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
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 organized
fibers 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
ii
MEFs on aligned and unaligned Phe PU scaffolds revealed both architectures supported adherent
and functionally contractile cells.
improved the organization and differentiation of mESCDCs suggesting these two parameters are
important for developing engineered myocardial constructs using mESCDCs and PU scaffolds.
iii
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.
Engineering and Applied Chemistry and OGSST was much appreciated. It has been a real
pleasure 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.
iv
Table of Contents
Chapter 1: Introduction...1
1.0.
Clinical Problem......1
1.1.
Hypothesis....2
1.2.
Research Objectives.3
1.3
References3
Introduction......5
2.1
Heart Tissue....5
2.1.1
Myocardial Cells.6
2.4.
References..58
Abstract..84
3.1.
Introduction85
3.2.
vi
Conclusions..112
3.5.
References113
Abstract116
4.1
Introduction..117
4.2.
Conclusions..141
vii
4.5.
References142
Abstract146
5.1.
Introduction..146
5.2.
Conclusions..166
5.5.
References166
Abstract170
6.1.
Introduction..171
6.2.
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.
viii
Conclusions..203
6.5.
References203
Chapter 7: Conclusions209
7.0.
Conclusions.209
7.1.
7.2.
Future Work.214
References217
ix
List of Figures
Figure 2.1.
Figure 2.2.
Figure 2.3.
Figure 2.4.
Figure 2.5.
Figure 2.6.
Figure 2.7.
Figure 2.8.
Figure 2.9.
Figure 3.1.
Figure 3.2.
Figure 3.3.
Figure 3.4.
Figure 3.5.
Figure 3.6.
Figure 3.7.
Figure 3.8.
Figure 3.9.
Figure 3.10.
Preparative column HPLC purification of chain extender using low and high
pH 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.
Figure 3.13.
Figure 3.14.
Figure 3.15.
Figure 4.1.
Figure 4.2.
Figure 4.3.
Figure 4.4.
Figure 4.5.
Figure 4.6.
Figure 4.7.
Figure 4.8.
Figure 4.9.
Figure 5.1.
Figure 5.2.
Figure 5.3.
Figure 5.4.
Figure 5.5.
Figure 5.6.
Figure 5.7.
Figure 5.8.
Figure 6.1.
Figure 6.2.
Figure 6.3.
Figure 6.4.
Figure 6.5.
Figure 6.6.
Figure 6.7.
Figure 6.8.
Figure 6.9.
Figure 6.10.
Figure 6.11.
Figure 6.12.
Figure A.1.
Figure A.2.
Figure A.3.
Figure A.4.
xii
List of Tables
Table 2.1.
Table 2.2.
Table 2.3.
Table 3.1.
Molecular weight averages for PUs containing Phe and Gly-Leu-based chain
extenders..107
Table 3.2.
Table 3.3.
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 varying
architecture...134
Table 4.3.
Table 4.4.
Table 6.1.
Table 6.2.
Table 6.3.
xiii
List of Abbreviations
3-D
three-dimensional
ACN
acetonitrile
ANP
APMA
4-aminophenylmercuric acetate
BMP
BV
blood vessels
CB
cardiac body
CDM
1,4-cyclohexane dimethanol
cTnT
Cx-43
connexin-43
DAPI
4',6-diamidino-2-phenylindole
DCM
dichloromethane
DMEM
Dnp
DSC
EB
embryoid body
ECM
extracellular matrix
EHT
ESC
ESI
electrospray ionization
FACS
FBGC
FBS
FS-6
FT-IR
G418
G-CSF
xiv
GFP
Gly
glycine
Gly-Leu PU segmented polyurethane composed of PCL of molecular weight 1250, LDI, and a
Gly-Leu-based chain extender
GPC
hESC
hESCDC
HPLC
HOCl
hypochlorous acid
LDI
lysine-based diisocyanate
Leu
leucine
LIF
Mca
MDM
monocyte-derived macrophage
mESC
mESCDC
MHC
MHC-neor
MI
myocardial infarction
MLC-2v
MMP
matrix metalloproteinase
NMR
ONOO-
peroxynitrite
PBS
PCL1250
PGA
poly(glycolic acid)
PGS
poly(glycerol sebacate)
Phe
phenylalanine
Phe PU
xv
poly (N-isopropylacrylamide)
PLA
poly(lactic acid)
PLGA
poly(lactic-co-glycolic acid)
PMN
neutrophils; polymorphonucleocytes
PTSA
PU
polyurethane
SDS
TCPS
TFA
trifluoroacetic acid
Tg
TGF
TIPS
Tm
melting temperature
xvi
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 $448.5
billion 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.
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.
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.
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) containing
biodegradable 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.
1.3 References
1.
Heart and Stroke Foundation of Canada, Statistics and Background Information Incidence of Cardiovascular Diseases. 1998.
2.
3.
American Heart Association, Heart disease and stroke statistics - 2008 update. American
Heart Association, 2008.
4.
Kumar, V., R.S. Cotran, and S.L. Robbins, Basic Pathology. 7th ed. 2003, Philadelphia:
Saunders. xii, 873.
5.
6.
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 the
International Society for Heart and Lung Transplantation: Third Annual Report - 2005.
Journal Of Heart And Lung Transplantation, 2005. 24(9): p. 1182-1187.
8.
Burkhoff, D., S. Klotz, and D.M. Mancini, LVAD-Induced reverse remodeling: Basic and
clinical implications for myocardial recovery. Journal Of Cardiac Failure, 2006. 12(3): p.
227-239.
9.
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.
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
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.
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.
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.
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.
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.
10
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.
11
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].
Synthetic
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)
12
chain and Gly775-Leu776 in the 2(I) chain, collagen type II at Gly775-Leu776 in the 1(II) chain,
and collagen type III at Gly775-Leu776 in the 1(III) chain [44, 45].
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 in
biomaterial 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-ProGly-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
13
design of biomaterials for cardiac tissue engineering that seek to exploit the presence of these
enzymes.
treatment of heart disease and warrants extensive investigation, but only the approaches that may
lead to true myocardial regeneration will be discussed here.
14
models independent of cell fusion [54]. Oh et al. [55] demonstrated the presence of Sca-1+ (ckitNeg) 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 vitro as 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.
subsequently been investigated including fetal and neonatal cardiomyocytes, bone marrowderived 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].
Easily
Highly
Cardiac
Clinical
Cell Source
Autologous
Safety
Obtainable
Expandable
Myogenesis
Trial
Somatic Cells
Fetal
Cardiomyocytes
No
No
No
Yes
No
No
Skeletal Myoblasts
Yes
Yes
Depends on
age
No
Yes
Yes,
arrhythmias
Smooth Muscle
Cells
Yes
Yes
Yes
No
No
No
Fibroblasts
Yes
Yes
Yes
No
No
No
Mesenchymal
Stem Cells
Yes
No
Depends on
age
Debated
No
Yes, fibrosis
calcification
Endothelial
Progenitor Cells
Yes
Yes
Depends on
age
Debated
No
Yes,
calcification
Crude Bone
Marrow Cells
Yes
Yes
Depends on
age
Debated
Yes
Yes,
calcification
Umbilical Cord
Cells
No
Yes
Yes
Debated
No
No
Hematopoietic
Stem Cells
Yes
Yes
Yes
Debated
No
Yes
Embryonic Stem
Cells
No
No
Yes
Yes
No
Yes, potential
teratomas
Stem and
Progenitor Cells
16
[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.
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].
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 the
infarct scar [72, 73]. The low cell engraftment number may be due to cardiomyocyte death
caused by ischemia [73].
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.
17
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
18
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.
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, which
mimics 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 ~2045 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 vivo with the expression of GATA-4 and Nkx2.5
observed prior to ANP, myosin light chain-2v (MLC-2v), and -myosin heavy chain, Na+-Ca2+
exchanger, and phospholamban [89]. Sarcomeric protein expression in mESCDCs also follows a
19
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 purkinjelike cells in the intermediate and atrial and ventricular cells later on [93].
Functionally, the
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].
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].
20
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.
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
21
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-hygror mESCs 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
22
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].
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.
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
23
24
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.
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.
tissue engineering field using this approach has come from Zimmermann, Eschenhagen and
colleagues. The system they developed uses liquid collagen type I and various species of
cardiomyocytes or cardiac cells for making what they termed engineered heart tissue (EHT). In
early work, Eschenhagen et al. [128] showed spontaneous remodeling of liquid collagen by
embryonic chick cardiomyocytes and the formation of a spontaneously and coherently beating
cardiomyocyte populated matrix.
26
formed by the collagen and in secreting new ECM for improved organization and function of the
EHT [134]. Furthermore, the enhanced number of vascular-like structures with the native heart
cell population may also facilitate faster vascularization of the EHT during transplantation into
the heart. Taken together, this study may help resolve some immunological issues associated
with EHTs and highlights the importance of using non-myocyte cells alongside cardiomyocytes
for the formation of myocardial tissue.
In an attempt to move away from neonatal cells, Guo et al. [136] used the EHT
technology to form cardiac tissue with mouse embryonic stem cell-derived cardiomyocytes. In
this study, an enriched population of mESCDCs was achieved by a Percoll separation strategy
and fibroblasts, neurons, and vascular endothelial cells were identified throughout the engineered
tissue with no undifferentiated ESCs. After mechanical stretching, the tissue beat spontaneously
and synchronously, generated contractile force, and responded to pharmacological manipulation.
The degree of cardiomyocyte differentiation and maturity varied, but some cells had highly
organized myofilaments and formed adherens junctions, desmosomes, and gap junctions with
neighboring cells. Implantation of the engineered tissue into nude mice demonstrated survival
and vascularization of the engineered tissue with no teratoma formation [136]. This important
early study identifies the potential of using ESCDCs for cardiac tissue engineering and suggests
the techniques developed for neonatal cardiac cells may be translated to ESC-derived cells.
Work is currently being conducted using the EHT technique along with iPS cell-derived
cardiomyocytes [137]. Still, it has not been determined if implanting EHT into an infarcted heart
improves systolic and diastolic function in a malfunctioning myocardium and is needed before
the potential of this technique is fully understood.
Several studies have demonstrated the feasibility and beneficial effect of using injectable
biomaterials in this cardiac tissue engineering approach. Christman et al. [138] demonstrated
that fibrin glue preserves ventricular geometry and improves cardiac function suggesting the
fibrin glue acts as a scaffold that prevents infarct expansion and provides space and/or signaling
for blood vessel ingrowth. Huang et al. [139] found fibrin, collagen I, and Matrigel each on their
own significantly enhance angiogenesis and blood flow in an infarcted myocardium with
collagen I showing a significantly higher number of myofibroblasts in the scaffold. Dai et al.
[140] similarly injected collagen into an infarcted myocardium and reported a thickening of scar
tissue due to the collagen scaffold that improved left ventricular stroke volume and ejection
fraction. Neovascularization and improvement to cardiac function has also been observed when
injecting alginate [141] and self-assembling peptides [142] on their own into injured hearts.
Although injectable biomaterials on their own show promising results, the combination of
these materials with cells leads to a more significant improvement to cardiac function.
Christman et al. [143] injected fibrin glue and skeletal myoblasts into infarcted rat hearts and
showed the fibrin glue significantly improved the survival of myoblasts, increased the
engraftment size, significantly reduced infarct scar size, and improved blood flow compared to
skeletal myoblast transplantation or fibrin alone. Similar beneficial effects on cardiac function
with neovascularization in the infarcted region has been demonstrated with fibrin glue and bone
marrow-derived cells [144]. In addition to fibrin, other biomaterials have also been used as
injectable scaffolds with cells for cardiac tissue engineering.
ventricular cardiomyocytes with collagen and Matrigel, similar to the EHT technique, and
injected it into an infarcted rat heart before it solidified into a gel.
transplanted cardiomyocytes survived, formed condensed tissue, and expressed the gap
junctional protein connexin-43 (Cx-43). Moreover, the transplanted cells and matrix preserved
left ventricular wall thickness and cardiac function better than either the cells or matrix alone, or
sham control. Matrigel has also been used to deliver mESCs into an intramural left ventricular
pouch and into infarcted myocardium with an observed improvement to heart function compared
to appropriate controls [146, 147].
The benefits afforded to injecting biomaterials alone or with cells suggest biomaterials
play an important role in repairing infarcted hearts. The mechanisms behind the beneficial
effects, however, still need to be elucidated. A current limitation with this approach is the types
28
of biomaterials that may be used. Injectable polymers must be liquid or of reasonable viscosity
to be injected into the heart and must form a solid matrix quickly once at the appropriate site.
The development of new temperature-responsive and other injectable biomaterials will certainly
hold an exciting future for in situ cardiac tissue engineering.
microtextured polystyrene substrates that were structurally organized with an array of parallel
grooves [154]. These grooved surfaces led to the formation of cell sheets that had cells highly
aligned in the direction of the grooves and this aligned cell orientation was maintained when
detached from the PIPAAm and further cultured on tissue culture polystyrene dishes. Although
this study was conducted using smooth muscle cells, this technique should work well in the
alignment of cardiomyocytes, an important criterion for developing myocardial tissue.
In
addition, cell sheet engineering can be used to form sheets that incorporate multiple cell types,
such as cardiomyocytes, endothelial cells, and fibroblasts, and may contribute important
structural and functional benefit to engineered tissue [155, 156].
The potential of this unique tissue engineering approach has been further demonstrated
through many in vivo studies. Shimizu et al. [157] showed that layers of cardiomyocyte sheets
29
could survive long-term after being implanted into rats with the implanted tissue being well
vascularized, contractile, force producing, and maintaining an elongated and well differentiated
cell structure. When implanted into an infarcted myocardium, cardiomyocyte cell sheets two
layers thick appeared homogenously integrated with host myocardium, were vascularized,
expressed Cx-43, and significantly ameliorated cardiac performance compared to cell sheets
from fibroblasts or untreated controls [158]. Cardiomyocyte cell sheets formed from fibrincoated dishes, instead of PIPAAm, showed bidirectional action potential propagation between
host hearts and transplanted tissue demonstrating similar functional integration with infarcted
hearts [159]. Furthermore, forming cell sheets by co-culturing cardiomyocytes with endothelial
cells promotes neovascularization with the transplanted endothelial cells contributing to new
vessel formation [156, 160]. Increasing the endothelial cell to cardiomyocyte ratio enhances
neovascularization
and
improves
cardiac
function
of
infarcted
hearts.
Although
neovascularization within the cell sheets occurs quickly, especially when they contain
endothelial cells, diffusion limitations leading to hypoxia, insufficient nutrient supply, and
accumulation of waste prevent the transplantation of engineered tissue greater than three cell
layers thick (~80 m) without getting necrotic cells in the middle of the tissue. To resolve this
issue, Shimizu et al. [161] performed a multistep transplantation of 3 layer thick cell sheets once
every few days allowing each transplant time to become vascularized before adding the next
layer. With this technique, engineered tissue ~ 1 mm in thickness was obtained in vivo without
tissue necrosis and became well vascularized and completely synchronized. This polysurgical
procedure may provide a method for overcoming one of the fundamental limitations with
myocardial tissue engineering - obtaining thick, vascularized engineered tissue in vivo while
preventing necrosis. Similarly, the lack of a suitable cell source for cardiomyocytes limiting
cellular cardiomyoplasty plays an equally large barrier in tissue cardiomyoplasty. As a result,
cell sheet engineering has formed tissue using mesenchymal stem cells [162], autologous skeletal
myoblasts [163], and the coculture of fibroblasts and endothelial progenitor cells [155], all of
which have improved cardiac function when implanted in infarcted myocardium. Despite the
drawback of significant macroscopic strength associated with cell sheet engineering, this
technique holds tremendous potential as a regenerative approach to repairing an infarcted heart.
30
These biomaterial
scaffolds should have the appropriate physical, chemical, mechanical and degradation properties
to promote cell attachment, proliferation, differentiation, organization, vascularization in vivo,
integration with host tissues, and the replacement of the scaffold by newly formed ECM at a rate
appropriate for the gradual transfer of mechanical load from biomaterial to new tissue. The
biomaterials used in making the scaffolds must be non-toxic and non-immunogenic and the same
should hold true with the materials degradation products. One of the main advantages of using
pre-formed 3-D scaffolds is the ability to tailor the structural and functional properties of the
scaffolds to meet these requirements for site-specific applications. Typically, this approach
utilizes biomaterial scaffolds either in the development of cardiac tissue in vitro for subsequent
implantation or as a transplantation vehicle of cells for in situ myocardial formation. Either way,
there are four main design components that determine the success of this tissue engineering
method; 1) biomaterial choice, 2) scaffold fabrication technique, 3) cell type, and 4) seeding and
cultivation methods.
components, an ideal scaffold and tissue formation method may be established leading to
successful regeneration of myocardial tissue.
31
Table 2.2: Summary of biomaterials and their application in cardiac tissue engineering. Used with permission from
Chen et al. [62].
Biomaterials
Physical State
Tissue Engineering Approach
Naturally-derived
Collagen
Gel or 3-D
porous mesh
Fibrin glue
Injectable gel
Peptide nanofiber
Injectable gel
Collagen glycosaminoglycans
3-D porous
mesh
Gelatin mesh
3-D porous
mesh
Alginate mesh
3-D porous
mesh
Poly(lactic acid)
3-D porous
mesh
3-D porous
mesh
3-D porous
mesh
Poly(glycerol sebacate)
3-D porous
mesh
Segmented Polyurethanes
3-D porous
mesh
Poly(ethylene terepthalate)
Knitted mesh
Polypropylene
Solid sheet
Solid sheet
Poly(N-isopropyl acrylamide)
Solid sheet
Synthetic Biodegradable
Synthetic Non-degradable
32
selecting biomaterials for this application are beginning to become more established.
Similar
neovascularization was reported and the transplanted engineered tissue attenuated left ventricular
dilatation and heart failure. While these early studies demonstrate the potential of naturallyderived materials, constraints of their use include limited control over the mechanical properties
and degradation rates, large scale production, and batch-to-batch variability [171].
(PLGA) and have been applied to cardiac tissue engineering. Some successes have been made
using these scaffolds for generating engineered cardiac tissue in vitro. Bursac et al. [172], for
example, seeded primary neonatal rat ventricular cells onto porous PGA scaffolds and cultured
the constructs in a bioreactor for 1 week. The samples formed a peripheral tissue-like region 5070 m thick containing differentiated cardiomyocytes organized into multiple layers in a 3-D
configuration. In this study, the engineered tissue had homogenous electrical properties and
continuous impulse propagation and was deemed a good model for electrophysiological studies.
A follow up study found that coating the PGA scaffolds with laminin improved the molecular,
structural, and electrophysiological properties of the constructs that closer mimicked native
ventricular tissue [173]. The use of these polyesters in developing in vitro models of myocardial
tissue warrants their investigation, but it has been recognized that these polymers are
inappropriate for implanting in the body for cardiac repair. Acidic degradation products from
these polyesters affect cell viability and may lead to an intense inflammatory response and an
adverse tissue reaction after implantation [174]. Upon degradation, the mechanical integrity of
these polyester scaffolds is lost rapidly and may not provide enough time for the engineered
tissue to buildup the appropriate structure to support itself [175]. In addition, these polymers are
stiff and possess inappropriate mechanical properties for cardiac tissue engineering. As a result
of the downfalls associated with PGA, PLA, and PLGA, other synthetic biomaterials have been
developed that are more promising for this application.
Cardiomyocytes cultured on hard matrices that mimic post-infarcted scar tissue (E ~ 35-70 kPa)
overstrain themselves, lack a striated appearance, and stop beating after a few days in culture.
Cells on soft matrices (E ~ 1 kPa) remain contractile for several days in culture but produce little
work. This study clearly identifies the influence elastic modulus has on cardiomyocyte structure
and function and suggests that understanding the mechanical properties of the native
myocardium may help to establish the appropriate parameters for developing biomaterials to be
used in the heart. Towards this goal, the ultimate tensile strength (), under static loading, of
human cardiac muscle taken from the middle layer of left ventricular myocardium is
approximately 115 mN/mm2 (115 kPa) in the direction parallel to contraction and about 38 kPa
in the transverse direction [177]. The ultimate percentage elongation () of the myocardial tissue
is 66% in the parallel direction and 86% in the transverse direction [177]. This tissue has a
Youngs modulus of ~20-500 kPa [177, 178]. In addition, a normal resting heart undergoes 75
beats/min [4] with a stress of at least 60 kPa generated by the myocardium for ~0.3 s/beat [179].
Taken together, mechanical properties of biomaterials should mimic those of the native
myocardium and are important design criteria in biomaterial development.
The importance of soft, flexible and elastic scaffolds suggests elastomeric biomaterials
may
be
appropriate
for
use
in
the
heart.
Several
biodegradable
polyesters,
determined, the construct showed improved cellularity, expression of cardiac markers and
contractile function compared to controls of collagen I and PLGA alone. Importantly, the
authors attributed the improved contractile function to the elastic mechanical properties of the
35
composite scaffold which promoted mechanotransduction within the construct and the formation
of electromechanical coupling of cells [190]. Scaffold stiffness associated with the PLGA
scaffold prevented construct contraction despite cell attachment and expression of cardiac
markers.
soft
segments
of
polycaprolactone-polyethylene
oxide-polycaprolactone,
1,4-
butanediisocyanate, and either putrescine or peptide chain extenders that show great promise in
cardiac tissue engineering [51, 195, 196]. This group demonstrated these polyurethanes have the
appropriate mechanical and biocompatibility properties for use in the cardiac environment and
improved cardiac function when implanted on infarcted rat hearts [197, 198]. One of the
advantages of using segmented polyurethanes for this work is the flexibility in chemistry used to
synthesize these polymers allows the ability to tune the mechanical properties and degradation
rates and incorporate chemical moieties to form bio-mimetic materials, discussed in greater
detail in section 2.5.
biomaterials that will be used in the heart. These materials promote mechanotransduction within
engineered constructs, help prevent detachment from surfaces, aid in transmitting forces during
contraction, and may contribute to elastic recoil during relaxation. As a result, elastomeric
biomaterials are going to play a major role in identifying ideal biomaterial scaffolds for cardiac
tissue engineering.
considered when forming scaffolds for tissue engineering include: a high surface area-to-volume
ratio to achieve a high density of electromechanically coupled cells and efficient transport of
oxygen, nutrients, and waste; pore sizes that allow cell migration; surface chemistries and
features that promote cell adhesion, growth, and differentiation; and the appropriate architecture
to organize the tissue [171, 199]. While a scaffold that meets all these requirements may be
obtained by decellularizing a cadaveric heart [200], this is not an option for synthetic
biomaterials.
Several polymer processing techniques can be used to form synthetic polymeric scaffolds
that meet many of the requirements for tissue engineering applications, including thermally
induced phase separation, molecular self assembly, laser ablation, electrospinning, solvent
casting and particulate leaching, polymer extrusion, gas foaming, phase inversion, rapid
prototyping, and others [171, 201]. Not all of these, however, can be easily used to form
scaffolds with the appropriate architecture that promotes anisotropic organization of
cardiomyocytes. The anisotropic organization of cardiomyocytes is critical for the structure and
function of the native myocardium and is increasingly being recognized as an important design
consideration for engineered myocardial tissue. Recent studies have identified different methods
of producing scaffolds with architectures that promote anisotropic cardiac tissue formation.
Bursac et al. [202] melted sucrose and extruded it to form a fibrous template with which to form
anisotropic fibrous and foamy scaffolds from PLGA through a solvent casting and particulate
leaching method. Neonatal cardiac cells cultured on the scaffolds aligned along the polymeric
fibers, were interconnected, and supported macroscopically continuous, anisotropic impulse
propagation as determined by optical mapping. Englemayr et al. [189] used a laser excimer
ablation technique to create accordion-like honeycomb scaffolds from PGS. These scaffolds
were physically and mechanically anisotropic, closely mimicked the mechanical properties of the
right ventricular myocardium of adult rats, and promoted the anisotropic alignment of cardiac
cells that beat in synchrony when electrically stimulated. Guan et al. [203] applied a thermal
gradient during the thermally induced phase separation (TIPS) process to form physically and
mechanically anisotropic scaffolds from an elastase-sensitive segmented polyurethane.
The
oriented scaffolds supported muscle-derived stem cell growth and were completely degraded in 8
weeks after subcutaneous implantation in rats.
37
PU onto a rotating collection mandrel [204]. Using this technique, Rockwood et al. [193]
cultured neonatal rat ventricular cells on aligned and unaligned electrospun PU scaffolds. Both
scaffold architectures supported the culture of the cardiac cells, but the aligned scaffold
promoted cardiomyocyte alignment and significantly decreased expression of atrial natriuretic
peptide (ANP), indicative of a more mature ventricular phenotype. Others have similarly used
electrospinning to form anisotropic scaffolds for culturing cardiac cells [205] and this approach
appears to have much potential in cardiac tissue engineering, discussed in greater detail in
section 2.6. The combination of the appropriate elastomeric biomaterial and scaffold fabrication
technique for an anisotropic organization may ultimately lead to ideal tissue engineering
scaffolds that have the appropriate physical, chemical, mechanical, and degradation properties
required to promote the regeneration of an infarcted heart.
directed at the development of functional cardiac tissue in vitro for subsequent implantation or as
models for studying development, disease, and regeneration, but some may also apply to
transplantation of cells for in situ tissue formation. In both of these approaches, biomaterial
scaffolds are used as transport vehicles to deliver cells into the body to help regenerate tissues.
High scaffold porosity is a tissue engineering requirement to achieve the transportation of a high
density of cells but if the seeding technique isnt conducive of promoting cell infiltration, then
this wont be realized. Towards this goal, it was determined that a high cell seeding density
39
(>1x105 cells/mm3) in combination with medium perfusion promotes a high density of uniformly
distributed cells in the scaffolds [168, 210].
centrifugation, pulling a vacuum, or applying pressure to push cells in the scaffolds, may also
work in achieving this goal [165, 194].
Once the cells have been seeded, if they are not to be implanted directly, bioreactors and
other cultivation conditions may aid in formation of the tissue. Medium perfusion (0.5-1.5
ml/min) during cultivation promotes the distribution of cardiac cells, oxygen, and nutrients
throughout scaffolds resulting in thick functional tissue ~1.5-2 mm thick [168, 210-213].
Perfusion of culture medium provides convective and diffusive transport of oxygen and nutrients
to all parts of the construct allowing for aerobic metabolism to take place in the scaffolds away
from the peripheral surface layer, >200 m [211-213]. The ability for cells to undergo aerobic
metabolism throughout the scaffold promotes spatially uniform cell distributions, cell viability,
expression of cardiac markers, and cardiomyocyte function [211-213]. Cultivation of tissue
engineered cardiac constructs under cyclic mechanical strains has also been shown to improve
tissue engineered constructs [129, 131, 134, 214]. As mentioned above, external mechanical
stimuli trigger intracellular signaling pathways and are critical for the development of cardiac
tissue.
electromechanical coupling, cell hypertrophy, tissue compaction and orientation, and improves
contractile function of engineered tissue [129, 131, 134, 214].
Interestingly, in a study
performed with cultured fibroblasts, it was found that anisotropic tissue constructs may
overcome contact guidance cues and remodel in response to mechanical signals [215], and it
would be interesting to see if this is true of cardiac constructs as well. Electrical pacing of
cardiac constructs has also been shown to improve tissue formation and function [216]. In
contrast to mechanical signals, though, physical guidance cues from surface topography
dominate in directing cell organization and elongation compared to electrical field stimulation
[217].
While these different construct cultivation methods are important for the development of
engineered tissue, one caveat of their use for in vitro formation and subsequent transplantation is
the increased susceptibility to ischemia due to thicker tissues and greater cell maturity. The issue
of quick vascularization of implanted tissue remains a great challenge in the tissue engineering
field and must be resolved if approaches other than polysurgery are going to be used in the
40
clinical setting. Advances in ischemic preconditioning and oxygen microcarriers hold potential
in helping to resolve this issue [218, 219], but timely vascularization will still be required for
successful transplantation of the engineered tissue.
41
segment regularity, rigidity, inter-segment interactions, and the presence or absence of bulky side
groups all have a major effect on hard segment interconnectivity and phase segregation [223].
The segregated morphology is also contingent on hard and soft segment length, polarity,
crystallinity, overall composition, and mechanical and thermal history [224-226].
from reaching labile bonds within the semicrystalline domains and reduces the rate of
degradation compared to amorphous domains [232]. As a result, high phase segregation may
lead to increased PU stability and relatively slow degradation rates. Biodegradable elastomeric
PUs designed for tissue engineering applications should therefore promote phase segregation for
achieving elastic mechanical properties while still allowing access to labile bonds for
degradation to occur at the appropriate rate. In an aqueous tissue environment where hard
segment surface enrichment occurs, the introduction of degradable sequences into the chain
extender chemistry is a logical approach in achieving the balance between elastic mechanical
properties and reasonable degradation rates. Ultimately, the reactant chemistries must be chosen
to obtain the appropriate properties for the site specific applications.
43
Figure 2.5: Standard two-step segmented polyurethane reaction. Step 1: Prepolymer formation.
Step 2: Chain extension.
During polyurethane synthesis, several side reactions may occur leading to branching,
crosslinking, or changes to the stoichiometric balance of reactants [220]. The presence of water
causes isocyanate groups to form unstable carbamic acids, which subsequently decompose to
amines with the liberation of CO2 gas. These newly formed amines may then quickly react with
isocyanates, thus changing reactant stoichiometry and leading to lower molecular weight
polymers. Isocyanates may also react with carboxylic acids, amides, urethanes, and ureas.
Undesirable branching and crosslinking may occur at elevated temperatures between isocyanates
and urethanes and/or ureas of the growing polymer chains. To minimize these side reactions,
PUs are synthesized under anhydrous conditions at temperatures below 100C.
properties. In general, a few points can be made regarding the structure of reactants, the effects
they have on polymer properties, and the choice of chemistries for obtaining an appropriate
polymer. Aliphatic reactants have the most molecular flexibility, then cycloaliphatic groups, and
then aromatic groups, which are more regular and rigid. While some chain mobility is required
to achieve a regular, ordered structure, too much flexibility prevents the formation of crystalline
structures, inter-chain interactions, and domain cohesion [233].
As a result, polyurethanes
composed of aromatic groups in the backbone structure generally have enhanced mechanical
properties due to improved phase segregation and domain cohesion, while aliphatic PUs are
generally softer and weaker [220, 234, 235]. Similar to high molecular flexibility, asymmetric
reactants and bulky side chains prevent the alignment of polymer segments thus reducing
interchain interactions, increasing phase mixing, and leading to reduced mechanical properties
[235,
236].
As
mentioned
above,
additional
hydrogen-bond
forming
groups
in
commonly
used,
commercially
available
diisocyanates
for
synthesizing
biodegradable polyurethanes are shown in Figure 2.6. Most of the diisocyanates are aliphatic or
cycloaliphatic, with the exception of 4,4-diphenylmethane diisocyanate (MDI). Our laboratory
has been particularly interested in synthesizing biodegradable PUs using 2,6 diisocyanto methyl
caproate, a lysine-based diisocyanate (LDI). LDI is an aliphatic molecule that has an asymmetric
methyl ester side chain and polyurethanes created using this diisocyanate reflect its aliphatic
asymmetric nature. Segmented PUs containing LDI had lower molecular weights and no hard
segment glass transition temperature, indicative of reduced hard segment cohesion, when
compared to an equivalent PU composed of the symmetric aliphatic 1,6-diisocyanatohexane
(HDI) [238, 239]. Predictably, Caracciolo et al. [236] recently demonstrated that the mechanical
properties of PUs formed with LDI were weaker than those made with HDI and was contributed
to reduced phase segregation due to LDI asymmetry.
45
Figure 2.6: Diisocyanates used to synthesize biodegradable PUs. Image used with permission from Guelcher [237].
mechanical properties of LDI-based PUs, the major appeal of it comes from the non-toxic
degradation products that are associated with its liberation in the body. This reagent is derived
from the amino acid L-lysine and if hydrolysis of the urethane linkages generate LDI in vivo, this
diisocyanate will react with water to form L-lysine methyl ester, a non-toxic product [240].
Biodegradable PUs formed using LDI found no toxic or tumorigenic responses to these materials
upon implantation into animal models [241-243]. In addition, the methyl ester side chain of LDI
provides a functional group that may be modified to attach biological agents to the surfaces of
the PUs. Ernsting et al. [244] utilized the side chain of LDI in attaching the natural anti-oxidant
vitamin E to surface modifying macromolecules as a model system for bringing bioactive
molecules to the surfaces of PUs. This technique was developed in an attempt to promote PU
stability, but has potential in conjugating biological agents relevant to tissue engineering, such as
the cell adhesion peptide RGD or various growth factors.
The polyols generally used as the soft segment of biodegradable PUs are polyethers,
polyesters, polyalkyl diols, or a combination of these. Figure 2.7 shows the chemical structure of
some common soft segments for biodegradable PUs. Polyols are responsible for the flexibility
and elongation limit of the PUs and given that they make up roughly 50-75% of the material, the
choice of soft segment often dictates the properties of the overall PU. Degradable PUs made
from polylactide, for example, are generally stiff, have a high strength, and are not very
46
Figure 2.7: Polyols often used in biodegradable PU synthesis. Image used with permission from Guelcher [237].
extensible, similar to polylactide homopolymers [245, 246]. For elastomeric biodegradable PUs,
polycaprolactone (PCL) soft segments lead to the formation of highly extensible, soft, elastic
polymers [239, 245, 246] and are particularly attractive for soft tissue applications. Skarja and
Woodhouse [239, 247] formed PUs with PCL and PEO soft segments of varying molecular
weight and found the mechanical and degradation properties of the PUs were directly related to
soft segment composition and molecular weight. All the PCL-based PUs were elastomeric, but
an increase in crystallinity, hydrophobicity, initial modulus, ultimate tensile stress and strain, and
resistance to degradation was observed with an increase in starting PCL molecular weight. PEObased PUs, on the other hand, were amorphous, soft, and tacky polymers that were much more
hydrophilic and had faster degradation rates than the PCL-based PUs [239, 247]. To take
advantage of good elastomeric mechanical properties of PCL and degradation characteristics of
PEO, several groups have developed a series of biodegradable segmented PUs with triblock soft
segments of PCL-PEO-PCL [51, 196, 248, 249]. A wide range of elastomeric PUs with different
mechanical properties and degradation characteristics were observed in these triblock soft
segment-based PUs with similar trends in PU properties with changes in PCL and PEO
molecular weights as observed by Skarja and Woodhouse. Thus, PUs with varying elastomeric
mechanical properties and degradation characteristics may be formed by adjusting the chemistry
and molecular weight of soft segment polyols.
A wide range of difunctional reactants can be used as the chain extender in synthesizing
biodegradable PUs, but the majority of these are diols and diamines. While commercially
47
available diisocyanates and polyols are typically used, several groups have developed novel
chain extenders as a method of introducing degradable chemical moieties into the hard segment
of these polymers [51, 196, 238, 239, 247, 250-252]. Using this approach allows the choice of
soft segment polyols for achieving specific physical and mechanical properties while still
promoting overall polymer degradation. Surface enrichment of the hard segment in aqueous
tissue environments also promotes accessibility of labile bonds to various hydrolytic agents, thus
enabling polymer degradation.
incorporated as chain extenders either directly, by adding a lysine residue on the end of desired
peptide sequences, or using linker molecules to create diamine reactants [51, 238, 251-253]. As
a result, PUs can be developed that are susceptible to cell-secreted proteases or exhibit other
unique biological functionality. The ability to create synthetic bio-mimetic PUs with very
diverse physical, chemical, mechanical, and degradation properties provides much promise for
segmented PUs in tissue engineering applications.
oxidative agents, calcification, mechanical stresses, metallic ions, salts, acids, and cells and is a
function of PU chemistry, morphology, and processing [255]. The complex interplay of all the
different factors makes a complete mechanistic understanding of PU degradation difficult, but
insight can be gained by understanding the contribution of the individual components on their
own.
Figure 2.8: Model for environmental biodegradation of PUs. Image used with permission from Santerre et al. [255].
structural morphology and phase segregation of the polymer [255]. This may expose labile
bonds to various hydrolytic agents that may have otherwise been protected, thus promoting
degradation. Strain induced crystallization may also occur in the heart through the organization
of polymer chains under stress [220]. Although crystallization may reduce PU degradation, it
may also alter the mechanical properties making it stiffer and potentially unable to withstand
subsequent stresses leading to structural failure. Environmental stress cracking through the
formation and propagation of cracks in the PU may also occur in the mechanically active heart.
Environmental stress cracking and PU degradation result from the combination of mechanical
stresses from the biological environment or residual effect of processing and several other factors
49
such as oxidative agents from inflammatory cells, the presence of foreign body giant cells
(FBGC) and monocyte-derived macrophages (MDM), polyether soft segments, PU morphology,
and various proteins including 2-macroglobulin [256, 257]. Polyether-based PUs and polymers
with inappropriate mechanical properties, such those that are not elastomeric, are too hard or too
soft, or show significant hysteresis, may have increased susceptibility to mechanical-based
degradation in the heart and may lead to inappropriate degradation rates for specific applications.
Chemical degradation of polyurethanes is mediated by oxidation and hydrolysis. One of
the most important processes associated with chemical degradation of polyurethanes in vivo is
inflammation. Inflammatory cells, such as neutrophils (polymorphonucleocytes, PMNs) and
MDMs, are recruited to the site of biomaterial implant. During the initiation of inflammation,
PMNs are the first cell type to arrive, but shortly after MDMs and FBGCs, formed by the fusion
of macrophages, take over and are the main cell types involved in biomaterial degradation and
chronic inflammation [258]. Inflammatory cells contain several reactive oxygen species that are
released during FBGC formation and chronic inflammation, including superoxide, hydrogen
peroxide, hypochlorous acid (HOCl), and peroxynitrite anion (ONOO-) [29, 259, 260].
Sutherland et al. [257] investigated the effects of neutrophils, HOCl, and ONOO- on
polyetherurethanes. It was found that all three oxidative factors induced polymer degradation,
with HOCl reacting with urethane-aliphatic ester linkages and ONOO- targeting aliphatic ether
groups.
Other studies have similarly shown these reactive oxygen species released from
50
[266, 267] and that the relative amount of hard segment correlates with the degree of
biodegradation [230].
proportional to hydrolytic degradation in three polyether-urea urethanes even though the hard
segments contained hydrolysable groups [230]. Similarly, the level of enzymatic hydrolysis is
inversely proportional to hydrogen bonding between hydrolysable bonds in the hard and soft
segments due to domain cohesion and the inability of enzymes to reach the labile bonds [268].
Other MDM-released esterases have also been shown to play a role in PU degradation adding to
the important role of MDMs in understanding the mechanisms behind environmental
biodegradation of PUs [255]. For segmented PUs used as biomaterial scaffolds for cardiac
tissue engineering, a combination of cyclic stresses, inflammatory cells, oxidative agents,
inflammatory and injury-related enzymes, as well as other factors will likely contribute to the
environmental biodegradation of these synthetic polyurethanes.
The knowledge gained in elucidating environmental biodegradation of PUs allows the
rational design of novel biomaterials for biomedical applications. MDM-induced degradation of
PUs, for example, has been exploited by Santerre and colleagues in the development of novel
antimicrobial-containing PUs for medical devices [255, 269]. The antimicrobial agents are
incorporated into the backbone structure of the PUs through labile bonds that have been shown
to be susceptible to MDM-secreted enzymes and are released as a direct consequence of polymer
degradation through inflammatory cells recruited during device implantation and infection. As a
consequence, the drug release profiles are dictated by the tissue in a time-course that responds to
the tissue environment and directly corresponds with tissue healing [255, 269]. By incorporating
specific chemical moieties into the backbone structure of PUs, a similar tissue-responsive
degradation mechanism may be exploited by cell-secreted proteases that are not traditionally
associated with PU degradation and is the subject of the next section.
While these
mechanisms will play a role in the resorption of PU scaffolds, PU degradation can be further
promoted by exploiting the presence of cell-secreted proteases that are not traditionally involved
in PU degradation but are present in the microenvironment in which biomaterial scaffolds may
be beneficially employed.
elastomeric segmented polyurethanes by incorporating amino acids and peptides into the
polymer backbone and has much potential as bio-mimetic biomaterials in soft tissue engineering.
Skarja and Woodhouse developed a novel family of enzyme-sensitive biodegradable
polyurethane elastomers using an L-phenylalanine-based diester chain extender [238, 239]. In
this work, 1,4-cyclohexane dimethanol was used to link phenylalanine (Phe) residues to form a
diamine, diester chain extender. It was conceived that this approach would produce non-toxic
degradation products that could be readily metabolized in vivo and the choice of amino acid or
peptide could be tailored for hydrolysis by specific enzymes. Phenylalanine was incorporated
into the polyurethane backbone to introduce enzymatic susceptibility to the polymer by
chymotrypsin-like serine proteinases found in the body, such as cathepsin G and chymase [247].
Segmented PUs synthesized with the Phe-based chain extender showed enhanced degradation in
the presence of chymotrypsin, and to a lesser extent trypsin, and could achieve a range of
physical, chemical, mechanical, and degradation properties by changing the soft segment
chemistry [239, 247]. One polymer from this family, synthesized from PCL of molecular weight
1250, LDI, and the Phe-based chain extender (Phe PU), has been successfully used in cardiac
tissue applications. Films of this PU supported a differentiated cardiac phenotype and contractile
function using neonatal rat cardiomyocytes [191]. Similarly, mESCDCs maintained a normal
cardiomyocyte morphology, expressed the cardiac specific markers myosin heavy chain, desmin,
and -sarcomeric actinin, and were contractile on ECM-coated Phe PU films [192]. Recently,
porous 3-D scaffolds formed by thermally induced phase separation and electrospinning were
used with mESCDCs and neonatal rat cardiac cells with similar positive results [193, 194].
Extensions of this work, described in this thesis, may provide further evidence of the potential of
the Phe PU and a newly developed dipeptide-containing PU for cardiac tissue engineering.
Biomaterial scaffolds that seek to mimic the biological structure and function of the
native ECM should also degrade and reorganize through the same mechanisms. Synthetic
hydrogels were developed to target polymer degradation by ECM degrading cell secreted
proteases [50]. Guan and Wagner [51] extended this work in the formation of elastomeric
segmented polyurethanes that were susceptible to degradation by the ECM degrading enzyme
elastase. In this work, the tri-peptide Ala-Ala-Lys (AAK) was used as a chain extender to
facilitate enzyme-mediated degradation of hard segments. Elastase is known to cleave peptides
between alanine residues and the lysine residue was added to generate a diamine structure [51].
52
PUs were synthesized using the peptide chain extender, 1,4-butanediisocyanate, and either PCL
or PCL-PEO-PCL triblock soft segments of varying block molecular weights. The resulting PUs
were flexible elastomers with a range of mechanical properties, breaking strains of 670-890%
and tensile strengths of 15-28 MPa, and showed enhanced degradation in the presence of elastase
through incorporation of the Ala-Ala cleavage site [51]. In subsequent work by this group, an
elastase-sensitive PU was formed into anisotropic and randomly oriented 3-D scaffolds by
thermally induced phase separation [203]. Scaffold architecture and physical and mechanical
properties were a function of the fabrication conditions.
anisotropic mechanical properties with a much higher breaking strain and ultimate tensile
strength in the longitudinal direction compared to the transverse direction or random scaffolds
and better supported muscle-derived stem cells than random scaffolds [203]. Of particular note
were the degradation characteristics of this elastase-sensitive PU scaffold. In vitro degradation
demonstrated the PU scaffold had similar passive hydrolysis as films of the same material after 8
weeks (~12% mass loss), but showed significantly enhanced mass loss in the presence of elastase
(~42% for scaffold vs. 27% for film), likely due to increased surface area and accessibility of
elastase to labile Ala-Ala peptide bonds [203]. Significantly, subcutaneous implantation of the
elastase-sensitive PU scaffold showed almost no signs of the scaffold after 8 weeks, whereas a
comparable PU scaffold with the elastase insensitive chain extender putrescine was still present,
albeit with considerable degradation [203]. Although the exact mechanism behind this in vivo
degradation work is difficult, it highlights a few critical aspects of PU degradation.
The
significant difference in degradation seen from the 8 week in vitro study compared to that done
in vivo suggests the in vitro models with enzymes alone, while providing useful mechanistic
information, cannot accurately replicate the complex in vivo environment. A higher elastase
concentration in vivo than was used in the in vitro studies may have contributed to enhanced
degradation, but more likely reflects the multifaceted mechanisms of environmental
biodegradation discussed above.
53
Wagner lab has shown the putrescine extended PUs have favorable properties for cardiac tissue
engineering applications [197, 198] and it will be exciting to see how the enzyme-sensitive PUs
function in the heart.
This
relatively simple, cost effective, and versatile method allows the processing of polymers into
fibers with diameters on the nanometer and micrometer scale, often with control of the specific
range. The resulting scaffolds have a topography and porosity that mimics the native ECM and
has been shown to influence cell behavior [270, 271]. The electrospinning process may also be
used to form anisotropic scaffolds with fibers oriented in one direction [272], an important
design criterion for engineering anisotropic tissues such as that found in the myocardium.
Electrospinning therefore is a promising technique in forming anisotropic 3-D scaffolds from
biodegradable elastomeric biomaterials for cardiac tissue engineering.
porosities, fiber diameters, pore sizes, fiber morphologies, thicknesses, and architectures [270,
54
272, 274]. If electrospinning parameters are not appropriate for fabricating continuous fibers,
such as inappropriate polymer molecular weight, concentration, or viscosity, polymer droplets
are deposited instead in a process called electrospraying [275].
Figure 2.9: Schematic of electrospinning apparatus. Image used with permission from Kenawy et al. [277].
Table 2.3
summarizes the effect that several of these parameters have on fiber morphology. Adjusting the
various parameters can lead to polymer morphologies such as droplets, beads-on-a-string, and
round, flat, porous, or fused fibers [270, 272, 274]. As well, the fibers may have diameters
ranging from a few hundred nanometers to several microns and may be randomly oriented or
aligned to various degrees. The diverse polymer morphologies that may be formed by altering
the electrospinning parameters allows the formation of scaffolds that exhibit a variety of
physical, mechanical, and degradation properties that will influence the performance of the
scaffold in the presence of cells and in vivo.
55
Table 2.3: Summary of electrospinning parameters and effects on fiber morphology. Used with permission from
Murugan and Ramakrishna [272].
Processing Parameter
Effect on Fiber Morphology
Concentration/viscosity
Low concentrations/viscosities yield beads or beads-on-a-string
morphology; increasing concentration/viscosity reduces beaded defects
Fiber diameters increase with increasing concentration/viscosity
Conductivity/solution charge
Increasing conductivity aids in production of uniform bead-free fibers
density
Higher conductivities yield smaller fibers with a few exceptions
Polymer molecular weight
Increasing molecular weight reduces number of beads and droplets
Dipole moment and dielectric
Successful spinning can be achieved in solvents with high dielectric
constant
constants
Flow rate
Lower flow rates yield fibers with smaller diameters
High flow rates produce fibers that are not dry upon reaching collection
plate
Field Strength/voltage
At high voltages, beading is observed
No direct correlation with field strength and fiber diameter
Distance between needle tip
A minimum distance is required to obtain dry fibers
and collector
At distances too close or too far, beading is observed
Needle tip design
Using a coaxial, 2-capillary spinneret, hollow fibers are produced
Multiple needle tips are used to increase throughput
Collector composition and
Smooth fibers result from metal collectors; porous fiber structures are
geometry
obtained using porous collectors
Aligned fibers are obtained by using a conductive frame, rotating drum,
or wheel-like bobbin collector
Ambient parameters
Increasing temperature reduces viscosity and leads to smaller fiber
diameters
Increasing humidity causes circular pores to appear on fibers
Importantly, thermoplastic
segmented PUs may be processed by conventional solvent-based methods and can be used with
the electrospinning technique [237]. As a result, several different biodegradable PUs have been
made into scaffolds using this process [276, 282-287]. Rockwood et al. [286], for example,
successfully electrospun the enzyme-susceptible, elastomeric Phe PU to form scaffolds with a
range of fiber sizes from several hundred nanometers to tens of microns.
electrospinning process did not affect the molecular weight averages, thermal properties, or
chemical composition of the polymer, but enhanced degradation compared to films due to
increased surface area. In the Wagner lab, Stankus et al. [282] used a biodegradable, elastomeric
PU mixed with type I collagen to electrospin elastic matrices. The resulting mats had randomly
56
oriented fibers ranging from 100 to 900 nm in diameter and supported smooth muscle cells when
seeded on the scaffolds. Interestingly, investigations into the mechanical properties of the
electrospun PU matrices suggested this processing technique did not compromise the materials
mechanical properties and had a tensile strength and distensibility similar to films formed with
the same material [282]. These results indicate that several of the favorable PU properties are
maintained when processed into scaffolds via electrospinning.
Although there are many benefits to forming tissue engineered scaffolds by
electrospinning, it has been recognized that one limitation with this is the fiber mats have
relatively small pore sizes that inhibit cellular infiltration into the scaffold [270]. Nanofibrous
meshes in particular, have small pore sizes and these scaffolds behave more as a 2-D sheet on
which cells can attach, grow, and migrate along rather than a 3-D scaffold where cells can grow
and migrate into [288]. To address this issue, a few studies have been conducted to improve the
ability of attaining cells within a 3-D electrospun scaffold.
electrospinning with salt leaching to create thick scaffolds that had deliberate 100-200 m gaps
that promoted cellular infiltration.
infiltrated as far as 4 mm into the scaffold with a cell density of ~70% within the delaminations.
In another approach, Pham et al. [290] observed that cells attached to fibers of different size, but
cell spreading was enhanced by nanofibers whereas cellular infiltration was promoted by
microfibers, which had pore sizes ranging from 20 45 m. To exploit the benefits of each fiber
size, bilayered constructs composed of a layer of nanofibers followed by a layer of microfibers
were utilized in this study with positive results on cell infiltration and spreading. In a novel
study by Stankus et al. [276], smooth muscle cells were electrosprayed concurrently with
electrospinning a biodegradable PU to achieve a high cellular density and infiltration within the
elastic matrix during scaffold formation. It was determined that electrospraying the cells did not
affect viability or proliferation either alone or while electrospinning the polyurethane matrix.
This cellular integration technique followed by culturing in a perfusion bioreactor resulted in
significantly higher smooth muscle cell numbers compared to static cultures and a uniform
distribution and an elongated cell morphology [276]. In addition, by altering the electrospinning
parameters, scaffolds with aligned fibers were formed and cells electrosprayed with these
scaffolds were oriented parallel to fiber orientation, which was independent of perfusion flow
direction [276]. The physical cues provided by fibrous electrospun scaffolds may play an
57
important role in the formation of engineered tissue and the techniques developed for promoting
cellular infiltration may contribute to the success of these scaffolds.
In cardiac tissue engineering, cellular alignment is critical to the structural organization
and functionality of the engineered tissue. Physical cues provided by biomaterial scaffolds may
help in the proper organization of cardiac cells. The ability to form aligned scaffolds from
electrospinning is an important consideration in choosing a scaffold fabrication technique for use
in the heart. Different collection plate designs in the electrospinning apparatus or post-scaffold
processing may lead to electrospun scaffolds with aligned fibers [205, 272]. Using a rotating
mandrel collection plate, Courtney et al. [204] electrospun an elastomeric biodegradable PU into
scaffolds that exhibited a direct relationship between mandrel rotational velocity and fiber
alignment. The aligned scaffolds were both physically and mechanically anisotropic with low
breaking strains in the preferred direction of fiber orientation and high breaking strains in the
cross-preferred direction [204].
processed them by heating and uniaxial stretching to achieve aligned scaffolds. Cardiac cells on
the aligned PLA scaffolds were highly aligned, developed mature sarcomeric structures and
intercalated discs, and were contractile. Several other studies have successfully demonstrated the
efficacy of electrospun scaffolds for culturing cardiac cells and highlight its potential in cardiac
tissue engineering [194, 291, 292].
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Large sections of this chapter have been accepted for publication [1]: Parrag IC, and KA
Sections of this chapter also contributed to the publication [2]: Rockwood DN, RE Akins, IC
3.0 Abstract
Biodegradable segmented polyurethanes (PUs) are promising biomaterials for soft tissue
engineering applications. The inherent flexibility of PU chemistry allows the incorporation of
specific chemical moieties into the back bone structure conferring unique biological function to
these synthetic polymers. Previous work has shown that chain extenders incorporating an amino
acid or short peptide sequence exhibit enhanced degradation in the presence of specific enzymes.
Building on this, the cleavage site of several matrix metalloproteinases, a Gly-Leu dipeptide, was
introduced into a chain extender through the reaction with 1,4-cyclohexane dimethanol. The
Gly-Leu-based diester chain extender was purified by high performance liquid chromatography
and successful synthesis and purification was confirmed by several techniques.
PUs were
84
a significantly higher initial modulus, yield stress and ultimate stress compared to the Phe PU,
suggesting the Gly-Leu-based chain extender allowed for better hard segment packing and
hydrogen bonding leading to enhanced mechanical properties. The newly developed Gly-Leu
PU had several properties that were promising for soft tissue engineering applications and
warranted further investigation into scaffold fabrication and biomaterial testing.
3.1 Introduction
The goal of tissue engineering scaffolds is to mimic the native ECM of tissues by
providing a temporary material that plays both a structural and functional role in guiding the
development of tissue constructs for regenerative medicine [3]. The appropriate biomaterial for
these applications must meet several requirements; the material and its degradation products
must be non-toxic and non-immunogenic, the mechanical properties should be appropriate for
the tissue under investigation, degradation should take place at a rate that allows newly secreted
ECM to replace the biomaterial, and physiochemical properties should promote cell adhesion,
growth, and differentiation. In mechanically active soft tissues, such as the heart, mechanical
forces play a critical role in the development and maintenance of the structural organization and
function of the tissue [4]. Tissue engineering scaffolds must therefore have the appropriate
mechanical properties to promote mechanotransduction within the tissue and degradation
characteristics that allow the gradual transfer of mechanical load from biomaterial to newly
formed ECM. Elastomeric segmented polyurethanes are important biomaterials for use in the
heart where soft, flexible and elastic mechanical properties are requisite. The inherent flexibility
in PU chemistry also allows the incorporation of amino acid and peptide sequences into the
backbone structure of the polymer making them susceptible to cell-secreted proteases [5, 6].
This may lead to degradation characteristics that are responsive to the tissue environment and are
therefore more appropriate for cardiac and other soft tissue engineering applications than
synthetic polymers that degrade predominantly by passive hydrolysis.
Building on previous work in our lab of enzyme-sensitive segmented polyurethanes, the
goal of this work was to develop a family of polyurethanes that incorporate either a Gly-Leu or
Gly-Ile dipeptide into the backbone structure of the polymer. The peptide bonds between
glycine and leucine and glycine and isoleucine residues are the cleavage sites of several MMPs
that have been found to play a critical role in heart disease [7-10]. Incorporation of the Gly-Leu
85
or Gly-Ile dipeptide was suspected to confer enzyme susceptibility to the PUs, thereby improving
the degradation characteristics by making them more responsive to ECM remodeling proteases.
This chapter describes the successful synthesis and characterization of a Gly-Leucontaining polyurethane (Gly-Leu PU) that may hold potential as a new biomaterial in cardiac
tissue engineering. Several synthesis and purification methods were initially investigated for
incorporating the Gly-Leu and Gly-Ile dipeptides into an amine terminated chain extender.
Ultimately, the Gly-Leu dipeptide was reacted with a cycloaliphatic diol linker to form a
symmetric diamine diester chain extender. A Gly-Leu PU was subsequently synthesized and
structure-property comparisons were made using a phenylalanine-containing polyurethane (Phe
PU) previously developed in our laboratory. The two PUs only differed structurally by the
presence of the amino acid phenylalanine in the chain extender instead of the Gly-Leu dipeptide.
Consequently, the PUs exhibited very similar physical, chemical, and thermal properties with
only subtle differences in mechanical behavior due to structural differences between the two
polymers.
investigate the synthesis methods and are illustrated in Figure 3.1. The first system (system 1)
utilized a round bottom reaction vessel, a Dean-Stark water trap, water condenser, stir bar, drying
tube, heating mantle, and magnetic stir plate. This setup was used to test either toluene alone or
toluene and dimethyl formamide (DMF) as solvents, the Gly-Leu and Gly-Ile dipeptides, the
catalysts p-toluene sulfonic acid (PTSA) and methane sulfonic acid (MSA), and the linkers 1,4cyclohexane dimethanol (CDM) and 1,6-hexane diol. Reaction contents were added to the
reaction vessel at a 2:1 molar ratio of dipeptide to diol linker in an excess of acid catalyst. The
reactions were carried out under reflux conditions for periods between 2 and 48 h. Any water
that formed during the reaction was collected in the trap to drive the reaction in favor of the
86
diester product. After the reaction, crude products were collected directly or by allowing the
solvents to evaporate in a fume hood. Collected products were dried in a fume hood at room
temperature for 24 h, then in a vacuum oven at 60C for 48 h, and were stored in a desiccator
until analysis.
In the second system (system 2), an addition funnel, round bottom reaction vessel, tygon
tubing, sparge, spin bar, 3-neck round bottom flask, and drying tube were employed. Using this
setup, a hydrogen chloride gas was generated by slowly dripping concentrated sulfuric acid into
a reaction vessel containing sodium chloride. The hydrogen chloride gas acted as an acid
catalyst and was sparged into the 3-necked reaction vessel containing the reactants with the 2:1
molar ratio of dipeptide to diol linker. This system was used to test the hydrogen chloride gas
catalyst and tert-amyl alcohol solvent with the Gly-Ile dipeptide and CDM linker.
After
allowing the reaction to occur for ~4 h, the product was recovered by vacuum drying at 60 C
using a rotary evaporator for 6-12 h. The product was further dried in a vacuum oven at 60C for
48 h and was stored in a desiccator until analysis.
Figure 3.1: Chain extender reaction system setups. a) System 1 was used to test the solvents toluene and
toluene/DMF, the catalysts PTSA and MSA, the dipeptides Gly-Leu and Gly-Ile, and the CDM and hexane diol
linkers. b) System 2 was used for hydrogen chloride gas catalyzed synthesis using t-amyl alcohol as a solvent.
Ultimately, a Gly-Leu-based chain extender was synthesized with system 1 using the
method by Huang et al. [11] for amino acid-based diester products. A Fischer esterification
reaction was used to react the Gly-Leu dipeptide and CDM at a 2:1 molar ratio (Figure 3.2). The
reaction was carried out in excess p-toluene sulfonic acid catalyst in toluene under refluxing
87
conditions and was driven to completion using a water trap. The reaction was stopped when no
more water appeared to evolve (approximately 3 h). The crude chain extender, which appeared
as a clear viscous product, was collected and residual solvent was allowed to evaporate in a fume
hood for 24 h and subsequently in a vacuum oven at 60C for 48 h. The resulting product was
ground into a fine powder and stored in a desiccator until purification.
Figure 3.2: Synthesis scheme for Gly-Leu-based diester, diamine chain extender.
The first method developed used a low pH aqueous mobile phase made up of 0.1%
trifluoroacetic acid (TFA) in ultrapure deionized water (pH ~2.1) and acetonitrile (ACN) as the
organic phase. The series of steps used to isolate and collect peaks on the preparative column
corresponding to the Gly-Leu-based diester chain extender product included: a hold at 15%
organic phase (85% aqueous phase) for 4 min; gradient step from 15-40% ACN in 10 min;
88
gradient step from 40-60% organic phase in 1 min; hold at 60% organic phase for 3 min;
gradient step from 60-15% ACN in 1 min; and re-equilibration hold at 15% organic phase for 7
min prior to injecting the next sample. The second method used a higher pH aqueous mobile
phase composed of 10 mM ammonium bicarbonate in ultrapure deionized water (pH 8.0) and the
same ACN organic phase. The higher pH profile on the preparative column had a hold at 32%
organic phase for 6 min; gradient step from 32-50% ACN in 6 min; 50% ACN hold for 2 min;
gradient step from 50-100% organic phase in 1 min; hold at 100% for 4 min; a gradient step from
100-32% ACN in 1 min; and a re-equilibration hold at 32% organic phase for 9 min. In both
methods, the raw chain extender was dissolved in aqueous phase at 75 mg/ml and 1 ml of
solution was injected into the column running at a flow rate of 18 ml/min using a Waters HPLC
system (Waters Chromatography, Mississauga, ON). Solvents were kept in an ice bath to reduce
hydrolysis of the desired product.
Chromatography) at a wavelength of 214 nm. Two prominent peaks were collected between
approximately 14.5-16.0 min with the low pH method and between approximately 12.5-15.5 min
with the high pH method. The purified chain extender was recovered following lyophilization
and the dry product was stored in a desiccator until use. After initial analysis of purified
products, the low pH method was chosen for subsequent use.
89
and assign spectral peaks. FT-IR samples were prepared as KBr crystals. Approximately 1 mg
of sample was added to ~300 mg of KBr powder, the mixture was homogenized, and crystals
were formed by high pressure using a hydraulic press (Carver Inc, Wabash, IN) at 15,000
pounds. Samples were analyzed using a Spectrum 1000 FT-IR spectrometer (PerkinElmer,
Waltham, MA) from 4000 - 400 cm-1 at a resolution of 4 cm-1 for 64 scans using PerkinElmer
Spectrum software.
Section was published in [1] and the Phe PU synthesized and characterized here was used in [2].
90
temperature for 72 h. The resulting thin films were dried under vacuum at room temperature for
24 h and stored in a desiccator until use.
(Varian, Sunnyvale, CA) ranging from 2,980 to 706,000 g/mol was used along with Waters
Empower 2 software (Waters Chromatography) to produce retention time data, number and
weight average molecular weights, and polydispersity.
Thermal properties of the PUs were characterized by differential scanning calorimetry
(DSC) using a Thermal Analyst 2100 thermal analyzer (DuPont Canada, Mississauga, ON)
Section was published in [1] and the Phe PU synthesized and characterized here was used in [2].
91
performed at the Brockhouse Institute for Materials Research (McMaster University, Hamilton,
ON). Scans were conducted at a rate of 15C/min from -100 to 200C. One sample from three
separate synthesis trials was subjected to one scan to erase the thermal history, and were
subsequently quenched to room temperature and exposed to a second scan. Results from both
scans were used to identify the glass transition temperature (Tg) and melting temperature (Tm)
of the polymer. Crystallinity of the PUs was calculated assuming an enthalpy of fusion of 32.4
cal/g for 100% crystalline PCL [14] and the theoretical mass fraction of PCL1250 expected from
a 2:1:1 molar ratio of either LDI:PCL1250:Phe chain extender or LDI:PCL1250:Gly-Leu chain
extender.
FT-IR analysis was carried out by dissolving the PU samples in chloroform at 5% w/v
and placing the solution directly onto NaCl plates.
temperature under vacuum for 30 min and stored in a desiccator until analysis. Spectra were
generated from 256 scans using parameters described above for the chain extender.
Uniaxial tensile properties of the PU films were determined according to ASTM D1708
using an Instron 4301 testing machine (Instron, Norwood, MA). Samples were cut from films
that were 70-90 m thick using an ASTM D1708 die.
approximately 24C and 55% relative humidity for 48 h. Testing was carried out using a 50 N
load cell running at a crosshead speed of 100 mm/min. Samples were stretched to break and
stress-strain curves were generated from force-displacement data. Stress and strain at yield,
stress and strain at break, and initial modulus were inferred from the stress-strain graphs.
Statistical comparisons were made using a two-tailed independent t-test using the SPSS Statistics
17.0 statistical software package (SPSS Inc, Chicago, IL).
92
included: 1) the direct use of amino acids and dipeptides, producing asymmetrical chain
extenders with urea and amide functional groups being formed during PU synthesis [15]; 2) by
adding a lysine residue on the end of the desired peptide sequences to form an asymmetric
diamine chain extender [5]; or 3) using a diol linker to create symmetric diamine reactants [16].
In regards to the last approach, several aliphatic and cycloaliphatic diols, including ethylene
glycol, 1,3-propane diol, 1,4-butane diol, 1,6-hexane diol, dodecane diol, and 1,4-cyclohexane
dimethanol, have been used as linkers to attach the amino acids phenylalanine, alanine, glycine,
and lysine and fabricate amine-terminated diester products [16-21]. Continuing with work that
was done in our laboratory using diol linkers, this approach was utilized to produce a dipeptidebased amine terminated diester chain extender.
corresponded to these molecular weights suggesting side reactions and the formation of
undesirable side products were not significantly detected. The monoester and diester products
were formed to some extent indicating the esterification reaction took place. The desired diester
product, however, had a lower relative peak intensity (~30%) in comparison to the monoester
product (~42%) and unreacted dipeptide peaks (~28%) suggesting the reaction did not go to
completion. As a result, reaction time was varied from ~2 - 48 hrs to achieve better diester
product yields, but this had little affect on the outcome and the monoester product and unreacted
dipeptide were consistently higher than the diester product.
explanation for this observation was due to a mixing issue within the reaction vessel. Once the
93
reaction started, a viscous product formed on the bottom that limited the stir bar from effectively
mixing the vessel contents.
progressing to completion and may lead to the higher monoester product observed.
Figure 3.4: Mass spectrum of raw Gly-Ile-CDM-PTSA product. Relevant peaks are highlighted. A higher
proportion of the monoester product was observed over the diester product suggesting the reaction did not go to
completion.
In an attempt to obtain a higher Gly-Ile-based diester product, two other solvent systems
were investigated. These solvents were explored to test if reaction heterogeneity and reactant
mixing influenced successful esterification. The first attempt used system 1 and the same
reactants with a 95% toluene/5% DMF co-solvent instead of toluene alone. Adding DMF to the
reaction vessel led to a more homogenous solution and better mixing in this system. The second
attempt used tert-amyl alcohol as a solvent in system 2. This solvent was tested in combination
with the HCl gas catalyst and led to a homogenous reaction mixture. Raw products from the two
alternative synthesis trials were analyzed by ESI mass spectroscopy and are shown in Figure 3.5.
The diester product in both of these systems was very low in comparison to the monoester
products and side reactions. As a result, the toluene-based solvent in system 1 appeared to be the
94
Figure 3.5: Mass spectra of raw Gly-Ile-based chain extender products synthesized in different solvent systems. a)
Raw Gly-Ile-CDM-PTSA product in toluene/DMF co-solvent and b) raw Gly-Ile-CDM-HCl product in t-amyl
alcohol. Very little diester product was observed in both systems.
95
best synthesis method tested for producing the desired Gly-Ile-based diester product. Similarly,
the PTSA catalyzed system produced better results than the HCl catalyzed system and therefore
the HCl gas catalyzed system was not further explored.
suggested a dipeptide-based chain extender could be synthesized with a reaction system that
favored the diester product. Purification strategies were subsequently investigated before PUs
were synthesized with this new chain extender.
Figure 3.6: Mass spectrum of crude product from Gly-Leu-CDM-PTSA. The diester product dominates the
spectrum at 243 and 485 amu.
Other systems that were investigated for the Gly-Leu-based chain extender include a
methane sulfonic acid catalyst and 1,6-hexane diol linker. MSA is a liquid catalyst and its use
was suspected to improve mixing within the reaction vessel if reaction scale-up was necessary.
The aliphatic diol linker was tested as an alternative to CDM as an option for influencing PU
properties. Results from these reactions are shown in Figure 3.7 and both show similar success
in forming a Gly-Leu-based diester product. Although the hexane diol linker showed positive
esterification results in this system, CDM was chosen for subsequent work due to the increased
rigidity associated with this molecule. This was anticipated to reduce hard segment mobility,
promote phase separation, and improve mechanical properties in comparison to the aliphatic diol,
especially in light of using the aliphatic, asymmetric side chain-containing diisocyanate LDI.
More importantly, PUs created with dipeptide-based chain extenders employing CDM as a linker
allowed a direct comparison to the Phe-containing PUs to test the effects chain extender
chemistry had on PU properties. The hexane diol linker and MSA were not investigated further,
97
Figure 3.7: Mass spectra of Gly-Leu-based chain extender using different catalysts and diol linkers. a) Raw GlyLeu-CDM-MSA and b) raw Gly-Leu-HD-PTSA. High relative peak intensities corresponding to diester product
were observed. Note: diester peaks with hexane diol linker are 230 and 459 amu and monoester peak is at 289 amu.
98
but offer alternative options for future work in scaling up and synthesizing alternative Gly-Leubased chain extenders.
No
significant differences were observed between the reactants and raw product so an alternative
approach was undertaken.
separate peptides and was investigated for purifying the dipeptide-based diester chain extender.
An analytical Gemini C-18 column was used to establish a method for purifying the chain
extender and was subsequently scaled up using a preparative column for large scale purification.
The Gemini C-18 column is a silica-based column containing covalently bonded hydrophobic C18 ligands (n-octadecyl alkyl chains). An advantage of using the Gemini column is the working
pH can range from 1-12, thus allowing the flexibility of developing and optimizing a separation
scheme for purifying the diester product in either a protonated or neutral state.
Two methods for purifying the Gly-Leu-based diester product were established on an
analytical scale. The first method involved an aqueous mobile phase consisting of ultrapure
deionized water with 0.1% TFA and an organic phase of ACN. The aqueous mobile phase
maintained a low pH (~2.1) to keep the analytes protonated and allowed separation to be based
on charge and relative hydrophobicity. A solvent flow rate of 1 ml/min was used along with a
UV detector at a wavelength of 214 nm. Using a gradient elution profile of 0-50% ACN in 30
min and 50-100% ACN in 5 min, peaks associated with the desired diester product were isolated
and collected. Figure 3.8 shows a representative chromatogram with collected peaks highlighted
and the resulting mass spectroscopy analysis. The mass spectrum indicated a pure Gly-Leubased diester chain extender. No peaks corresponding to the monoester product, unreacted
dipeptide, or other contaminants were observed suggesting successful product purification.
99
Figure 3.8: HPLC separation of Gly-Leu-based diester product using analytical column and low pH aqueous mobile
phase. a) Chromatogram of run going from 0-50% ACN in 30 min and 50-100% ACN in 5 min with collected
peaks highlighted. b) Mass spectrum of peaks identified in a). A pure diester product was identified.
The elution peaks in the TFA-based profile described above corresponding to the diester
product were relatively close to other peaks and there was a concern about peak broadening and
successful separation when scaling up to a prep column. As a result, a second method was also
developed on the analytical scale. A higher pH aqueous mobile phase was used in this system
composed of 10 mM ammonium bicarbonate (pH~8.0). One advantage to using this method was
the retrieved product would be neutralized, thus eliminating a potential step in the purification
scheme. Using liquid chromatography-mass spectroscopy in tandem, peaks corresponding to the
desired product were identified. Subsequent method development attempted to separate the
desired peaks by using a series of mobile phase gradient and hold steps. A typical chromatogram
from this method is shown in figure 3.9 along with the corresponding mass spectrum of collected
peaks. The high relative proportion of diester product and relatively few other peaks suggested
this method to be promising for purifying the chain extender with the prep column.
100
Figure 3.9: HPLC separation of Gly-Leu-based diester product using analytical column and high pH aqueous
mobile phase. a) Chromatogram from an elution profile of gradient-hold steps with collected peaks highlighted. b)
Mass spectrum of peaks identified in a). Both the diester and monoester products are observed using this method.
On the analytical scale, mass spectrometry results suggested the two purification schemes
developed may be used to separate out the Gly-Leu-based diester chain extender. These two
methods were then scaled up to a prep column system to obtain enough of the purified product
for further characterization and use in PU synthesis. Adjustments were made to the elution
profiles using a combination of gradient and hold steps to isolate desired peaks. Representative
chromatograms from the two methods and corresponding mass spectra are shown in Figure 3.10.
The spectra from both methods appeared similar with relatively high levels of the diester product
along with the presence of some monoester product and few other peaks. Using peak intensities
from the spectra, the relative percent of monoester and diester products in the low pH method
was approximately 10% monoester and 90% diester product indicating a relatively pure
population of the desired diester chain extender. The high pH method had approximately 30%
monoester and 65% diester product. The higher monoester product with the high pH method
may have resulted from increased hydrolysis of the diester product during separation despite
keeping the mobile phases on ice during the HPLC runs. Although the hydroxyl-terminated end
of the monoester product may still react with the isocyanates during PU synthesis, this product
was not desirable due to its asymmetric structure, different end reactivities, and reduced potential
for hydrogen bonding. As a result, the low pH method that produced a higher relative percent of
diester product was preferred for chain extender purification.
101
Figure 3.10: Preparative column HPLC purification of chain extender using low and high pH aqueous mobile
phases. a) Chromatogram of low pH method with collected peaks identified. b) Mass spectrum of low pH method.
c) Chromatogram of high pH method with collected peaks. d) Mass spectrum of high pH method. The low pH
method had a higher relative intensity of desired diester product suggesting this was preferred over high pH method.
Using the prep column system, enough product was recovered from the two HPLC
purification methods to further characterize the chain extender and provide further evidence for
the preferred purification scheme. C13 NMR was conducted to help characterize the products
and the corresponding C13 NMR spectra are shown in Figure 3.11. Using the low pH method,
the spectrum revealed the presence of 10 prominent non-identical carbon peaks in the purified
product. This corresponds well with the number of non-identical carbons found in the theoretical
102
structure of the Gly-Leu-based diester chain extender and provided further proof of successful
purification using this method. Analysis of the reactants and theoretical predictions using ACD
i-Lab software (Appendix A) were used to assign the chemical shifts in the spectrum to the
corresponding carbons in the chemical structure of the chain extender. The unshielded carbonyl
carbon from the ester functional group was assigned downfield at the highest chemical shift of
172 ppm in relation to the unaffected carbonyl carbon of the amide functional group found at 167
ppm. A slight change in chemical shift was noted for the methylene carbon from CDM, which
shifted downfield following esterification, but the unreacted form of this carbon present in the
monoester product was not observed.
monoester product compared to diester product as well as the low signal to noise ratio with
obtained spectra. Importantly, the aromatic carbons normally found in the spectrum of the
unpurified product (Appendix A) between 120-140 ppm are absent, indicating separation from
the acid catalyst p-toluene sulfonic acid. The large unassigned peaks centered at approximately
49 ppm correspond to the deuterated methanol solvent.
The high pH method was similarly able to separate the product from the PTSA catalyst,
but this spectrum had several additional non-identical carbons in the collected product. Four
carbonyl peaks were identified in the region from 160-220 ppm. Although only a speculation,
the additional carbonyl carbons may be carboxyl functional groups from the Gly and Leu amino
acids that would be formed through hydrolysis of the amide and ester linkages in the diester and
monoester products. The Gly and Leu amino acids have molar masses of 75 and 131 g/mol
respectively and would not show up in the mass spectroscopy range investigated. These results
suggested that the low pH method was able to achieve a relatively pure population of the desired
Gly-Leu-based diester product whereas the high pH method was not. The low pH method was
therefore chosen for purifying the chain extender for subsequent studies. Approximately 30 mg
of product was recovered for each 30 min run when injecting 75 mg of raw product giving an
approximate 70% yield. Despite the limitation of being time consuming, this system produced
enough of the purified chain extender for synthesizing the Gly-Leu PU.
103
Figure 3.11: C13 NMR spectra of products collected from preparative column HPLC using the two developed
methods of separation. a) C13 NMR spectrum of low pH HPLC method. This method led to the same number of
chemical shifts as non-identical carbons in the theoretical structure of chain extender and was the preferred method
of separation. The chemical structure of chain extender along with corresponding carbon peak assignments is
provided. * Corresponds to the large unassigned solvent peaks at ~49 ppm. b) C13 NMR spectrum of high pH
HPLC method. This method led to more than 10 non-identical carbons suggesting this method was not preferred for
the purification of the chain extender.
104
Once the preferred method of separation was chosen, FT-IR analysis was conducted on
the purified products to identify functional groups within the chain extender.
The FT-IR
spectrum of the purified chain extender is shown in Figure 3.12. The formation of an ester peak
at 1736 cm-1 confirms successful esterification between the carboxyl group of the Gly-Leu
dipeptide and the hydroxyl groups of the 1,4-cyclohexane dimethanol. Amide I and amide II
functional groups are indicated by the peaks at 1675 cm-1 and 1560 cm-1 respectively indicating
the peptide bond between the glycine and leucine residues was not disrupted during chain
extender synthesis or purification. A protonated amine group is indicated by the peak at 2960
cm-1, suggesting the need for chain extender neutralization prior to PU synthesis. This was
performed by adding triethylamine at twice the molar concentration of chain extender in DMF
before adding the mixture to PU synthesis reaction.
Figure 3.12: FT-IR spectrum of purified Gly-Leu-based chain extender. Key functional groups are identified.
105
Figure 3.13: The chemical structure of the Phe and Gly-Leu-based chain extenders.
Section was published in [1] and characterization of the Phe PU contributed to [2].
106
Gly-Leu-based monoester product that may not have been separated during purification or
formed from hydrolysis of the diester product during purification did not greatly impact the
ability to obtain a high molecular weight PU. The molecular weight averages obtained for both
polyurethanes are well within the range to impart elastomeric mechanical behavior to polymer
films, Mn > 25,000 g/mol [23].
Table 3.1: Molecular weight averages for PUs containing Phe and Gly-Leu-based chain extenders. Mean
standard deviation. n=3
Polymer
Gly-Leu PU Films
Phe PU Films
Mn (g/mol)
82700 6000
76100 4300
Mw (g/mol)
126600 9400
128600 1800
PD (Mw/Mn)
1.531 0.159
1.696 0.099
A low soft segment glass transition temperature (Tg) of approximately -51C was
observed, indicating a soft, flexible polymer at room and body temperature. Importantly, this
low Tg suggests the Gly-Leu-based chain extender promoted good phase segregation for the
formation of a biphasic morphology with hard and soft segment domains. As a result, the
polymer was semi-crystalline with a soft segment melting temperature of approximately 42C
and a soft segment crystallinity of ~28%. No hard segment glass transition temperature or
melting temperature was observed out to the tested temperature of 200C. Although it can not be
ruled out that hard segment thermal transitions may occur above 200 C, the bulky methyl ester
side chain of LDI as well as the pendant side chains on the leucine residue within the chain
extender may limit hard segment packing thus preventing hard segment crystallinity. The Phe
PU also had no hard segment transitions, which may also be contributed to the bulky side chains
within the hard segment [24].
Table 3.2: Thermal properties of the Phe and Gly-Leu PUs as determined by DSC. Mean standard deviation. n=3
Polymer
Gly-Leu PU Films
Phe PU Films
Tg (C)
-51.5 1.1
-50.6 1.2
Soft Segment
Tm (C)
Crystallinity (%)
42.0 1.5
28.4 2.6
43.0 0.7
27.4 1.6
Polymer
Crystallinity (%)
16.5 1.5
16.0 0.9
PU centered at 3373 cm-1 corresponding to the urethane and urea functional groups. The slight
difference in N-H stretching peaks between the Phe and Gly-Leu PU may result from a higher
degree of hydrogen bonding within the Gly-Leu PU, which causes a shift to a lower wavenumber
[25]. A broad ester band centered around 1732 cm-1 was observed for the Gly-Leu PU along
with a shoulder band at 1736 cm-1 corresponding to a hydrogen bonded and free functional
groups respectively. The ester peaks are predominantly contributed to the PCL soft segment,
however, some contribution may also be made from the ester linkages within the chain extender.
Similar bands were seen with the Phe PU. Although the ester functional group dominated the
carbonyl stretching region from 1650-1800 cm-1, a shoulder band centered at 1654 cm-1 and 1648
cm-1 was observed for the Gly-Leu and Phe PUs respectively. These bands correspond to the
amide I carbonyl stretching within the amide, urea, and urethane groups in the Gly-Leu PU and
the urea and urethane groups in the Phe PU. Similarly, an amide II band caused by N-H bending
and C-N stretching was observed at approximately 1549 cm-1 for both PUs. No unreacted
isocyanate groups were found for either PU due to the absence of a peak at approximately 2267
cm-1.
Figure 3.14: FT-IR analysis of Phe and Gly-Leu PUs with functional groups highlighted.
108
Figure 3.15: Representative stress-strain curves for Phe and Gly-Leu PU films.
109
extender structure may be one factor causing the observed differences in mechanical properties.
Although DSC scans revealed hard segment cohesiveness was not sufficient to see any thermal
transitions, the bulkier aromatic side chain in the Phe-based chain extender may reduce mobility
and limit hard segment packing to a larger extent than the side chain in the Gly-Leu-based chain
extender. As a result, the hard segment domains within the Gly-Leu PU may be more organized
to promote hydrogen bonding. The Gly-Leu PU had greater hydrogen bonding within the N-H
stretching region as suggested by FT-IR and further supports this notion. As well, amide groups
within the Gly-Leu-based chain extender add additional hydrogen bond forming groups and may
also play a role in the enhanced tensile properties of the Gly-Leu PU.
Table 3.3: Summary of mechanical properties of PU films. Mean standard deviation. n=9. a Statistical difference
between means at p<0.05 using two-tailed independent t-test. *not in accordance with ASTM standard
Polymer
Gly-Leu PU
Phe PU
Initial
Modulus
(MPa)
50.9 7.5*a
44.1 4.8*a
Stress at
Yield (MPa)
Strain at
Yield (%)
Stress at
Break (MPa)
Strain at
Break (%)
4.6 0.3a
3.5 0.2a
19.4 2.2a
22.5 0.6a
12.9 1.2a
8.0 0.9a
908.2 64.5
871.9 80.1
enhances hard segment cohesion. In general, though, the Gly-Leu PU and Phe PU exhibited very
similar polymer properties based on the analytical techniques employed here.
In synthesizing the PUs, PCL1250 was the only soft segment investigated. Previous
work demonstrated a family of PUs using LDI and the Phe-based chain extender as the hard
segment could be formed that exhibit a wide range of physiochemical, thermal, mechanical, and
degradation characteristics by altering the soft segment chemistry and soft segment molecular
weights [6, 28]. Increasing PCL molecular weight to 2000 g/mol resulted in elastomers that had
an increase in crystallinity, hydrophobicity, initial modulus, ultimate tensile stress and strain, and
resistance to degradation. Decreasing PCL molecular weight to 530 had the opposite effect on
PU properties. Polyethylene oxide (PEO)-based PUs were amorphous, soft, and tacky, were
much more hydrophilic, and had faster degradation rates than the PCL-based PUs [6, 28]. These
trends have been verified by several other groups using triblock soft segments of PCL-PEO-PCL
and different hard segments [5, 29-31]. This work suggests that polymer properties using the
Gly-Leu-based chain extender may also be adjusted by altering the soft segment chemistry and
molecular weights as required for particular applications. Ultimately, a family of PUs with
diverse characteristics may be achieved that contain a Gly-Leu dipeptide.
The method of incorporating the Gly-Leu dipeptide into the chain extender was chosen
based on previous work in our laboratory. A few other methods have been demonstrated to
introduce peptides into the backbone structure of segmented PUs. A direct comparison to other
amino acid and peptide containing PUs is difficult due to the differences in soft segments and
diisocyanates used in these studies but a few points may be made about these other techniques.
In the study by Lipatova et al. [15], amino acid and dipeptides were used directly as chain
extenders in the production of segmented PUs. Isocyanates can react with several different
functional groups and in this situation, reacted with the amine end to form urea groups and the
carboxylic acid end to make amide linkages. Although details of the polymer synthesis were not
provided in this report, the PUs were all of lower molecular weight, ~10,000 20,000 g/mol
[15], which is in the range where molecular weight averages have a large impact on overall
polymer properties [23]. Typical elastomeric PUs have number average molecular weights
ranging from 20,000 100,000 g/mol [23], and this approach may not be desirable for achieving
high molecular weight polymers that exhibit good elastic mechanical properties.
Chain
extenders with different end groups may lead to lower molecular weights and high
111
polydispersities due to different end group reactivities and irregular polymerizations [32, 33].
An important finding made by Lipatova et al. [15], however, was that increased phase separation
was observed when moving from a Phe amino acid chain extender to a Phe-Phe dipeptide and
was attributed to the addition of the peptide linkage. In comparing the Phe and Gly-Leu PUs, a
similar degree of phase segregation was identified between the two PUs, but the presence of the
amide bonds in the Gly-Leu PU may account for the enhanced mechanical properties.
A second method for introducing peptides into the chain extender involves the addition of
a lysine residue at the C-terminus of a specific peptide sequence to create a diamine structure.
Guan and Wagner [5] used this approach with an Ala-Ala-Lys sequence to make PUs that
display elastase sensitivity.
butanediisocyanate, and the AAK chain extender (AAK PU) is structurally the closest polymer
of this family to the Phe and Gly-Leu PUs. Although the AAK PU had lower molecular weight
averages than the Phe and Gly-Leu PUs, other properties were fairly similar. The AAK PU was
a semicrystalline phase segregated elastomer that had a comparable soft segment Tg, -54C, and
breaking strain, 830%, to the Phe and Gly-Leu PUs, but did have a higher ultimate tensile
strength, 28 MPa. The difference in ultimate tensile strength between the AAK PU and the Phe
and Gly-Leu PUs may be attributed to the higher PCL molecular weight. PUs made using
PCL2000, LDI, and the Phe-based chain extender showed a similar high ultimate tensile
strength, ~30 MPa [28]. It is important to note, though, that the AAK chain extender, which has
relatively small side chains, was used with a symmetric, linear diisocyanate, which may have
allowed for good phase segregation and mechanical properties. The symmetric diol linker
method used with the Gly-Leu PU may have helped to achieve adequate PU properties in
conjunction with the asymmetric, bulky side chain containing LDI. Based on the knowledge of
structure-property relationships, one might speculate that a PU made from a Gly-Leu-Lys chain
extender, PCL1250, and LDI may not have resulted in the favorable properties observed with the
Gly-Leu-based chain extender developed here.
3.4 Conclusions
Enzyme-susceptible segmented polyurethanes may be made through amino acid and
peptide-containing chain extenders. In an attempt to create MMP-sensitive PUs, a synthesis and
purification method was developed to successfully fabricate an amine terminated, Gly-Leu-based
diester chain extender.
averages and were phase segregated polymers. The PUs were soft, flexible elastomers that
exhibited a high breaking strain and high ultimate tensile strength. The Gly-Leu PU had very
similar physical, thermal, and chemical properties compared to the Phe PU. Chain extender
chemical structure reflected the subtle differences in mechanical properties observed between the
two PUs. Previous success with the Phe PU as biomaterial scaffolds and the comparable
properties of the Gly-Leu PU suggest this synthetic polymer may also hold promise as a
biomaterial in soft tissue engineering applications.
3.5 References
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Atala, A., Engineering tissues, organs and cells. Journal Of Tissue Engineering And
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Skarja, G.A. and K.A. Woodhouse, In vitro degradation and erosion of degradable,
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Elliott, S.L., J.D. Fromstein, J.P. Santerre, and K.A. Woodhouse, Identification of
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Guan, J.J., M.S. Sacks, E.J. Beckman, and W.R. Wagner, Biodegradable poly(ether ester
urethane)urea elastomers based on poly(ether ester) triblock copolymers and putrescine:
synthesis, characterization and cytocompatibility. Biomaterials, 2004. 25(1): p. 85-96.
32.
Shi, F.Y., L.F. Wang, E. Tashev, and K.W. Leong, Synthesis And Characterization Of
Hydrolytically Labile Poly(Phosphoester Urethanes). Acs Symposium Series, 1991. 469:
p. 141-154.
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Wirpsza, Z. and T.J. Kemp, Polyurethanes: chemistry, technology, and applications. Ellis
Horwood series in polymer science and technology. 1993, Chichester; New York: E.
Horwood. xv, 517 p.
115
Sections of this chapter have been accepted for publication [1]: Parrag IC, Woodhouse KA.
Sections of this chapter also contributed to the publication [2]: Rockwood DN, Akins RE,
Parrag IC, Woodhouse KA, Rabolt JF. Culture on Electrospun Polyurethane Scaffolds
Decreases Atrial Natriuretic Peptide Expression by Cardiomyocytes In Vitro. Biomaterials,
2008, 29(36): p. 4783-4791.
4.0 Abstract
Electrospinning was used to form porous three-dimensional PU scaffolds.
The
concentration of the Phe and Gly-Leu PUs in the electrospinning system was systematically
adjusted to form scaffolds with different structural features. Consistent with the literature, the
PUs went through concentration-dependent structural transitions with the formation of beads, to
beads-on-a-string, to good fibers lacking beaded features with an increase in concentration.
Average fiber diameter showed a direct correlation to electrospinning concentration. The Phe
and Gly-Leu PUs formed from a 14% and 10% w/v concentration respectively both had
randomly organized fibers, an average fiber diameter of approximately 3.6 m, similar molecular
weight averages and thermal properties, and were chosen for subsequent degradation and cellbased studies. In addition, the Phe PU was electrospun into scaffolds of varying architecture to
investigate how fiber alignment influences the orientation response of cardiac cells. The Phe PU
was electrospun into physically aligned and unaligned scaffolds. Uniaxial tensile testing of the
scaffolds was conducted in the preferred and cross-preferred direction of fiber orientation. The
unaligned scaffold was soft, flexible, and highly extensible in both directions while the aligned
scaffold was much stiffer, not very extensible, and had a high ultimate tensile stress when
stretched in the direction of fiber orientation.
116
properties, and average fiber diameter were similar between the Phe PU substrates of different
architecture and were used for subsequent cell-based studies.
4.1 Introduction
Tissue engineering scaffolds act as a temporary ECM for the attachment, organization,
and delivery of a high density of cells to an injured or diseased tissue. Some of the parameters
which must be considered when forming scaffolds for tissue engineering include: a high surface
area-to-volume ratio to achieve a high density of appropriately coupled cells and efficient
transport of oxygen, nutrients, and waste; pore sizes that allow cell migration; surface
chemistries and features that promote and guide cell adhesion, growth, and differentiation; and a
suitable architecture to organize the engineered tissue [3]. In addition, the combination of
starting biomaterial and polymer processing method will dictate if the scaffold has the proper
mechanical properties and degradation rates for the tissue under investigation.
Elastomeric segmented polyurethanes are thermoplastic polymers and may be processed
by conventional solvent-based methods. Two of the more promising techniques used in making
PU scaffolds for cardiac and other soft tissue engineering applications are electrospinning and
thermally induced phase separation (TIPS). Fujimoto et al. [4, 5] demonstrated PU scaffolds
formed by TIPS have the appropriate physical, mechanical, and biocompatibility properties for
use as a patch in normal and infarcted hearts. In another study using TIPS, Guan et al. [6] used
an elastase-sensitive PU to form porous anisotropic scaffolds that may be used to guide
anisotropic tissue formation. These scaffolds completely degraded in vivo in 8 weeks by passive
hydrolysis, enzyme-mediated cleavage, and other mechanisms and may have appropriate
degradation rates that allow the transfer of structural and mechanical functionality to the
engineered tissue [6]. Previous work in our laboratory by Fromstein et al. [7] described the
formation of Phe PU scaffolds by both electrospinning and TIPS and investigated how the
different scaffold morphologies influenced murine embryonic stem cell-derived cardiomyocytes
(mESCDCs). Cardiac cells on both scaffold types were contractile and expressed the proteins
sarcomeric myosin heavy chain and Cx-43 but exhibited markedly different morphologies. In
contrast to the round cells found on TIPS scaffolds, mESCDCs were more elongated on the
electrospun PU fibers and were characteristic of more differentiated and mature mESC-derived
cells [7]. As a result, while both TIPS and electrospinning may be used to form anisotropic PU
117
scaffolds that may help to control cell organization, electrospinning may be preferred in
promoting a differentiated and mature cardiomyocyte phenotype.
Electrospinning is a relatively simple, cost effective, and versatile method for processing
polymers into fibers with diameters on the nanometer or micrometer scale [8]. The non-woven,
3-D scaffolds that are formed from electrospinning have a topography and porosity that mimics
the native ECM and has been shown to positively influence cell behavior [8, 9]. This polymer
processing technique provides control over fiber diameters, structural features, and fiber
orientation and has become widely used for tissue engineering [8, 9].
Previous experience in our lab with scaffold fabrication techniques suggested
electrospinning as a very promising approach to scaffold formation in the development of a
cardiac patch [7].
electrospinning parameters, scaffolds with good fiber formation were developed with the Phe
and Gly-Leu PUs. Characterizing the morphology and fiber diameter allowed the selection of
Phe and Gly-Leu PU scaffolds that shared similar structural features and were deemed
appropriate for subsequent degradation and cell-based studies. The Phe PU was also used to
form scaffolds with aligned and unaligned fibrous structures. The scaffolds developed here were
used for cell-based studies with mESCDCs and MEFs, discussed in chapter 6, and primary
cardiac cells, which contributed to the publication by Rockwood et al. [2].
The Phe and Gly-Leu PU scaffolds electrospun from 14% and 10% w/v were used in [1] and the conditions
established here for producing aligned and unaligned Phe PU scaffolds contributed to the scaffolds used in [2].
118
Figure 4.1: Illustration of electrospinning apparatus. Image used with permission from Kenawy et al. [12].
119
Aligned and unaligned scaffolds were made using the Phe PU.
dissolved in DCM at 14% w/v and was electrospun using the conditions described above.
Randomly oriented fibers were obtained by using a collection mandrel rotating and translating at
5 cm/s. To achieve fiber alignment, the mandrel rotational velocity was increased to 270 cm/s
and the translation velocity was decreased to 3 cm/s. The electrospinning process was carried
out under ambient conditions until the deposited polymer reached approximately 100 m in
thickness (~8 h) when measured with a micrometer. The resultant polymer mats were placed in a
vacuum chamber overnight to remove residual solvent and stored in a desiccator until use.
Statistical
comparisons of fiber diameters were made using either a two-tailed independent t-test or oneway analysis of variance (ANOVA) with Bonferroni or Dunnett post-hoc analysis using the
SPSS Statistics 17.0 statistical software package (SPSS Inc, Chicago, IL). Comparison of fiber
Characterization of the Phe and Gly-Leu PU scaffolds was published in [1] and part of the characterization of the
aligned and unaligned Phe PU scaffolds was published in [2].
120
angles was performed with a two-tailed independent t-test using the absolute value of fiber
angles.
Molecular weight averages and thermal properties of the electrospun scaffolds were
determined by GPC and DSC analysis respectively as described in chapter 3. The mechanical
properties of the aligned and unaligned electrospun scaffolds were determined by uniaxial tensile
testing in the preferred and cross-preferred direction of fiber orientation. All parameters during
testing were conducted according to ASTM D1708 with the exception of sample dimensions.
Rectangular samples 3 cm long and 1 cm wide were used instead of the dumbbell-shaped
specimens to conserve material. Scaffolds were preconditioned at ~24C and ~55% relative
humidity for 48 h. Testing was carried out using an Instron 4301 testing machine with a
crosshead speed of 13 mm/min and a 10 N load cell. Samples were placed between grips 2 cm
apart, were stretched to break, and stress-strain curves were generated from force-displacement
data. All sample failure occurred away from the grips. Stress and strain at break and initial
modulus were inferred from the stress-strain graphs. Statistical comparisons were made with a
one-way ANOVA with Bonferroni post hoc analysis.
In the study by
Rockwood el al. [11], the Phe PU was electrospun using 18% and 20% w/v concentrations in
DCM, a 23 gauge needle, a positive 10 kV applied voltage, and a 25 cm distance from needle tip
to collection plate. The resulting scaffolds generally lacked the beaded formations that occur at
lower polymer concentrations, but exhibited extensive fiber fusion and melting that masked the
porosity and fibrous nature of the scaffolds [11].
electrospun using the same conditions but with a 15% w/v concentration and 20 cm distance to
collection plate [7].
structures that were missing the melted morphology observed previously. As a result, these new
conditions were initially investigated to electrospin the Phe PU with the custom built
121
electrospinner in our laboratory. Representative SEM images of the scaffolds formed from the
same conditions using the electrospinner in the Rabolt lab and the one in our lab are shown in
Figure 4.2.
A markedly different scaffold morphology was obtained when using the two
different systems. Both electrospinners produced fibers without any beads but the fibers formed
with our system appeared much smaller and had a more melted appearance than the mat
fabricated in the Rabolt lab. These results indicated that the Phe PU could be successfully
electrospun using our apparatus but suggested that differences between the two setups required
some optimization of parameters to reduce this melted scaffold appearance.
Figure 4.2: A comparison of electrospun Phe PU mats formed in a) the Rabolt laboratory and b) our laboratory
under the same conditions. A distinct difference in scaffold morphology resulted from the different electrospinners.
To optimize the Phe PU scaffolds and better understand our electrospinning system,
several parameters were adjusted including concentration, solvent type, flow rate, distance from
needle tip to collection mandrel, solution temperature, and needle gauge. Although a systematic
investigation of all these parameters was not conducted, many of the conditions tested led to
ubiquitous fiber fusion and a melted scaffold morphology (Figure 4.3a). Fiber fusion may result
from insufficient solvent evaporation or could be due to polymer chain rearrangement, as occurs
with PU chains in an attempt to reduce interfacial free energy when placed in different
environments [13]. Ultimately, conditions were identified for achieving a highly porous scaffold
with a fibrous morphology (Figure 4.3b).
122
concentration in DCM, a 22 gauge needle, a 3 ml/hr flow rate, a +12 kV applied voltage, and a
30 cm distance from needle tip to collection plate under ambient conditions (~25C and ~60%
relative humidity). Although concentration was varied, these other conditions were held constant
in forming subsequent PU scaffolds.
Figure 4.3: Comparison of Phe PU scaffolds formed before and after optimizing electrospinning parameters. a)
unoptimized parameters with scaffolds displaying ubiquitous fiber fusion and a film-like morphology. b) optimized
scaffold parameters leading to good fiber formation with reduced fiber fusion.
123
Figure 4.4: SEM images of Phe (top) and Gly-Leu (bottom) PU scaffolds electrospun from different concentrations.
The diameters of 100 individual fibers from 3 different images were measured. Average fiber diameter standard
deviation is given for defect-free fibers. A significant difference in average fiber diameter was observed between all
concentrations for Phe PU (ANOVA, p<0.05) with the exception of the 14-16% comparison. Similarly, a significant
difference in average fiber diameter was observed for all concentrations quantified for the Gly-Leu PU (p<0.05).
124
increases in concentration led to fibers lacking the beaded formations. This trend of morphology
transitions from electrospraying, to beads-on-a-string, to good fiber formation with increasing
concentration is consistent with other electrospun polymers in the literature [14].
More
measuring 100 different fibers and is quantified in Figure 4.4. A trend of increasing average
fiber diameter was observed with increasing concentration and is consistent with the literature on
electrospun polymers [14, 16]. A significant difference in average fiber diameter was observed
between all concentrations investigated for the Phe PU (ANOVA, p<0.05) with the exception of
the 14-16% comparison (p>0.05). Similarly, a significant difference in average fiber diameter
was observed between all concentrations quantified for the Gly-Leu PU (p<0.05). Therefore,
altering PU concentration has a significant impact on average fiber diameters that are formed.
Characterizing the fiber diameters further identifies a range of microfibers formed under each
polymer concentration tested. The distribution of fiber diameters within each concentration are
shown in Figure 4.5. The range of fibers obtained may be the result of polymer splaying, or fiber
splitting, observed while electrospinning the two PUs at different concentrations. The splaying
125
Figure 4.5: Fiber diameter distributions of the Phe and Gly-Leu PU scaffolds electrospun from varying
concentrations. a) Phe PU electrospun in concentrations from 12-16% w/v in DCM and b) Gly-Leu PU electrospun
in concentrations from 10-14% w/v in DCM. A range of microfibers are observed under all conditions.
126
phenomenon is caused by high repulsive forces on the polymer chains due to an increase in
surface charge density as the polymer jet travels through the electric field [17]. Deitzel et al.
[16] identified that polymer splaying occurred at higher concentrations in the electrospinning
range with a polyethylene oxide/water system and led to a bimodal fiber diameter distribution.
Although a bimodal distribution was not seen here, polymer splaying likely contributed to the
range of fiber diameters obtained with the two PUs. Fibers at all concentrations fused to other
fibers at points of intersection, which may be due to incomplete solvent evaporation or polymer
chain rearrangement. The different electrospun PU scaffolds had a porous, 3-D structure and
were considered promising for future experiments.
Fiber diameter has been shown to affect the physical, mechanical, and degradation
properties of biomaterial scaffolds [18] and also plays a role in influencing cell behavior [19,
20]. Therefore, the Phe and Gly-Leu PU concentrations that yielded scaffolds with the most
similar physical morphology and average fiber diameters were identified to reduce the variables
that may influence scaffold degradation and cell-response to the PUs. A comparison of fiber
diameter means was conducted by a one-way ANOVA with Dunnett post hoc analysis using the
different Gly-Leu PU concentrations as a control to compare to the Phe PU concentrations. The
Gly-Leu PU scaffolds electrospun at 12 and 14% w/v concentrations had average fiber diameters
that were significantly different than average fiber diameters of all the Phe PU scaffold
concentrations measured (p<0.05).
significantly different than the Phe PU scaffolds electrospun at 12 and 14% w/v (p>0.05).
Importantly, this Gly-Leu PU scaffold had approximately the same average fiber diameter as the
Phe PU formed at 14% w/v (3.6 1.2 m and 3.6 1.4 m for the Gly-Leu and Phe PUs
respectively).
morphology (Figure 4.6a) and a similar fiber diameter distribution (Figure 4.6b). The structural
similarities between the Gly-Leu and Phe PUs electrospun at 10 and 14% respectively provided
justification for their use in subsequent studies.
127
Figure 4.6: Comparison of structural features of the Phe and Gly-Leu PU scaffolds used for degradation and cellbased studies. a) SEM images of Phe and Gly-Leu PU scaffolds formed from 14% and 10% w/v concentrations
respectively. Data represents average fiber diameter standard deviation. No statistical difference in average fiber
diameter was observed between the two scaffolds. b) Fiber diameter distribution of Phe and Gly-Leu PU scaffolds.
128
The electrospun PU
scaffolds were analyzed by GPC and DSC to help identify how the electrospinning process
affected these polymer properties. Table 4.1 summarizes the GPC and DSC results of the preprocessed PU films and post-processed PU scaffolds. No major deviations in molecular weight
averages or thermal properties were noted for the electrospun scaffolds when compared to the
PU films, consistent with previous findings with the Phe PU [11]. The high molecular weight
averages observed with the PU scaffolds suggested the shear stresses associated with this
processing technique did not lead to degradation of the polymer. Similarly, no changes in the
thermal transitions indicated that the PUs remain as soft, flexible polymers at physiological
temperature and were phase segregated, semi-crystalline materials that may exhibit good
elastomeric mechanical properties. A slight decrease in overall percent crystallinity was noted
for the electrospun Gly-Leu PU scaffolds. This may be a result of the polymer chains not having
enough time to form ordered 3-D crystal structures before solidifying during solvent evaporation
[16, 18]. The slight change in crystallinity (~3%), however, was not suspected to have a major
impact on scaffold performance. These results suggested that the PUs retained several essential
polymer properties of the base material.
Table 4.1: GPC and DSC results of Phe and Gly-Leu PU films and scaffolds. Data is given as mean standard
deviation. n=3.
Mn
(kDa)
76.1
4.3
79.5
7.0
82.7
6.0
82.3
1.9
Mw
(kDa)
128.6
1.8
135.8
6.3
126.6
9.4
125.4
4.6
Polydispersity
1.696 0.099
1.709 0.171
1.531 0.159
1.523 0.066
Differential Scanning
Calorimetry
Tm
Crystallinity
Tg (C)
(C)
(%)
-50.6 43.0
16.0 0.9
1.2
0.7
-49.5 42.5
15.8 2.5
1.4
1.9
-51.5 42.0
16.5 1.5
1.1
1.5
-52.4 40.3
13.6 0.8
0.3
0.1
those with nanofibers. Henry et al. [21] formed electrospun PU scaffolds with fiber diameters
ranging from 3-10 m and found a higher number and improved morphology of fibroblasts
cultured on the porous scaffolds compared to films of the same material. Fromstein et al. [7]
successfully cultured mESCDCs onto electrospun Phe PU scaffolds that had fiber diameters
ranging from 2-10 m. The mESCDCs on these scaffolds were adherent and had a sarcomeric
phenotype that appeared more mature than cells on TIPS scaffolds. Increased surface area
associated with the Phe PU microfiber scaffolds also led to significantly higher enzyme-mediated
degradation than films of the same material [11]. These results suggest that the Phe and Gly-Leu
PU microfiber-based scaffolds formed here with fibers ranging from 1-7 m may also be
successfully employed in degradation and cell-based studies for which they were developed.
The focus on producing nanofibers as a means of mimicking the size of fibril-forming
proteins found in the native ECM has had a positive influence on cell attachment, morphology,
and proliferation [8, 9]. While micron-sized fibrous scaffolds were formed from the two PUs for
this work, electrospinning parameters can be adjusted that may allow the formation of
nanofibrous scaffolds using these polymers. Changing the solvent is one method that has been
shown to successfully move from microfiber to nanofiber-based scaffolds. Bolgen et al. [18], for
example, found that electrospinning pure PCL in chloroform produced microfibrous scaffolds
but switching to a co-solvent of chloroform and DMF caused a shift to nanofiber-based
scaffolds. Adjusting the relative percent of DMF in this co-solvent system allowed the formation
of fibers with a variety of diameters on the hundreds of nanometer scale range [18]. Stankus et
al. [22] electrospun a biodegradable PU elastomer in hexafluoroisopropanol at 5% w/v and
achieved good fiber formation with diameters from 100-900 nm. Cha et al. [23] reported a
tetrahydrofuran-DMF co-solvent system for electrospinning PU scaffolds with a range of fibers,
several of which were on the submicron scale. Others have similarly reported electrospinning
submicron fibers with biodegradable PUs by various solvent systems and electrospinning
parameters [24].
parameters for achieving submicron-sized fibers using the Phe and Gly-Leu PUs.
4.3.2
aid in the development of anisotropic tissues suggests aligned scaffolds may be preferred over
random scaffolds in the anisotropic myocardium [25-27]. Physical cues provided by underlying
130
substrates have a strong influence over cellular organization in cardiac tissue engineering [28]
and this organization is critical to the functionality of the tissue [29]. Elastic biodegradable
biomaterials with an aligned orientation may yield a material with the appropriate mechanical
properties and organizational cues for the development of myocardial constructs. To further
investigate the potential of electrospun PU scaffolds for cardiac tissue engineering, the Phe PU
was formed into scaffolds with aligned and unaligned orientations. The physical, thermal, and
mechanical characteristics of the scaffolds were determined to identify how fiber alignment
affects these biomaterial scaffold properties.
By
adjusting the speed of the electrospinning collection mandrel, scaffolds were formed into two
different morphologies with a general trend of either aligned or unaligned polymer fibers. The
unaligned scaffold was formed at a low rotational and translational mandrel velocity (~5 cm/s for
both motions). Fiber alignment was achieved by increasing the rotational mandrel velocity to
270 cm/s and decreasing the translational velocity to 3 cm/s. Representative SEM images of the
scaffolds formed under these conditions are shown in Figure 4.7. Qualitatively, electrospinning
the PU at a low mandrel rotational speed appeared to result in polymer fibers being randomly
oriented on the collection plate. The fibers collected at a high rotational speed showed a general
alignment in the direction of mandrel rotation. This was achieved at a somewhat lower mandrel
rotational velocity than has been reported in the literature with other electrospun polymers (~1218 m/s) [25, 30, 31]. Image analysis software was used in conjunction with the SEM images to
quantify the angle of fiber axis and is presented in Figure 4.8a. The observation of randomly
oriented fibers at a low rotational speed and predominantly aligned fibers at a high speed was
confirmed by this analysis. A fairly uniform distribution of fiber angles was observed with the
unaligned scaffold whereas the aligned scaffold had a more normal distribution with the majority
of fibers within 20 of the reference line. The average absolute value of fiber angle was 16.7
19.9 for the aligned scaffold and 37.2 27.9 for the unaligned scaffold with a significant
difference between the two (t-test, p<0.05). In this analysis, a value of 0 theoretically represents
131
perfectly aligned fibers while a value of 45 theoretically represents perfectly random fibers,
supporting the trend of fiber alignment at high mandrel rotational speeds and random fibers at
low rotational speeds.
Figure 4.7: SEM images of aligned and unaligned Phe PU scaffolds. a) Phe PU electrospun at a high mandrel
rotational speed and b) at a low mandrel rotational speed. Average absolute fiber angle and average fiber diameter
standard deviation were calculated from measuring fiber angle and diameter from 150 individual fibers from 6
different images. * Indicates statistical difference in average values using a two-tailed independent t-test (p<0.05).
132
Figure 4.8: Characteristics of aligned and unaligned Phe PU scaffolds. a) Quantification of fiber angle. The
aligned scaffold had the majority of fibers within 20 of a reference angle while the unaligned scaffold had a more
uniform distribution. b) Fiber diameter size distribution. A similar fiber diameter distribution was observed for the
two scaffold architectures. Fiber diameters and angles were measured from 150 individual fibers from 6 images.
133
mandrel and this discrepancy may reflect the different electrospinner setups, which was
demonstrated above to affect scaffold features. The aligned and unaligned scaffolds formed
using the dynamic collection mandrel had an average fiber diameter of 2.9 1.7 m and 2.7
1.5 m respectively with no statistical difference between the two (t-test, p>0.05). A high
mandrel rotational speed used to align fibers has been reported in the literature to decrease fiber
diameters due to additional stretching during deposition on the rotating mandrel [31], but this
was not observed here. The lower mandrel rotational velocity (2.7 vs.13 m/s) that induced fiber
alignment may not have been sufficient to see this phenomenon. The formation of aligned and
unaligned scaffolds with a similar average fiber diameter prevented the need to optimize
electrospinning conditions and allowed further investigations into how fiber alignment affected
scaffold properties.
Mn
(kDa)
76.1
4.3
79.5
7.0
75.4
4.2
Mw
(kDa)
128.6
1.8
135.8
6.3
131.6
6.2
Polydispersity
1.696 0.099
1.709 0.171
1.746 0.128
134
Differential Scanning
Calorimetry
Tm
Crystallinity
Tg (C)
(C)
(%)
-50.6 43.0
16.0 0.9
1.2
0.7
-49.5 42.5
15.8 2.5
1.4
1.9
-48.3 43.7
16.7 1.0
0.7
2.2
to increased stretching and molecular chain orientation was not observed here [25, 32]. The high
molecular weight averages and thermal transitions of soft, flexible semi-crystalline polymers
suggest the potential of good PU performance at physiological temperatures in further studies.
scaffolds. The unaligned scaffold exhibited similar mechanical behavior in both directions with
a relatively high ultimate tensile strain, ~300%, but a low ultimate tensile stress, ~1 MPa, and
initial modulus, ~8 and ~4 MPa when stretched in the preferred and cross-preferred directions
respectively. In contrast, the aligned scaffold exhibited a significant difference in all tensile
properties when stretched in the preferred and cross-preferred directions of fiber orientation
(ANOVA, p<0.05).
When stretched in the preferred direction, the scaffold was stiff and
incapable of withstanding high deformations. Samples stretched in this direction had a relatively
high initial modulus, ~24 MPa, and ultimate tensile stress, ~4 MPa, but exhibited a relatively low
ultimate elongation, ~60%. When stretched in the cross-preferred direction, the aligned scaffold
was more similar to the unaligned scaffold with a high ultimate tensile strain and a low ultimate
stress and modulus.
mechanical properties when comparing Phe PU films to the different scaffold architectures and
stretch directions. The PU films had a significantly higher initial modulus, ultimate stress, and
strain at failure compared to the electrospun scaffolds (ANOVA, p<0.05) suggesting the
electrospinning process had a major impact on resulting mechanical properties of the scaffolds.
135
Figure 4.9: Representative stress-strain curves for aligned and unaligned PU scaffolds stretched in preferred and
cross-preferred directions of orientation.
Table 4.3: Summary of mechanical properties of aligned and unaligned Phe PU scaffolds stretched in
preferred and cross-preferred directions of orientation. Data is given as mean standard deviation. N=8 unless
otherwise stated. One-way ANOVA with Bonferroni post hoc analysis showed statistical difference (p<0.05)
compared to a) aligned scaffold stretched in preferred direction, b) unaligned scaffold stretched in preferred
direction and c) Phe PU films.
Scaffold Architecture
Cast Phe PU Films
(N=9)
Aligned Stretched in
Preferred Direction
Aligned Stretched in
Cross-Preferred Direction
Unaligned Stretched in
Preferred Direction
Unaligned Stretched in
Cross-Preferred Direction
(N=5)
Initial Modulus
Ultimate
Strain at
(MPa)
Tensile Stress (MPa) Failure (%)
44.09 4.77a,b
8.01 0.94a,b
872 80a,b
24.42 5.04b,c
3.94 1.04b,c
61 11b,c
0.60 0.11a,b,c
0.29 0.02a,c
263 25a,c
8.36 1.86a,c
1.07 0.11a,c
298 70a,c
4.12 0.59a,c
0.94 0.04a,c
345 29a,c
136
A mandrel
rotational velocity of 3 m/s began to lead to the alignment of fibers and further increases in
rotational speed induced higher degrees of physical anisotropy. As fiber alignment became
higher, scaffolds became increasingly stiff in the preferred direction of fiber orientation and had
the opposite affect in the cross preferred direction. Other groups similarly demonstrated aligned
scaffolds exhibit high strengths and low elongations when stretched in the preferred direction of
orientation and have much higher strains and lower strengths when stretched in the perpendicular
direction [30, 31]. Importantly, a study by Johnson et al. [30] used pure electrospun PCL to
investigated the microstructure of scaffolds under strain to identify the response of polymer
fibers to mechanical stretch. It was determined that electrospun fibers initially not aligned in the
direction of stretch rearrange and undergo a strain-induced orientation of fibers parallel to
applied force [30]. In this situation, applied loads are carried by weak inter-fiber interactions and
structural reorganization events. As more fibers become aligned with increasing strain, the
applied stress is efficiently transferred to unstrained areas of the scaffold where fibers have yet to
be aligned [30]. This leads to scaffolds that have low strengths and high elongations due to the
weak load-bearing features and high strains needed to align the fibers. In contrast, the aligned
scaffold stretched in the preferred direction of orientation has little capacity for fiber
reorganization in the direction of applied force. The applied load is therefore carried directly by
the strong fibers leading to high strengths and relatively low elongations [30].
What is less known about the mechanical properties of aligned and unaligned PU
scaffolds is the microstructural organization of hard and soft segments within the electrospun
fibers and their contribution to the mechanical properties. GPC and DSC results suggested the
electrospinning process did not affect molecular weight averages or thermal properties of the PU,
so the polymer retained a high molecular weight, phase segregated, and semi-crystalline
structure.
polyurethane films [13] but it is unclear what their contribution is to the mechanical properties of
the electrospun fibrous scaffolds. High strain rates and polymer fiber stretching experienced
during the electrospinning process can lead to highly oriented molecular chains within the
polymer fibers and a correspondingly stiff polymer behavior [32, 33]. Further mechanical
testing of individual PU fibers and determination of the molecular structure within the fibers is
required to elucidate this microstructure-property relationship. Understanding this behavior may
137
be an important consideration for determining if the mechanical properties of these scaffolds are
suitable for cardiac applications.
environment exposes scaffolds to repetitive stresses and the scaffolds should be able to withstand
these forces without losing mechanical integrity. Moreover, mechanical forces play a critical
role in the formation of normal cardiac structure and function. Tissue engineering scaffolds
should therefore have the appropriate mechanical properties to promote mechanotransduction
between cells and the external cardiac or cardiac-mimicked environment [36]. Fujimoto et al. [5]
provided evidence that biodegradable PU elastomers may be formed into scaffolds with the
appropriate physical, mechanical, and biocompatibility properties for use as a patch in improving
the function of infarcted hearts.
biodegradable PU elastomer used in that in vivo study and the Phe PU used in this work suggests
that the electrospun Phe PU scaffolds may also have success in the heart [5, 37, 38].
The exact mechanical properties required for cardiac tissue engineering scaffolds have
not been explicitly defined, but recent work in the field is moving towards mimicking the
mechanical properties of the native myocardium [39-41]. Engler et al. [35] identified that
cardiomyocytes develop force, organize a striated sarcomeric structure, and sustain a contractile
phenotype long-term when cultured on substrates that have an elastic modulus similar to that of
myocardial tissue. Cardiomyocytes on hard matrices do not form striated sarcomeres or remain
contractile for long periods whereas these cells cultured on very soft substrates remain
contractile but do little work [35]. Human myocardium has a Youngs modulus of ~20-500 kPa,
a tensile strength of ~3-115 kPa, and elongations from ~60-90% [41, 42]. The aligned and
unaligned electrospun PU scaffolds formed here have mechanical properties that generally
exceed those of the native myocardium, with the exception of properties of the aligned scaffold
stretched in the cross-preferred direction. The higher ultimate percent elongations (~60-300%)
may be beneficial to PU scaffold performance in the heart, whereas the high ultimate stresses
(~1-4 MPa) and initial modulus (~4-24 MPa) may be more problematic. In particular, scaffold
stiffness is an important determinant of cell organization and function [35] and the higher
stiffness of these scaffolds may limit these properties in engineered tissue. To date, this has not
been observed in in vitro studies with cardiac cells on either the electrospun Phe PU scaffolds or
Phe PU films, which have a higher modulus than the scaffolds [2, 7, 43, 44]. It is important to
point out that the tensile testing of these scaffolds was conducted under ambient conditions and
the mechanical properties may be altered in the physiological environment where water may
disrupt the hydrogen bonding within these materials.
139
experience may be quite different than the modulus of overall scaffold where mechanical
properties were a function of numerous individual fiber strengths, inter-fiber interactions, and
fiber alignment events.
The initial modulus and ultimate tensile strength of several synthetic biomaterials that
have been used or hold potential in cardiac tissue engineering are shown in Table 4.4 and are
ranked based on their similarity to the modulus of native myocardial tissue.
It has been
suggested that biomaterials stiffer than heart tissue may be used as a heart patch to reduce heart
wall stress and attenuate cardiac deterioration following a myocardial infarction [45]. Diastolic
dysfunction, however, will occur if the biomaterial is too stiff [45]. Until the mechanical
properties of scaffolds for cardiac tissue engineering become strictly defined, comparing the
modulus of biomaterials to native heart tissue is one approach to develop biomimetic matrices
for use in the heart.
myocardium than some biomaterials, such as the traditional tissue engineering synthetic
polymers PGA and PLA, but is not as close as other materials, such as PGS. The electrospun
Phe PU scaffolds had reduced mechanical properties that were closer to the native myocardium
than the films, but there still may be room for improvement. Since unaligned scaffolds have a
lower modulus than those with fibers aligned in one direction, a bi-layered scaffold with a thin
layer of aligned fibers over unaligned fibers may provide physical cues to cells while decreasing
scaffold stiffness. This may not address the issue of the modulus the cells experience, but may
have implications as a cardiac patch. Alternatively, one of the major advantages of using
polyurethanes in tissue engineering is the ability to tailor the properties for site specific
applications. Therefore, the soft segment of the Phe PU may be changed to lower the modulus
and more closely mimic the mechanical properties of the heart. Synthesizing the Phe PU with a
lower PCL diol molecular weight (e.g. 530 Da) or introducing PEO of different molecular
weights by using tri-block PCL-PEO-PCL soft segments may help to achieve this goal [37, 38].
Further cell-based and in vivo studies with the Phe PU scaffolds are required to test whether
these modifications are necessary.
140
Table 4.4: Initial modulus and ultimate tensile strength for films of investigated or potential synthetic biomaterials
in cardiac tissue engineering. Polymers are ranked from top to bottom based on similarities in modulus to human
myocardium [37, 38, 45, 46].
Polymer
Human Myocardium
0.02-0.5 MPa
3-115 kPa
Poly(glycerol sebacate)
0.04-1.2 MPa
0.2-0.5 MPa
Poly(1,8-octandiol-co-citric acid)
1-16 MPa
6.7 MPa
Poly(1,3-trimethylene carbonate)
5-6 MPa
2-12 MPa
5-75 MPa
8-20 MPa
Phenylalanine-based Polyurethanes
12-30
7-80 MPa
16 MPa
10 MPa
Polycaprolactone
320 MPa
32 MPa
Poly(p-dioxanone)
600 MPa
12 MPa
Polylactic Acid
1-5 GPa
30-80 MPa
Polyglycolic Acid
7-10 GPa
70 MPa
4.4 Conclusions
The ability to form porous 3-D scaffolds with the appropriate physical and mechanical
properties is critical to the success of tissue engineering scaffolds.
Electrospinning is an
important scaffold fabrication technique that allows the formation of fibrous polyurethane
scaffolds that are promising for soft tissue applications.
scaffolds were formed via electrospinning with randomly organized structures and a similar
average fiber diameter. The similarities in structural, physical, and thermal properties warranted
their use in subsequent degradation and cell based studies. Phe PU scaffolds were processed into
different fibrous architectures by electrospinning the polymer at varying mandrel rotational
speeds. The aligned and unaligned PU scaffolds were fabricated for subsequent coculture studies
with mESCDCs and MEFs. The different scaffolds developed and characterized in this chapter
have promising properties for cardiac and other soft tissue engineering applications and warrant
their further investigation in degradation and cell-based studies.
141
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Degradation
experiments conducted with the Phe and Gly-Leu PUs in the presence of active MMP-1, active
MMP-9, or a buffer solution were carried out over a 28 day period. Mass loss and structural
assessment suggested that neither PU experienced significant hydrolysis to observe degradation
over the course of the experiment.
enhanced degradation in the presence of the MMPs. The lack of MMP-mediated Gly-Leu PU
degradation may be due to enzyme concentration, time frame of study, enzyme adsorption onto
PU surfaces, length of peptide sequence in PU, and accessibility of enzymes to labile bonds.
5.1 Introduction
Degradation rates and characteristics are an important consideration for the success of
tissue engineering scaffolds. One criterion for biomaterial scaffolds is that the temporary matrix
must degrade at a rate that is appropriate for tissue regeneration. In the case of heart tissue,
degradation should simultaneously occur as ECM proteins are synthesized, allowing the gradual
transfer of mechanical load from the biomaterial to the newly secreted ECM [1, 2]. This enables
the cells and ECM to respond to the dynamic cardiac environment by promoting proper tissue
organization and remodeling that is critical to heart function [3]. Moreover, the scaffolds should
be present long enough to guide integration with the host myocardium but should not last so long
as to interfere with electromechanical coupling of cells [4]. Similarly, degradation should occur
in a time frame that prevents fibrous tissue encapsulation that may inhibit proper integration with
the host.
146
Normal ECM degradation and tissue remodeling is carried out by MMPs. MMPs play a
critical role in the wound healing events that occur in the heart following a MI and continue to
participate in ECM degradation during detrimental ventricular remodeling in the progression to
heart failure [5-7]. Specifically, MMP-1, MMP-2, and MMP-9 have been shown to have major
roles in the heart after injury. Disrupting the normal expression of these proteases attenuates
ventricular remodeling and improves cardiac performance following an infarction [8-10]. A
priori knowledge of the critical MMP involvement in an injured heart and their sites of cleavage
in ECM proteins allow the design of biomimetic synthetic polymers that may exploit the
presence of these cell-secreted proteases to degrade the material. An MMP-sensitive synthetic
biomaterial may degrade at a rate that is more appropriate for promoting ECM production, tissue
remodeling, and vascularization as required by the host tissue than other synthetic polymers that
degrade predominantly by passive hydrolysis.
degradation rates that are appropriate for cardiac tissue engineering and would be an appealing
property of candidate biomaterials for use in the heart.
The cleavage site of several MMPs was built into the backbone structure of PUs through
the formation of a Gly-Leu-based diester chain extender. The Gly-Leu PU and chymotrypsinsensitive Phe PU were synthesized, characterized, and formed into electrospun scaffolds as
discussed in previous chapters. In this chapter, the degradation of the PU scaffolds by passive
hydrolysis and enzymatic mechanisms using the proteases MMP-1 and MMP-9 is described.
The degradation experiments were conducted to provide a better understanding of how altering
PU hard segment chemistry and incorporating a Gly-Leu dipeptide into the chain extender
affected PU properties. The ability to obtain activated MMPs was initially confirmed and the
activity of these enzymes was tested. Subsequently, a PU scaffold degradation study was carried
out and changes in mass and physical structure were assessed.
147
the MMPs. The organomercurial agent (4-aminophenyl)mercuric acetate (APMA) was used in
the activation studies.
Laemmeli sample buffer (0.125 M Tris-HCl of pH 6.8, 4% w/v SDS, 20% v/v glycerol, and
0.04% w/v bromophenol blue) at final dilution of 240 ng per 10 l solution with the exception of
active MMP-9 (40 ng per 10 l solution).
approximately 45 min. The gel was incubated with zymogram renaturing buffer (Invitrogen,
Carlsbad, CA) for 30 min, then with zymogram developing buffer (Invitrogen) for ~4 h, and
finally with SimplyBlue SafeStain (Invitrogen) for ~1 h all at room temperature with ultrapure
148
deionized water rinse steps in between. A standards lane of pre-stained calibrated molecular
weight proteins (Bio-Rad Laboratories, Mississauga, ON) was used as a reference to help
identify the molecular weights of the MMP species.
An activity study was conducted to identify the duration the MMPs remain active in free
solution. Active MMP-9 and proMMP-1 activated by APMA for 24 h were diluted in buffer
(200 ng/ml) and were incubated with PU scaffolds for 6, 12, and 24 h. Enzyme solution was
removed at each time point and protease activity was measured using the fluorogenic substrate
assay. Percent activity was calculated by comparing to initial fluorescent measurements prior to
incubation with scaffolds.
The
difference in mass before and after incubation in water was used to calculate percent water
uptake. Enzyme inhibition was performed by measuring MMP activity using the fluorogenic
substrate assay in the presence of various concentrations of the Gly-Leu dipeptide. Fluorescence
levels in the presence of Gly-Leu dipeptide were compared to those without the dipeptide to test
if enzyme binding to the Gly-Leu dipeptide inhibited cleavage of the fluorogenic peptide
substrate.
149
150
demonstrated protease activity after incubation with APMA at all time points while MMP-9
showed little protease activity after incubation with APMA. MMP-1 displayed a high level of
fluorescence, 5337 451 units, after only 15 min of incubation with APMA. This fluorescence
decreased slightly at 30 min and slowly increased over the next few time points with the highest
level observed at 24 hr, 5619 413 units. In contrast, low levels of fluorescence were observed
at all time points with MMP-9 incubated with APMA. Given the proven ability of APMA to
activate proMMP-9 [14, 17, 18], the problem with activating MMP-9 was not likely due to the
method of activation but rather reflected a problem with the MMP-9 enzyme. As a result, MMP9 was supplied in its active 67 kDa form (Calbiochem) and this enzyme showed high protease
activity after incubation with the fluorogenic substrate (Figure 5.1). Therefore, active forms of
both MMP-1 and MMP-9 were obtained that exhibited protease activity for use in subsequent
studies.
Figure 5.1: Activation of MMPs using APMA. Fluorescence was normalized to buffer background with FS-6.
MMP-1 showed protease activity at all time points after incubation with APMA while MMP-9 showed little activity.
Active MMP-9 (Calbiochem) demonstrated protease activity. Error bars represent standard deviation. n=3, N=3.
The results from the fluorogenic substrate assay were supported by zymography of the
different MMP solutions.
through a polyacrylamide gel containing gelatin under denaturing and non-reducing conditions.
151
Following separation, the resolved proteins are renatured and proteolytic activity is detected
through degradation of gelatin. A 10% polyacrylamide gel was used and the gel was stained
using a Coomassie blue staining solution after the enzymes were renatured. The resulting gel is
shown in Figure 5.2 with clear bands indicating proteolytic activity against the non-degraded
blue gelatin background.
proMMP-9 and MMP-9 solution incubated with APMA indicating a mixed protein solution.
MMP-9 forms complexes with several proteins after secretion from cells and these bands may
reflect a complexed form of MMP-9 [17]. No difference in band patterns was observed between
the proMMP-9 and APMA-incubated MMP-9 solution suggesting the absence of an active form
of this enzyme. In contrast, MMP-9 supplied in an active form exhibited additional bands
not present in the other MMP-9 lanes. Gelatin degradation was observed at all points within the
Figure 5.2: Zymogram of MMP activation solutions. No differences in proMMP-9 and MMP-9 incubated with
APMA were observed. Active MMP-9 resulted in two additional lower molecular weight bands, one corresponding
to the 67 kDa active species. Incubation of MMP-1 with APMA led to a loss of the 52 kDa pro-enzyme band with
the 44 kDa intermediate and 42 kDa active species observed in both lanes.
152
lane above a certain cutoff indicating either a mixed protein solution similar to proMMP-9 or the
presence of active intermediate products that were not completely denatured during
electrophoresis. In addition, two lower molecular weight bands with distinct gelatinase activity
were observed. The enzyme was supplied in the 67 kDa form and the bands likely corresponded
to this active species and a further processed form. An active 55 kDa MMP-9 species has been
reported [18] and could account for this lower molecular weight species. MMP-1 was supplied
as a mixture of both the inactive zymogen and activated species. As a result, the proMMP-1 lane
displayed three bands corresponding to the 52 kDa pro-enzyme, the 44 kDa intermediate species,
and the 42 kDa active form. After activation by APMA for 24 h, the 52 kDa pro-enzyme was no
longer present while the 44 kDa intermediate and 42 kDa active species remained. This is
consistent with the activation mechanism of APMA reported in the literature [16-18]. These
results support the fluorogenic substrate assay for MMP activation and subsequent experiments
were performed using MMP-1 activated by APMA for 24 h and the active MMP-9 directly.
153
Figure 5.3: Activity of MMPs after incubation with PU scaffolds. a) MMP-1 activity remains at initial level for at
least 24 h. b) MMP-9 activity drops to less than 50% its initial level after 6 h. Error bars represent standard
deviation (n=3, N=3).
154
the enzymes onto the PU scaffolds are required to help elucidate these mechanisms. For the
degradation study, fresh enzyme solutions were added to PU scaffolds each day to replenish any
lost activity.
155
Figure 5.4: Mass remaining of PU scaffolds over 28 day degradation study. a) Gly-Leu PU scaffolds and b) Phe PU
scaffolds after incubation with MMP-1, MMP-9, and buffer solutions over a 28 day period. No appreciable
difference in mass was observed over the 28 day period. Error bars represent standard deviation (n=3, N=1).
156
Figure 5.5: SEM images of PU scaffolds after 28 day incubation period in various solutions. A) Gly-Leu PU in
buffer. B) Gly-Leu PU in MMP-1. C) Gly-Leu PU in MMP-9. D) Phe PU in buffer. E) Phe PU in MMP-1. F)
Phe PU in MMP-9. No changes in surface or scaffold morphology were observed.
The results from the initial degradation study of the PU scaffolds in MMP and buffer
solutions suggested that little or no observable degradation occurred over the 28 day time period.
The Phe PU has been shown to be relatively resistant to passive hydrolysis while being more
susceptible to enzyme-mediated degradation by chymotrypsin and to a lesser extent by trypsin
[19]. Phe PU films exhibited ~1%, 3.5%, and 4.5% mass loss after 28 days in the presence of
buffer, trypsin, and chymotrypsin solutions respectively and enzymatic degradation occurred
through a surface-mediated mechanism [19]. Electrospun Phe PU scaffolds were shown to have
higher mass loss (~10%) over 28 days in chymotrypsin due to increased surface area [20].
Increased fiber surface roughness and breaking of PU fibers were observed with increasing
chymotrypsin exposure and is consistent with the surface-mediated degradation mechanism [20].
157
In the current work, the Phe and Gly-Leu PU scaffolds did not exhibit changes in mass or surface
features that would be expected if the scaffolds were susceptible to passive hydrolysis or
enzymatic degradation. Although previous work with PCL-based PU films suggested little
passive hydrolysis of the PU scaffolds would occur, the increased surface area of the electrospun
mats may be anticipated to cause a higher mass loss in the buffer solution (>1%) compared to
films [19, 20]. Nonetheless, Bolgen et al. [21] found that electrospun scaffolds made from pure
PCL were more hydrophobic than PCL films, thereby reducing the degradation rates. A similar
phenomenon may be observed here with the PCL-based PUs. It was not anticipated that the Phe
PU would be susceptible to degradation by the MMPs. The lack of visible degradation with this
polymer provides further support that the Phe-based chain extender confers specific and
enhanced degradation in the presence of certain enzymes.
More importantly though is the lack of observable MMP-mediated degradation with the
Gly-Leu PU. The Gly-Leu dipeptide was incorporated into the chain extender as a means of
introducing the MMP cleavage site into the polymer backbone. Surface enrichment of hard
segments as is commonly observed with PUs was anticipated to present the labile bonds to the
MMPs and lead to PU degradation. Unfortunately, in the degradation study conducted here, the
Gly-Leu PU did not exhibit enhanced degradation in the presence of MMP-1 or MMP-9.
There are several potential explanations for the absence of any enzyme-mediated GlyLeu PU degradation observed in this study. One may be related to the concentration and time
period investigated. The degradation experiment was conducted by incubating the PU scaffolds
with active MMPs at a concentration of 200 ng/ml, which is similar to the concentration found in
the plasma of patients with congestive heart failure [22]. This concentration was chosen in an
attempt to carry the degradation study out under physiologically-relevant conditions, but MMP
concentrations found in the plasma may not necessarily correlate to the actual enzyme level
scaffolds will be exposed to when implanted in the heart. MMP expression following a MI may
become elevated in the presence of biomaterial scaffolds due to the bodys inflammatory
response and the increased number of MMP-secreting cells recruited to implant site [6, 23].
Moreover, dose-dependent PU degradation has been demonstrated with polycarbonate-based
PUs using cholesterol esterase whereby some PUs were stable at low enzyme concentrations but
exhibited significant degradation at higher concentrations [24]. The MMP concentration used in
this study may have been too low to observe PU degradation in the time period investigated.
158
Increasing the enzyme levels and length of the study may lead to substantial and observable PU
degradation.
Enzyme adsorption and activity may also have been a factor in explaining the absence of
PU degradation. Santerre et al. [25] provided a mechanistic model for degradation of PUs by
hydrolytic enzymes: 1) PU surface contact with aqueous environment and structural
rearrangement of polymer chains; 2) enzyme adsorption/desorption with the surface; 3) active
adsorbed enzymes react with susceptible bonds; 4) degradation products are released from main
polymer chain into solution exposing new polymer surfaces; 5) water, electrolytes, and enzymes
establish interactions with newly exposed surface; and 6) process is propagated over time to
degrade the polymer. Therefore, enzymes must adsorb onto the PUs and must remain active in
the surface-bound state for polymer chain cleavage to occur. Enzyme adsorption onto the PU
scaffolds was difficult to determine from the activity study conducted here and may have been a
limiting step in the degradation study.
A third explanation for the lack of Gly-Leu PU degradation could be that introducing the
Gly-Leu dipeptide alone without any flanking amino acid sequences into the PU structure was
not sufficient for recognition by the MMPs for binding and substrate cleavage.
It was
hypothesized that this minimalistic approach of incorporating the Gly-Leu cleavage site may
enhance degradation rates in PUs, but it was not known conclusively whether the dipeptide alone
would confer enzyme-susceptibility to the biomaterial. While most enzymes have very specific
substrates in which they bind to and act upon, all the MMPs have broad substrate specificities
and are able to cleave several ECM and non-ECM proteins [17, 26]. An early MMP-1 study
suggested that specificity with this enzyme was largely independent of substrate conformation
and reflected the amino acids surrounding the cleavage site [27].
Much work was subsequently conducted on short peptide sequences to identify substrate
specificities of the MMPs.
approximately the peptide length that can fit into the active site of the enzymes [26]. One group
systematically altered the peptide sequence Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, known to be
cleaved by several MMPs between the Gly and Ile residues, to investigate the effects amino acid
residues surrounding the cleavage site had on substrate specificity [17]. Hydrolysis rates were
dependent on both the specific amino acid and position within the peptide sequence for different
MMPs [17]. In addition, successively removing amino acids from the C and N-terminus of this
159
reference peptide led to rapid decreases in hydrolysis rates once the peptide was smaller than six
amino acids long [17]. While this would suggest that a hexapeptide is the minimum length for
high rates of hydrolysis, it does not necessarily indicate that peptide cleavage by MMPs will not
occur with shorter peptide lengths.
Hubbell and West [28] incorporated the peptide Ala-Pro-Gly-Leu into synthetic
hydrogels that rendered the biomaterial susceptible to collagenase-mediated degradation.
Hydrogel discs 5 mm in diameter and 2 mm thick were completely degraded after 5 and 7 days
of incubation with 2 mg/ml and 0.2 mg/ml collagenase solutions, respectively. Recently, Guan
et al. [29] used the same sequence with a lysine residue on the end (Ala-Pro-Gly-Leu-Lys) as a
diamine chain extender in the development of segmented polyurethanes that were also
susceptible to collagenase. Although the details of this work have not been fully published, PU
films showed approximately 20% and 30% mass loss after 4 and 8 weeks in the presence of
collagenase, respectively. The exact peptide requirements for achieving MMP susceptibility
with biomaterials are not known. However, the literature would suggest that peptide length and
sequence will affect the rates of hydrolysis. It is possible that additional amino acids flanking
the Gly-Leu cleavage site are required for degradation by the MMPs. The lack of observed
degradation may also be due to low rates of hydrolysis with the dipeptide alone. Higher enzyme
concentrations as well as longer time periods may be necessary to detect any Gly-Leu PU
degradation by the MMPs.
In an attempt to further elucidate the mechanisms behind PU degradation, a preliminary
competitive substrate assay was carried out. This was conducted to test if the Gly-Leu dipeptide
alone is a substrate for MMPs and therefore if it can act as a site for potential cleavage by the
proteases. If the Gly-Leu dipeptide is a binding substrate for the MMPs, then introducing it into
the fluorogenic substrate assay may lead to a competitive reaction and lower fluorescence
(Figure 5.6). Therefore, active MMPs were incubated with FS-6 and different concentrations of
the Gly-Leu dipeptide were introduced into the solution. Fluorescence levels were compared to
those with the MMPs incubated with FS-6 alone to get a relative percent of FS-6 cleavage
(Figure 5.7). No change in FS-6 cleavage was observed when the Gly-Leu dipeptide was
introduced at 100x the molar concentration of FS-6 but a drop of approximately 10% occurred at
1000x molar concentration. Although repeated trials are necessary to confirm these results, this
160
Figure 5.6: Reaction scheme for enzyme activity assay and competitive substrate enzyme activity assay.
Figure 5.7: Inhibition of FS-6 cleavage using the Gly-Leu dipeptide. No difference in FS-6 cleavage was observed
with 100x molar concentration of dipeptide but a drop of ~10% occurred at 1000x. Error bars represent standard
deviation. n=3, N=1.
161
preliminary study suggested that FS-6 is a much better substrate than the Gly-Leu dipeptide. The
unchanged FS-6 cleavage rates in the presence of 100x excess of Gly-Leu dipeptide may reflect
a much higher binding efficiency to the length and sequence of the FS-6 peptide substrate
compared to the dipeptide alone. This would be consistent with the trend of higher rates of
hydrolysis with peptides longer than 6 amino acids in length and dramatically reduced rates with
shorter peptides [17]. The drop in FS-6 cleavage at 1000x molar excess of dipeptide, however,
may suggest that while having much lower binding kinetics than FS-6, the Gly-Leu dipeptide
may still act as a substrate for the MMPs. This would support the notion that the Gly-Leu
dipeptide is susceptible to MMPs, but hydrolysis rates were too slow to be observed in the study
conducted here.
The exact interpretation of the competitive substrate enzyme activity results, however,
remains difficult. This competitive substrate enzyme inhibition study may be better validated by
comparing the peptide Ala-Pro-Gly-Leu, which is susceptible to collagenase within PUs and
other synthetic polymers [28, 29]. Moreover, it is important to consider the length of the
dipeptide as a substrate as this may influence MMP binding to the molecule. Inhibition of
MMPs has been observed with a short sequence consisting of Pro-Leu-Gly-NHOH [18], but it is
unclear if a dipeptide alone is long enough to see similar behavior. It has been suggested that the
active site of MMPs can accommodate peptide sequences up to 8 amino acids in length [26].
Shorter peptide sequences may lead to reduced rates of hydrolysis because these peptides are not
stabilized in the active site of the enzymes as well as the longer sequences. When the Gly-Leu
dipeptide is contained within a polymer chain, the MMPs may have a larger substrate to bind to
allowing a more stabilized complex to form between the enzyme and potential cleavage site. A
better model for this system may be to replace the Gly-Leu dipeptide with the Gly-Leu-based
chain extender, thus providing a longer chain for the MMPs to bind to. Similarly, using the GlyLeu-based chain extender as a linker to make a short polymer chain may provide a model
polymer segment to test both competitive reaction enzyme inhibition and cleavage of polymer
chains at the Gly-Leu peptide bond. Guan and Wagner [30] verified the sensitivity of the AlaAla-Lys chain extender to elastase by synthesizing a methyl-PEG flanked peptide conjugate
(mPEG-Ala-Ala-Lys-mPEG) and identifying the molecular weight of products formed after
exposure to the enzyme. This may better indicate if the Gly-Leu dipeptide is sufficient to get
chain cleavage by the proteases or if additional amino acids are required.
162
A fourth possible explanation for the Gly-Leu PU scaffold stability may be due to
polymer hydrophobicity and hard segment packing within the PU.
Enzyme-mediated PU
degradation is surface limited [19] and polymer hydrophobicity and hard segment packing may
limit accessibility of the MMPs to potentially susceptible cleavage sites. Work by Santerre and
colleagues found that increased hard segment content was indirectly proportional to hydrolytic
degradation of polyurethanes even though the hard segments contained hydrolysable groups [31].
In addition, the level of enzymatic hydrolysis was inversely proportional to hydrogen bonding
between hydrolysable bonds in the hard and soft segments due to domain cohesion and the
inability of enzymes to reach the labile bonds [32]. A similar phenomenon may account for the
stability of the Gly-Leu PU to enzyme-mediated hydrolysis. Characterization of the Gly-Leu PU
indicated a phase segregated polymer with enhanced mechanical properties compared to Phe PU
and increased hydrogen bonding within the hard segment. These results suggested the Gly-Leu
PU had some hard segment interactions but the microphase was amorphous with no observed
hard segment thermal transitions due to the bulky side chains within the LDI and chain extender.
Hard segment surface enrichment can also occur with segmented PUs in aqueous environments,
suggesting that the labile peptide bonds may have been at the surface where enzymes would have
access. Water uptake by the PU scaffolds (Figure 5.8) revealed that both PU scaffold types
absorbed some water but the Gly-Leu PU scaffolds absorbed more water than the Phe PU
scaffolds. This trend suggests that the Gly-Leu PU was not as hydrophobic as the Phe PU and
that MMPs may indeed have had access to polymer fiber surfaces.
Interestingly, cleavage of polymer chains by the MMPs requires that the susceptible
bonds not only be accessible but also presented in a conformation that is suitable for binding to
the active site of the enzymes [17]. Santerre et al. [25] demonstrated that similar levels of
adsorbed cholesterol esterase to different PU surfaces did not necessarily correlate to the same
amount of polymer degradation. If the Gly-Leu dipeptide alone is sufficient to be cleaved by
MMPs at a rate that would have been observed in this study, then polymer chain organization
and hard segment interactions may have played a role in preventing hydrolysis due to the
conformation of the labile bonds.
163
Figure 5.8: Water uptake by Phe and Gly-Leu PU scaffolds. A trend of higher water uptake was observed with the
Gly-Leu PU compared to the Phe PU suggesting the Gly-Leu PU was not as hydrophobic as the Phe PU. n=9, N=1.
The cleavage site of MMPs was incorporated into the PUs as a means of developing a
MMP-sensitive PU. It remains unclear what contribution the Gly-Leu-based chain extender had
on polymer degradation. Subtle differences in mechanical properties and polymer processing
were previously identified between the Phe and Gly-Leu PUs, but the majority of the polymer
properties were very comparable. It was anticipated that one of the main differences in polymer
properties between the two PUs would come from their degradation characteristics. This was not
observed in the degradation study conducted here. Little or no evidence of degradation for either
PU scaffold occurred over the 4 week time period from passive hydrolysis or MMP-mediated
cleavage. This would suggest that introducing the Gly-Leu dipeptide into the backbone structure
of the PUs did not confer specific and enhanced degradation to the PU scaffolds.
To better understand the degradation properties of the PU scaffolds, in vitro and in vivo
cell-based degradation studies are required. The PU degradation system using C14-labeled hard
segments extensively utilized by Santerre and colleagues [33-35] may be a useful model for
elucidating the mechanisms of Phe and Gly-Leu PU degradation by inflammatory cells,
164
including PMNs and MDMs, or other MMP-secreting cells found in the heart such as cardiac
fibroblasts. MMP-mediated PU degradation in this in vitro model could be determined by
measuring MMP activity and corresponding PU degradation and then inhibiting MMP activity
and again quantifying PU degradation. In vivo degradation testing should also be performed in
animal models to determine the residence times for the Phe and Gly-Leu PU scaffolds in the
cardiac environment.
synergistically in degrading PUs in vivo make it difficult to fully appreciate the performance of
the PU scaffolds by in vitro studies alone and are required if these materials will ever move to
the clinical setting.
oxidative agents, calcification, mechanical stresses, environmental stress cracking, metallic ions,
salts, acids, and cells will all contribute to the environmental biodegradation of PU scaffolds
[36]. Guan et al. [37] demonstrated an elastase-sensitive PU scaffold exhibited ~42% mass loss
over an 8 week in vitro enzyme-based degradation study but was completely degraded after the
same time period when implanted subcutaneously in rats.
degradation rates between the in vitro and in vivo studies highlights the multifaceted mechanisms
of environmental biodegradation that may be difficult to predict using enzyme-based in vitro
studies alone. The work presented here further identifies the need to move to in vitro and in vivo
cell-based studies to obtain a better appreciation of the degradation properties of the Phe and
Gly-Leu PU scaffolds.
If after additional cell-based testing the degradation rates for the Phe and Gly-Leu PU
scaffolds are not appropriate for use in the heart, several modifications can be made to the PU
chemistry to alter these properties. Previous work has demonstrated that the degradation rates of
the Phe-containing family of PUs by both passive hydrolysis and enzymatic means may be
significantly increased by lowering the PCL soft segment molecular weight or by using PEO of
various molecular weights [19]. A similar approach may be used to increase degradation rates
with the Gly-Leu PU.
scaffolds. If the Gly-Leu dipeptide alone is not sufficient to confer MMP susceptibility to the
PUs, then numerous other peptide sequences may be used to tune the degradation properties of
the PUs [17, 26]. Altering the length and peptide sequence can dramatically alter rates of peptide
hydrolysis and could lead to synthetic polymers with a large repertoire of degradation
characteristics for specific applications [17, 28]. The method of incorporating larger peptides
165
into the PUs will be important because of the higher molecular weights associated with the
additional amino acid residues. If the diol linker method used in developing the Phe and GlyLeu-based diester chain extenders were used with larger peptide sequences, the ratio of soft
segment to hard segment would consequently decrease. As a result, several of the favorable
properties conferred to the PUs by soft segment choice may be lost due to the large hard segment
domains. The method used by Wagners group of adding a lysine residue at the C-terminal of a
desired peptide sequence may work better with larger peptide sequences without allowing the
chain extender molecular weight to get too big [29, 30]. Alternatively, it may be interesting to
characterize PU properties of hexa or octapeptide-based diester chain extender. Further testing
of the Phe and Gly-Leu PU scaffolds will help identify whether modifications to the chemistry
are necessary to achieve appropriate degradation properties for use in the heart.
5.4 Conclusions
The degradation characteristics of PU scaffolds are critical to their success in tissue
engineering applications. MMP activation and activity studies were conducted to confirm active
enzymes could be obtained for degradation trials.
5.5 References
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169
Sections of this chapter have been accepted for publication [1]: Parrag IC, and KA
6.0 Abstract
Cell-based studies were conducted to investigate the cellular response to PU scaffolds
and identify the potential of these biomaterials in soft tissue engineering applications. The Phe
and Gly-Leu PU scaffolds were seeded with a high density of mouse embryonic fibroblasts
(MEFs).
constructs indicated that both PUs could support a high density of viable cells out to at least 28
days. Cells were adherent and spread out with no regular organization on the randomly oriented
substrates. For cardiac applications, the Phe PU was electrospun into scaffolds with aligned and
unaligned architectures and two culture conditions were investigated: 1) murine embryonic stem
cell-derived cardiomyocytes (mESCDCs) seeded alone onto the scaffolds or 2) mESCDCs
seeded onto the PUs pre-seeded with MEFs. In both culture conditions, viable mESCDCs
attached to the PU scaffolds and were functionally contractile out to at least 28 days post seeding
the mESCDCs. Importantly, the aligned scaffolds led to the anisotropic organization of rodshaped cells, improved sarcomere organization, and increased mESCDC aspect ratio (length to
diameter ratio) when compared to cells on the unaligned scaffolds. In addition, pre-seeding the
scaffolds with MEFs improved sarcomere formation, increased cell alignment and aspect ratio,
and led to a mESCDC morphology that was more extended on the PU scaffolds than the
mESCDCs cultured alone. These results suggest that both fiber alignment and pre-treatment of
scaffolds with fibroblasts improved the differentiation and organization of mESCDCs. The
results of this work are very promising for cardiac tissue engineering and further characterization
of the PU constructs will help to understand the potential of the PU scaffolds for use in the heart.
170
6.1 Introduction
Tissue engineering scaffolds that seek to mimic the native ECM should provide a
temporary polymer matrix that plays a structural and functional role in guiding tissue
development. Studying the cellular response to biomaterial scaffolds can help demonstrate if the
scaffolds meet several of the properties required in developing functional constructs for
regenerating injured or diseased tissues. Some of these requirements include the biomaterial and
degradation products being non-cytotoxic, appropriate physiochemical properties to promote cell
adhesion, growth, and differentiation, and a suitable architecture for proper tissue organization
and cell-cell coupling [2]. In vitro cell-based studies will help to understand if scaffolds elicit a
favorable response from the cells and may help in better defining criteria needed for specific
tissue engineering applications.
Previous work has demonstrated that the Phe PU has favorable properties to support cells
for cardiac tissue engineering. Phe PU films were successfully used to culture neonatal rat
cardiomyocytes and murine embryonic stem cell-derived cardiomyocytes (mESCDCs) while
maintaining normal phenotypic and contractile properties [3, 4]. Using thermally induced phase
separation (TIPS) and electrospinning, 3-D Phe PU scaffolds supported contractile mESCDCs
with cells on the electrospun fibrous scaffolds having a striated sarcomeric phenotype that
appeared more mature than cells on TIPS scaffolds [5]. Recently, the aligned and unaligned
electrospun Phe PU scaffolds developed for this work were used to culture primary neonatal
cardiac cells [6]. The aligned scaffolds provided physical cues for the anisotropic organization
of cardiac cells resulting in a more similar organization to native cardiac tissue. In addition, the
aligned cardiac constructs were associated with a decrease in atrial natriuretic peptide compared
to unaligned constructs suggesting a more mature, ventricular-like cardiac phenotype [6].
To further investigate the use of PUs for cardiac and other soft tissue engineering
applications, cell-based studies were conducted with electrospun PU scaffolds. Phe and Gly-Leu
PU scaffolds were seeded with mouse embryonic fibroblasts (MEFs) to test if the Gly-Leu PU
could support a high density of adherent cells and whether the biomaterial or any degradation
products released during the culture period were cytotoxic. Results from these experiments
suggest that both the Phe and Gly-Leu PU scaffolds could support a high density of adherent and
morphologically extended cells out to at least 28 days.
applications, embryonic stem cells were differentiated into cardiomyocytes and seeded onto
171
aligned and unaligned Phe PU scaffolds on their own or in coculture with MEFs.
The
mESCDCs attached to the electrospun scaffolds, formed striated sarcomeric structures, expressed
the gap junctional protein Cx-43, and were functionally contractile on the scaffolds for at least 28
days. In addition, cells were organized in arrangements that were dictated by the underlying
fibrous scaffold substrate and helped to form constructs that were physically anisotropic,
resembling the organization in the native myocardium. Future work will help to characterize and
optimize the cardiac constructs but the results from this study support the use of PU scaffolds for
cardiac tissue engineering applications.
172
immunostaining. AlamarBlue analysis was conducted on the cell-seeded constructs after 7, 14,
21, and 28 days in culture. The alamarBlue dye (Invitrogen, Carlsbad, CA) was diluted with
fresh culture medium at 10% v/v prior to analysis. The constructs were incubated with the
solution for 4.5 h and were analyzed at 570 nm and 600 nm with a spectra thermo microplate
reader (SLT Labinstruments, Salzburg, Austria). Percent reduction of the alamarBlue was
calculated according to the manufacturers instructions to give relative cell viability of MEFs on
the PU scaffolds. At least 5 samples (n>5) were analyzed at each time point and the experiment
was repeated three times (N=3).
independent t-test or one-way analysis of variance (ANOVA) with Bonferroni post hoc analysis
using the SPSS Statistics 17.0 statistical software package (SPSS Inc, Chicago, IL).
A Live/Dead assay (Invitrogen, Carlsbad, CA) was used to visualize live and dead cells
on the scaffolds after 28 days. Three samples in three separate trials (n=3, N=3) were stained
with calcein AM and ethidium homodimer-1 at concentrations of 2 M and 20 M in PBS
respectively for 20 min at 37C. Cells were examined with a confocal microscope (Carl Zeiss
Canada, Toronto, ON) at the Advanced Optical Microscopy Facility (AOMF; Princess Margaret
Hospital, Toronto, ON) equipped with Zeiss LSM software using excitation/emission spectrum
of 494/517 and 528/617 for calcein and ethidium homodimer-1 (in the presence of DNA)
respectively.
173
Molecular Probes) for 1 h, and subsequently with DAPI (0.5 g/ml) for 10 min. The constructs
were washed 3X in blocking buffer between each step. Three labeled samples from three
separate trials (n=3, N=3) were imaged using a two-photon confocal microscope with Zeiss LSM
software.
Undifferentiated
ESCs were cultured as described for the CM 7/1 cell line [3] on 0.2% v/v gelatin coated tissue
culture polystyrene dishes in DMEM supplemented with 15% ESC-screened FBS (Hyclone,
Logan, UT), 1% L-glutamine,
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Figure 6.1: Illustration of experimental details for cardiomyocyte production and cell seeding. Large scale cell
growth, differentiation, and selection was performed in spinner flasks. Cardiac bodies were dissociated into
individual mESCDCs and were seeded onto the PU scaffolds on their own or pre-seeded with MEFs.
175
placed in a Falcon tube with Hanks Balanced Salt Solution containing 2% FBS at approximately
1 million cells per tube. Ethidium monoazide (1 g/ml) was added to the samples and were
placed on ice under bright light for 30 min. An Intraprep permeabilization kit was then used to
fix and permeabilize the cells according to kit instructions. Samples were subsequently labeled
with either an anti-cardiac isoform of troponin-T (clone 13-11; 2 g/ml; Lab Vision, Fremont,
CA) or an anti-Oct 4 (2.5 g/ml; BD Biosciences, San Jose, CA) primary antibody and a rabbit
anti-mouse IgG FITC-conjugated secondary antibody (1:100 dilution; Invitrogen Corporation,
Carlsbad, CA). Rinses with Hanks Balanced Salt Solution with FBS were performed between
steps. Samples were analyzed at the Faculty of Medicine Flow Cytometry Facility (University of
Toronto, Toronto, ON) using a BD FACS Calibur analyzer with an Argon-488 nm blue laser
excitation. FITC was detected with a 530/30 nm band pass filter and EMA was detected using a
670 nm long pass filter.
cardiomyocytes (cardiac bodies, CBs) were removed from the spinner flask, allowed to settle,
and excess medium was removed. Cardiac bodies were dissociated to obtain single cells by
incubation with collagenase type IV-DNAse and trypsin-EDTA steps as outlined above.
Following dissociation, the cells were filtered with a 40 m cell filter (Becton Dickenson,
Mississauga, ON) and approximately 2 million ESCDCs were seeded on the same side of the
scaffolds as the MEFs. MEFs or ESCDCs alone on aligned and unaligned scaffolds were also
prepared. MEFs or ESCDCs alone or in coculture on TCPS served as controls. Constructs were
cultured in differentiation medium with 75% of the medium exchanged everyday until analysis.
nuclei, and either a sarcomeric structural protein or gap junctional protein were stained using
TRITC-conjugated phalloidin (Chemicon), DAPI (Chemicon), and either an anti--actinin or
anti-connexin-43 (Cx-43) monoclonal antibody respectively.
immunostaining MEFs on Phe and Gly-Leu PU scaffolds described above was used here with the
aligned and unaligned constructs. Samples were labeled with either anti--actinin or anti-Cx-43
at a 1:1500 dilution for both primary antibodies and the same dilutions for TRITC-conjugated
phalloidin, Alexa 488 anti-mouse or anti-rabbit secondary antibodies, and DAPI as previously
outlined. Labeled samples were imaged using a two-photon confocal microscope with Zeiss
LSM software. Prior to obtaining images, polymer auto-fluorescence was used to help orient the
constructs so the major axis of fiber alignment was parallel with the top to bottom plane of each
image. Image Pro Express image analysis software (Media Cybernetics, Bethesda, MD) was
used to quantify the angle of cell axis and give qualitative measurements of cell length and
diameter, aspect ratio (length/diameter), and cell area (length*diameter).
dimensions were measured manually using the image analysis software from at least 200 cells
from 6 to 9 different images. Image Pro Analyzer 6.3 software (Media Cybernetics, Bethesda,
MD) was used to determine the orientation index and standard cell area as a second method of
quantifying cell orientation and area respectively. The orientation index was determined by
performing a Fast Fourier Transform (FFT) on 6 to 9 different immunostained images as
described by Nichol et al. [8]. Subtracting the minor to major axis ratio of the thresholded FFT
image from 1 resulted in the orientation index. A value of 0 corresponded to a completely
random orientation whereas a value of 1 corresponded to a perfectly aligned orientation. The
standard cell area was calculated by quantifying the area of f-actin expression and dividing by
the number of cell nuclei quantified in the same image. Statistical comparisons were made using
either a two-tailed independent t-test or a one-way ANOVA with Bonferroni post hoc analysis.
Beating constructs were captured by video microscopy using an Olympus microscope
equipped with a Sony EXwaveHAD colorwide digital camera. Image J software was used to
record cell contraction and produce the videos. Samples were assessed after 6 and 14 days in
culture.
177
178
reduction while the Gly-Leu PU exhibited a slight decrease. Day 21 to day 28 showed a
significant decrease in alamarBlue reduction for both PUs (ANOVA, p<0.05) potentially
indicating the occurrence of cell death. Interestingly, despite similar trends over the 28 day
culture period, the Phe PU had a significantly higher percent reduction compared to the Gly-Leu
PU for all time points investigated (t-test, p<0.05) suggesting the Phe PU was able to support a
higher number of viable cells. TCPS controls were conducted alongside the PU scaffolds but the
relative percent reduction with the control was always 100% due to increased culture area
associated with the 48-well TCPS plates. A TCPS control group containing a similar number of
cells as observed on the PUs may help to determine if the drop in relative cell number at day 28
was a function of the culture substrates.
Figure 6.2: AlamarBlue analysis of MEFs on unaligned Phe and Gly-Leu PU scaffolds over 28 day period. A twotailed independent t-test indentified a statistical difference in means between PU types for each time point.
* represents a statistical difference in means using a one-way ANOVA with Bonferroni post hoc analysis (p<0.05)
when looking at the time progression of each PU. Error bars represent standard deviation. n>5, N=3.
To further investigate the cell seeded constructs, cells were visualized by Live/Dead
staining (Figure 6.3a) and immunostaining (Figure 6.3b). The results from the Live/Dead
staining of the cell seeded constructs identified a high density of viable cells on both scaffold
types and TCPS controls after 28 days post seeding. Despite the alamarBlue results suggesting
some cell death from day 21 to day 28 on the PU scaffolds, there was little evidence of any dead
179
cells on the scaffolds at 28 days. Although a decrease in percent reduction may indicate cell
death, it could also be a consequence of cell metabolism slowing down. In addition, dead cells
may be washed off the scaffolds during medium changes or washes performed during assays and
this may account for their absence in the obtained images.
Figure 6.3: Staining of MEFs on unaligned Phe and Gly-Leu PU scaffolds and TCPS by a) Live/Dead staining
(green = live cells, red = dead cells, blue = PU scaffold) and b) Immunostaining (green = vinculin, red = f-actin, blue
= cell nuclei). n=3, N=3.
Immunostaining the actin cytoskeleton, cell nuclei, and focal adhesions confirmed the
Live/Dead results of a high density of cells on the PU scaffolds. Cells appeared attached and
extended on all substrates with no apparent orientation. A fairly confluent layer of cells was
identified for the culture surfaces with cells in close contact or on top of each other in many
spots. This may be a result of cell overgrowth if MEFs are not contact inhibited. However, this
phenomenon more likely reflects the high cell density used for seeding as a similar result was
observed when immunostaining the cells after 7 days in culture. Interestingly, the Phe PU
scaffolds had a confluent layer of cells on both sides of the scaffolds even though cells were
seeded on only one side. In contrast, the Gly-Leu PU scaffolds had a confluent layer on the cell-
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seeded side but only high density patches on the non-seeded side. This observation correlates
with the alamarBlue results that suggested a higher relative number of viable cells seen on the
Phe PU scaffolds compared to the Gly-Leu PUs. The non-cell-seeded side of the Phe PU may
have been in closer contact with the bottom of the culture wells during and after seeding and may
have allowed for better cell attachment and migration than the Gly-Leu PU scaffolds. Seeding
both sides of the PU scaffolds may help to resolve this difference, as it may not accurately reflect
the Phe PUs ability to support a greater number of cells.
A reduced cell number after 28 days in culture compared to earlier time points may have
occurred, but the results from this cell-based study suggest both PU scaffolds are capable of
supporting a high density of MEFs out to at least 28 days. The ability of the electrospun PU
scaffolds to support the attachment and viability of cells was not surprising. Electrospun fibrous
scaffolds with random architectures and micron-sized fibers have been shown to promote cell
attachment [14, 15]. Moreover, mESCDCs seeded on electrospun Phe PU scaffolds attached to
the material and maintained a phenotype typical of this cell type [5]. Previous cytotoxicity
testing of the Phe-based family of PUs in the presence of keratinocytes suggested the PUs were
not cytotoxic but that PU degradation was associated with a decrease in cell viability [16]. The
chemical reactants used in synthesizing the Phe and Gly-Leu PUs were chosen because they are
non-toxic [17, 18]. A systematic investigation of chymotrypsin-mediated cleavage sites in the
Phe PU similarly suggested that non-toxic products are released from the material during
degradation [19]. The large similarities between the Phe and Gly-Leu PU, including polymer
chemistry and properties, scaffold architecture, and fiber diameters, would suggest a similar
ability of the Gly-Leu PU to support the attachment and viability of cells and this is supported by
the studies conducted here.
and pure population of cardiac progenitors or fully differentiated cardiomyocytes. The Zandstra
laboratory has been particularly interested in achieving this goal by developing and optimizing
bioreactor parameters for the generation of large quantities of ESCDCs [7, 22, 23]. The systems
developed by this group utilize genetically modified mESCs (MHC-neor/pGK-hygror) that allow
for the selection of cardiomyocytes to obtain a pure population of desired cells. In one study,
Zandstra et al [7] generated ~14 x 106 mESCDCs in a 250 ml spinner flask that were
spontaneously beating and expressed characteristic markers of this cell type.
The
cardiomyocytes were produced by aggregating mESCs in static culture for 4 days followed by
the additional growth and differentiation in a spinner flask system. On day 9 after initiating
differentiation, medium was supplemented with G418 and retinoic acid to select for and drive
differentiation towards cardiomyocytes. On day 18 the relatively pure mESCDC population was
harvested from the spinner flasks. In collaboration with the Zandstra group, a similar system to
generate mESCDCs was previously performed in our lab towards the development of a cardiac
patch using the Phe PU [3, 5] and was used again for the work described here.
Several methods have been used to form EBs including suspension culture in bacterialgrade dishes, culture in methylcellulose semisolid media, the hanging drop method, aggregation
in a 96-well round bottom plate or conical tube, and methods for the scalable production of EBs
[24]. The large scale production of mESCDCs for tissue engineering applications requires the
scalable production of these cells in spinner flasks and bioreactor systems. Towards this goal,
EBs have been formed by suspension culture with bacterial-grade dishes for 4 days before
transfer to spinner flasks or the static suspension culture for 1 day followed by encapsulation of
the EBs in size-specific agarose hydrogel capsules [7, 22]. This was performed to prevent
undesirable EB agglomeration using spinner flasks with paddle-type impellers. Other systems
have been developed that allow the direct formation of EBs within the spinner flasks or
bioreactors thereby eliminating this 2-step process [24]. Schroeder et al. [25] compared the
paddle-type and glass bulb-shaped impeller spinner flasks and found that the bulb-shaped
impellers reproducibly formed homogenous EBs by directly inoculating mESCs whereas the
paddle impellers caused EB agglomeration. Adjusting impeller speed affected the shear stress on
cells and influenced EB size, formation, and subsequent cardiomyocyte differentiation [25].
A culture period of 18 days in spinner flasks was used to generate mESCDCs for
subsequent investigations with the Phe PU scaffolds. The D3 MHC-neor/pGK-hygror mESC line
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was grown on gelatin-coated TCPS dishes in culture medium containing LIF and 12.5 million
cells were inoculated in 250 ml glass bulb-shaped impeller spinner flasks in the absence of LIF
(day 0). Cell aggregation occurred over the first 24 h to form embryoid bodies and subsequent
cell growth and differentiation took place within EBs. Starting on day 9, G418 and retinoic acid
were added to the culture medium to select for and drive differentiation towards cardiomyocytes
respectively. Cells were harvested from the spinner flasks on day 18 for the cell-based studies.
Cell growth and differentiation were monitored during the 18 day period by counting cells and
EBs and flow cytometry for expression of cell markers.
Cell number in the spinner flasks was measured by dissociating EBs and counting the
cells at various time points during the 18 day differentiation and selection period. Results are
shown in Figure 6.4. High cell expansion within the EB structures was observed during the first
6 days of culture reaching a maximum number of approximately 1 x 109 total cells on day 6.
From day 6 on, a steady drop in total cell number occurred out to day 18 when the cells were
harvested from the spinner flasks. The drop in cell number from day 9 on was expected due to
the heterogeneous cell population generated during EB differentiation and the start of antibiotic
selection of cardiomyocytes. The drop in cell number from day 6 to day 9, however, occurred
prior to the addition of G418 and was not an observation previously recorded in the large-scale
production of mESCDCs in spinner flasks [7, 23]. Diffusion limitations as EB size increased
most likely affected cell viability.
accumulation of toxic waste products due to the high cell population could account for the drop
in cell numbers. Monitoring EB size and glucose/lactate levels may help identify the cause of
cell death prior to selection and may indicate potential methods for optimizing this protocol.
Despite cell death prior to selection, a large number of cells were harvested from the spinner
flasks after 18 days. The mESCDC output to mESC input ratio using similar systems for
mESCDC production have recorded values from ~1.5-3.5 [22, 23]. Although some differences
exist between those studies and the one conducted here, the actual viable cardiomyocyte yields
likely correspond to these numbers more closely than the total cell number may suggest (~12
mESCDC/input mESC).
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Figure 6.4: Total cell number in spinner flasks during differentiation of mESCs into cardiomyocytes. Cell growth
was observed for 6 days followed by a drop in cell number for additional 12 days in suspension. A drop in cell
number was observed prior to the start of selection indicating room for optimization of culture parameters. Error
bars represent standard deviation. n=4.
In addition to total cell number, EB number and cells/EB were monitored during the first
9 days of culture in the spinner flasks. As seen in Figure 6.5a, a high and variable EB number
was observed on day 2 followed by a lower and more constant EB number for the remainder of
the time period investigated. The large deviation on day 2 indicated that the EB formation in the
spinner flasks was not as reproducible as previous studies have shown [25].
In addition,
although shear stresses in the glass bulb-shaped impeller spinner flasks limit EB agglomeration
[25], some smaller EBs may still have aggregated during the first 4 days in suspension. This is
consistent with the expression of E-cadherin and EB agglomeration seen in other spinner flask
and bioreactor systems [26]. The number of cells per EB during the first 9 days in the spinner
flask is shown in Figure 6.5b. A similar trend as total cell number was observed with a steady
increase in number of cells per EB from inoculation to day 6 followed by a decrease from day 6
to day 9. This was expected given the trend of total cell number and the relatively constant EB
number. The maximum number of cells per EB on day 6 was ~9,000 1,800 and may suggest a
maximum size before diffusion limitations to the center of the EBs became a limiting factor to
cell viability. Niebruegge et al. [23] recorded a higher number of cells per EB with the CM7/1
cell line, ~25,000 cells per EB, before cell number began to drop. This could reflect differences
184
in cell line or that it was not EB size but rather total number of cells and culture medium
limitations that decreased cell viability. Interestingly, the trials that had fewer EB numbers on
day 2 and a correspondingly higher number of cells per EB produced a higher number of cells on
day 18 and had a higher frequency of beating EBs and total cardiomyocytes. Starting EB size
has been identified as an important determinant of differentiation towards specific lineages and
higher mesoderm and cardiac induction has been associated with larger EB sizes [27].
Controlling the starting EB size has therefore emerged as a way to increase cardiomyocyte yields
from ESCs [28]. The results here suggest that the larger EB sizes similarly produced a higher
number of cardiomyocytes. Further characterization of ESC differentiation in the spinner flasks
should be conducted to better understand the production of cardiomyocytes using the D3 mESC
line and the glass bulb-shaped impeller spinner flasks. Specifically, characterizing EB size,
glucose/lactate levels, frequency of beating EBs, and better quantifying viable cardiomyocytes
will help determine the cause of cell death prior to selection and may identify potential
parameters in the process that need to be optimized.
Figure 6.5: EB characteristics during differentiation of mESCs into cardiomyocytes. a) EB number and b) cells/EB
for first 9 days of culture in spinner flasks. Error bars represent standard deviation. n=4.
The cell populations that were used to inoculate the spinner flask and were harvested
after 18 days of culture were identified by flow cytometry. Oct 4 is a transcription factor
expressed by undifferentiated mESCs that is down regulated during differentiation and is used as
a marker of pluripotent mESCs [29]. Oct 4 has been commonly used to identify undifferentiated
mESCs in cell populations for the scalable production of cardiomyocytes [7, 22] and was used to
identify mESCs in the cell populations before and after differentiation in spinner flasks. Cardiac
cells were identified by expression of the cardiac isoform of troponin T (cTnT). Troponin T is a
component of the troponin complex involved in regulating actin-myosin interactions during
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cardiomyocyte contraction and has been used as a marker to identify mESCDCs [30, 31]. Flow
cytometry analysis of the cell populations at day 0 and day 18 is shown in Figure 6.6. Ethidium
monoazide was used to indicate cell viability and the data was gated to remove dead cells. The
cell population used to inoculate the spinner flasks on day 0 expressed high levels of Oct 4
(>80%) indicating a high percentage of undifferentiated ESCs. Cardiac cells were not identified
in the starting cell population. Following 9 days of cell growth and differentiation and an
additional 9 days of selection (day 18), EBs were contracting spontaneously (referred to as
cardiac bodies, CBs) indicating the presence of cardiomyocytes. Cardiac-specific troponin T
staining of cells at day 18 identified that the viable cells in the CBs were composed of a
relatively pure population of cardiomyocytes (~97% cTnT positive). In addition, no Oct 4
positive cells were observed demonstrating the absence of any undifferentiated ESCs. The
selection of mESCDCs by antibiotics using genetically modified mESCs has been shown to
result in a pure population of cardiac cells [20, 32] and is currently the best method of purifying
cardiomyocytes from a heterogeneous cell population [33]. The potential of teratoma formation
associated with the transplantation of undifferentiated ESCs in the heart is a major concern of
using ESCs for cardiac repair [13, 34-36]. Ensuring no undifferentiated ESCs are present in cell
populations used in developing engineered myocardial constructs is therefore of utmost
importance.
Although repeated trials are needed to confirm the results, a relatively pure
186
Figure 6.6: Flow cytometry of cells before and after differentiation in spinner flasks. Cells were analyzed at a) day 0
and b) day 18. A high Oct 4 expression was observed on day 0 indicating a high proportion of undifferentiated
ESCs. On day 18, a high population of cardiac troponin T (cTnT) positive cells with no Oct 4 positive cells was
observed consistent with a pure population of cardiomyocytes and no undifferentiated ESCs. n=1.
Similarly, coculturing
188
Figure 6.7: AlamarBlue analysis of cell-seeded PU constructs of varying architecture and TCPS controls. a)
coculture, b) MEFs alone, and c) mESCDCs alone. No statistical difference was observed in alamarBlue reduction
between the aligned and unaligned PU scaffolds for any time point or any cell type (ANOVA, p>0.05). Cells
cultured on TCPS were significantly different than both aligned and unaligned scaffolds for all cells and all time
points investigated (p<0.05). n>3, N=3 (except mESCDCs alone, N=2).
189
scaffolds for all cells and time points (p<0.05). Although it is unclear the reason for this,
possible explanations may include: a higher number of seeded cells adhered to the TCPS than
those seeded on PU scaffolds; the increased culture area with TCPS wells allowed for
proliferation of cells; or the cells on TCPS expressed a phenotype that was more metabolically
active than cells on PU. A direct comparison of alamarBlue reduction between different cell
groups was difficult due to differences in metabolic activity for each cell type. A higher percent
reduction, however, was observed for coculture group compared to MEFs alone suggesting that
some mESCDCs adhered to the PU scaffolds.
To visualize viable and non-viable cells on the PU scaffolds, cells were prepared by
Live/Dead staining.
formation of non-cardiac myocytes and selecting for the cardiomyocytes leads to cell death in the
non-myocyte population. No attempt was made to separate out the dead cells prior to mESCDC
seeding and it was of interest to determine if any dead cells remained attached to the scaffolds or
if they were washed away during media changes. Cytokines released from dead cells may affect
cell behavior and can elicit a strong inflammatory response in vivo [39] that could adversely
affect function and integration with the host if these constructs are implanted in animal models.
Figure 6.8 shows images from the Live/Dead staining of MEFs and coculture cell groups on the
aligned and unaligned scaffolds. In these images, green corresponds to viable cells, red spheres
to dead cells, and red and blue fibers to the PU scaffold due to auto-fluorescence of the polymer
at the investigated wavelengths. All of the images show a fairly high number of viable cells on
the PU scaffolds. MEFs on the scaffolds had a fairly uniform cell distribution but cell-cell
contact was limited. There was no sign of dead MEFs on the scaffolds. The coculture group
similarly had viable cells over most of the scaffolds but the culture surface had several large high
density patches of viable cells that were in close contact. A few small round red spots were
observed on the coculture constructs indicating the presence of some dead cells. Although some
dead cells were identified, the frequency was not high suggesting the majority of the dead cells
were washed away following seeding or during medium changes. As a result, there was no
motivation to change the seeding protocol to separate viable and dead cells by Percoll gradient
prior to seeding.
organization on the aligned and unaligned scaffolds. Cells elongated along the PU fibers and
appeared aligned with the orientation of fibers in the aligned scaffold whereas they were more
190
randomly oriented on the unaligned PU. This was consistent with cells on other electrospun
polymers [37] and suggested that the underlying fibrous substrates were providing physical cues
for cellular organization. The results from the Live/Dead staining confirmed the ability to
successfully seed a high number of viable cells on the PU substrates and that the cells appeared
to be interacting with PU fibers.
Figure 6.8: Live/Dead staining of cells on Phe PU scaffolds of varying architecture at day 18+6. Green = viable
cells, Blue = PU scaffold, Red = dead cells (round spots) and PU scaffold (fibers). High density patches of viable
cells were observed for coculture constructs with the presence of some dead cells. MEFs on PU matrices were
viable and had a more scattered cell distribution with no dead cells. n=3, N=3.
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Figure 6.9: Immunostaining of cells on aligned and unaligned PU scaffolds. Red = cytoskeleton (f-actin), Green =
sarcomere (-actinin), Blue = cell nuclei. n=3, N=3.
192
cultured alone and with MEFs on both scaffold architectures exhibited different degrees of
differentiation as indicated by cell shape and the appearance of a striated sarcomeric structure.
Early stages of mESCDC differentiation within EBs are typically characterized by small round
cells with no evidence of sarcomere development [40, 41]. With increased time, mESCDCs
become elongated, rod-shaped cells with highly developed and organized sarcomeric structures,
Figure 6.10: Immunostaining of cardiac constructs with mESCDCs showing varying levels of differentiation. a)
coculture on unaligned PU, b) coculture on aligned scaffolds, c) mESCDCs alone on unaligned PU, and d)
mESCDCs alone on aligned scaffolds. Red = cytoskeleton (f-actin), Green = sarcomere (-actinin), Blue = cell
nuclei, Yellow arrows = round cardiomyocytes with poorly defined sarcomeric structures, White arrows = elongated
cardiomyocytes with well defined and organized sarcomeres.
193
consistent with cardiomyocyte development in vivo [40, 41]. The mESCDCs on PU scaffolds
varied from immature cells that were round or triangular shaped with -actinin staining in the
cytosol and no evidence of sarcomere assembly, to more mature cells that were rod-shaped with
highly defined and organized sarcomeric structures (Figure 6.10). Importantly, ranking cell
shape and the appearance of striated structures within the immunostained images by blinded
observers identified a trend that both fiber alignment and coculture with fibroblasts improved the
differentiated state of the mESCDCs (Table 6.1). A higher percentage of rod-shaped mESCDCs
were found on the aligned scaffolds compared to the unaligned scaffolds.
In addition,
coculturing with MEFs reduced the percentage of circular cells and increased the striated cell
appearance compared to mESCDCs cultured alone.
Table 6.1: Assessment of cell shape and sarcomere formation for mESCDCs cultured alone and in coculture with
MEFs on PU scaffolds. Three blinded observers ranked 60-200 cells from 6-9 different images for cell shape and
appearance of striated structure within each culture group. Values are given as mean standard deviation.
Striated
Appearance (%)
Cylindrical
67.0 28.2
26.3 28.8
70.7 25.6
36.7 29.2
Triangular
10.7 12.5
41.3 20.7
15.7 18.7
32.0 23.9
Circular
22.3 15.6
32.5 8.2
13.5 6.9
31.3 5.2
47.3 0.8
48.6 6.6
73.0 0.7
71.5 12.9
The trend that fiber alignment and coculture with MEFs improved the differentiation of
mESCDCs was further supported by measuring cell dimensions. Qualitative cell dimensions of
mESCDCs alone and in coculture on the PU scaffolds were measured using image analysis
software and the -actinin expressing immunostained samples (Table 6.2). Typical mESCDCs
range in size from neonatal (diameter ~7-9 m and length ~20-45 m) to adult dimensions
(diameter ~10-30 m and length ~80-150 m) [41] and was similarly observed with the
mESCDCs on PU scaffolds. The mESCDCs alone ranged from approximately 30-85 m and
20-60 m in length, 7-21 m and 8-21 m in diameter, and had an average aspect ratio
(length/diameter ratio) of 4.5 1.4 and 2.8 0.8 and area (length*diameter) of 540 201 m2
and 604 176 m2 on aligned and unaligned scaffolds respectively. In coculture, the mESCDCs
were approximately 40-100 m and 30-75 m in length, 9-18 m and 9-24 m in diameter, and
had an average aspect ratio of 4.7 1.4 and 3.3 0.9 and area (length*diameter) of 717 237
m2 and 818 198 m2 for the aligned and unaligned scaffolds respectively. The standard
194
average cell area resulted in a similar area as that calculated from length and diameter
measurements. Importantly, the aligned architecture led to a more elongated cell morphology
with a higher length and aspect ratio, more similar to adult cardiomyocytes (aspect ratio of 5.3
for adult and 2.9 for neonatal cardiomyocytes [41]), than the unaligned architecture for both
culture groups. In addition, coculturing the mESCDCs with MEFs increased cell length on both
architectures and led to a higher aspect ratio on the unaligned scaffold compared to mESCDCs
alone.
Table 6.2: Assessment of mESCDC dimensions. Cell dimensions were measured from at least 200 cells from 6 to 9
different immunostained images. Values given as mean standard deviation.
Architecture
Aligned
mESCDCs
alone
Unaligned
mESCDCs
alone
Aligned
Coculture
Unaligned
Coculture
Length
(range,
m)
Average
Length
(m)
Diameter
(range,
m)
Average
Diameter
(m)
Aspect
Ratio
(L/D)
Area
(L*D,
m2)
Standard
Area (m2)
~30-85
49 11
~7-21
11 2
4.5 1.4
540
201
583 112
~20-60
41 9
~8-21
15 3
2.8 0.8
604
176
706 254
~30-100
58 14
~8-18
12 2
4.7 1.4
~30-75
52 10
~9-24
16 3
3.3 0.9
717
237
818
198
724 185
702 188
Assessments of cell shape, sarcomere formation and cell dimensions suggested that both
fiber alignment and coculturing with MEFs improved mESCDC differentiation and maturity
compared to cells on unaligned scaffolds or mESCDCs cultured alone. This is consistent with
previous work in the literature using primary neonatal cardiac cells. We recently demonstrated
that culturing neonatal cardiac cells on the aligned PU scaffolds led to a more mature ventricularlike phenotype compared to the same cells on unaligned PU scaffolds or TCPS controls [6]. In
addition, recent studies have shown that pre-seeding cardiac fibroblasts alone or with endothelial
cells improved cardiomyocyte elongation, viability, compaction, and functional properties
compared to simultaneous seeding of cells or enriched cardiomyocyte cultures [11, 38]. Thus,
scaffold architecture and culturing with non-myocytes affects both primary cardiomyocyte and
mESCDC phenotypic maturity. Future work should be conducted to assess the mESCDC-based
constructs by ultrastructural analysis using transmission electron microscopy to confirm the
results obtained here.
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Initial results from Live/Dead staining suggested that cells on aligned scaffolds were
elongated and organized parallel to PU fibers whereas cells on unaligned scaffolds showed no
regular organization. This trend was much more pronounced by immunostaining the cytoskeletal
and sarcomeric structures and further indicated that fiber alignment had a major influence on the
organization of cells. Using auto-fluorescing PU fibers as a reference, all cell types on the
aligned scaffolds had the major cell axis oriented parallel to the PU fibers while no general trend
of cellular organization appeared to occur with cells on the unaligned scaffolds. Measuring the
angle of cell axis for mESCDCs alone (Figure 6.11a) and when cocultured with MEFs (Figure
6.11b) on the two PU architectures confirmed the qualitative assessment regarding cellular
organization. In both culture groups, the majority of the cells on the aligned PU were within 20
of the reference angle while cells on the unaligned scaffold were found at nearly all orientations.
Using the absolute value of the angle of cell axis, the average cell angle was calculated for the
mESCDCs alone and coculture on PU scaffolds and is shown in Table 6.3. A significant
difference in average angle of cell axis was observed between mESCDCs on aligned and
unaligned scaffolds both on their own and in coculture (ANOVA, p<0.05) suggesting that fiber
architecture significantly influences cell organization.
determining the orientation index (Table 6.3), where cells on the aligned scaffolds were
significantly higher, and therefore more aligned, than cells on unaligned scaffolds (ANOVA,
p<0.05). This observation was also made with neonatal primary cardiac cells cultured on the
aligned and unaligned Phe PU scaffolds [6]. The parallel anisotropic organization of mESCDCs
observed here with the aligned constructs is more similar to the organization of cardiomyocytes
found in native cardiac tissue than on the unaligned scaffolds [42]. Anisotropic cardiomyocyte
organization is critical to myocardial function and is increasingly being identified as an
important criterion for generating engineered cardiac tissue [6, 43-45].
Interestingly, a
significant difference in absolute cell axis angle was also observed between mESCDCs alone and
in coculture on unaligned PU scaffolds (p<0.05), with the coculture group showing more
alignment than the mESCDCs alone. This may suggest that in the absence of physical alignment
cues from the underlying scaffold, the presence of MEFs promoted some degree of alignment of
the mESCDCs. Nichol et al. [8] found that coculturing primary neonatal cardiomyocytes with
cardiac fibroblasts induces cardiomyocyte alignment via a MMP-2 mediated mechanism and
limits apoptosis compared to enriched cardiomyocyte cultures. The improved cardiomyocyte
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Figure 6.11: Quantifying the alignment of a) mESCDCs alone and b) coculture of mESCDCs and MEFs on PU
scaffolds. The majority of cells on aligned scaffolds were within 20 of reference angle consistent with the
alignment of the underlying PU fibers. Cells on the unaligned scaffolds exhibited no general organization. Angles
were measured from at least 200 cells taken from 6 to 9 different images.
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elongation, compaction, electrical connectivity and contractile function that has been observed
with pretreatment of scaffolds with non-myocytes [11, 38] may also play a role in promoting
mESCDC alignment.
Table 6.3: Average angle of cell axis and orientation index (mean SD). Cell angles were measured from at least
200 cells from 6 to 9 different images. Orientation index was determined from 6 to 9 different images. a Significant
difference for same culture group on different architecture. b Significant difference for different culture group on
same scaffold architecture (ANOVA, p<0.05).
Orientation Index
Aligned mESCDCs
9.4 10.1a
0.63 0.04a
Unaligned mESCDCs
37.5 24.7a,b
0.26 0.12a
Aligned Coculture
9.0 7.4a
0.65 0.06a
Unaligned Coculture
29.1 22.5a,b
0.30 0.11a
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Figure 6.12: Gap junction staining of mESCDCs and MEFs in coculture on a) aligned and b) unaligned PU
scaffolds. Cells on both scaffold architectures expressed gap junctional proteins indicating cell-cell contacts and
potential electrical connectivity. n=3, N=1.
The mESCDCs cultured alone or with MEFs were contracting on the PU scaffolds
indicating a contractile phenotype was retained on the PU substrates. Moreover, the mESCDCs
were contracting synchronously and with enough force to observe PU scaffold movement at the
biomaterial edges. The contracting constructs were captured by video microscopy indicating an
important functional component of the engineered cardiac tissue. This was consistent with
previous findings that suggested the Phe PU may have appropriate elastic mechanical properties
to allow cell contraction while being attached to PU surfaces [3-6]. PU movement associated
with cell contraction was initially observed on day 4 post cardiomyocyte seeding and continued
out to at least day 28. Engler et al. [46] determined that cardiomyocytes cultured on surfaces
with an elastic modulus that mimics native cardiac tissue (E ~ 10-15 kPa) is optimal for staining
long-term rhythmic contractions whereas cells on hard matrices (E ~ 35-70 kPa) stop beating
after a few days in culture. Although the aligned and unaligned PU scaffolds had an initial
modulus much greater than 35-70 kPa and were therefore stiffer surfaces, the cells remained
contractile for at least 4 weeks. The mechanical properties of the scaffolds were not measured in
a hydrated state nor were individual PU fibers measured, so it is not known what modulus the
cells experience. The ability of the PU scaffolds to maintain cellular contraction for several
weeks while being attached to the PU substrate provides additional evidence of the potential of
the Phe PU for cardiac applications. In addition, scaffold movement became higher over time
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suggesting increased strength of cell contraction and cell maturity with increasing time in
culture. No observable differences were made between aligned and unaligned scaffolds, but the
anisotropic organization of cells on the aligned scaffold would suggest cells were pulling on the
PU in one direction. There was no apparent difference in contraction between mESCDCs
cultured alone or with MEFs. The presence of high density patches of cardiac cells made it
difficult to quantify construct beating because a higher degree of PU movement may not
necessarily correlate to higher strength of contraction but rather a higher number of cells at the
edge of scaffolds contributing to the movement between samples and groups. Achieving a
confluent layer of cells on scaffolds would allow quantification of beating and may help to better
identify how fiber alignment affected contractile properties of the mESCDCs and the
contribution coculturing mESCDCs with MEFs had on functional properties.
electrospun fibers suggesting that the physical cues provided by the fibers could be used to direct
cell orientation and promote the alignment of cardiac cells [37, 48]. Anisotropic organization is
critical to the structure and function of native myocardium and is an important criterion of
cardiac tissue engineering. Electrospun scaffolds with fiber alignment is one technique for
achieving anisotropic organization of cardiac cells and is promising in developing myocardial
tissue constructs.
The results presented here provided evidence of the potential of developing a cardiac
patch using PU scaffolds and mESCDCs. A higher density of mESCDCs was cultured on the
PU scaffolds than previous work in our lab and a distinct cellular organization was achieved by
200
aligning PU fibers in the underlying substrate. This system identified that fiber alignment and
coculture with fibroblasts improved the differentiation, maturity, and organization of the
mESCDCs on the PU constructs but more work is necessary to optimize and characterize the
constructs to fully understand the influence these factors have on cell behavior and engineered
tissue function. There are several optimization and characterization methods that should be
carried out in the future. First, the seeding protocol should be adjusted to achieve a high density
of cardiomyocytes uniformly distributed on the electrospun substrates as opposed to having high
density patches. This would promote electromechanical coupling of cells critical to tissue
function and cell-cell signaling that may influence tissue formation. A Percoll gradient could be
used to isolate only the viable cardiomyocytes thereby limiting any adverse signaling from the
presence of dead cells during seeding. Perfusion or other dynamic seeding method has been
shown to improve the distribution of cardiac cells in polymeric matrices [49, 50] and may be
another method for obtaining a high density of uniformly distributed cells.
Second, constructs should be characterized by ultrastructural analysis to verify cell
dimensions and mESCDC differentiation and maturity. Cell size, sarcomere organization and
intercalated disc formation identified by transmission electron microscopy may be used as
markers of mESCDC differentiation and maturity [41].
mESCDCs exhibited a higher level of differentiation and organization on aligned scaffolds and
in coculture with MEFs, but this analysis should be confirmed by other methods.
Third, cell maturation should be tested using quantitative polymerase chain reaction to
look at differences in gene expression of atrial natriuretic factor, the - and -myosin heavy
chain isoforms, and skeletal and cardiac troponin I. Early mESCDCs express slow skeletal
muscle troponin I and a greater proportion of -myosin heavy chain [51-53]. More mature
mESCDCs shift from these fetal protein isoforms to cardiac troponin I and -myosin heavy chain
characteristic of more mature neonatal and adult cardiomyocytes [51-53]. In addition, it was
found that primary neonatal cardiac cells cultured on aligned PU scaffolds had a significant
decrease in ANF expression compared to cardiac cells on unaligned PU or TCPS [6]. A lower
expression of ANF is associated with maturation of ventricular myocardium suggesting the
anisotropic organization of cardiac cells led to more mature ventricular-like engineered tissue
than isotropic cellular organization. Alignment of mESCDCs may similarly lead to more mature
and highly differentiated cells compared to cells with no general organization.
201
202
important for cardiac tissue function. Ultimately, if further studies continue to identify PU
scaffolds may be used in cardiac tissue engineering applications, clinically relevant cardiac
constructs may be developed by seeding PUs with human ESCDCs or induced pluripotent stem
cell-derived cardiomyocytes.
engineering and allow the development of tissue constructs for regenerating the myocardium.
6.4 Conclusions
Polyurethanes are promising biomaterials for soft tissue engineering applications. Phe
and Gly-Leu PUs were formed into scaffolds that have several similar physical, chemical, and
thermal properties. A high density of MEFs was seeded onto the PU substrates and several
analytical techniques were used to characterize the PU constructs. The results from this study
indicated that the scaffolds could support a high density of viable cells out to at least 28 days and
suggested that the Phe and Gly-Leu PUs meet some fundamental requirements of tissue
engineering scaffolds. For cardiac applications, the Phe PU was electrospun into scaffolds with
varying architecture. Embryonic stem cells were differentiated in a spinner flask system to
generate a large and pure population of cardiomyocytes. The mESCDCs were seeded onto the
aligned and unaligned Phe PUs alone or onto the PUs pre-seeded with MEFs. Viable cells
attached to both architectures and the substrates supported a contractile phenotype. Importantly,
the aligned scaffolds led to the anisotropic organization of rod-shaped cells, improved sarcomere
organization, and increased mESCDC aspect ratio when compared to cells on the unaligned
scaffolds. In addition, pre-seeding the scaffolds with MEFs improved sarcomere formation,
increased cell alignment and aspect ratio, and led to a mESCDC morphology that was more
extended on the PU scaffolds than the mESCDCs cultured alone. These results suggest that both
fiber alignment and pre-treatment of scaffolds with fibroblasts improves the differentiation and
organization of mESCDCs and are important parameters for developing engineered myocardial
tissue constructs using ESC-derived cardiac cells and PU scaffolds.
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208
Cellular
cardiomyoplasty has emerged as a method of introducing cells into the heart to improve cardiac
function but to date, clinical trials using skeletal myoblasts and bone marrow-derived cells have
produced mixed results [1-12]. Cellular cardiomyoplasty has been limited by the delivery,
engraftment, and survival of a sufficient number of cells into the heart [1, 13, 14]. This may be
improved through the use of the appropriate delivery vehicle, such as biomaterial scaffolds.
Cardiac tissue engineering employs the use of biomaterials in combination with cells to
develop viable tissue constructs to improve or regenerate injured or diseased myocardium.
Significant achievements have been made in the last several years towards developing cardiac
tissue in vitro but scaffold requirements for this application have not been well defined.
Biodegradable segmented polyurethanes (PUs) are excellent biomaterials for research in the
cardiac tissue engineering field due to its flexible chemistry. This provides an opportunity to
develop new biomaterials that incorporate specific chemical moieties that confer unique
biological functionality to the synthetic polymers.
The work conducted in this thesis involved the development and testing of new
biodegradable PU scaffolds to better identify important scaffold properties for in vitro cardiac
tissue formation. This was broken up into two closely related parts. The first involved the
synthesis, characterization, processing, and in vitro testing of a segmented PU that incorporates
the Gly-Leu cleavage site of several matrix metalloproteinases (Gly-Leu PU). Most synthetic
polymers degrade predominantly by passive hydrolysis, so the development of an MMPsensitive segmented PU may help to tailor the degradation properties to the cardiac environment.
The results presented in Chapter 3 demonstrated the successful synthesis and characterization of
the Gly-Leu PU using a previously developed chymotrypsin-sensitive phenylalanine-based PU
(Phe PU) as a comparison.
minimalistic means of incorporating the Gly-Leu cleavage site. This allowed flexibility in the
209
choice of the soft segment which in turn may be used to obtain diverse polymer properties with
this family of PUs. This also allowed a direct comparison to the Phe PUs to investigate how
changing the chain extender chemistry from the phenylalanine amino acid to the Gly-Leu
dipeptide affected polymer properties.
characterization of the Gly-Leu-based diester chain extender, the Gly-Leu PU was synthesized
using a soft segment composed of polycaprolactone diol of molecular weight 1250. This soft
segment was chosen because the Phe PUs incorporating this have been very promising in cardiac
applications [15-18]. As may have been predicted by the chemistry, the Gly-Leu PU had several
properties that were very similar to the Phe PU. Both PUs had high molecular weight averages,
were phase segregated, semi-crystalline polymers, and were soft, flexible elastomers with high
breaking stresses and strains. Interestingly, despite similarities in stress-strain curves, the GlyLeu PU had a significantly higher initial modulus, yield stress and ultimate stress compared to
the Phe PU.
Once the Gly-Leu PU had been synthesized and characterized, it was processed into
porous, 3-D scaffolds for further characterization and testing. Electrospinning was used to
process the PUs because it forms fibrous matrices that meet several criteria of biomaterial
scaffolds including; a high surface area-to-volume ratio, structural features for promoting cell
adhesion, growth and differentiation, and an architecture to help organize cells. In Chapter 4, it
was demonstrated that both the Phe and Gly-Leu PUs could be formed into scaffolds with a
variety of structural features, including beads, beads-on-a-string, and defect-free fibers of
varying diameters, by adjusting the polymer solution concentration during electrospinning. The
structural features obtained were similar to those found with other synthetic polymers in the
electrospinning literature [19]. The Phe and Gly-Leu PU scaffolds fabricated from a 14% and
10% w/v concentration respectively were chosen for subsequent studies due to their similarities
in structural features. Both scaffolds had a randomly organized fiber structure, an average fiber
diameter of approximately 3.6 m, and similar fiber diameter distributions. In addition, the
electrospinning process did not affect the molecular weight averages or thermal properties of
either PU suggesting the electrospun PUs retained several important properties of the base
material.
The remaining experiments for this part of the thesis were conducted to test the
performance of the electrospun Phe and Gly-Leu PU scaffolds for tissue engineering
210
applications.
hydrolysis of the two PU scaffolds and by assessing the viability of cells cultured on the two PUs
in vitro. In the degradation study in Chapter 5, changes in mass and structural features of the PU
scaffolds following incubation in either MMP-1, MMP-9, or buffer solutions were investigated.
Neither the Phe nor Gly-Leu PU scaffolds exhibited any detectable passive hydrolysis or MMPmediated chain cleavage over the 28 day experiment. This result does not support the original
hypothesis and provides evidence against the rational for developing the Gly-Leu PU. Several
mechanisms may account for the Gly-Leu PU stability but the minimalistic approach of
incorporating the Gly-Leu dipeptide alone without any flanking amino acid sequences along with
the phase segregated nature of the PUs is suspected to have the most influence. A segmented PU
was recently developed that incorporates a Pro-Ala-Gly-Leu-Lys sequence in the chain extender
chemistry and exhibits enhanced degradation in the presence of collagenase [20]. This supports
the need for a longer peptide sequence to achieve MMP-susceptibility.
Despite the degradation results, the cell-based studies with the Gly-Leu PU were more
promising. The experiments in Chapter 6 using the Gly-Leu PU were conducted to test whether
the PU scaffolds could support the attachment of cells and whether the material or its
degradation products were cytotoxic. These fundamental criteria of biomaterial scaffolds were
assessed by seeding mouse embryonic fibroblasts (MEFs) onto the PUs at a high seeding density
and evaluating cell viability and morphology during a 28 day culture period. MEFs were
specifically chosen as the cell type for these experiments because fibroblasts are involved in
tissue remodeling through the secretion of matrix metalloproteinases. As well, fibroblasts play
an important role in the development of engineered myocardial tissue constructs [21-24].
Therefore, establishing conditions to culture MEFs on the PU scaffolds provided a starting point
for subsequent coculture studies with murine embryonic stem cell-derived cardiomyocytes
(mESCDCs). Characterizing the MEFs on the Phe and Gly-Leu PU scaffolds demonstrated that
both PUs could support a high density of viable cells out to at least 28 days. Cells were adherent
and spread out with no regular organization on the randomly oriented substrates.
These
encouraging results suggested the scaffolds meet some fundamental requirements for tissue
engineering applications. Further work is required to fully characterize the Gly-Leu PUs,
particularly testing the degradation properties using in vitro cell-based and in vivo animal
models.
This may help to identify whether the Gly-Leu PUs may be used for cardiac
211
applications or if further modifications to the chemistry are needed to tailor the PUs to this
unique environment.
While the first part of this thesis was motivated by the degradation characteristics of the
PUs, the second part focused on physical properties of elastomeric PUs and their influence on
cardiac cells. The anisotropic organization of cardiac cells is critical to the structure and function
of the native myocardium and achieving this is an important goal in engineering myocardial
tissue constructs. The electrospinning process allows control over parameters such as the size
and organization of the fibrous scaffold structure. Therefore, the conditions for achieving PU
scaffolds with varying architecture were explored in Chapter 4 as a means of investigating how
fiber alignment influences the organizational response of mESCDCs and MEFs. Consistent with
previous reports using other polymers [25, 26], the Phe PU was electrospun into scaffolds with
aligned and unaligned architectures by adjusting the rotational speed of the collection mandrel in
the electrospinning system. A high mandrel rotational speed was used to achieve fiber alignment
whereas a low rotational speed was used to obtain randomly organized fibers. Electrospinning
the PU at different mandrel rotational speeds did not influence the molecular weight averages or
thermal properties of the resulting scaffolds but significantly affected their mechanical
properties. Specifically, the unaligned PU scaffold exhibited similar mechanical behavior when
stretched in the two perpendicular directions with a relatively low initial modulus and ultimate
tensile stress and high ultimate elongation.
anisotropic with a relatively high initial modulus and ultimate tensile stress and low elongation
when stretched in the preferred direction of fiber orientation.
Most studies in the cardiac tissue engineering field have been conducted using primary
cardiac cells as a proof-of-principle in establishing the potential of various techniques and
parameters needed for developing engineered myocardial tissue. Work performed with our
collaborators at the University of Delaware demonstrated that culturing primary cardiac cells on
the aligned Phe PU scaffold significantly influenced the phenotype of the cells when compared
to either the unaligned PU scaffold or a tissue culture polystyrene control [18]. Specifically, we
found that the aligned scaffolds provided physical cues for the anisotropic organization of
cardiac cells that resembled native cardiac tissue. In addition, the aligned cardiac constructs
were associated with a decrease in atrial natriuretic peptide compared to unaligned constructs
suggesting a more mature, ventricular-like cardiac phenotype [18].
212
Embryonic stem cell-derived cardiomyocytes are currently the best source of de novo
cardiomyocytes and are therefore the best candidates for achieving true regeneration of
myocardial tissue. To be used in cardiac tissue engineering, scaffolds that promote ESCDC
differentiation and functionality must be identified. Relatively few studies have been conducted
using ESCDCs on pre-formed scaffolds and even less work has looked at the coculture of
ESCDCs with other cells found in the myocardium on 3-D scaffolds. Thus, the cell experiments
carried out in Chapter 6 using the aligned and unaligned electrospun scaffolds were designed to
test the response of mESCDCs alone and in coculture with MEFs on the two scaffold
architectures. Murine ESCs were differentiated in a spinner flask system to generate a large and
pure population of cardiomyocytes. The mESCDCs were seeded onto the aligned and unaligned
Phe PUs on their own or pre-seeded with MEFs. In both culture conditions, viable mESCDCs
attached to the PU scaffolds and were functionally contractile. Importantly, the aligned scaffolds
led to the anisotropic organization of rod-shaped cells, improved sarcomere organization, and
increased mESCDC aspect ratio when compared to cells on the unaligned scaffolds. In addition,
pre-seeding the scaffolds with MEFs improved the differentiated phenotype of the mESCDC as
observed by an increase in sarcomere formation, elongation, alignment, and aspect ratio of
mESCDCs compared to this cell type alone. These results build on previous work in our
laboratory that showed unaligned electrospun PU scaffolds supported contractile mESCDCs with
a striated sarcomeric phenotype and appeared more differentiated than cells on scaffolds formed
by thermally induced phase separation [16]. The current work showed that both fiber alignment
and coculturing the mESCDCs with MEFs further improved the organization and maturity of the
differentiated cells.
fibroblasts are important considerations for the formation of cardiac constructs using PU
scaffolds and mESCDCs. Future work should further characterize the cell-seeded PU constructs
to determine if the organization and mature differentiated phenotype associated with the aligned
cocultured constructs has an influence on the functional behavior of the engineered tissue. If so,
this aligned coculture system may be used as a platform to better define critical scaffold
properties for cardiac tissue engineering by altering the mechanical, degradation, and structural
characteristics of the PU scaffolds and testing how each affects the functional properties of
cardiac constructs.
213
Developed a system for coculturing mESCDCs with MEFs and demonstrated that both
scaffold architecture and coculture with fibroblasts improved cell orientation,
morphology, and differentiated phenotype.
differentiated phenotype were achieved with the mESCDCs than previous work in our
laboratory and in the literature.
Adjust the soft segment chemistry of the Gly-Leu PU to PCL of different molecular
weights or tri-block soft segments of PCL-PEO-PCL of varying molecular weight to
expand the available polymer properties with the Gly-Leu-containing family of PUs. Try
to obtain a PU with mechanical properties that more closely mimics the mechanical
properties of the native myocardium.
Systematically adjust electrospinning parameters to achieve nanofibers with Phe and GlyLeu PUs and characterize scaffold properties to further contribute to knowledge of how
fiber size influences scaffold properties and performance.
214
Test the mechanical properties of individual fibers to understand what cells will
experience. Compare the mechanical properties of individual fibers of different sizes and
PUs synthesized with different soft segments. Investigate the response of cardiac cells to
PU fibers of different size and elastic modulus.
Determine the mechanical properties of Gly-Leu PU scaffolds and perform all testing in a
hydrated state.
Implant the PU scaffolds in a normal and infarcted heart to determine if scaffolds have
appropriate biocompatibility properties for this application.
7.2.3 PU Degradation
Carry enzyme-based degradation experiments out to longer time points and with higher
concentration of MMPs.
Validate the competitive substrate enzyme inhibition assay with the peptide Ala-Pro-GlyLeu.
Synthesize a short polymer chain using the Gly-Leu-based chain extender and look at
cleavage of Gly-Leu dipeptide by MMPs.
Perform in vivo degradation studies to identify residence times and foreign body reaction
to Phe and Gly-Leu PUs in an infarcted rat heart.
Optimize mESCDC seeding to obtain a high and uniform density of cells on the aligned
and unaligned PUs. This may be achieved by removing dead cells and using a perfusion
seeding technique.
Characterize the ratio of mESCDCs and MEFs in coculture on PU matrices and optimize
this ratio as needed.
Implant PU scaffolds seeded with mESCDCs and MEFs into infarcted mouse hearts as a
model system for investigating coupling with host myocardium, improvements to cardiac
function, and potential regeneration of myocardial tissue.
Culture cardiac progenitor cells on PUs and allow differentiation to occur as a method for
achieving different myocardial cells in one seeding procedure. Identify if the scaffolds
direct differentiation towards a particular cell type and alter scaffold properties to test if
this may lead to different ratio of cell types.
Test the ability to form cardiac constructs using PU scaffolds and human ESCDCs or
iPSC-derived cardiomyocytes.
216
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references to assign peaks to the corresponding chemical structure of the chain extender.
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Figure A.3: Theoretical predictions of Gly-Leu-based diester chain extender using ACD i-Lab software.
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Figure A.4: C13 NMR spectrum of raw Gly-Leu-CDM-PTSA. The prominent peaks found around 120-140 ppm
correspond to the aromatic carbons of p-toluene sulfonic acid.
222