure 3.6. Mass spectrum of crude product from Gly-Leu-CDM-PTSA...97

Figure 3.7. Mass spectra of Gly-Leu-based chain extender using different catalysts and

diol linkers.....98

Figure 3.8. HPLC separation of Gly-Leu-based diester product using analytical column

and low pH aqueous mobile phase..100

Figure 3.9. HPLC separation of Gly-Leu-based diester product using analytical column

and high pH aqueous mobile phase.101

Figure 3.10. Preparative column HPLC purification of chain extender using low and highpH aqueous mobile phases..102

Figure 3.11. C13

NMR spectra of products collected from preparative column HPLC using

the two developed methods of separation104

Figure 3.12. FT-IR spectrum of purified Gly-Leu-based chain extender ...105

Figure 3.13. The chemical structure of the Phe and Gly-Leu-based chain extenders..106

Figure 3.14. FT-IR analysis of Phe and Gly-Leu PUs.108

Figure 3.15. Representative stress-strain curve for Phe and Gly-Leu PU films..109


11/238

xi

Figure 4.1. Illustration of electrospinning apparatus..119

Figure 4.2. A comparison of electrospun Phe PU mats formed in the Rabolt laboratoryand in our laboratory using conditions established in the Rabolt laboratory...122

Figure 4.3. Comparison of Phe PU scaffolds formed before and after optimizing

electrospinning parameters..123Figure 4.4. SEM images of Phe and Gly-Leu PU scaffolds electrospun from different

concentrations.....124

Figure 4.5. Fiber diameter distributions of the Phe and Gly-Leu PU scaffolds electrospun

from varying concentrations126

Figure 4.6. Comparison of structural features of the Phe and Gly-Leu PU scaffolds usedfor degradation and cell-based studies.128

Figure 4.7. SEM images of aligned and unaligned Phe PU scaffolds132

Figure 4.8. Characteristics of aligned and unaligned Phe PU scaffolds.133

Figure 4.9. Representative stress-strain curves for aligned and unaligned PU scaffolds

stretched in preferred and cross-preferred directions of orientation136

Figure 5.1. Activation of MMPs using APMA..151

Figure 5.2. Zymogram of MMP activation solutions.152

Figure 5.3. Activity of MMPs after incubation with PU scaffolds154

Figure 5.4. Mass remaining of PU scaffolds over 28 day degradation study.156

Figure 5.5. SEM images of PU scaffolds after 28 day incubation period in various

solutions...157

Figure 5.6. Reaction scheme for enzyme activity assay and competitive substrate

enzyme activity assay..161

Figure 5.7. Inhibition of FS-6 cleavage using the Gly-Leu dipeptide....161

Figure 5.8. Water uptake by Phe and Gly-Leu PU scaffolds.164

Figure 6.1. Illustration of experimental details for cardiomyocyte production and cell

seeding.175

Figure 6.2. AlamarBlue

analysis of MEFs on PU scaffolds over 28 day period.....179

Figure 6.3. Staining of MEFs on PU scaffolds and TCPS.....180

Figure 6.4. Total cell number in spinner flasks during differentiation of mESCs intocardiomyocytes....184

Figure 6.5. EB characteristics during differentiation of mESCs into cardiomyocytes..185

Figure 6.6. Flow cytometry of cells before and after differentiation in spinner flasks..187

Figure 6.7. AlamarBlue

analysis of cell-seeded PU constructs of varying architecture

and TCPS controls...189


12/238

xii

Figure 6.8. Live/Dead

staining of cells on Phe PU scaffolds of varying architecture at

day 18+6..191

Figure 6.9. Immunostaining of cells on aligned and unaligned PU scaffolds192

Figure 6.10. Immunostaining of cardiac constructs with mESCDCs showing varying

levels of differentiation....193Figure 6.11. Quantifying the alignment of cells on PU scaffolds in coculture constructs...197

Figure 6.12. Gap junction staining of mESCDCs and MEFs in coculture on aligned andunaligned PU scaffolds199

Figure A.1. C13

NMR spectrum of Gly-Leu dipeptide220

Figure A.2. C13

NMR spectrum of CDM221

Figure A.3. Theoretical predictions of Gly-Leu-based diester chain extender using

ACD i-Lab software.221

Figure A.4. C

13

NMR spectrum of raw Gly-Leu-CDM-PTSA...222


13/238

xiii

List of Tables

Table 2.1. List of cell types considered for cardiac repair..16

Table 2.2. Summary of biomaterials and their applications in cardiac tissue engineering.....32

Table 2.3. Summary of electrospinning parameters and effects on fiber morphology...56

Table 3.1. Molecular weight averages for PUs containing Phe and Gly-Leu-based chain

extenders..107

Table 3.2. Thermal properties of the Phe and Gly-Leu PUs as determined by DSC107

Table 3.3. Summary of mechanical properties of PU films..110

Table 4.1. GPC and DSC results of Phe and Gly-Leu PU films and scaffolds.....129

Table 4.2. GPC and DSC results for Phe PU films and electrospun scaffolds of varyingarchitecture...134

Table 4.3. Summary of mechanical properties of aligned and unaligned PU scaffolds

stretched in preferred and cross-preferred directions of orientation136

Table 4.4. Mechanical properties of films of investigated or potential synthetic

biomaterials in cardiac tissue engineering...141

Table 6.1. Assessment of cell shape and sarcomere formation of mESCDCs..194

Table 6.2. Assessment of mESCDC dimensions......195

Table 6.3. Average angle of cell axis and orientation index.198


14/238

xiv

List of Abbreviations

3-D three-dimensional

ACN acetonitrile

ANP atrial natriuretic peptide

APMA 4-aminophenylmercuric acetate

BMP bone morphogenic protein

BV blood vessels

CB cardiac body

CDM 1,4-cyclohexane dimethanol

cTnT cardiac isoform of troponin T

Cx-43 connexin-43

DAPI 4',6-diamidino-2-phenylindole

DCM dichloromethane

DMEM Dulbeccos modified eagles medium

Dnp fluorescence-quenching group; 2,4-dinitrophenyl

DSC differential scanning calorimetry

E initial modulus; Youngs modulus; elasticity; stiffness

EB embryoid body

ECM extracellular matrix

EHT engineered heart tissue

ESC embryonic stem cell

ESI electrospray ionization

FACS fluorescent activated cell sorting

FBGC foreign body giant cell

FBS fetal bovine serum

FS-6 fluorogenic substrate for MMPs; Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2

FT-IR Fourier transform infrared

G418 geneticin; a neomycin analog

G-CSF granulocyte colony stimulating factor


15/238

xv

GFP green fluorescent protein

Gly glycine

Gly-Leu PU segmented polyurethane composed of PCL of molecular weight 1250, LDI, and a

Gly-Leu-based chain extender

GPC gel permeation chromatography

hESC human embryonic stem cell

hESCDC human embryonic stem cell-derived cardiomyocyte

HPLC high performance liquid chromatography

HOCl hypochlorous acid

LDI lysine-based diisocyanate

Leu leucine

LIF leukemia inhibitory factor

Mca fluorescent molecule; (7-methoxycoumarin-4-yl)acetyl

MDM monocyte-derived macrophage

mESC mouse embryonic stem cell

mESCDC mouse embryonic stem cell-derived cardiomyocyte

MHC myosin heavy chain

MHC-neor transgene carrying neomycin resistance gene driven by -myosin heavy chain

promoter

MI myocardial infarction

MLC-2v myosin light chain-2v

MMP matrix metalloproteinase

NMR nuclear magnetic resonance

ONOO-

peroxynitrite

PBS phosphate buffered saline solution

PCL1250 polycaprolactone diol of molecular weight 1250 g/mol

PGA poly(glycolic acid)

PGS poly(glycerol sebacate)

Phe phenylalanine

Phe PU segmented polyurethane composed of PCL of molecular weight 1250, LDI, and a

Phe-based chain extender


16/238

xvi

pGK-hygror transgene carrying hygromycin resistance gene driven by phosphoglycerate kinase

promoter

PIPAAm poly (N-isopropylacrylamide)

PLA poly(lactic acid)

PLGA poly(lactic-co-glycolic acid)

PMN neutrophils; polymorphonucleocytes

PTSA p-toluene sulfonic acid

PU polyurethane

SDS sodium dodecyl sulfate

TCPS tissue culture polystyrene

TFA trifluoroacetic acid

Tg glass transition temperature

TGF transforming growth factor

TIPS thermally induced phase separation

Tm melting temperature


17/238

1

Chapter 1: Introduction

1.0 Clinical Problem

Heart disease is one of the leading causes of disability and death in industrialized nations.

In the most recent study in Canada in 1998 (with updates in 2004), it was found that

cardiovascular diseases affect a quarter of the Canadian population accounting for more than a

third of the deaths and placing an estimated $18 billion burden on the Canadian economy [1, 2].

Topping the list of cardiovascular diseases was coronary heart disease, which leads to ischemic

heart disease, acute myocardial infarctions and congestive heart failure. Similarly, the

prevalence of cardiovascular diseases in the United States in 2005 was 80.7 million, or

approximately 37% of the population. These cases cost the U.S. health care system $448.5billion and resulted in 869,700 deaths (36.3% of all deaths) [3]. Furthermore, 8.1 million

individuals in the U.S. suffer from the debilitating affects of a myocardial infarction with more

than 920,000 new or recurring cases and 156,800 fatalities in 2004. Interestingly, successes in

treating myocardial infarctions and other cardiac diseases have allowed individuals with

damaged hearts to live longer, but is leading to an increase in the prevalence of congestive heart

failure [2]. In the U.S. alone, 5.3 million people suffer from congestive heart failure with

284,400 deaths in 2004. As a consequence, these studies indicate the huge health care burden of

heart disease and identify the need for effective treatments to combat it.

The heart has a limited capacity to regenerate on its own. Cardiomyocytes that are lost

due to a myocardial infarction (MI), if not fatal, are replaced by the formation of scar tissue, an

adaptive response leading to the loss of contractile function [4]. Subsequent remodeling events

occur in the heart to compensate for this loss of contractile function in an attempt to maintain

cardiac output. Some of these events include changes in cell type, extracellular matrix

composition and organization, ventricular size and architecture, neurohormonal signaling, gene

and protein expression, and paracrine signaling, to name but a few [5]. In the short term, these

remodeling events attempt to maintain cardiac performance but inevitably become destructive to

the heart causing congestive heart failure and ultimately death.

There currently exists several treatment options following a MI and in congestive heart

failure. Pharmacological agents, such as thrombolytic agents, antithrombotics, nitrates, -


18/238

2

blockers, Ca+2 channel blockers, angiotensin converting enzyme inhibitors, statins, and

adrenoceptor antagonists are typically used to increase blood flow, limit the ventricular

remodeling events, and increase cardiac output [6]. Although this therapy may be effective in

temporarily warding off heart failure, it is generally used to manage patients and offers little in

the way of treating the root of the condition. A second form of treatment employs the use of

mechanical devices, such as the left ventricular assist device. This treatment option has

traditionally been used as a bridge-to-transplantation, but has gained wider use as a destination

therapy and as a bridge-to-recovery option [7]. By reducing the workload of the injured heart,

LVAD therapy allows reverse remodeling to occur, whereby the destructive changes occurring in

the heart during remodeling are reversed [8]. This is an exciting new treatment option with some

patients undergoing sufficient recovery for the mechanical device to be removed, but the number

of patients eligible for this therapy remains low. A third treatment option, which remains the

gold standard because the recipient often regains full cardiac function, is heart transplantation.

This option, however, is limited by the lack of suitable donors and has motivated the field of

regenerative medicine to find alternatives that repair, replace, or augment the heart to restore

cardiac functionality.

There are many promising new approaches that are currently being investigated to

regenerate injured myocardial tissue and help fight heart disease. Some of these approaches

include pharmacological strategies, protein and peptide-based methods, gene therapy, cell-based

techniques, and tissue engineering [9]. Cardiac tissue engineering, in particular, offers the

advantage of combining several of these beneficial regenerative techniques along with novel

biomaterials and holds tremendous potential in the treatment of heart disease. As research in

cardiac tissue engineering continues to move forward, so to does the potential of easing the

enormous social and economic burden of this disease.

1.1 Hypothesis

The project hypothesis is defined in two parts: 1) glycine-leucine (Gly-Leu) containingbiodegradable segmented polyurethanes can act as temporary scaffolds that support cells; and 2)

fiber alignment within polyurethane scaffolds influences the orientation response of murine

embryonic stem cell-derived cardiomyocytes and mouse embryonic fibroblasts seeded on the

constructs.


19/238

3

1.2 Research Objectives

1) Synthesize and characterize a family of biodegradable, segmented polyurethanes using a

Gly-Leu-based diester chain extender, lysine-based diisocyanate, and polycaprolactone

diol

2)

Develop and characterize porous, three-dimensional biodegradable polyurethane

scaffolds by electrospinning and investigate the effects electrospinning has on scaffold

and polymer properties

3) Evaluate the in vitro degradation of amino acid and dipeptide-containing polyurethane

scaffolds in the presence of matrix metalloproteinase-1 and matrix metalloproteinase-9

4) Characterize the in vitrocellular response of cells seeded on polyurethane scaffolds

a) Assess the viability of mouse embryonic fibroblasts seeded on Phe and Gly-Leu-

containing polyurethane scaffolds

b) Characterize the response of murine embryonic stem cell-derived cardiomyocytes

and mouse embryonic fibroblasts on aligned and unaligned Phe-containing

polyurethane scaffolds

1.3 References

1. Heart and Stroke Foundation of Canada, Statistics and Background Information -Incidence of Cardiovascular Diseases.1998.

2. Heart and Stroke Foundation of Canada, Statistics. 2008.

3. American Heart Association,Heart disease and stroke statistics - 2008 update.AmericanHeart Association, 2008.

4. Kumar, V., R.S. Cotran, and S.L. Robbins,Basic Pathology. 7th ed. 2003, Philadelphia:Saunders. xii, 873.

5. Swynghedauw, B.,Molecular mechanisms of myocardial remodeling.PhysiologicalReviews, 1999. 79(1): p. 215-262.

6. Gelfand, E.V. and C.P. Cannon,Myocardial infarction: contemporary management

strategies.Journal Of Internal Medicine, 2007. 262(1): p. 59-77.

7. Deng, M.C., L.B. Edwards, M.I. Hertz, A.W. Rowe, B.M. Keck, R. Kormos, D.C. Naftel,J.K. Kirklin, and D.O. Taylor,Mechanical circulatory support device database of theInternational Society for Heart and Lung Transplantation: Third Annual Report - 2005.Journal Of Heart And Lung Transplantation, 2005. 24(9): p. 1182-1187.


20/238

4

8. Burkhoff, D., S. Klotz, and D.M. Mancini,LVAD-Induced reverse remodeling: Basic andclinical implications for myocardial recovery.Journal Of Cardiac Failure, 2006. 12(3): p.227-239.

9. Puceat, M., Pharmacological approaches to regenerative strategies for the treatment ofcardiovascular diseases.Current Opinion In Pharmacology, 2008. 8(2): p. 189-192.


21/238

5

Chapter 2: Literature Review

2.0 Introduction

The high prevalence and economic burden of myocardial infarctions, congestive heart

failure, and other heart diseases has motivated researchers and clinicians to develop new

strategies to treat patients. Tissue engineering is a field that seeks the development of tissue

constructs to repair, replace, or augment damaged or diseased tissues. This field has already had

some clinical successes that demonstrate how tissue engineering may revolutionize the way

clinicians approach disease management and therapy [1-3]. The first tissue engineered trachea

transplant, for example, was recently performed using a decellularized human donor trachea

combined with the patients epithelial and mesenchymal stem cell-derived chondrocytes [1].This novel procedure not only prevented the need to remove the diseased lung, the conventional

treatment choice, but also eliminated the need for immunosuppression therapy and drastically

improved the quality of life compared to the pre-operation condition and lung-resection option.

The heart is a complex organ composed of many critical components that give rise to its

unique function but also renders it susceptible to various injuries and diseases. Cardiovascular

tissue engineering on a whole explores tissue substitutes for the various components of the heart,

such as blood vessels, heart valves, and cardiac muscle. Advances in cardiovascular tissue

engineering have been made for each of the different components of the heart, but the

development of fully functional cardiac muscle remains one of the most challenging aspects of

this field.

2.1 Heart Tissue

The heart is a muscular organ responsible for circulating blood throughout the body. It is

composed of four muscular chambers, the right and left atria, which pump blood to the

ventricles, and the right and left ventricles, which pump blood to the pulmonary and systemic

circuits respectively. Critical to this pumping function is the heart wall. The heart wall is

composed of three distinct layers, the endocardium, myocardium, and epicardium, respectively

located from the lumen of each cardiac chamber out, all surrounded by the pericardium [4].

While each layer plays a critical role for normal cardiac function, the myocardium is the

contractile portion that generates the necessary forces to pump blood to the body and constitutes


22/238

6

the bulk of the heart wall. The myocardium consists of multiple interlocking layers of cardiac

muscle tissue and the associated blood vessels, connective tissue, and nerves (Figure 2.1). Cells

within each layer of cardiac muscle tissue are anisotropically organized parallel to each other and

each layer is subsequently oriented at different angles depending on chamber type and location

within each chamber [4]. Due to the high energy requirements associated with contraction, the

myocardium is a highly vascularized structure [4]. The high demand for oxygen within this

muscular layer, however, renders it susceptible to ischemic injury. Disruption of the normal

tissue composition and organization of this portion of the heart wall is observed in many diseases

leading to a loss of contractile function. As a result, regenerative medicine techniques target the

myocardium in an attempt to restore contractility to the tissue. Understanding the cellular

components and tissue organization in the myocardium is therefore a requisite for the design of

engineered myocardial constructs.

2.1.1 Myocardial Cells

The myocardium is composed of several cell types including vascular endothelial cells,

vascular smooth muscle cells, fibroblasts, neurons, and cardiomyocytes [5]. Cardiomyocytes are

the contractile cells taking up the bulk of the space in the myocardium. Mature adult ventricular

cardiomyocytes are rod-shaped, typically 10-30 m in diameter and 80-150 m in length [5], and

contain a high number of mitochondria and myoglobin to meet the energy requirements of

contraction [4]. Cardiomyocytes are composed primarily of bundles of myofibrils. Myofibrils

consist of a long repeated chain of sarcomeres, the basic contractile unit that give the cells a

striated appearance, composed of actin, myosin, tropomyosin, the troponin complex, and other

associated proteins [6]. In a resting state, the troponin complex and tropomyosin prevent myosin

from interacting with actin filaments. In response to a propagating action potential, the

excitation-contraction coupling mechanism causes an increase in intracellular Ca2+

concentration, the removal of the tropomyosin protein barrier, and, in the presence of ATP,

allows myosin to bind to actin leading to sarcomere shortening [6]. The excitation-contractioncoupling mechanism is made possible by the unique plasma membrane within these cells, the

sarcolemma, along with the transverse tubular system, the sarcoplasmic reticulum, and numerous

protein pumps, ion channels, and regulatory proteins. All working in a coordinated fashion, the

action potential, initiated independently of the nervous system, triggers this complex mechanism

ultimately leading to cardiomyocyte contraction.


23/238

7

Individual cardiomyocytes contracting on their own, however, does little in generating

the required forces to pump blood to the body. A coordinated effort is required and as such,

cardiomyocytes are organized into multiple interlocking layers connected to neighboring cells at

intercalated discs unique to cardiac muscle (Figure 2.1) [4]. At intercalated discs, cells are

electromechanically coupled by desmosomes, fascia adherens junctions, and gap junctions [7].

Because of the mechanical, chemical, and electrical connections between cardiomyocytes, the

cardiac tissue acts as a functional syncytium providing synchronous contraction and effective

force production to pump blood from the heart chambers.

Figure 2.1: The structure of the myocardium: a) histology image showing multiple interlocking layers of

cardiomyocytes with arrows indicating intercalated discs. b) Schematic identifying organization of bundles of

cardiomyocytes, fibroblasts, blood vessels (BV), and extracellular matrix. Images used with permission from Dr.

Caceci [8].

Cardiac fibroblasts are the most numerous cells in the myocardium, and they play a

pivotal role in regulating tissue organization and function [9]. Fibroblasts are organized adjacent

to groups of myocytes where they interact with other fibroblasts, myocytes, and extracellular

matrix (ECM) macromolecules (Figure 2.1b) [10]. Cardiac fibroblasts are electrically connected

to adjacent fibroblasts and cardiac myocytes via gap junctions that aid in signaling between cells

[10]. The predominant role of cardiac fibroblasts, however, is to regulate the structure and

function of the ECM through deposition of its constituents and secretion of enzymes that degrade

them. The extracellular matrix acts as a mechanical support for tissues and transmits information

from the extracellular environment to regulate cell shape and function. Fibroblasts synthesize

and deposit the majority of the cardiac ECM, especially fibrillar collagen types I and III, elastin,

the proteoglycans laminin and fibronectin, and glycosaminoglycans [5, 11, 12]. ECM

remodeling and turnover is carried out by matrix metalloproteinases secreted by the fibroblasts in


24/238

8

both physiological and pathological states. Several growth factors, cytokines, and other

bioactive molecules are also produced by cardiac fibroblasts highlighting their regulatory role in

the heart [9]. In light of their important function in the myocardium, there is an increasing body

of evidence suggesting the critical role of cardiac fibroblasts and other non-myocytes in the

development of engineered cardiac tissue. This will be discussed in further detail in section

2.4.3.

2.1.2 Extracellular Matrix Organization and Function

The myocardial extracellular matrix is made up of a fibrillar collagen network, basement

membranes, elastic fibers, proteoglycans, glycosaminoglycans and a variety of bioactive

signaling molecules (Figure 2.2a) [13]. This ECM is subdivided into three groups: the

endomysium, which surrounds individual myocardial cells; the perimysium, which surrounds

groups of myocytes; and the epimysium surrounding the entire muscle [14]. The specific

organization of the ECM layers aid in proper function of the tissue and relay important signaling

cues to the cardiac cells during normal physiology and disease.

Figure 2.2: Cardiac extracellular matrix: a) components and b) organization of endomysium and perimysium.

Images used with permission from MacKenna et al. [15] and Goldsmith and Borg [16] respectively.

The endomysium contains the basal lamina, encompassing individual cardiomyocytes,

and fibrillar collagens that form lateral connections between cells (Figure 2.2b). The function of

the endomysium is to support and align myocytes, aid in cell attachment, bring cardiomyocytes

together, and keep blood vessels close to cells for short diffusion distances of nutrients and


25/238

9

oxygen [17]. The perimysium is composed of fibrillar collagens, types I and III, in a weave that

connects the basal lamina of the endomysium to the large collagen fibers of the epimysium. The

thick collagen fibers of the epimysium are organized parallel to myofibrils protecting sarcomeres

from overstretch during relaxation [14]. In addition, this parallel organization allows forces to be

transmitted across the tissue layer during contraction to pump blood and aids in tissue elasticity

by pulling back on cardiomyocytes during relaxation. The unique organization of the

endomysium, perimysium, and epimysium, therefore, imparts mechanical integrity to the

myocardium necessary for the dynamic cardiac cycle.

Aside from the structural and functional role of the ECM during contraction and tissue

organization, the ECM also plays a critical role in transmitting signals to cardiac cells during

myocardial development, normal physiology, and in disease. The ECM, for example, provides

micro-structural cues to differentiating cardiomyocytes that regulate sarcomere self-assembly

and guide myofibrillogenesis [18, 19] and influences the rate of maturation of neonatal

cardiomyocytes [20]. Importantly, much of the regulatory information conveyed to cardiac cells

by the ECM is transmitted in the form of physical forces [21]. Cell attachment to the ECM is

primarily carried out by the transmembrane glycoprotein receptors, integrins, present at the cell

surface. Cardiac cells use integrins as mechanotransducers to sense mechanical stimuli within

the tissue leading to intracellular signaling and therefore a cellular response to stresses associated

with normal physiology and in pathological overload [21]. Mechanical forces can help maintain

normal cell shape and an oriented myofibrillar architecture, alter ECM production, gene

expression, cell size, phenotype, and expression and release of paracrine factors, increase

sensitivity to other signaling molecules, and upregulate cell-cell contacts important for the

electrical and mechanical properties of the tissue [15, 22-28]. The response of different cardiac

cells to mechanical forces is very complex and depends on the specific cell type and physical

state of the tissue. Taken together, the ECM is far more than a passive component of the

myocardium but rather an active structural, functional and regulatory component of this tissue.

2.1.3 Reparative Response of the Heart to Myocardial Infarctions

Cardiac tissue has a high demand for oxygen due to the high energy consumption

associated with muscle contraction. To ensure that active cardiomyocytes obtain a sufficient

supply of oxygen required to maintain aerobic respiration, the cells are in close proximity to

blood vessels of the coronary arteries. Reduced blood flow in the coronary arteries renders the


26/238

10

heart muscle susceptible to ischemic injury. Coronary artery disease refers to degenerative

changes in the coronary circulatory supply resulting in a reduction in the blood flow to the tissue

[29]. Sudden occlusion of the coronary arteries, or a myocardial infarction (MI), occurs in

severe cases of coronary artery disease leading to myocardial necrosis [29].

Several phases characterize the hearts wound repair process after a myocardial

infarction: cardiomyocyte death, acute inflammation, formation of granulation tissue, ventricular

remodeling, and the formation of organized collagen-rich scar tissue (reparative fibrosis) [30].

In reparative fibrosis, fibroblasts and myofibroblasts, both resident and recruited to the infarct

region, synthesize and deposit ECM proteins including collagen types I, III, and V [28]. While

ECM constituents are being produced, proteases are continuously degrading the ECM to allow

cell migration and remodeling to take place [30]. The end result of the reparative fibrosis is an

adaptive response that maintains the structural integrity of the ventricle, but replaces the injured

myocardial tissue with a dynamic non-contractile scar tissue [31]. Reactive fibrosis, which

occurs in the absence of cell loss around the insulted region, occurs alongside the reparative

fibrosis leading to enhanced myocardial stiffening, arrhythmias, and reduced systolic function

[28, 31]. The hearts response to myocardial insult, therefore, is a reparative one characterized by

a loss of contractile function and myocardial fibrosis. The initial myocardial injury and

secondary effects of the hearts reparative process leads to disruption of the normal cellular and

extracellular composition and organization and the progression to heart failure.

2.2 Matrix Metalloproteinases and their Role in Heart Remodeling andDisease

The matrix metalloproteinases (MMPs) are a family of zinc-binding endoproteinases that

are the driving force behind myocardial matrix remodeling. All MMPs share several functional

features; they degrade ECM components, are activated when zinc is removed from the active

site, need calcium for stability, function at neutral pH, and are inhibited by specific tissue

inhibitors of metalloproteinases (TIMPs) in a 1:1 stoichiometric ratio [32]. MMPs are localized

in the cardiac interstitial space as latent pro-enzymes requiring activation by autoproteolysis, the

serine protease plasmin, oxidized glutathione, or other activated MMPs [33, 34]. Several MMPs

and TIMPs have been identified in the heart and help to maintain normal ECM turnover. In the

developing mouse heart, Nutell et al. [35] found that different levels of MMP-2, MMP-3, MMP-

8, MMP-9, MMP-11, MMP-12, MMP-13, MMP-15, MMP-19, MMP-23, MMP-24, and MMP-


27/238

11

28 along with TIMP-1, TIMP-2, TIMP-3, and TIMP-4 are all expressed. Similarly, MMP-1,

MMP-2, MMP-3, MMP-9, MMP-13, and MMP-14 have all been found in human myocardium

[36], and it is likely that others are also expressed. The interplay between these different

proteases and inhibitors creates a balance that contributes to normal myocardial ECM structure

and function. Cardiac pathologies, however, cause an imbalance in these enzymes and play a

role in myocardial collagen accumulation, collagen fibril disruption, myocyte loss, and altered

spatial orientation of cells and intracellular components.

2.2.1 MMP Expression Following Myocardial Infarctions and in HeartFailure

Following a myocardial infarction and in the progression of heart failure, myocardial

fibrosis and remodeling occur due to an imbalance in ECM production, MMP activity, and TIMP

expression [37]. This dysregulation in ECM turnover is a response of cardiac and inflammatory

cells triggered by many different factors including various inflammatory cytokines, growth

factors, and mechanical stresses associated with myocardial injury and pressure overload [37-

39]. Although the exact expression profiles of MMPs and TIMPs depends on the cause, severity,

and stage of heart disease (Figure 2.3), significant increases in MMP-1, MMP-2, MMP-9, MMP-

13, and MMP-14 and reduced levels of TIMP-1, TIMP-3, and TIMP-4 have been observed in

human patients with heart disease [37]. In a study by Webb et al. [40], temporal profiling of

various plasma MMP and TIMP levels was performed on patients following a MI demonstratingthe dynamic changes in MMP and TIMP expression patterns over time. Although in this study

an elevation of MMP-9 levels was linked to left ventricular dilation and adverse myocardial

remodeling months after the initial insult, the exact contribution of the different MMPs and

TIMPs expressed in the progression of heart failure is difficult to determine. Genetic mouse

models, however, have revealed important insight into the role of some of these MMPs and

TIMPs in heart disease. Kim et al. [41] constitutively expressed MMP-1 in the heart and found

compensatory myocyte hypertrophy at 6 months and a loss of cardiac interstitial collagen

concurrent with a marked deterioration of systolic and diastolic function at 12 months. This

study directly demonstrated that disruption of the extracellular matrix in the heart reproduces the

changes observed in the progression of heart failure. Similarly, the targeted deletion of MMP-2

[42] and MMP-9 [43] in knockout mice after an induced myocardial infarction attenuated left

ventricular dilation and ventricular remodeling and improved cardiac function compared to wild


28/238

12

type controls. These studies clearly implicate the role of MMP-1, MMP-2, and MMP-9 in

adverse myocardial remodeling in the progression to heart failure following a MI and are being

investigated as potential targets for pharmaceutical intervention [38].

Figure 2.3: Alterations in MMP and TIMP levels in human heart disease. Italic lower case letters depict mRNA

levels, capital letters indicate protein levels, , and represent increase, decrease, and no change, respectively.

* denotes circulating plasma levels. Image used with permission from Kassiri and Khokha [37].

2.2.2 Cleavage Sites of ECM Proteins, Peptides and Biomaterials by MMPs

A priori knowledge of the expression profiles and key proteases involved in ECM

remodeling following a myocardial infarction and in heart failure not only identifies key targets

for therapeutic intervention but also allows the development of techniques that exploit the

presence of those enzymes to achieve a particular goal. In tissue engineering, specific sequences

have been incorporated into biomaterial scaffolds that make them susceptible to degradation by

MMPs expressed in various events, such as ECM remodeling, cell migration, angiogenesis, and

wound healing. Biological materials derived from the ECM inherently have these sequences

contained within them and therefore may naturally be degraded by the MMPs. Synthetic

materials, however, may also be developed to incorporate specific MMP-sensitive sequences

conferring unique biological function to these synthetic polymers. When designing novel

enzyme-degradable biomaterials for cardiac tissue engineering, MMP-1, MMP-2, and MMP-9

are rational targets due to their role in heart disease.

After being activated, MMP-1 functions by cleaving various collagens at specific sites in

the native triple helical structure. MMP-1 cleaves collagen type I at Gly775-Ile776 in the 1(I)


29/238

13

chain and Gly775-Leu776in the 2(I) chain, collagen type II at Gly775-Leu776in the 1(II) chain,

and collagen type III at Gly775-Leu776 in the 1(III) chain [44, 45]. This specific and

characteristic cleavage of collagens at approximately the length of the collagen fiber from the

N-terminus leads to two fragments that lose stability and unfold to produce single -chains

called gelatins. Further breakdown of the gelatins and short peptides is not as specific as in the

intact collagen but is traditionally carried out by the gelatinases, MMP-2 and MMP-9, and may

occur at other Gly-Leu and Gly-Ile sites in the polypeptide chains [45]. In addition to MMP-1

breaking down collagens and MMP-2 and MMP-9 degrading gelatins, each of these enzymes are

able to cleave a broad range of substrates, including various collagens, gelatins, elastin,

proteoglycans, regulatory molecules, and other ECM proteins [45-48]. An important result of an

early MMP-1 study was that specificity with this enzyme is largely independent of substrate

conformation and reflects the cleavage site and surrounding amino acid sequences in the native

proteins [49]. As a result, much information has been generated on the specificity requirements

of MMPs by measuring the kinetics of cleaving various short peptide sequences [45]. It was

determined that the recognition of MMPs is based on short peptide sequences up to seven amino

acids in length, three or four amino acids on either side of the scissile bond, and the rate of

cleavage by specific MMPs is determined by the sequence chosen [45]. Several peptides cleaved

by many MMPs do so at Gly-Leu and Gly-Ile sites, mimicking sequences cleaved in native ECM

proteins [45], thus identifying potential sequences and target bonds that may be used inbiomaterial design.

Pioneering work by West and Hubbell [50] incorporated short peptide sequences into

synthetic hydrogel systems making them susceptible to degradation by the cell secreted

proteases, collagenase and plasmin. Hydrogels were developed using the sequences Ala-Pro-

Gly-Leu, with cleavage between the Gly and Leu residues, and Val-Arg-Asn, with cleavage

between the Arg and Asn residues, for degradation by collagenase and plasmin respectively [50].

Subsequent work by Guan and Wagner [51] involved the formation of an elastase sensitive

segmented polyurethane by using the tri-peptide Ala-Ala-Lys in the backbone structure of the

polymer. A critical finding in this study was incorporation of the cleavage site of elastase alone

(Ala-Ala) was enough to confer biological function to the material. Taken together, these

findings suggest that targeting the Gly-Leu and Gly-Ile cleavage sites of MMP-1, MMP-2, and

MMP-9 may confer protease-sensitivity to synthetic biomaterials and may aid in the rational


30/238

14

design of biomaterials for cardiac tissue engineering that seek to exploit the presence of these

enzymes.

2.3 Regenerative Approaches to Repair the Heart

The need for new therapeutic options to treat an infarcted or failing heart has motivated

researchers to establish techniques to repair, replace, or augment the function of the diseased or

injured tissue. Current techniques being investigated to achieve this fall into a few broad

categories: 1) induction or stimulation of endogenous mechanisms of cardiac repair and

regeneration; 2) the direct transplantation of cells into the damaged tissue; or 3) the use of

biomaterials on their own or in combination with 1 and/or 2 to engineer cardiac tissue either ex

vivofor subsequent transplantation or in situ. Each of these different approaches has potential in

the treatment of heart disease, but it is important to distinguish improvements in physiological

function that occur due to myocardial regeneration and those that occur due to other

mechanisms, such as improved vascularization, reduced scar size, and enhanced cell survival.

Murry et al. [52] recommended that to prove heart regeneration has been achieved, structural,

physiological, and molecular end points must be used to demonstrate the technique has resulted

in newly created cardiomyocytes that are electromechanically connected to host myocardium and

contribute to cardiac function. Any improvement to cardiac function is important for the

treatment of heart disease and warrants extensive investigation, but only the approaches that may

lead to true myocardial regeneration will be discussed here.

2.3.1 Inducing Endogenous Mechanisms in Heart Repair

Adult cardiomyocytes have traditionally been considered terminally differentiated cells

that are incapable of proliferating to any significant degree and are therefore unable to regenerate

an injured or diseased heart. Although the inability of the heart to regenerate considerably on its

own appears acceptable, work performed in this field has challenged the view that the heart does

not regenerate at all and stem and progenitor cells that have cardiomyogenic potential have been

identified in the adult heart. Beltrami et al. [53] isolated Lin- c-kitPos cells from the adult rat

heart that were self renewing, clonogenic, and multipotent, giving rise to cardiomyocytes,

smooth muscle cells, and endothelial cells. This same group subsequently identified and isolated

similar c-kitPos

cardiac stem cells from human hearts, which mimicked the properties of those

from the rat heart, and demonstrated these cells could form new myocardium in infarcted animal


31/238

15

models independent of cell fusion [54]. Oh et al. [55] demonstrated the presence of Sca-1+(c-

kitNeg

) cardiac progenitor cells in the adult murine heart that can differentiate into cells

expressing several cardiac-specific markers in vitro. In response to a myocardial infarction,

endogenous Sca-1+ cells were not mobilized but transplanted Sca-1

+ cells homed and

differentiated into cardiac cells in the infarct border zone, half of which fused with host

cardiomyocytes [55]. Matsuura et al. [56] similarly isolated Sca-1+cells from the adult murine

heart and differentiated these cells in vitro into cardiomyocytes that expressed cardiac

transcription factors and contractile proteins, displayed sarcomeric structures, and contracted

spontaneously. Sca-1+ cardiac progenitor cells have also recently been isolated from adult

human hearts and demonstrate the same potential for deriving cardiomyocytes in vitroas their

mouse equivalents [57]. Martin et al. [58] used an ATP-binding cassette transporter, Abcg2, as a

marker for cardiac side population cells found in the developing and adult murine heart that may

function as a progenitor cell population in developing, maintaining, and repairing the heart. In

addition, Isl1+cardiac progenitor cells that give rise to cardiomyocytes, smooth muscle cells and

endothelial cells have also been identified in embryonic and postnatal hearts, but it remains

unclear what potential they have in the adult heart [59, 60]. Taken together, several studies have

identified different resident cardiac stem and progenitor cells in an adult heart that may have

potential in regenerating injured myocardium. Evidence has been presented that suggests some

of these cells are activated by injury and inherently contribute to heart regeneration [61], but if it

is occurring, it is not significant on its own to regenerate the tissue. Thus, the next step in

achieving myocardial repair using the bodys endogenous regenerative capacity is determining

how to induce these cells to regenerate significant portions of the injured heart.

2.3.2 Cellular Cardiomyoplasty

Cellular cardiomyoplasty, or cell transplantation, is a technique that seeks to promote

cardiac regeneration by introducing cells either directly to the site of injury or to the blood

supply for subsequent homing and integration. This cell-based approach attempts to directlyaddress the fundamental consequence of a myocardial infarction and a critical component of

heart failure progression; the loss of cardiomyocytes. By introducing cells into the injured heart,

it was hypothesized that the new cells could adapt to the unique myocardial microenvironment

and replace the function of the dead cardiomyocytes. Skeletal myoblasts were the first cell type

to be chosen for transplantation into an infarcted heart and numerous cell types have


32/238

16

subsequently been investigated including fetal and neonatal cardiomyocytes, bone marrow-

derived stem cells, endothelial progenitor cells, resident cardiac stem cells, and both mouse and

human embryonic stem cells. Table 2.1 provides a full list of potential cells for myocardial

repair along with some advantages and disadvantages of each for this application. Although

several of these cell types have been shown to improve cardiac function when transplanted in an

infarcted heart and have prompted clinical trials, relatively few of them result in true myocardial

regeneration. Using the recommended guidelines for defining heart regeneration by Murry et al.

Table 2.1: List of cell types considered for cardiac repair. Used with permission from Chen et al. [62].

Cell Source AutologousEasily

Obtainable

Highly

Expandable

Cardiac

Myogenesis

Clinical

TrialSafety

Somatic Cells

FetalCardiomyocytes

No No No Yes No No

Skeletal Myoblasts Yes YesDepends on

ageNo Yes

Yes,

arrhythmias

Smooth Muscle

CellsYes Yes Yes No No No

Fibroblasts Yes Yes Yes No No No

Stem and

Progenitor Cells

Mesenchymal

Stem CellsYes No

Depends on

ageDebated No

Yes, fibrosis

calcification

EndothelialProgenitor Cells

Yes YesDepends on

ageDebated No

Yes,calcification

Crude Bone

Marrow CellsYes Yes

Depends on

ageDebated Yes

Yes,

calcification

Umbilical CordCells

No Yes Yes Debated No No

Hematopoietic

Stem CellsYes Yes Yes Debated No Yes

Embryonic Stem

CellsNo No Yes Yes No

Yes, potential

teratomas


33/238

17

[52], the ideal cell source for cellular cardiomyoplasty should meet the following criteria: easy to

isolate and expand in vitro; achieve electrical and mechanical integration with host myocardium;

contribute to the structural organization and contractile performance of the heart; and be able to

attain an adult cardiomyocyte phenotype. The literature on cellular cardiomyoplasty is quite

large, so only a brief discussion of the cell types that may lead to true myocardial regeneration

will be presented.

2.3.2.1 Fetal and Neonatal Cardiomyocytes

To replace the lost cells associated with a MI, cardiomyocytes are the logical cell choice

for cell-based therapies. Early proof-of-principle work demonstrated transplanted fetal and

neonatal cardiomyocytes can form stable grafts in the myocardium that are electromechanically

connected to host cells via intercalated discs in normal and infarcted hearts [63-65]. Stable

integration of these cells into injured hearts resulted in decreased scar tissue formation, increased

angiogenesis and vascularization, reduced dilatation, and improved ventricular function as

measured using several different techniques [66-71]. Fetal and neonatal cardiomyocytes,

therefore, appear to meet several of the criteria as an ideal cell type for cellular cardiomyoplasty:

electromechanical coupling with host; structural and functional contribution to myocardium; and

the potential of an adult cardiomyocyte phenotype. Importantly, these transplantation studies

provide significant evidence to support the hypothesis that true myocardial regeneration may be

achieved in an infarcted heart and offers continued hope for the development of regenerative

therapeutic options for myocardial repair. Unfortunately, there are a few caveats associated with

the transplantation of fetal and neonatal cardiomyocytes preventing their use in the clinical

setting. First and foremost, human fetal and neonatal cardiomyocytes cannot be used due to

ethical considerations associated with their origin and consequences of harvesting. While

meeting most of the criteria of an ideal cell source for this work, they fall short on being easy to

isolate and expand. Second, only a limited number of transplanted cardiomyocytes engrafted

and survived in the injured heart resulting in the replacement of only a small fraction of theinfarct scar [72, 73]. The low cell engraftment number may be due to cardiomyocyte death

caused by ischemia [73]. These results suggest that a fundamental limitation in cellular

cardiomyoplasty is the delivery, engraftment, and survival of a sufficient number of cells into the

heart to replace the lost cardiac function. Methods to overcome this obstacle may be provided

through the use of biomaterials and will be discussed in greater detail below.


34/238

18

2.3.2.2 Embryonic Stem Cell-Derived Cardiomyocytes

Embryonic stem cells (ESCs) have the unique advantage over adult stem cells in that they

have the potential of providing a potentially unlimited source of new cardiomyocytes.

Embryonic stem cells are derived from the inner cell mass of the blastocyst stage developing

mammalian embryo [74, 75]. ESCs derived from mice (mESCs) are pluripotent cells capable of

long term undifferentiated proliferation in vitrowhile retaining the developmental potential of

forming all three embryonic germ layers; endoderm, mesoderm, and ectoderm [76]. Human

ESCs (hESCs) have similar capabilities as mESCs but have the unique advantage of also being

able to give rise to the trophoblast, an extra-embryonic tissue [75, 77]. Being able to give rise to

mesodermal cells, mESCs and hESCs are capable of differentiating into cardiomyocytes with

similar characteristics as those found in vivo and therefore provide a potential source of new

cardiomyocytes for cardiac repair [78, 79]. The potential use of ESCs in myocardial

regeneration, however, requires several criteria be met: 1) a sufficient number of starting ES

cells; 2) efficient and directed differentiation into cardiac progenitor cells or cardiomyocytes; 3)

high production of ESC-derived cardiac progenitors or myocytes; 4) a highly pure population of

desired cells; and 5) resulting phenotype and function similar to adult cardiomyocytes. To be

used in the clinical setting, these criteria must be proven with hESCs. Information and

established techniques for culturing, differentiating, and genetically manipulating murine ESCs,

however, make them a useful model for studying the potential of embryonic stem cell-derived

cardiomyocytes (ESCDCs) in cardiac regeneration. In relation to work conducted in this thesis,

this discussion will mostly focus on mESCs.

To help identify the potential of using ESCs for cellular cardiomyoplasty, several

investigators looked at directly injecting undifferentiated mESCs into the myocardium to test if

the unique microenvironment can drive the differentiation of the cells towards the cardiac

lineage without any adverse affects. Behfar et al. [80] found that transplanted undifferentiated

mESCs differentiated into cardiomyocytes and became functionally integrated into normal and

infarcted myocardium. This work suggests that the host myocardium creates an environment

that can commit undifferentiated mESCs to a specific cardiac lineage and the functionally

integrated cells can lead to an improvement in cardiac function. Similar studies by Min et al.

[81, 82] demonstrated the survival, engraftment, and differentiation of mESCs into mature

cardiac myocytes that attenuated left ventricular hypertrophy, reduced infarct size, improved left


35/238

19

ventricular contractility, and increased angiogenesis within the infarcted region. While these

results seem promising for the use of undifferentiated ESCs directly, Nussbaum et al. [83] found

that normal or infarcted hearts do not provide the appropriate cues to guide undifferentiated

mESCs towards a cardiomyocyte fate, but rather leads to teratoma formation and subsequent

rejection in immunocompetent mice. Other groups have similarly found teratoma formation is

the consequence of transplanting undifferentiated mESCs in the heart and other tissues and

represents a major concern for their clinical use [84-86]. As a result of this controversy, a more

restricted ESC-derivative may be more appropriate for use in these studies.

2.3.2.2.1 Differentiation of Murine Embryonic Stem Cells intoCardiomyocytes

Murine ESCs are maintained in an undifferentiated state by coculturing with an

embryonic fibroblast (MEF) feeder layer or by the soluble factor leukemia inhibitory factor (LIF)

and can potentially lead to indefinite self renewal and the generation of a large number of these

cells [87]. Removal of LIF from the culture medium induces differentiation into multiple cell

types and is correlated with a change in expression of the transcription factor Oct-4, a marker of

undifferentiated cells [88]. In vitro differentiation is accomplished via the formation of

aggregated ESCs, called embryoid bodies (EBs), in the absence of LIF leading to the formation

of a number of specialized cells, including cardiomyocytes [89].

Cardiomyocytes derived from mESCs exhibit varying levels of development, whichmimics in vivo differentiation, and appears to be a function of time in culture [89]. In early

beating EBs, mESCDCs may appear as small, round cells with sparse and irregular myofibrils or

more rod-shaped with parallel bundles of myofibrils and A and I bands [78]. As culture time

progresses, cell size increases, ranging greatly from neonatal (diameter ~7-9 m and length ~20-

45 m) to adult dimensions (diameter ~10-30 m and length ~80-150 m), myofibrils become

densely packed and well organized, and sarcomeres have defined A, I, and Z bands [78]. In

addition, the more developed cells form nascent intercalated discs, with desmosomes, fascia

adherens junctions and gap junctions, and the gap junctions are functional as demonstrated

through dye transfer studies [78]. The cardiac gene expression pattern of mESCDCs follows the

developmental pattern of cardiomyocytes in vivowith the expression of GATA-4 and Nkx2.5

observed prior to ANP, myosin light chain-2v (MLC-2v), and -myosin heavy chain, Na+-Ca

2+

exchanger, and phospholamban [89]. Sarcomeric protein expression in mESCDCs also follows a


36/238

20

progressive developmental pattern observed in vivo [89]. In addition, early mESCDCs express

slow skeletal muscle troponin I, a greater proportion of -myosin heavy chain, and have a high

sensitivity to calcium similarly seen in embryonic cardiomyocytes [90-92]. Increased culture

time leads to a shift from these fetal isotypes to cardiac troponin I, -myosin heavy chain, and

decreased sensitivity to Ca2+

more characteristic of mature neonatal and adult cardiomyocytes.

The specialized cardiomyocyte types undergo a shift from pacemaker-like cells early to purkinje-

like cells in the intermediate and atrial and ventricular cells later on [93]. Functionally, the

mESCDCs spontaneously contract, exhibit many features of the excitation-contraction coupling

mechanism found in isolated fetal and neonatal cells, express all major cardiac-specific ion

channels, and may respond to pharmacological agents at later stages of development [89, 93].

Taken together, mESCs can differentiate into cardiomyocytes that initially appear as embryonic-

like cells, but with increased time in culture mature and express more neonatal and adult-like

phenotypes.

Cardiomyocytes spontaneously form in differentiating EBs, but the actual yield of

cardiomyocytes derived from mESCs depends on a number of factors including starting EB size,

culture medium and conditions, ES cell line being used, and time of EB plating [94]. EBs

resemble early post-implantation embryos and the signaling events that drive differentiation into

the different specialized cell lineages loosely mimic those that occur during normal development.

As a result, several extrinsic factors that play a role in cardiomyogenesis in vivo similarly

promote cardiomyocyte formation within EBs. Numerous growth factors and signaling proteins

have been identified that help drive differentiation towards the cardiac lineage in a concentration

and temporal manner including TGF-1, bone morphogenic protein (BMP)-2, BMP-4, insulin-

like growth factor-1, fibroblast growth factor, hepatocyte growth factor, platelet-derived growth

factor, activin, oxytocin, Wnt/-catenin inhibition, and erythropoietin [95, 96]. Similarly,

synthetic compounds, such as dimethyl sulfoxide, 5-azacytadine, ascorbic acid, retinoic acid,

opioid, and dynorphin, and free radicals and reactive oxygen species have also been shown to

stimulate cardiomyogenesis [95, 96]. In addition, the physical microenvironment that the

mESCs are placed in may contribute to driving differentiation towards the cardiac lineage.

Features such as matrix composition, topography, 3-D structure, rigidity, and mechanical

stimulation may influence mESCDC yields [97]. Coculture with various cells, such as visceral

endoderm-like cells, may be another method for promoting cardiomyocytes from mESCs [98].


37/238

21

Taken together, the identified factors may help drive cells towards the cardiac lineage and

increase the percentage of cardiomyocytes, but have not been able to yield a large and pure

population of ESC-derived cardiac progenitor cells or cardiomyocytes, a requirement for use in

many regenerative applications.

2.3.2.2.2 Large-scale Production of a Pure Population of Embryonic StemCell-Derived Cardiomyocytes

One requirement for the successful implementation of ESCs for regenerating the

myocardium is a large and pure population of cardiac progenitors or fully differentiated

cardiomyocytes. As discussed above, any undifferentiated ESCs used in these applications may

lead to undesirable teratoma formation. Similarly, even with the addition of factors to drive cells

towards the cardiac lineage, ESC differentiation results in a mixture of cell types that could have

deleterious consequences when transplanted into the heart.

The recognized need for obtaining a pure population of cells has led to a few novel

techniques for selecting ESCDCs. The first approach involves genetic manipulation of the ESCs

to introduce antibiotic resistance to ESCDCs. Fields group developed a simple system that

inserted a fusion gene carrying two transcriptional units into mESCs [99]. The fusion gene

contained a phosphoglycerate kinase promoter driving hygromycin resistance gene (pGK-hydror)

to select for mESCs that were stably transfected and a -myosin heavy chain promoter driving an

aminoglycoside phosphotransferase gene (MHC-neor

), which allowed for the selection ofmESCDCs by adding the neomycin analog geneticin (G418) to culture medium. The mESCDCs

selected by this method were >99% pure and expressed markers of highly differentiated cardiac

cells [99]. Kolossov et al. [85] used a similar approach to get a highly purified mESCDC

population (>99%) by having the -myosin heavy chain promoter drive both a puromycin

resistance gene and a green fluorescent protein (GFP) gene for purification and identification of

the cells. Other groups have also used this genetic selection method for purifying

cardiomyocytes from mESCs and hESCs [100-102]. A second approach to purifying ESCDCs is

to label cell surface markers with fluorescent or magnetic tags and to use fluorescence activated

cell sorting (FACS) or magnetic-activated cell sorting. If appropriate cell surface markers

become available, this approach is advantageous as it avoids any need to genetically modify the

cells. Cell surface markers for cardiac progenitor cells or cardiomyocytes are currently not

known [103], but the molecular signature of ESCDCs is being explored [104] and may lead to


38/238

22

potential markers to be used in this purification scheme. Still, the proof of concept for purifying

mESCDCs by FACS has been demonstrated by Muller et al. [105] using a transgenic mESC line

expressing GFP under the cardiac chamber-specific promoter MLC-2v. A Percoll gradient

separation followed by FACS resulted in a >97% pure cardiomyocyte population and

electrophysiological tests identified the cells were preferentially ventricular-like [105]. Hidaka

et al. [106] also used FACS to purify Nkx2.5 positive cardiac progenitor cells from mESCs and

showed the resulting cells differentiated into sinoatrial node, atrial, or ventricular-like cells.

Transgenic GFP expression has been used to identify other cardiac progenitor cells or specialized

cardiomyocyte types including pacemaker, atrial, and ventricular cells [80, 107, 108], suggesting

these cells may also be separated by the FACS approach. Other methods to help purify ESCDCs

are a Percoll gradient separation and manually picking out beating cells, but both methods only

lead to an enriched culture of mESCDCs and these heterogeneous cell populations may inhibit

clinical acceptance [103].

The large number of cells required for use in cell-based regenerative strategies for

myocardial repair has motivated researchers to develop systems for the large-scale production of

ESCDCs. The Zandstra group has been particularly interested in developing and optimizing

bioreactor parameters for the generation of large quantities of ESCDCs. In an early study,

Zandstra et al [109] aggregated MHC-neor/pGK-hygro

rmESCs in static culture for 4 days and

transferred the EBs to a spinner flask system for subsequent growth and differentiation. On day

9 after initiating differentiation, medium was supplemented with G418 and retinoic acid to select

for and drive differentiation towards cardiomyocytes. A relatively pure mESCDC population

was harvested from the spinner flasks on day 18 with no undifferentiated mESCs and the cells

were spontaneously beating and expressed characteristic markers of mESCDCs [109].

Importantly, this system allowed the generation of ~1.4 x 107 cells in a 250 ml spinner flask

using the CM7/1 mESC line, thus identifying the large-scale production of mESCDCs.

Subsequent work by this group optimized cell and bioreactor conditions to produce even greater

numbers of ESCDCs. Bauwens et al. [110] used a similar approach but encapsulated the EBs in

a hydrogel to prevent aggregation and generated nearly 24 times more ES cell-derived

cardiomyocytes (~3.15 ESCDC per input mESC) than in unencapsulated controls (~0.15 ESCDC

per input ESC) after 9 days of differentiation and 5 days of selection using the D3 mESC line.

These mESCDC yields were increased even further (~3.77 mESCDC/ESC) by perfusion feeding


39/238

23

the bioreactor system and culturing under hypoxic conditions (~4% O2 compared to normoxic

levels of ~20%). Recently, Niebruegge et al. [111] directly inoculated bulb-shaped glass spinner

flasks with 2 x 105 CM7/1 mESCs per ml of culture medium for EB formation within the

bioreactor system. Optimization of several parameters including addition of retinoic acid at day

7 instead of day 9, starting selection at day 11 not day 9, and changing 50% of medium every

other day instead of every day resulted in a 4.3 fold increase in number of mESCDCs (~7.6

ESCDC/input mESC for optimized conditions vs. ~1.8 for unoptimized method) with a total of

19 x 107 cardiomyocytes in the 250 ml spinner flask [111]. Similar optimization efforts have

been reported by this group for the large scale generation of human ESCDCs. Factors such as a

homogenous starting EB size, a stirred suspension bioreactor, and hypoxic culture conditions are

more conducive of cardiomyocyte generation and improve hESCDC yields [112, 113].

Ultimately, the ability to derive a large and pure population of cardiomyocytes or cardiac

progenitor cells from ESCs is a critical step in identifying a cell source for regenerating the

myocardium.

2.3.2.2.3 Transplantation of Murine and Human ESC-derived Cells into theHeart

Several studies have been conducted to transplant murine and human ESCDC or cardiac-

committed ESCs for cellular cardiomyoplasty. In a study by Klug et al. [99], a highly pure

population of mESCDCs were transplanted into the heart and formed stable intracardiac graftsout to at least 7 weeks with a similar frequency of engraftment as fetal murine cardiomyocytes.

Menard et al. [114] transplanted cardiac-committed mESCs into infarcted sheep myocardium and

demonstrated the cells successfully engrafted into the infarct region, differentiated into mature

cardiomyocytes that were electrically connected to host myocardium, improved left ventricular

ejection fraction, and may avoid immune rejection. Kolossov et al. [85] injected purified

mESCDCs either alone or with an equal number of mouse embryonic fibroblasts into an

infarcted heart. It was determined that the mESCDCs had very low engraftment frequency when

injected on their own but was significantly increased when transplanted together with the

fibroblasts. The engrafted mESCDCs formed mature sarcomeric structures, electrically coupled

with host cardiomyocytes, did not develop into teratomas, contributed to ventricular force

contraction, and improved left ventricular function [85]. Thus, using mESCs to form cardiac


40/238

24

committed cells or a pure population of cardiomyocytes avoids the concern of teratoma

formation and may contribute to true myocardial regeneration.

While mESCs provide a good model for studying development, disease, and the potential

of ESCs in regenerative medicine, ultimately hESCs must be used if this technology is ever

going to transfer to the clinical setting. Several studies have been conducted using hESCs for

cellular cardiomyoplasty in animal models. Although the transplantation of undifferentiated

hESCs into an infarcted animal myocardium may help drive differentiation towards the

cardiomyogenic lineage without teratoma formation [115], the lessons learned from mESCs

along with the finding that these cells do form teratomas [116, 117] suggests hESCs must be at

least somewhat committed if they are going to gain clinical acceptance. As a result, recent work

has investigated transplanting hESC-derived cardiomyocytes (hESCDCs) or cardiac committed

hESCs into normal and infarcted animal hearts. Evidence has been provided that hESCDCs

survive, proliferate, form mature contractile structures, and electrically couple to host cells

following transplantation into healthy hearts of immunodeficient mice and rats [116, 118, 119].

In infarcted rodent myocardium, cardiac-committed hESCs and hESCDCs similarly appear to

survive and form a mature cardiomyocyte phenotype without any teratoma formation [116, 117,

119-122]. In addition, several of these investigations report an improvement to cardiac function

due to the engraftment of the cells [116, 117, 119, 121].

A few limitations associated with hESCDC transplantation studies include an inefficient

hESC differentiation into cardiomyocytes, a heterogeneous cell population, poor cell survival

after transplantation, and a transient contribution to cardiac function. In an attempt to overcome

some of these limitations, Laflamme et al. [121] treated a high density monolayer of

undifferentiated hESCs with two cytokines to drive differentiation towards the cardiac lineage

and enriched the hESCDCs by a Percoll gradient to get a ~83% pure cardiomyocyte culture (3:1

ratio of generated cardiomyocyte to input hESC). Transplantation of these cells along with a

mixture of prosurvival reagents into infarcted rat hearts resulted in large muscular grafts of

human myocardium in the central infarct region that coupled to host tissue and significantly

improved ventricular structure and contractile function compared to appropriate controls [121].

Despite long-term survival of the transplanted cells, the exact contribution they have on cardiac

function and their long-term benefit remains unclear [119, 123].


41/238

25

While ESC appear to be the best candidate for regenerating the myocardium, ethical

considerations associated with harvesting human ESCs has limited wide-spread funding and use

of these cells. Induced pluripotent stem (iPS) cells may offer an alternative source of

cardiomyocytes [124] that may reduce ethical concerns, but this technology is still in a very early

stage with many obstacles to overcome before this option is viable. Regardless of the cell type

or source, cellular cardiomyoplasty on its own is limited by the delivery, engraftment, and

survival of cells in the heart. Interestingly, the prosurvival reagents used by Laflamme et al.

[121] for achieving improved long-term engraftment of hESCDCs included Matrigel, a

gelatinous protein mixture rich in structural extracellular matrix proteins and growth factors. In

this situation, Matrigel acted as a supportive matrix for the cells to adhere to, thus increasing cell

survival. The use of biomaterial scaffolds as a delivery vehicle for cells may overcome the

limitations of cellular cardiomyoplasty and may help to contribute to long-term clinically

relevant cardiac repair.

2.3.3 Cardiac Tissue Engineering

Cardiac tissue engineering is a technique that employs the use of biomaterials in

combination with cells and/or various signaling agents towards the development of viable tissue

constructs for regenerating the myocardium. Research in this area has expanded tremendously

over the past several years and continues to grow, but the goal remains the same and to date, a

few promising strategies have emerged. These approaches include using cells along with: 1)

biodegradable synthetic and natural-based scaffolds with pre-formed three-dimensional

structures; 2) biodegradable synthetic and natural-based materials with undefined structures; 3)

injectable biomaterials for in situ myocardial regeneration; and 4) temperature-responsive

biomaterials that act as a substrate for cell sheet formation. The focus of this thesis is on the first

of the four approaches, but a brief discussion of the other three will first be presented.

2.3.3.1 Myocardial Tissue Engineering Using Biomaterials with Undefined

StructuresCardiac cells have an endogenous ability to organize into native-like car

Date post:	24-Feb-2018
Category:	Documents
Upload:	adga-rwerwe
View:	223 times
Download:	0 times

Parrag Ian C 201006 PhD Thesis.pdf

Documents