peptide-modified polymer for endothelialization
TRANSCRIPT
Peptide-Modified Polymer For Endothelialization
A Thesis
Submitted to the Faculty
Of
Drexel University
By
Jamie Lyn Ostroha
In partial fulfillment of the degree
Of
Masters of Science
In
Materials Engineering
December 2001
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Dedications I would like to dedicate this book to my husband and my parents for their support and encouragement.
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Acknowledgements I would like to thank my advisor, Dr. T. Twardowski, for providing me with the opportunity to work on this project. It has been an interesting and enjoyable project. I would like to thank John Kemnitzer and Integra LifeSciences Corporation for the donation of polymer. Special thanks are due to Dr. James San Antonio from Thomas Jefferson University for providing the materials and lab space necessary for most of my work and for valuable discussions on the direction of this project. Special thanks to Dr. Markus Germann, also of Thomas Jefferson University, for the valuable NMR education and providing resources to run NMR. This work was funded in part by a joint Thomas Jefferson University and Drexel University grant.
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Table of Contents
List of Tables .................................................................................................................... vii
List of Figures .................................................................................................................. viii
Abstract ............................................................................................................................... x
1 Introduction................................................................................................................. 1
2 Background ................................................................................................................. 3
2.1 Cardiovascular Diseases ..................................................................................... 3
2.1.1 Atherosclerosis................................................................................................ 3
2.1.2 Stenosis ........................................................................................................... 4
2.1.3 Thrombosis ..................................................................................................... 4
2.2 Biology................................................................................................................ 5
2.2.1 Artery and Vein Biology................................................................................. 5
2.2.2 Luminal Endothelial Cell Biology.................................................................. 6
2.2.2.1 Endothelial Cell Surface ......................................................................... 7
2.2.2.2 Endothelial Cell Injury............................................................................ 7
2.3 Current Treatments of Cardiovascular Disease .................................................. 8
2.3.1 Non-Surgical Therapy..................................................................................... 8
2.3.2 Minimally Invasive Surgical Methods............................................................ 9
2.3.2.1 Angioplasty ............................................................................................. 9
2.3.2.2 Stents..................................................................................................... 10
2.3.3 Invasive Surgical Methods............................................................................ 11
2.3.3.1 Grafting ................................................................................................. 11
2.3.3.2 Synthetic Grafts .................................................................................... 13
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2.3.3.3 Biodegradable Synthetic Grafts ............................................................ 14
2.3.3.4 Alternative Materials ............................................................................ 15
2.4 Polymer Modification ....................................................................................... 16
2.4.1 RGD Containing Peptides............................................................................. 17
2.4.2 Endothelial Seeding ...................................................................................... 18
3 Design Concept ......................................................................................................... 19
3.1 Material Selection ............................................................................................. 19
3.2 Heparin-Binding Peptides................................................................................. 20
4 Experimental ............................................................................................................. 23
4.1 Polymer Derivatization ..................................................................................... 23
4.2 NMR Spectroscopy........................................................................................... 25
4.3 Cell Culture....................................................................................................... 26
4.4 EC Attachment.................................................................................................. 26
4.5 EC Growth ........................................................................................................ 28
5 Results and Discussion ............................................................................................. 30
5.1 NMR ................................................................................................................. 30
5.2 Cell Attachment ................................................................................................ 36
5.3 Cell Growth....................................................................................................... 38
6 Conclusions............................................................................................................... 40
6.1 Recommendations: Characterization ................................................................ 41
6.1.1 Peptide Attachment....................................................................................... 41
6.1.2 Peptide Structure........................................................................................... 42
6.1.3 Cell Affinity .................................................................................................. 42
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6.1.4 Polymer Selection ......................................................................................... 42
6.2 Recommendations: Fabrication ........................................................................ 43
6.2.1 Processing ..................................................................................................... 43
6.2.2 Sterilization................................................................................................... 44
6.2.3 Storage .......................................................................................................... 44
List of References ............................................................................................................. 46
Appendix A- Tables.......................................................................................................... 58
Appendix B- Figures......................................................................................................... 66
Appendix C- Nomenclature .............................................................................................. 82
Appendix D- Abbreviations.............................................................................................. 84
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List of Tables 1. Patency rates for ASV and IMA grafting of the coronary artery of varying
diameters, from [142].................................................................................................. 58
2. Patency data for representative vascular grafts, adapted from Ku [51]. ..................... 59
3. Improving hemocompatibility of artificial biomaterials [73]. .................................... 60
4. Properties of native tissue and several synthetic polymers......................................... 61
5. NMR peak integration values for p(DTEC-c-X%DTC), X=0, 13, and 35, peaks...... 62
6. Integration values corresponding to polyalanine peaks for attachment to p(DTEC-c-X%DTC), X=0, 23, 50............................................................................................. 63
7. The corresponding attachment percentages have been calculated.............................. 63
8. Cell attachment strength as assessed using a centrifugation assay. Note that the poly(DTE carbonate) shows no significant improvement in attachment over the negative control, but attachment increases with additional functional groups. .......... 64
9. Comparative cell growth data at 7 days for various experimental and common vascular implant materials. ......................................................................................... 65
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List of Figures
1. Cross-Section of a typical artery and vein [143]. ........................................................66
2. a) Molecular model of poly(DTEC) repeat unit showing bond angles and bond lengths. b) Repeat unit of poly(DTE-co-X%DT carbonate). When R = CH2CH3 the repeat unit is desaminotyrosil-tyrosine-ethyl ester (DTE); when R = H the free unit is desaminotyrosil-tyrosine (DT). X is the molar fraction of repeat units with pendant acid groups. (Note: H’s are labeled α, β, γ, δ, ε, for identification and quantification by NMR spectra. The hydrogens in the pendant R groups are labeled by proximity to the ester group. Thus, the hydrogen on the free acid is R1, while the hydrogens in the ethyl ester are labeled R2 and R3.) c) Repeat unit of poly(DTEC-co-X%DTC) and L-lysine ethyl ester. Arrows indicate both possible attachment locations.....................................................................................................67
3. 2D TOCSY of poly(DTEC) in CDCl3. Peaks are labeled as detailed in Figure 2. Solid lines indicate three different correlation systems with interactions between neighboring hydrogen. .................................................................................................68
4. 1D NMR spectra of a) P(DTEC-c-0%DTC) and b)P(DTEC-c-13%DTC) with peak integration values. The peak labels correspond to hydrogen as marked in Figure 2. .......................................................................................................................69
5. 1-D and 2-D COSY NMR spectrum of L-lysine ethyl ester. Solid lines indicate correlation systems. Below is the corresponding molecule. ........................................70
6. TOCSY spectrum of P(DTEC-c-13%DTC) with attached L-lysine ethyl ester. The original correlation systems from the poly(DTE-co-X%DT carbonate) system are indicated by lines. The solid lines indicate correlation systems from the L-lysine. Note that the system has ( ) ( ) two related correlation systems, corresponding to α– and ε–amine attachment of the lysine.........................................71
7. NMR spectrum of P(DTEC-c-13%DTC) with attached L-lysine ethyl ester. Integration values are below peaks. The ratio of the α poly(DTE carbonate)peak, which should be constant at a concentration of 1 across chemistries, to the lysine spectral group at ca. 4.38 shows an approximately 11.5±0.5% lysine attachment......72
8. TOCSY spectrum of p(DTEC-c-13%DTC) with attached L-lysine ethyl ester and polylysine. Note that there are no additional peaks, only intensification of existing peaks. ...........................................................................................................................73
9. a) 1D NMR spectrum of p(DTEC-c-50%DTC). Peak integration values are shown below select peaks. The integration value for the peak at 4.7 ppm corresponds to the α peak in the polymer. Characteristic polyalanine peaks are also indicated. b) Structure of polyalanine...............................................................................................74
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10. 1D NMR spectrum of p(DTEC-c-35%DTC) with peptide (ARKKAAKA)4 attached via a lysine bridge..........................................................................................75
11. Attachment data from Table 8 for functional polymers, p(DTEC-c-X%TDC) X=0, 13, 23, 35, and 50% and positive (FN) and negative (MeCl2) controls. Attachment increases with additional functional groups.................................................................76
12. Attachment data for functional polymers and positive and negative controls. P(DTEC-c-X%TDC) X=0, 13, 23, 35, and 50% are shown at both the low (300 g) and medium (600 g) force. FN (positive control) and Blank (negative control) are drawn as straight lines as they are not subject to the same functionalization. The lines for the FN and the Blank indicate the cell attachment at the specified force......77
13. Cell attachment data for p(DTEC-c-13%DTC) alone (13%), with attached lysine (13wL), attached polylysine (PolyL), and attached heparin-binding 32mer peptide (32mer PEP).................................................................................................................78
14. Optical microscope images of cell attachment and growth on 13% poly(DTE-co-X%DT carbonate) at a) 1 hour, b) 1 day, c) 3 days and d) 7 days. Note that at 1 hour, the cells show flattening indicative of attachment. The total number of cells increases through day 7. On day 7, there are regions of cell confluence.....................79
15. Optical microscope image of cell attachment at day 5 on a) poly(DTE carbonate), b) 13% poly(DTE-co-X%DT carbonate), c) 35% poly(DTE-co-X%DT carbonate), d) PLA, e) PET, f) PVC and g) glass. .......................................................80
16. Optical microscope image of stained cells at the end of the growth assay, a) 12X shows areas of semi-confluence b) 23X shows spread cells indicating attachment c) 46X shows spread cells with visible nuclei and cells in the process of dividing. ...81
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Abstract
Peptide-Modified Polymer For Endothelialization Jamie Lyn Ostroha
Thomas Twardowski
A functionalized polycarbonate material derived from tyrosine containing varying
levels of free acid functionality along the backbone, poly(DTE-co-X%DT carbonate), has
been functionalized with a peptide to promote endothelial cell attachment and evaluated
as a potential synthetic vascular graft material. Lysine derivatives have been produced to
promote the attachment of engineered peptides for promoting endothelialization.
NMR has been used to analyze the initial structure of the poly(DTEC-co-
X%DTC) and changes in the structure with the attachment of L-lysine ethyl ester and
polylysine, polyalanine, and the (ARKKAAKA)4 peptide. The polymer peak at 4.8 ppm
has been labeled the α-peak. This peak is the primary peak used to evaluate changes in
the spectra related to changing polymer functionality and the attachment of molecules to
the pendant acid group. Changes in the β-peak (3.05 ppm), the R2 peak (4.1 ppm) and the
R3 peak (1.3 ppm) are compared to the α-peak for quantification purposes. Key NMR
peaks have been identified and lysine attachment and quantification is discussed. A lysine
peak has been identified at 4.38 ppm. This peak is also compared to the polymer α-peak,
indicating 85-90% attachment efficiency. The use of polylysine, polyalanine, and
peptides establish that peptide binding is successful; qualitative and semi-quantitative
results are presented. Semi-quantitative results with polyalanine indicate a large degree of
attachment, although some interference with the spectral peaks has been noted.
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The effect of poly(DTEC-co-X%DTC) on human endothelial cell attachment and
growth is also studied and compared to current synthetic materials. Cell attachment has
been shown to increase significantly as the polymer becomes more hydrophilic. Non-
functionalized polymers show that less than five percent of cells remained attach at a
force of 300 g, while 50% functionalized polymers show 40% attachment at the same
force. Cell growth studies show an increase in cell affinity with increasing hydophilicity.
35% functionalized polymer shows over double the number of cells as the non-
functionalized material after seven days. The results for the p(DTEC) at increasing levels
of functionality bracket Dacron and PLA depending. Cell spreading, confluence, and
division have been confirmed by visual analysis.
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1
1 Introduction
Mundth et al predicted in 1978 [1] that there would be an increasing number of
cardiovascular problems due to the American lifestyle. In the late 1970s, deaths from
cardiovascular disease were around 750,000 a year. In 1998, cardiovascular diseases
claimed close to one million lives [2]. Though an increase in deaths from cardiovascular
disease has been observed, it has not increased as rapidly as predicted. This is mostly due
to rapid and continual advances in medicine and technology.
In 1949 the vascular graft was introduced [3]. The 1950s saw a great increase of
resources, both public and private, devoted to cardiovascular education and research [4].
The availability of synthetic grafts made it possible to prevent death from rupture of
arteriosclerotic aneurysms, to prevent loss of limbs in many patients with occlusive
diseases, and to improve the quality of life in others [5]. However, even though surgical
cardiovascular treatment has been in practice since the 1940s [4], there is an ongoing
need to develop improved vascular repair materials. According to 1998 estimates,
60,800,000 Americans have one or more forms of cardiovascular disease (CVD) [2], with
55,000 lower limb vascular reconstructions being necessary each year [6]. The current
materials and techniques used for cardiovascular repairs exhibit some problems, e.g.
inadequate long-term patency, especially in diseased blood vessels of the lower limbs [6-
9].
The focus of this research is the development of a material, modified to increase
patency and decrease the need for surgical revision. Native vessels have a structure that
resists thrombosis, intimal hyperplasia, and other causes of blockage. This is primarily
due to the inner lining of endothelial cells on the vessels. However, disease or surgery
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often impairs these cells, increasing the risk of blockage. A functionalized, biodegradable
polymeric material has been developed with the potential to enhance endothelial cell
attachment.
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2 Background
There are a variety of diseases that can disrupt blood flow and result in the need for
surgery. CVD, the biology of vasculature and some current treatments and synthetic
materials for vascular prostheses are reviewed.
2.1 Cardiovascular Diseases
Vascular diseases often result in a need to open, replace or bypass diseased or
damaged blood vessel segments. The primary CVD is atherosclerosis. Atherosclerotic
vascular disease, particularly coronary heart disease, is a leading cause of death
worldwide [10], and accounts for nearly three-quarters of all deaths from CVD.
2.1.1 Atherosclerosis
Atherosclerosis is a disease of the arterial wall. This disease often begins with
intima (inner lining) thickening, especially in locations corresponding to high shear
stress. Over time, lesions (scars) form and grow until the vessel becomes occluded [11].
It is caused primarily by environmental factors, although individuals may have an
increased or reduced susceptibility that is genetically predetermined. Such environmental
factors include a high-fat diet, cigarette smoking, and hypertension [2, 10, 12]. CVD
involves deposits of fatty substances, cholesterol, cellular waste products, calcium or
other substances on the inner lining of an artery. This build-up is called plaque, and
usually affects large and medium-sized arteries [2]. It has been shown that injured
endothelial cells lead to atherosclerosic lesions, which in turn lead to occluded vessels in
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a three-step process: (1) smooth muscle cells accumulate, (2) the smooth muscle cells
form a connective tissue matrix, and (3) lipids accumulate [13]. This process occurs
because the damaged endothelial cells can no longer carry out the multiple, complex
interactions involving blood and vessel wall components that maintain vascular
homeostasis, in which healthy endothelial cells are a key component [14]. When these
cells are damaged or removed, homeostasis can longer be maintained and cardiovascular
disease results.
2.1.2 Stenosis
The simplest local disturbance is a restricted lumina, known as stenosis [12]. The
artery may become clogged by the buildup of fat, cholesterol or other substances over
time [2]. This produces highly increased shear stresses that are capable of removing
endothelial cells from the luminal lining [12].
2.1.3 Thrombosis
Plaques that rupture form blood clots (thrombus) that can block blood flow or
break off and travel to another part of the body (embolus). If either of these events occurs
and blocks a blood vessel that feeds the heart, it causes a “heart attack”. If it blocks a
blood vessel that feeds the brain, it causes a “stroke”. If the blood supply to the arms or
legs is reduced, it can cause difficulty in proper functioning and, in extreme cases,
gangrene [2].
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2.2 Biology 2.2.1 Artery and Vein Biology
The vascular system is divided into two parts, the arteries and the veins. The
arteries consist of relatively thick-walled, viscoelastic tubes that undergo high pressure as
they carry blood away from the heart. On the other hand, veins are thinner, but have
larger diameter, elastic tubes that experience low-pressure conditions as they carry blood
toward the heart [15]. The arteries originate from the aorta and its branches, becoming
smaller and smaller as they branch out. As the cross-sectional area of the vessel
decreases, the blood velocity increases to maintain a constant total blood volume. Veins
collect blood from the capillaries and successively join together to form progressively
larger veins. They then return blood to the heart. Veins can accommodate much larger
volumes of blood with very slight changes in pressure [4]. Except for the differences in
thickness, the walls of the largest arteries and veins consist of the same three distinct,
well-defined layers (Figure 1). The innermost layer is the thinnest, tunica intima, a
continuous and well-developed lining consisting of a single layer of simple, flat
endothelial cells that are held together by VE cadherin [16]. The endothelial cells are
surrounded by a thin layer of subendothelial connective tissue interconnected with a
number of circularly arranged elastic fibers to form the subendothelium, and are
separated from the next adjacent wall layer by a thick elastic band called the internal
elastic lamina. The middle layer, tunica media, is the thickest and is composed of many
elastic fibers, a significant number of smooth muscle cells, some interlacing collagenous
tissue, and an intercellular mucopolysaccharide substance. These are separated from the
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next adjacent layer by another thick elastic band called the external elastic lamina. The
medium-sized tunica adventitia is the outer vascular sheath consisting entirely of
connective tissue [11, 15, 17]. Veins and arteries are traditionally divided into three
categories based on size: large (>6mm), medium (4-6mm) and small diameter vessels
(<4mm) [3, 18, 19]. The difference in the vessel size alters the flow characteristics of the
blood, which can in turn alter the vessel-blood interface. There is an increased surface to
volume ratio and a reduced blood flow volume in smaller-diameter vessels. This causes
an increase in the blood-surface contact time, which can result in increased activation of
surrounding blood elements if the endothelium becomes damaged. This may explain why
patency rates are significantly lower in medium and especially smaller synthetic vessels.
2.2.2 Luminal Endothelial Cell Biology
The endothelium is generally a single layer of tightly packed cells that line the
vascular lumen [20] to create the intima. The cells of the lining are dynamic partners in
multiple, complex interactions involving macromolecules, cellular blood components,
and vessel wall components, such as smooth muscle cells and extracellular matrix [21].
The luminal endothelium senses, transmits, and participates in the adjustments that are
necessary as a result of deviations in homeostasis [22]. Deviations in homeostasis can
include blood loss, reduced or elevated blood pressure, and constricted or dilated vessels.
Arterial endothelial cells form a continous, nonthrombogenic, metabolically active lining
for the vascular system [23]. One key function performed by the endothelial cells is
heparan sulfate synthesis [24, 25], which is known to have antithrombogenic and
anticoagulant properties [26, 27]. This coagulation/anticoagulation system is very
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important in the homeostasis of the circulatory system, promoting fluidity within and
rapid clot formation outside of the blood vessels. During homeostasis, anticoagulant
mechanisms dominate, while in a disturbed state coagulation mechanisms dominate [28].
So, any change in the normal structure and function of the endothelial lining will cause
coagulation. For example, following denudation injury, where endothelial cells are
stripped locally from a small path of vascular tissue, the cells in and around the wound
area are stimulated to spread, migrate, and proliferate to reconstitute a continuous lining
[23]. If the cells are unable to re-establish a continuous lining, platelet attachment, release
of platelet growth factor, and smooth muscle cell migration and proliferation are
stimulated instead [29]. The migration and adhesion of platelets at the site of vascular
injury depends on a combined interaction between the platelet cell surface receptors and
the adhesive matrix proteins on the exposed subendothelium [30, 31].
2.2.2.1 Endothelial Cell Surface
The complex interaction of endothelial cells and blood components is a result of
the complex surface composition of the endothelial cell. The endothelial cell surface is
made up of proteins, glycoproteins, glycolipids, and proteoglycans (PG). The prevalence
of anionic sites, predominately from the proteoglycans, gives the luminal surface a net
negative charge [22].
2.2.2.2 Endothelial Cell Injury
Endothelial cell injury can result from many causes, including hemodynamic
stress, mechanical trauma, hypercholesterolemia, infectious agents, oxygen, and chemical
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agents like homocysteine [32]. These forces play an important role in the occurrence of
cardiovascular disease [33] by causing what is known as endothelial cell dysfunction.
The best method for prevention of CVD is primarily to prevent damage to the endothelial
layer or secondarily to repair a damaged endothelial layer [23]. This has been an area of
great interest since the various functions of the endothelial layer have become more
clearly understood.
2.3 Current Treatments of Cardiovascular Disease
Treatments of CVDs include medical therapy with specific medication; minimally
invasive surgical treatments such as balloon angioplasty, laser angioplasty, atherectomy,
and stents; and invasive surgical methods, which includes the implantation of vascular
grafts.
2.3.1 Non-Surgical Therapy
There are several risk factors for vascular disease, as mentioned earlier, the most
notorious being high cholesterol levels. More specifically, elevated serum levels of total
cholesterol (TC) and low-density lipopotein cholesterol (LDL-C) lead to CVD [34, 35].
Dietary intervention and drug therapy have been shown to lower LDL-C levels. Another
non-surgical procedure in current practice is the use of LDL adsorption compounds [36].
Synthetic and natural anti-oxidants [37], like vitamin E and gene therapy [38], have been
examined for the prevention of atherosclerosis and have been shown to reduce the
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progression of this disease [39, 40]. Generally, however, these techniques are not
sufficient to treat acute or developing CVD.
2.3.2 Minimally Invasive Surgical Methods 2.3.2.1 Angioplasty
Angioplasty is a minimally invasive procedure and is now the customary
procedure for treating coronary artery disease: the common technique being percutaneous
transluminal coronary angioplasty (PTCA) [41]. During angioplasty, a balloon-tipped or
laser-tipped catheter is introduced into a diseased blood vessel, usually from a remote site
like the femoral artery. In balloon angioplasty, a balloon is inflated at the site of closure
and the vessel opens further, allowing for improved flow of blood [16].
Because of the complex nature of vascular tissue, angioplasty can result in several
complications. First, balloon dilation can cause a fracture of the atherosclerotic plaque.
Second, endothelial damage can occur that in turn can stimulate platelet adhesion.
Finally, the traumatic dilation can cause damage to the media, ranging from stretching to
tearing [41]. Angioplasty often results in early restenosis, due to shrinkage of the
muscular components of the arterial wall [41-43]. However, stenting after angioplasty has
been shown to reduce the likelihood of restenosis [41, 44]. The stent prevents shrinkage
of the vessel and maintains vessel patency mechanically. In 1998, 926,000 angioplasties
were performed in the United States. Of these, 539,000 were PTCAs [2].
Similar to angioplasty, atherectomy is a procedure for opening coronary arteries
blocked by plaque. Coronary atherectomy uses a laser catheter to vaporize the plaque, or
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a small rotating “shaver” to shave off the plaque. The catheter is inserted into the body
the same way as in angioplasty. Balloon angioplasty or stenting may then be used after
the atherectomy [2].
A primary disadvantage of these techniques is a limited capability in small vessel
applications and a high rate of restenosis.
2.3.2.2 Stents
Stents date back to 1912 when Carrel used a glass tube to open occluded canine
arteries [45]. Vascular stents were introduced into human vascular surgery in the 1960s as
a way of maintaining the patency of angioplasties [46]. During stenting a wire mesh stent
is used to support an artery that has recently been cleared by angioplasty. The stent is
collapsed to a small diameter, placed over an angioplasty balloon catheter and moved into
the blocked area. When the balloon is inflated, the stent expands, locks in place and
forms a scaffold to hold the artery open. The stent remains in the artery permanently to
hold it open, which improves blood flow to the heart muscle [2]. Stents can also be used
for opening occluded blood vessels and spanning small dissections [44].
The use of vascular stents to combat cardiovascular disease has, in many cases,
reduced the need for large incisions and long hospital stays that generally occur with
major vascular surgery. Today vascular stents are still being used to maintain patency
following angioplasty of occluded arteries and veins. The stent procedure is fairly
common, now representing 70-90% of CVD treatment procedures [47]. A stent may be
used as an alternative to, or in combination with, angioplasty.
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In recent years doctors have used new stents, some of which are covered with
drugs that help prevent the blood vessel from re-closing [48]. These new stents have
shown some promise for improving the long-term success of this procedure [2]. Also,
stent-grafts, in which synthetic material covers the interstices of the stent [49], have
become a possible solution to increase long-term stent patency. Again, difficulties arise
when using the stent in small-diameter vessels. The stent may not be flexible enough to
travel through the necessary vasculature to reach the occluded vessel. Significant damage
can also occur to the endothelial lining as the stent moves to the desired location. The
disturbances in flow caused by the expanded stent often bring about restenosis. This is
because of the different flow characteristics of the small-diameter vessel and can result in
increased activation of surrounding blood elements.
2.3.3 Invasive Surgical Methods 2.3.3.1 Grafting
When a blood vessel becomes damaged or occluded, it often must be replaced.
Venous autografts have been the ideal graft since the vascular graft was introduced in
1949 [3]. Autografting, generally using the autologous saphenous vein (ASV), is
preferred because of increased patency and fewer post-surgical complications. The ASV
is removed from one of a limited number of harvest sites and sutured above and below
the damaged site to redirect blood flow. After surgery the graft begins to remodel: a 60-
70% loss of endothelial cells within 48 hours, then re-endothelialization after 6-8 weeks
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[7]. Each year, 300,000 patients in the United States receive a venous autograft to bypass
an occluded or narrowed coronary or peripheral artery.
However, 10-30% of patients who need vascular bypass surgery do not have
suitable saphenous veins due to prior harvesting or disease damage [3, 50]. Alternative
biological grafts can occasionally be used. The internal mammary artery (IMA) is used
extensively for coronary artery bypass grafting and has better long-term patency rates
than ASV [51] (Table 1). Less desirable, but used as needed, are allografts and
xenografts. The allograft of choice is the gluteraldehyde-treated umbilical cord vein graft
[50]. Allografts have been able to reach patency rates similar to ASV in large diameter
applications. Xenografts can result in a high occurrence of thrombus formation, infection,
and rupture [3], but modified bovine heterografts are nevertheless infrequently used [50,
51].
ASV grafts are not completely successful. They typically have five-year patency
rates of only 30-75% for femoropopliteal bypasses [7]. One of the causes of saphenous
graft failure is graft disease [52], which follows the same progression of events discussed
earlier to reach atherosclerosis. One possible cause for graft disease is the damage done
to the endothelial cell lining during harvesting of the vein. The loss of this layer results in
accumulation of fibrin at the luminal surface and the activation of coagulation elements.
Graft occlusion occurs following a series of events: first trauma to the natural vessels
brings about the implantation procedure; then the implanted graft produces fluid dynamic
disturbances in the blood flow that cause endothelial cell damage. Because the graft is
also thrombogenic, blood derived mitogens act on exposed smooth muscle cells leading
to hyperplasia [53].
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2.3.3.2 Synthetic Grafts
Synthetic alternatives to autografts such as ePTFE and Dacron have the advantage
of being readily available. However, synthetic prosthetics suffer from decreased patency
due to factors including smooth muscle cell hyperplasia and thrombus formation. The
characteristics of an ideal prosthetic graft include biocompatibility with tissue and blood
elements, nonthrombogenicity, durability, proper interaction with surrounding connective
tissue, resistance to the formation of aneurysms, and acceptable mechanical properties.
Synthetic vascular grafts used in large diameter vessel applications have achieved
a relatively high degree of patency (Table 2). However, in medium and small-diameter
grafts patency remains greatly reduced [51]. Failure within 30 days of implantation is
primarily caused by thrombosis; failure within six months is generally attributed to
intimal hyperplasia [50]. The size difference has been observed to greatly affect the
performance of grafts. The first clinically used synthetic vascular implants, developed in
the 1950s, were made from woven or knitted Dacron, (polyethylene terephthalate, PET),
and Teflon. Despite possessing thrombogenic surfaces and demonstrating a lack of elastic
compliance, they functioned sufficiently well in large-vessel applications. The grafts
were incorporated in fibrous tissue and infection rarely occurred [7]. However, these
vascular grafts were inadequate when used in medium or small diameter applications.
There are several reasons why synthetic grafts function well in large but not in
small diameter applications. The increased surface to volume ratio in smaller-diameter
vessels results in an increased activation of surrounding blood elements, which leads to
thrombosis. A reduced blood flow volume also causes an increase in the contact time
with the luminal graft surfaces. For these reasons, synthetic grafts are rarely used for
14
bypass or reconstructive procedures in small diameter applications, including the
coronary artery or arteries below the knee. Additionally, compliance affects the function
of small-diameter grafts. Compliance, in this application, is generally a structural and not
a material property so it depends on the geometry (diameter and wall thickness) of the
blood vessel [54]. It is desirable that the compliance match the compliance of the vessel it
is replacing in order to avoid a discontinuity of blood-flow velocity and potential stagnant
regions [18]. The smaller grafts are unable to meet the compliance requirements for
small-diameter vessels.
Currently, the material with the best patency is expanded poly
(tetrafluoroethylene), ePTFE [7]. It exhibits satisfactory tensile strength,
thromboresistance, and resistance to neointimal hyperplasia in large-diameter
applications [55] and shows partial healing with endothelial cell coverage [56]. However,
both ePTFE and Dacron have unacceptable long-term patency rates in small-diameter
applications [8, 51, 57].
2.3.3.3 Biodegradable Synthetic Grafts
A new trend in biomaterials has been the use of biodegradable polymers [58].
Biodegradation generally implies a material that can be broken down by hydrolysis or
enzymatic mechanisms. This process has also been referred to as absorption, erosion, and
resorption in the literature [59]. In principle, cells attach and grow on a tissue scaffold;
the cells proliferate and are eventually able to adsorb and replace the scaffold.
Biodegradable materials have several advantages over non-biodegradable materials: they
do not elicit a permanent, chronic foreign-body response; they can be used for tissue
15
engineering; and they can be used to deliver drugs to the implantation site. However,
using degradable polymers in bio-application requires consideration of two inter-related
processes: (1) the degradation of the polymer due to blood contact, and (2) the interaction
of degradation products and the body. Poly (L-lactic acid), PLA, is especially useful due
to its slow biodegradation, useful mechanical properties, and especially, prior FDA
approval in other applications [60]. This family of polymers was first used for
biodegradable wound closures and now is being investigated for vascular applications.
However, the acidic degradation products have been shown to cause inflammation and
swelling [61] in vascular applications.
2.3.3.4 Alternative Materials
In very recent years a large variety of polymers have been and are being evaluated
as possible vascular materials. At the top of the list are many polyurethane based
materials like polyurethane with plasminogen [62], elastomeric polyurethanes [63], and
peptide modified gold-coated polyurethanes [64]. Others possible vascular materials
include poly(ether-amidoamines) and polyamidoamines [65], plasma surface modified
poly(ethylene terephthalate) [66], hydrogels [67], and many more. However, no polymer
or polymer combination has yet been able to meet all the requirements for a vascular
material. For instance, the adverse effects of polyurethane degradation products have
been observed to reduce patency [7] in vascular applications. In spite of this, several
common trends have been observed in potential vascular materials; surface or bulk
modification and the use of endothelial cell seeding to improve biological interactions.
16
2.4 Polymer Modification
In 1955 Edwards and Tapp first attempted to chemically modify a commercially
available polymer to develop a negatively charged coating promoting heparin attachment
[68]. There are many different ways to chemically modify a polymer to change the
properties (Table 3). Since Edwards and Tapp’s first attempt, many others have used
surface chemistry in an attempt to increase the patency of vascular grafts [62, 65, 66, 69-
75]. A great deal of work has focused on altering the surface chemistry of a substrate to
specifically affect cell functions like motility, proliferation, and adhesion [76].
Attachment of physiologically active substances such as fibronectin [71] and heparin
[77], to substrate surfaces has been investigated. A number of natural and synthetic
growth factors have been identified that are capable of stimulating endothelial growth
[78] or regulating the initial adsorption of proteins and deposition of platelets and blood
cells [79, 80]. For example, the synthetic surface can be treated with fibronectin (FN).
The FN has a high affinity for binding heparin, with two classes of affinities (with
binding sites located towards opposite ends of the molecule) Kd=100 nm to 1.0 nm [80].
FN-coated PTFE grafts retained more than six times the number of seeded endothelial
cells compared to untreated controls at 24 hours following the restoration of blood flow
[71]. Despite advances with the Teflon graft, intimal hyperplasia [53, 81], restenosis, and
occlusion remain severe in most small-diameter applications. To date, no clinically
applicable small-diameter vascular prostheses have been developed [82].
17
2.4.1 RGD Containing Peptides
A major objective in the design of functionalized polymer surfaces for tissue
engineering applications has been the covalent attachment of peptides that regulate cell
adhesion: specifically those that promote integrin-mediated cell attachment [83, 84].
Since the vast majority of mammalian cells are anchorage-dependent, they must attach
and spread on a substrate to proliferate and function normally [23, 76]. By controlling the
surface properties of the substrate, cell attachment and growth in vitro can be altered [85].
A great deal of work has focused on RGD containing peptides to regulate cell adhesion
[83, 86-88].
Peptides containing the RGD (R=arginine, G=glycine, D=aspartic acid) sequence
are considered important small cell-adhesive ligands [83, 86]. This cell-binding sequence
is present in adhesive proteins like fibrinogen, vitronectin, collagens, and fibronectin [89,
90]. Membrane proteins of blood platelets, endothelial cells, and several other cell types
can bind RGD-containing peptides whether the peptide is in solution or immobilized onto
a solid surface [87, 91]. For example, a 25-residue RGD-containing peptide binds to the
α4β1 integrin receptor with 40% of the activity of fibronectin [92].
In an effort to promote cell adhesion onto biodegradable implants, RGD peptides
have been covalently grafted onto poly(amino acid-lactic acid) copolymers [93] and
polystyrene [94]. Modification of both PET and PTFE arterial patches with an RGD-
containing cell adhesion peptide was reported to improve healing characteristics after
implantation in both dogs and sheep [88]. However, the mere presence of the RGD
sequence does not mean that the protein will possess adhesive properties [80].
18
Other potentially useful, non-RGD containing peptides have been identified for
vascular applications, which include consensus amino acid sequences such as
(XBBXBX) (X= hydrophobic residue, B=positively charged basic residue). These are
shown to be involved in heparin binding [84, 95, 96]. These peptides are discussed in
more detail later.
2.4.2 Endothelial Seeding
Endothelial seeding is a process where endothelial cells are placed on the luminal
surface of a vascular graft and allowed to proliferate and migrate to cover the surface
before implantation. The concept of endothelial cell seeding was initially conceived to
address the low patency rates resulting from using small-diameter vascular prosthesis [8].
The first successful attempt to seed a synthetic vascular graft with endothelial cells was
achieved in 1978 by Herring et al [97]. A great deal of research has been conducted on
the topic since this initial discovery. Results have shown that endothelial seeding
promotes endothelialization in some cases, which increases patency [98-100]. Animal
studies have demonstrated the resistance of seeded grafts to thrombosis and
pseudointimal hyperplasia [3, 8, 101]. One of the disadvantages of graft seeding is the
increased time required for the cells to multiply and cover the graft surface. In addition,
many cells are lost shortly after implantation and the cell functions are not easy to control
[55]. Despite successful endothelialization in animal studies, clinical evaluation of
endothelial cell seeded grafts has not been as successful; significant neoendothelialization
of vascular prostheses, beyond 1-2 mm, has not yet been observed in humans [8, 19].
19
3 Design Concept
The overall object of the project, of which this research is a part, is to engineer a
vascular substitute that can (1) be directly implanted without requiring secondary surgery,
and (2) increase patency. The specific goal of this part of the project was to modify a
polymer with a peptide to produce a surface that actively promotes endothelial cell
coating. Since surface chemistry affects protein adsorption, cell adhesion, and plays an
important role in blood compatibility, a polymer has been chosen that allows surface
modification. The functional groups on the surface are used for derivatization with
peptides having an affinity for endothelial cell PGs.
3.1 Material Selection
Poly(DTE-co-X%DT carbonate) is the polymer of choice for the development of
synthetic vascular graft material. Poly(DTE-co-X%DT carbonate) is a copolymer based
on the natural amino acid tyrosine, with a functional pendant group (Figure 2a) [102].
This polymer is termed a pseudo-poly(amino acid), because the amino acids are linked
together by non-amide bonds [103]. The use of these backbone-modified pseudo-
poly(amino acid)s was first investigated in 1984 by Kohn et al [104]. The polymerization
process controls the amount of free acid available. The amount of free acid available is
indicated by the addition or removal of the R protecting group (Figure 2b). When R =
CH2CH3 the repeat unit is desaminotyrosil-tyrosine-ethyl ester (DTE); when R = H the
free unit is desaminotyrosil-tyrosine (DT). X is the molar fraction of repeat units with
pendant acid groups. When the R group is absent, the polymer has 100% free acid
20
groups. The amount of free acid in turn controls the functionality and the degradation rate
[105]. In this project, the functional group is used to bind a peptide via a lysine bridge.
The attachment of the lysine bridge is illustrated in Figure 2c. The degradation rate can
vary from a few days to several years, depending on the free acid and environmental
conditions. With a DT content of X=0%, the polymer requires 366 days to reach 50% of
the starting molecular weight when implanted subcutaneously in rats [106]. The polymer
degrades by random hydrolytic chain cleavage that results in an increase in polydispersity
[106]. As the DT content increases, the polymer becomes more hydrophilic and anionic
in nature, and more susceptible to hydrolytic degradation [107]. In vitro cytotoxicity and
short term in vivo evaluations in rats have shown tyrosine-derived polycarbonates to be
generally biocompatible [58, 105, 108] and to support attachment and growth of various
cell types [109, 110]. The degradation products are non-acidic [111], especially when
compared with PLA [105]. The pendant functional groups provide a location to
covalently attach functional side groups, including peptides. Poly(DTEC-co-X%DTC) is
completely amorphous, with a Tg of approximately 80°C (176°F) [112], and is readily
formed [109] using common melt and solution techniques. The tensile strength (Table 4)
is also greater than that of thoracic tissue.
3.2 Heparin-Binding Peptides
Similar to the RGD containing peptides, heparin-binding peptides have been
designed to specifically bind PGs and can therefore be inferred to be useful in promoting
endothelialization [96]. These peptides are based on the heparin binding (XBBXBX) or
(XBBBXXBX) (X= hydropathic residue, B=positively charged basic residue) sequence
21
discovered by Cardin and Weintraub [95]. Instead of binding to an integrin, these
peptides target PGs. These peptides are less specific than RGD containing peptides and
are capable of binding to all of the PGs on the cell surface, including those carrying
chondroitin, dermatan, keratan, and heparan sulfates. RGD containing peptides, on the
other hand, are only capable of binding to specific integrin molecules. This is because the
RGD containing peptides are integrin receptor mediated [90], whereas the heparin
binding peptides attract PGs electrostatically [84]. In addition, RGD containing peptides
must use a spacer arm to achieve a suitable distance between the substrate and the RGD
end of the peptide for cells to attach. The RGD sequence must be available at the surface
of the substrate and its conformation must fit integrin receptors for attachment to occur.
In this particular aspect of the project the (ARKKAAKA) sequence, with alanine,
arginine and lysine (ARK), is used. The important aspects of peptides with regard to
binding are generally surface charge, structure, and attachment location [113]. The
predominant molecules on endothelial cells are PGs, which contain a core protein with
one or more covalently bound anionic glycoaminoglycan (GAG) chains [27]. The heparin
binding peptide provides positively charged sites to bind these PGs [95, 96]. GAGs
participate in the adhesion of endothelial cells to the extracellular matrix or another
substrate. Heparan Sulfates (HS) comprise 85% of the GAG on endothelial cells [114]. If
a peptide with a high affinity for heparin is designed, the same peptide should also have a
high affinity for endothelial cells.
Modeling these peptides predicts arrangement of the amino acids into an α–helix
[96], which has been confirmed by circular dichroism [115]. The helix structure gives a
three-dimensional arrangement of multiple heparin-binding consensus sites; it allows for
22
clustering of noncontiguous basic amino acids on one side of the helix, thus forming a
charged domain to which GAGs can bind. The formation of this structure has been found
to be critical to the binding efficiency [96]. To achieve this, the peptides contain multiple
copies of the eight-mer ARK consensus. In addition, the amino acids that make up the
peptide should be conducive to helix formation. Alanine is used because of its stabilizing
activity on α-helices [116] and the basic amino acids are chosen to represent those with
the highest probability of occurrence in each basic position in the heparin-binding
consensus sequences of native heparin-binding proteins [95]. For the peptide to take an
active role in binding, it must bind specifically at the end of the chain and posses
sufficient chain length to assume the helical structure. The total length of the peptide also
plays an important role in the binding efficiency; maximum binding of the peptide to low
molecular weight heparin reaches a plateau around 30 amino acid residues [96].
23
4 Experimental
Specific experiments designed to functionalize and characterize the polymer are
discussed. Preliminary tests for the effect of the polymer and functionalized polymer on
cell functions are also discussed.
4.1 Polymer Derivatization
The aim of the project was to produce a polymer surface with peptides covalently
attached. P(DTEC-c-X%DTC) polymers with 0, 5, 13, 23, 35, 50% free acid were
provided by Integra LifeSciences Corporation (San Diego, CA). The functional groups on
the surface are used for derivatization, via a lysine bridge, with peptides having an
affinity for endothelial cell PGs. By creating this functionalized polymer surface with
attached peptides, protein adsorption and cellular interactions may be controlled.
L-lysine ethyl ester (lysine) (Sigma, Lot #17H1152) was attached to the free acid
group on p(DTEC-c-X%DTC) (X>0%) as a linking agent to connect peptide chains to the
polymer. By using lysine as a bridge, connection to the polymer at the unique carboxylic
acid terminus of the peptide is assured. This leads to a basic surface to promote cell
interaction. Without the lysine bridge, binding could occur at any lysine residue
containing point along the peptide chain. The reaction was conducted in dilute solution
with excess lysine to inhibit multiply attached lysines and to drive the reaction to
completion.
The polymer was reacted in solution with excess lysine and 1,3-
dicyclohexylcarbodiimide (DCC) to catalyze the reaction. DCC has been shown to couple
24
amines and carboxylic acid groups with a high efficiency [60, 117, 118]. While pDTEC
is soluble in organic solvents including chloroform and methylene dichloride, the desired
attaching groups, including lysine, polyamino acids, and the peptide, are not. This
allowed for easy removal of unreacted molecules, which were filtered from the polymer
solution with a 0.45µm nylon filter. To ensure removal of all unreacted species, the
solvent was evaporated from the solution to make thin films. These films were then
washed with water and dried to further remove water-soluble lysine and peptides. This
process was repeated several times to be sure all unattached molecules were removed.
The process for attaching lysine was repeated with the other polyamino acids and the
synthesized peptide to attach them to the lysine functionalized polymer.
Polylysine (Sigma, Lot# 30K5906, Mr=9,000 g/mol) and polyalanine (Sigma,
Lot# 42H5546, Mr=5,000 g/mol) were used to simulate the attachment of the peptide.
Polylysine is a hydrophilic model for the charged heparin binding peptide. Polylysine
will not form an α-helix alone in physiological solution because of the density of charged
groups along the chain, but when attached to a substrate has a tendency to form the α-
helix. This is similarly true for the peptide [96]. Polyalanine, on the other hand, is a
hydrophobic model for the peptide. In addition, since the amino acid alanine is the first
and last amino acid found in the peptide, polyalanine simulates the specific attachments
possible by the ARK peptide. The peptide is also composed of 50% alanine, which has a
stabilizing effect on the α-helix. Polyalanine with this molecular weight has a chain
length similar to that of the peptide.
The specific peptide attached to the poly(DTEC-co-X%DTC) via the lysine
bridge is (ARKKAAKA)4. This peptide has sufficient length to form an α-helix and also
25
has been demonstrated to bind heparin (Kd≈50 nm) and endothelial cell PGs with a
Kd≈300 nm [96]. The general advantage to covalently binding a peptide to a substrate is
the strong chemical bond present to prevent desorption [117]. Peptides were
commercially obtained from the University of Virginia Biomolecular Research Facility
(Charlottesville, VA) or Genosys Biotechnologies (The Woodlands, TX). Peptides were
synthesized by standard FMOC solid phase synthesis. Peptide Mr was certified by mass
spectroscopy, and purity certified by HPLC.
4.2 NMR Spectroscopy
NMR was performed to confirm the attachment of the lysine and peptides and to
determine the efficiency of the attachment. The structure of the p(DTEC-c-X%DTC), and
the attachment of various molecules, was studied using both 1D and 2D NMR
spectroscopy. NMR measures the energy produced as a hydrogen proton’s spin returns to
the equilibrium state in an applied, pulsed magnetic field, providing information about
the chemical structure of the polymers.
NMR spectra were recorded on a Bruker AMX 600 NMR spectrometer (3 channel
MCI) operating at 600 MHz (1H) and a temperature of 300 K. Assignments of the spin
systems were based on double quantum filtered correlated spectroscopy, COSY, and total
correlated spectroscopy, TOCSY, experiments. These two-dimensional techniques
measure correlation in related spin systems, which were used to determine connectivity
between subunits. This allowed spectral peaks to be mapped to specific molecules in the
1-D spectrum. Peak integration from the 1-D spectrum indicated the number of hydrogen
protons producing a given peak. Peak integration values have a deviation of ±5.0% [119].
26
At least two samples were run at least twice (minimum of four points) to determine
integration values.
4.3 Cell Culture
Cell attachment and growth studies were conducted with human umbilical vein
endothelial cells (HUVEC). Cells were isolated from human umbilical cords [120] and
cultured [121] in medium 199 (Gibco BRL) with 10% FBS, 80 µg/mL endothelial growth
supplement (ECGS), 50-60 µg/mL heparin from pig intestinal mucosa (Sigma: Grade I-
A), penicillin, streptomycin and fungizone, on gelatinized tissue culture flasks (Falcon).
Cells were incubated at 37°C and 5% CO2. HUVEC from sub-confluent cultures of less
than passage seven are removed from the culture flask using trypsin/EDTA for five
minutes. When using passage greater than seven, the cells begin to de-differentiate and
may not function in the same manner as young cells [122].
4.4 EC Attachment
A modified cell detachment assay [123] was used to determine the degree or
strength of cell attachment to a given material. In this assay, cells are added to coated
wells, forced to adhere to the substrate at 40 g, then removed from the material by
varying centrifugal forces. The degree of force required to remove the cells corresponds
to the strength of attachment between the cells and the substrate.
Plates were prepared by coating the well bottoms of 96 well plates with p(DTEC-
c-X%DTC) X=0, 13, 23, 35, and 50%, and X=13% with lysine, lysine plus polylysine,
and lysine plus peptide attached. The materials were dissolved in dichloromethane at
27
≈0.05g/mL and 200 µL was applied to the well bottoms. The solvent was allowed to
evaporate in air. The plates were placed in a vacuum oven at 30 in Hg and room
temperature to draw off any residual solvent.
During assay preparation, the 96-well plates were kept on ice to reduce the
contribution of cytoskeletal and other intracellular components to cellular attachment.
15,000 HUVEC were added to each well with enough media 199 to give a positive
meniscus. The plates were sealed with tape and centrifuged using a microplate carrier at
40 g for three minutes at 4°C. This brings the HUVEC in contact with the test material.
The plates were then inverted and spun at an equal or greater force for three minutes,
again at 4°C. After centrifuging, the plates were kept inverted and incubated for two
hours to allow the attached cells to strengthen their attachment and spread on the well
bottom; unattached cells will not make contact and adhere to the test material. After
incubation, the media was removed from the inverted plates.
Because the various polymers were not transparent, spectrophotometry could not
be performed directly on these plates. The attached cells were removed to a transparent
surface for spectrophotometry. The plates were washed with 100 µL of PBS+ then 75 µL
of trypsin was added to each well and the plates were placed in the incubator for five
minutes. The trypsin was pipetted to a new tissue culture treated (TCT) plate. Cells are
able to attach and grow directly on TCT plates; they do not attach to non-TCT plates. The
original wells were washed twice with complete media and the media was added to the
wells containing trypsin in the new TCT plates. These plates were then inclubated for at
least two hours to allow cells to recover and attach. The media was gently removed and
the attached cells were fixed with 200 µL 1% glutaraldehyde and stained with crystal
28
violet [124]. Spectrophotometry was performed directly on both the wells coated with
fibronectin and the wells where the attached cells were transferred from the coated wells
to a new TCT plate using the above procedure. No significant variations in cell count
were seen between the two sets of plates.
To measure the amount of bound dye after the cells were fixed and stained, plate
wells were treated with 1% Triton X-100 for at least four hours to solubilize the cell
membranes and release the bound stain. The concentration of crystal violet is then
measured as A60015 using a spectrophotometer (Molecular Devices). A standard curve was
generated using cell quantities from 500 to 25,000/well on tissue culture treated wells.
Fibronectin was used as a positive control; cells show a linear decrease in attachment
with increasing force similar to that discribed in the literature [96, 115]. The maximum
force, 900 g, was chosen because it is large enough to remove most cells from
fibronectin. Forces were chosen at fairly even increments: 100, 300, 600, and 900 g.
MeCl2 washed wells were used as a negative control and show no cell attachment at any
force. The experiments were repeated at least three times for accuracy; the standard
deviation is ±4%.
4.5 EC Growth
Cell functions such as adhesion and growth depend on the nature of the substrate,
of the hydrophobic or hydrophilic character of its surface [74]. For this reason, polymers
with varying functional levels are studied to determine the effect of the surface on three
cell functions: attachment, viability, and division. The growth behavior of HUVEC on
P(DTEC-c-X%DTC) and several control materials was evaluated over seven days. Glass
29
slide cover slips (18mm dia., 0.15mm thick, Fisher Scientific) were solution coated with
P(DTEC-c-X%DTC) at X=0, 13, and 35%; PET (Eastpak 12270), PVC (Cognis), and
PLA (Sigma 33135-50-1), at a dry weight of roughly 0.005-0.010g. Samples were UV
sterilized for 20 minutes. The cover slips were then placed in the bottom of a six-well
plate (Falcon) with 50,000 cells suspended in media 199, and 2 mL of complete media.
This allows enough surface area for cells to grow and spread over the seven-day period.
Cells were incubated at 37°C and 5% CO2 and observed at 1, 3, 5, and 7 days under an
optical microscope at 100X.
After the seventh day, the cells attached to the coated slides were removed and
counted. The cells remaining in the well, attached to the well bottom adjacent to the glass
slide, were fixed and stained using a modified Wright-Giemsa staining protocol (Hema 3
stain set, Fisher Scientific). These cells were examined on an inverted Olympus phase
contrast microscope at various magnifications to evaluate signs of abnormal cell
functions. These experiments were conducted three times using three identical samples
per experiment. The cell counting accuracy is 95%.
The cell density was calculated by counting cells in four representative 1cm2
regions in the optical image at 23x. The accuracy of this method is ± 1.0x104 cells/cm2.
30
5 Results and Discussion 5.1 NMR
With NMR, the attachment points and the attachment efficiency of different
molecules can be determined. In 1-D 1H NMR, resulting peaks correspond to the number
of hydrogen protons. Cross peaks in 2-D correlated spectroscopy (COSY) and total
correlated spectroscopy (TOCSY) NMR show spin-spin coupling between hydrogens.
Both 1-D and 2-D techniques were used to determine binding efficiency. Once peaks
have been mapped to certain molecular subunits via 2-D NMR, ratios of 1-D peak heights
give the relative amounts of subunits present.
Peak assignments for p(DTEC), obtained using TOCSY (Figure 3), were shown
to be similar to those of Hooper et al [105, 106]. In the TOCSY spectrum peak
correlation systems are identified as indicated via the solid connector lines. This enables
the assignment of the spectral peaks. A key peak at 4.7 ppm is taken as the primary
hydrogen (α). This hydrogen is in a system with the γ (6.0 ppm) and β (3.0 ppm)
hydrogens, as seen in the TOCSY spectrum. To determine exactly which peak is from
which hydrogen, peak integration must be performed. Integration values indicate the
number of hydrogens responsible for an individual peak. Upon integration, the α-peak is
set to 100.0 and values of 198.0 (β), and 99.3 (γ) are obtained, giving values of 200 and
100 within experimental error. This indicates that the β-peak has double the hydrogens
and the γ-peak has equal the hydrogens as the α-peak, which leads to the identifications
shown in Figure 2. The ε (2.4 ppm) and δ (2.7 ppm) hydrogens are likewise correlated,
but separate from the α-β-γ system. These peaks give integration values of 198.7 (ε), and
31
201.5 (δ), which are both close to 200, indicating two hydrogens per peak. By the same
analysis, the peaks at 4.1 and 1.2 ppm correspond to the CH2-CH3, R2 and R3 hydrogens,
respectively, of the pendant group. The total intensity in the CH2-CH3 peaks should
integrate to two and three times the α-peak, as there are two and three protons in the R2
and R3 positions, respectively.
P(DTEC-co-X%DTC) has a more complex structure because both the free and
protected forms of the acid are present. Changes in several peaks are expected to occur
because of the heterogeneous nature of the structure. There is no longer a pendant group
for every α hydrogen. In addition, peak shifting is expected for the hydrogens in close
proximity to the pendant group. The 1D NMR spectra of both p(DTEC) and p(DTEC-co-
13%DTC) are shown in Figure 4. The integration values are shown below each peak.
When the α hydrogen integration is set to one hundred in the situation where the material
is p(DTEC-co-X%DTC), integration values of less then two and three hundred are
expected for the R2 and R3. Integration values of 172.1 and 269.2 are obtained for the
CH2 and CH3 peaks, respectively, corresponding to values of 14 and 11% DT. In
addition, there is a small shoulder at 3.1 ppm in the 13% DT spectrum. This corresponds
to a shift in the β-peak caused by the OH pendant group, and is 12.6% when integrated.
This confirms the samples have approximately 13% DT content. There is also a shoulder
on the γ-peak arising from the same interactions that cause the β-peak shift. This was not
used due to the difficulty in separating the two peaks. The α-, β-, and γ-peaks also should
be affected by the attachment of additional groups, including lysine. Analysis of
p(DTEC-co-35%DTC) confirms the same change in integration values (Table 5), giving
32
values for the CH2 and CH3 peaks corresponding to values of 37 and 33% DT,
respectively.
With the attachment of lysine to the polymer free acid, new peaks will be present
in the NMR spectrum. To understand the relation of these new peaks to the polymer it is
first useful to determine how the lysine peaks relate to one another in the absence of the
polymer. Figure 5 shows the 1-D and 2-D NMR spectrum for lysine and how these peaks
correspond to the lysine molecule. By comparing these coupled peaks to the spectrum of
the polymer with the lysine attached, several unique lysine peaks can be identified.
A 2D spectrum of p(DTEC-co-13%DTC) with lysine attached is shown in Figure
6. There is a large concentration of additional peaks between 0.8 and 4.5 ppm. These
have been determined to be lysine based on the previously discussed coupled systems.
The spectrum becomes more complex with the addition of lysine, both by the possibility
of α- and ε- terminus attachment of the lysine to the polymer backbone and by the
interactions between the lysine and the polymer. Multiple attachment locations produce
groups of similar peaks that are slightly shifted from one another. The two circled regions
on the graph are consistent with multiple attachment locations. There are a large number
of as yet unidentified peaks, as well. A corresponding 1D spectrum between 4.0 and 5.0
ppm (Figure 7) has integration values for peaks assigned to the α at 4.7 ppm and a yet
unassigned lysine peak at 4.38 ppm. The α-peak is a function of the polymer backbone
and can be considered independent of reaction and set to unity. The ratio of the α-peak to
the lysine peak gives approximately 11.5 ± 0.65% attachment of lysine. Attachment of
the lysine to p(DTEC-co-35%DTC) gives a ratio of the α-peak to the lysine peak of 30.2
33
± 1.5%. This corresponds to attachment efficiency between 85-90%. The NMR spectra
give clear evidence of lysine attachment.
Once the lysine attachment was verified, polylysine, a homo-polyamino acid, was
attached to the lysine amine terminus. Polylysine was chosen to simulate the attachment
of the peptide. Polylysine is a charged poly(amino acid) with similar chemical and
physical properties to the target peptide. TOCSY analysis (Figure 8) shows an increase in
intensity of the correlated peaks assigned to lysine. This is consistent with attachment to
the polymer, and further supports the original assignment of these correlation systems.
However, the polylysine peaks occurred at the same locations as the lysine peaks and
could not be quantified to determine attachment efficiency.
To further investigate attachment of peptides to the polymer via the lysine bridge,
polyalanine was attached. This molecule was also chosen to simulate the attachment of
the peptide; it is another polyamino acid chemically similar to the peptide, conserving the
hydrophobic nature and the tendancy to form an α helix. Unlike polylysine, polyalanine
shows unique peaks in the NMR spectrum. Figure 9 shows the 1D spectrum of the
polymer with the polyalanine attached. Peak integration values have been added for the α
polymer peak at 4.7 ppm and several peaks determined to be polyalanine peaks from a
TOCSY spectrum. The TOCSY spectrum is difficult to understand without more detailed
NMR analysis so it is not shown in this paper. The α polymer peak has been set to 1.0 so
the peak ratios can be calculated. The peaks at 4.0 and 3.5 ppm, uniquely, from
polyalanine do not overlap peaks from the polymer or lysine and can be used to
determine percent polyalanine attachment. However, the peaks at 3.2, 1.9, and 1.7 ppm
do not give clear attachment data. The integration values (Table 6) for these peaks are
34
more than would be expected from polyalanine alone because they contain areas from
other peaks occurring in the same location. The peaks at 1.9, and 1.7 ppm also
correspond to the lysine β- and δ-peaks shown in Figure 5. The peak at 3.2 ppm occurs
when the lysine has been attached to the polymer.
Table 6 shows integration values for the peaks at 4.0, 3.5, 3.2, 1.9, and 1.7 ppm.
The particular values of interest are the 4.0 and 3.5 peaks. These peaks are approximately
zero for X=0% polymer. As the functionality increases, the amount of polyalanine
increases significantly. The data in Table 6 has been normalized by subtracting the X=0%
value from the values for X=23 and 50%, and is shown in Table 7. The attachment is
30% for p(DTEC-c-23%DTC)and between 60-70% for p(DTEC-c-50%DTC). This
implies that 30% of the pendant groups on the polymer backbone of X=23% and between
60-70% of the pendant groups on the polymer backbone of X=50% have polyalanine
molecules attached. However, it would be expected that no greater amount of polyalanine
could be attached than the degree of functionalization. It is unclear what is causing the
increase in measured attachment of the polyalanine. One possibility is an increase in acid
functionality during the various treatments, caused by degradation of the polymer
backbone. However, an increase in attachment is not seen with the attachment of lysine,
as would be expected if this were the case because it uses the same chemical process for
attachment. Another possibility arises from potential subunit exchange when the
polyalanine chains come into close proximity with each other. If a subunit exchange
occurred it would likely change the molecular weight of the attached molecules. This
change in molecular weight may alter the spectral peaks. To assure proper quantification,
35
peaks must be identified that (1) do not overlap other polymer or lysine peaks and (2) are
not sensitive to molecular weight and side chain interactions.
While there appears to be some interference with the peaks used to measure
polyalanine attachment, Table 7 does show roughly a doubling of attached polyalanine
with a doubling of the functionality. In addition, even though the peaks at 3.2, 1.9, and
1.7 ppm do not give unambiguous attachment data, they still show increasing attachment
with increasing functionality. Even though quantification is possibly subject to some
interference, polyalanine is clearly being attached to the polymer in significant quantities.
Accurate quantification of the polyalanine or peptide attachment will require careful
selection of reference peaks.
The final NMR spectrum produced was p(DTEC-c-35%DTC) with the peptide
(ARKKAAKA)4 attached to the lysine bridge (Figure 10). From this spectrum the
addition of many new NH peaks (7-6.5 ppm) and methyl group peaks (2.0-1.2 ppm) can
be seen, as compared to Figure 6. This is expected given that multiple alanines and
arginines, in addition to lysine, make up the peptide. There also appears to be a great deal
of overlapping in peaks. This overlapping, the shifting in peaks due to multiple
attachment mechanisms, and the influence of conformational changes in the peptide
creates a spectrum that is very difficult to assign. From this spectrum, there is clear
evidence of peptide attachment. Detailed assignment will require more NMR work,
potentially using a spin labeled peptide.
36
5.2 Cell Attachment
This experiment is used to determine the strength of attachment between the
HUVECs and the polymer substrate. The attachment results in Table 8 show that the
HUVECs do not readily attach to p(DTEC-co-0%DTC). However, as the amount of free
acid groups on the polymer increases and the polymer becomes more hydrophilic the cell
attachment increases (Figure 11). In fact, from Figure 12, the most hydrophilic polymer
tested, p(DTEC-co-50%DTC), has attachment values close to the positive control (FN).
Since the cells do not generally attach to p(DTEC-co-0%DTC), any cellular attachment
seen with functionalization and with the addition of L-lysine ethyl ester and peptides
should be related only to the modification of the polymer.
A cell attachment assay was also performed on a variety of modified materials:
p(DTEC-c-13%DTC), with attached lysine, with polylysine attached, and with the
heparin-binding peptide attached (Figure 13). It is important to note that since the overall
attachment, for 15,000 initial cells, for this experiment is lower than expected based on
positive controls, and since the experimental error is high, the results are largely
inconclusive. There is some indication that the addition of lysine to the polymer may
increase attachment slightly while the addition of polylysine or peptide may decrease the
ability of the cells to attach to the polymer substrate. The lack of observed binding in
these systems may be caused by increased polymer solubility, unstable polylysine
attachment, or cell incompatibility.
The overall polymer-peptide-cell system may be producing a more soluble system
that the applied force can more easily remove from the well bottoms. This is supported by
other observations. In another experiment conducted in the San Antonio laboratory [122],
37
cells were tested for attachment to FN, blank and 8mer and 36mer peptides coated onto
well bottoms. The majority of the cells were removed from blank, 8mer and 36mer wells
but showed excellent binding to the FN. While there was no evidence of attachment in
the blank and 8mer wells, there were islands of superior cell attachment in the 36mer
wells. In separate observations, particularly rapid degradation or dissolution of the higher
functionalized polymers was observed, which is consistent with increased degradation of
the more hydrophilic p(DTEC-c-X%TDC) polymers [107]. The attachment of lysine and
peptides is expected to make the polymer system even more hydrophilic in nature. It is
reasonable to assume that this will also increase both the solubility and the degradation
rate. More work must be conducted in this area to understand any peptide changes that
occur upon binding to the polymer. If the cells are attaching to the peptide and then the
entire system is being pulled away from the well bottoms, then collecting and analyzing
the cells removed during the adhesion assay should show polymer still attached. Reacting
the polymer with less than stoichiometric quantities of lysine in order to allow the lysine
to cross-link the polymer slightly might solve the degradation problem.
Alternatively, the peptide may be changing shape when it is attached to the
polymer and this may be affecting cellular attachment functions. If the structure is not an
α-helix, the attachment locations may not be oriented for enhanced PG attachment. Steric
effects may also inhibit helix formation [117] or the peptide may not be attaching to the
polymer at the desired terminus, which would also cause reduced attachment. This type
of effect would most likely be observed as a modest instead of strong increase in binding.
This is not consistent with the observation that no significant change occurs at all.
38
5.3 Cell Growth
The effect of the surface on three cell functions have been studied on p(DTEC-c-
X%DTC): attachment, viability, and division. Figure 14 shows the results of the cell
growth study on p(DTEC-c-13%DTC). Table 9 shows the quantitative results of this
study. After 1 hour (Figure 14a), a number of cells have attached to the polymer. After 1
day, the cells change conformation, indicative of surface attachment and cell growth. The
cells multiply and continue to spread throughout the duration of the experiment (e.g. 3
days, Figure 14c). By day 7 (Figure 14d) there are regions of confluence.
Figure 15 shows the cell growth on a) p(DTEC), b) p(DTEC-co-13%DTC), c)
p(DTEC-co-35%DTC), d) PLA, e) PET, f) PVC (negative control), and g) blank glass
(positive control) at day 5. The cells grow more readily on the p(DTEC-co-X%DTC) as
the amount of functionalization increases. As the amount of copolymerized, non-
protected acid groups increases, both hydrophilicy and charge sites increase, either of
which may result in the observed increase. For example, cell proliferation and spreading
have been shown to increase with increasing surface hydrophilicity by Kottke-Marchant
et al [125]. The PLA and PET both exhibit moderate affinity for HUVEC growth. The
PVC shows no HUVEC affinity, as expected, because the PVC is expected to cause cell
death [3]. The heparin-coated glass shows strong affinity for cell growth, again as
expected. The final comparative, quantitative results are summarized in Table 9. The
results for the p(DTEC) bracket Dacron and PLA depending on the degree of
functionalization. All polymers are less supportive of cell growth than the heparin coated
blank glass.
39
Cells remaining in the six-well plate were fixed and stained and then evaluated for
signs of abnormal cell functions. Figure 16 shows the stained cells at increasing
magnifications. Figure 16a shows cells with semi-confluence, Figure 16b shows cells
spread on the plate surface, and Figure 16c shows cells in the process of dividing. Cells
spreading, confluence, and division are indicative of healthy, growing cells.
The endothelial cell lining in a healthy vessel is 105 cells/cm2 [114, 126]. Cell
density is calculated from Figure 14d. The HUVEC dimensions are roughly 50 x 10 µm,
consistent with the literature [122]. Calculations for four different regions give an
average cell density of 5.0x104 ± 1.0x104 cells/cm2. This is about half the density of cells
lining a healthy vessel. However, the relationship between the cell density in this in vitro
experiment and any in vivo applications is a matter of speculation at this point.
40
6 Conclusions The functionalization of a poly DTE carbonate polymer with a heparin binding
peptide has been accomplished. DTE Carbonate is a commercially available polymer that
has side groups available at varying levels for functionalization. Previous studies have
uncovered a peptide based on Cardin and Weintraub sequences that have high affinity for
EC PGs. A technique for attaching these peptides to the polymer has been developed. Use
of dilute solution insured no cross-linking between polymer chains. Excess lysine insured
maximum conversion during the attachment reaction. Since the reactants were insoluble
in organic solvents, excess reactant was efficiently removed using filtration and water
washing.
NMR has been used successfully to quantify available polymer functionality and
lysine bridge attachment and qualitatively or semi-quantitatively for additional
functionalization with long chain molecules. The peaks of the poly(DTE-co-%DT
carbonate) have been identified by NMR and attachment of the L-lysine ethyl ester to the
p(DTEC-co-X%DTC) has been verified. Lysine is shown to attach with an efficiency of
85-90%. Binding of polylysine, polyalanine and peptide establish that peptide binding
can be accomplished. Changes in the spectra confirm the attachment of polylysine,
polyalanine, and the (ARKKAAKA)4 peptide. Semi-quantitative results with polyalanine
indicate a large degree of attachment, although some interference among the spectral
peaks has been noted. As alanine provides the carboxylic acid terminus with which the
peptide will attach to the polymer lysine bridge, it is expected that the peptide will bind
similarly. Integration of polyalanine peaks shows increasing attachment that is directly
proportional to the increase in polymer functionality.
41
Cellular studies have confirmed that the material is non-cytotoxic and increasing
hydrophilicity increases the affinity of HUVECs. Preliminary results from experiments
with the peptide attached to the polymer were inconclusive. Additional work must be
completed to study the cell affinity and adherence to the attached peptide.
The NMR results demonstrate the feasibility of chemical functionalization of
DTE Carbonate, resulting in a peptide-modified polymer surface. The cell work
demonstrates that the materials are biocompatible and support cell growth. The premise
of making a vascular graft seems reasonable based on this data. A graft from this material
could result in a synthetic vascular graft that is much closer to the biology of native
vessels than currently available grafts. A graft from this material could have a significant
impact on the treatment of CVDs.
6.1 Recommendations: Characterization 6.1.1 Peptide Attachment
Quantifying the amount of peptide attached to the polymer via the lysine bridge
has yet to be determined. Quantification of attachment will be critical to determining the
efficiency of the cell attachment and regulating cell density. To quantify the attachment, a
spin- or radio-label containing peptide can be attached to the polymer. This should
provide a unique and easily identifiable peak to compare with known polymer peaks.
42
6.1.2 Peptide Structure
An α-helix secondary structure, or the ability to form it, may be important in
binding affinity of endothelial cells to the peptide [96]. Circular dichroism cannot be used
on materials that are not in solution, so a helix-sensitive peak in NMR or IR must be
located.
6.1.3 Cell Affinity
Tests of the affinity of HUVECs for peptide-modified polymer surfaces have been
inconclusive. One possible cause is degradation and subsequent removal of the entire
polymer-peptide-cell system with an applied force. One method to reduce degradation is
to cross-link the polymer. This can be accomplished by reacting the polymer with less
than stoichiometric amounts of lysine, in a more concentrated solution. This will allow
cross-linking between two polymer chains via the lysine bridge. Cross-linking the
polymer should increase the overall strength of the polymer-peptide-cell system.
In addition, variations in the cell attachment assay may be required. For example,
collection and analysis of the material removed from the well bottoms during
centrifugation may reveal polymer-peptide-cell systems.
6.1.4 Polymer Selection
Once the peptide-modified polymer has been completely characterized it will be
necessary to determine the cell density produced by seeding this surface with HUVECs.
The density can then be tailored by altering either the chemistry involved to attach the
43
lysine and peptide, or by using a polymer with more or fewer acid pendant groups. The
resulting cell density must then be optimized with the mechanical properties and
degradation rate produced from the polymer-peptide system. Cross-linking the polymer
via the lysine bridge can alter both the mechanical properties and degradation rate.
6.2 Recommendations: Fabrication 6.2.1 Processing
Once the peptide-polymer combination has been proven as a vascular material, it
must be made into vascular graft constructs. This can be accomplished by multi-layer,
two-dimensional braiding. This technique can make three-dimensional structures where
the braid parameters allow the tailoring of properties, like fiber volume fraction, porosity,
strength, and compliance.
Another possible technique to form the vascular graft construct is by extrusion.
This polymer has been extruded at a temperature of 80°C to 90°C above the glass
transition temperature [106]. However, the extrusion process would not allow for creating
and adjusting porosity.
Other possible techniques include compression mesh [127-129], solvent
casting/salt leaching [130, 131], emulsion freeze-drying [132], expansion from
pressurized carbon dioxide [133, 134], and phase separation [135-137]. These techniques
have all been shown to produce highly porous biodegradable polymer scaffolds [138].
Any technique chosen to fabricate the vascular graft construct would require
careful examination as to the effect of the processing on the attached peptides. Attaching
44
the peptide after the construct has been formed is a possible means of avoiding the
aforementioned problems.
6.2.2 Sterilization
Any surgical material used in the body must first be sterilized to prevent infection.
Sterilizability of biomedical polymers is important because polymers have lower thermal
and chemical stability than metals and ceramics [139]. Sterilizing techniques include dry
heat, autoclave, radiation, and ethylene oxide gas [140]. In dry heat sterilization the
temperature ranges between 160-190°C, which is above the melting and softening
temperatures and can cause deformation of the pDTEC construct or destruction of the
peptide. Autoclaving is performed under high pressure and higher temperatures ranging
between 125-130°C [75]. This technique would also cause significant degradation and
deformation of the pDTEC construct because of water vapor attack. Either ethylene oxide
gas [106] or radiation is the suggested method for sterilization. Both have been shown to
be effective with pDTEC. Both are used at low temperatures. Radiation can sometimes
cause degradation, but has not been found to do so significantly in pDTEC. However, no
experiments have been conducted as to the affects of ethylene oxide gas and radiation on
the L-lysine bridge and the peptide attached to the polymer surface. These techniques
must be evaluated for racemization[141] and degradation of the pendant molecules.
6.2.3 Storage
One final concern for the polymer/peptide construct is the result of short- and
long-term storage. The polymer itself showed no loss in molecular weight when stored at
45
0°C with dessication for more than one year. Sterilized polymer devices can be safely
stored at room temperature for one year, but storage over longer periods of time requires
dessication and cooling [106].
46
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Appendix A- Tables
Table 1- Patency rates for ASV and IMA grafting of the coronary artery of varying diameters, from [142]
Patency rates >1 year (%) Coronary Artery Size
(mm) ASV IMA 3.0 100 100 2.5 97 94 2.0 91 96 1.5 87 93 1.0 56 91
59
Table 2- Patency data for representative vascular grafts, adapted from Ku [51].
Graft Diameter (mm) Conduit 5-year patency (%)
Dacron 90 Aortobifemoral >6 ePTFE 90 Dacron 80 Femorofemoral <6 ePTFE 80 ASV 70
Dacron 50 Femoropopliteal <4 ePTFE 40
60
Table 3-Improving hemocompatibility of artificial biomaterials [73].
Modifications to Achieve Hemocompatibility
Bulk Material Surface Modification • Crystalline, amorphous, conformation
• Functional Groups
• Hydrophilic/hydrophobic balance
• Charge
• Block copolymers
• Hydrogel
• Charge
• Hydrogel
• Topology
• Cell seeding endothelial cells
• Immobilization of biologically active or passive biopolymers or low molecular weight substances
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Table 4- Properties of native tissue and several synthetic polymers.
Material Tensile Modulus MPa (ksi)
Tensile Strength MPa (ksi)
Thoracic Tissue - 3.8 (0.6) DTE Carbonate 1600-2000 (232-290) 67 (9.7)
Reinforced PTFE - 330 (47.9) Dacron 2800-4100 (406-595) 48-72 (7.0-10.4)
PLA 2700 (392) 50 (7.3)
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Table 5- NMR peak integration values for p(DTEC-c-X%DTC), X=0, 13, and 35, peaks.
Degree of Functionality Peak (ppm)
0% 13% 35%
α (4.8) 100.0 100.0 100.0
β (3.05) 197.7 185.4 163.2
γ (6.0) 96.4 99.7 98.2
δ (2.9) 202.5 198.0 201.4
ε (2.4) 198.7 205.6 204.3
Shoulder (3.1) - 12.6 35.3
R2 (4.1) 202.0 172.1 126.2
R3 (1.3) 305.0 269.2 201.2
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Table 6- Integration values corresponding to polyalanine peaks for attachment to p(DTEC-c-X%DTC), X=0, 23, 50.
Integration values Peak location (ppm) 0% 23% 50%
4.0 0.03 0.32 0.64
3.5 0.04 0.34 0.72
3.2 0.35 0.59 0.94
1.9 0.96 2.0 3.08
1.7 1.07 2.14 3.51
Table 7- The corresponding attachment percentages have been calculated.
Attachment of polyalanine Peak location
(ppm) 23% 50%
4.0 29 % 61 %
3.5 30 % 69 %
3.2 24 % 59 %
1.9 104 % 212 %
1.7 107 % 244 %
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Table 8- Cell attachment strength as assessed using a centrifugation assay. Note that the poly(DTE carbonate) shows no significant improvement in attachment over the negative control, but attachment increases with additional functional groups.
Cells attached (%) at g force Material 100 g 300 g 600 g 900 g
MECL2 0 0 0 0
0% DT < 5 0 0 0
13% DT 10 5 0 0
23% DT 15 12 10 2
35% DT 35 30 20 3
50% DT 40 35 25 6
FIBRONECTIN 80 60 30 5
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Table 9- Comparative cell growth data at 7 days for various experimental and common vascular implant materials.
Material Average cells per slide
X=0 4950 X=13 5500
poly(DTEC-co-
X%DTC) X=35 12100 PLA 9350 PET 6050 PVC 0 Blank 18700
66
Appendix B- Figures
Figure 1- Cross-Section of a typical artery and vein [143].
67
Figure 2- a) Molecular model of poly(DTEC) repeat unit showing bond angles and bond lengths. b) Repeat unit of poly(DTE-co-X%DT carbonate). When R = CH2CH3 the repeat unit is desaminotyrosil-tyrosine-ethyl ester (DTE); when R = H the free unit is desaminotyrosil-tyrosine (DT). X is the molar fraction of repeat units with pendant acid groups. (Note: H’s are labeled α, β, γ, δ, ε, for identification and quantification by NMR spectra. The hydrogens in the pendant R groups are labeled by proximity to the ester group. Thus, the hydrogen on the free acid is R1, while the hydrogens in the ethyl ester are labeled R2 and R3.) c) Repeat unit of poly(DTEC-co-X%DTC) and L-lysine ethyl ester. Arrows indicate both possible attachment locations.
O
Carbon Oxygen Hydrogen Nitrogen
c)
b)
a)
α-terminus ε-terminus
68
Figure 3- 2D TOCSY of poly(DTEC) in CDCl3. Peaks are labeled as detailed in Figure 2. Solid lines indicate three different correlation systems with interactions between neighboring hydrogen.
69
Figure 4- 1D NMR spectra of a) P(DTEC-c-0%DTC) and b)P(DTEC-c-13%DTC) with peak integration values. The peak labels correspond to hydrogen as marked in Figure 2.
70
Figure 5- 1-D and 2-D COSY NMR spectrum of L-lysine ethyl ester. Solid lines indicate correlation systems. Below is the corresponding molecule.
71
Figure 6- TOCSY spectrum of P(DTEC-c-13%DTC) with attached L-lysine ethyl ester. The original correlation systems from the poly(DTE-co-X%DT carbonate) system are indicated by lines. The solid lines indicate correlation systems from the L-lysine. Note that the system has ( ) ( ) two related correlation systems, corresponding to α– and ε–amine attachment of the lysine.
72
Figure 7- NMR spectrum of P(DTEC-c-13%DTC) with attached L-lysine ethyl ester. Integration values are below peaks. The ratio of the α poly(DTE carbonate)peak, which should be constant at a concentration of 1 across chemistries, to the lysine spectral group at ca. 4.38 shows an approximately 11.5±0.5% lysine attachment.
polymer
73
Figure 8- TOCSY spectrum of p(DTEC-c-13%DTC) with attached L-lysine ethyl ester and polylysine. Note that there are no additional peaks, only intensification of existing peaks.
74
Figure 9- a) 1D NMR spectrum of p(DTEC-c-50%DTC). Peak integration values are shown below select peaks. The integration value for the peak at 4.7 ppm corresponds to the α peak in the polymer. Characteristic polyalanine peaks are also indicated. b) Structure of polyalanine.
α polymer Polyalanine Polyalanine
a)
b)
75
Figure 10- 1D NMR spectrum of p(DTEC-c-35%DTC) with peptide (ARKKAAKA)4 attached via a lysine bridge.
NH peaks
Methyl group peaks
76
0%
20%
40%
60%
80%
100%
0 300 600 900 1200
Force (g)
Cel
ls A
ttach
edFibrin50%35%23%13%0%MeCl2
Figure 11- Attachment data from Table 8 for functional polymers, p(DTEC-c-X%TDC) X=0, 13, 23, 35, and 50% and positive (FN) and negative (MeCl2) controls. Attachment increases with additional functional groups.
77
0%
10%
20%
30%
40%
50%
60%
70%
0% 10% 20% 30% 40% 50%
Polymer Functionalization (%)
Cel
ls A
ttach
ed (%
)300 g
600 g
FN 300
FN 600
Bl 300
Bl 600
Figure 12- Attachment data for functional polymers and positive and negative controls. P(DTEC-c-X%TDC) X=0, 13, 23, 35, and 50% are shown at both the low (300 g) and medium (600 g) force. FN (positive control) and Blank (negative control) are drawn as straight lines as they are not subject to the same functionalization. The lines for the FN and the Blank indicate the cell attachment at the specified force.
78
0%
3%
5%
8%
10%
13%
15%
0 300 600 900 1200
Force (g)
Cel
ls a
ttach
ed13%
13wL
PolyL
32mer PEP
Figure 13- Cell attachment data for p(DTEC-c-13%DTC) alone (13%), with attached lysine (13wL), attached polylysine (PolyL), and attached heparin-binding 32mer peptide (32mer PEP).
79
a) b)
c) d)
Figure 14- Optical microscope images of cell attachment and growth on 13% poly(DTE-co-X%DT carbonate) at a) 1 hour, b) 1 day, c) 3 days and d) 7 days. Note that at 1 hour, the cells show flattening indicative of attachment. The total number of cells increases through day 7. On day 7, there are regions of cell confluence.
200 µm
80
a) b)
c) d)
e) f)
g)
Figure 15- Optical microscope image of cell attachment at day 5 on a) poly(DTE carbonate), b) 13% poly(DTE-co-X%DT carbonate), c) 35% poly(DTE-co-X%DT carbonate), d) PLA, e) PET, f) PVC and g) glass.
200 µm
81
Figure 16- Optical microscope image of stained cells at the end of the growth assay, a) 12X shows areas of semi-confluence b) 23X shows spread cells indicating attachment c) 46X shows spread cells with visible nuclei and cells in the process of dividing.
100 µm 50 µm
200 µm a)
b) c)
82
Appendix C- Nomenclature Allografts- a graft from one species to another Angioplasty- a balloon angioplasty is a noninvasive procedure where a balloon-tipped catheter is introduced into a diseased blood vessel. As the balloon is inflated, the vessel opens further allowing for improved flow of blood. Atherosclerosis- a disease of the blood vessels characterized by thickening of the vessel wall and eventually occlusion of the vessel Autograft- a graft from one individual to another Compliance- structural property of a tube that expresses dimensional change in response to a change in intraluminal pressure Denudation- the act of stripping off, or removing the surface, in this case removal of ECs Endothelium/Endothelial cell lining- flat cells that line the innermost surfaces of blood and lymphatic vessels and the heart Homeostasis- a tendency to uniformity or stability in an organism by maintaining within narrow limits certain variables that are critical to life Hydrogel- polymer that can absorb water to 30% or more of its weight Integrin- cellular transmembrane proteins that act as receptors for adhesive extracellular matrix proteins such as fibronectin. The tripeptide RGD is the sequence recognized by many integrins. Neointima- newly formed intimal surface Neointimal hyperplasia- growth of a new intimal surface formed by fibroblasts or smooth muscle cells Occlusion- closed vein or artery Patency- the time a repair remains functional Platelet- one of the formed elements of blood responsible for blood coagulation
83
Stenosis- tissue ingrowth into vessel causing a narrow lumen and reduction of blood flow Thrombosis- the formation of an aggregation of blood factors, primarily platelets and fibrin with entrapment of cellular elements, frequently causing vascular obstruction. VE Cadherin- integral membrane proteins involved in calcium dependent cell adhesion. Formed of a 600 amino acid extracellular domain, containing 4 repeats believed to contain the Ca binding sites, a transmembrane domain and a 150 amino acid intracellular domain.
84
Appendix D- Abbreviations ASV autologous saphenous vein
COSY correlated spectroscopy
CD circular diochroism
CVD cardiovascular disease
DCC 1,3-Dicyclohexylcarbodiimide
EC endothelial cell
ePTFE expanded poly(tetrafluoroethylene)
FDA Federal Drug Administration
FMOC 9-fluorenylmethoxycarbonyl
FN fibronectin
GAG glycosaminoglycan
HUVEC human umbilical vein endothelial cell
IMA internal mammary artery
MeCl2 methylene dichloride
NMR nuclear magnetic resonance
PBS phosphate buffered saline
PET poly(terephthalate)
PG proteoglycan
PLA poly(L-lactic acid)
PTCA percutaneous transluminal coronary angioplasty
PVC poly(vinyl chloride)
TOCSY total correlated spectroscopy