introduction & review of literature -...
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Chapter 1
Introduction & Review of Literature
Amrita Centre for Nanosciences and Molecular Medicine Page 2
1.1 Introduction
Coronary heart disease (CHD) is one of the leading causes of death worldwide
1,2,3. Atherosclerosis is the commonest form of CHD characterized by arterial
narrowing (stenosis) due to the accumulation of fatty deposits beneath the
endothelium (i.e. the layer of cells forming the inner lining of a blood vessel), mainly
affecting the coronary arteries owing to its smaller diameter. This buildup can cause
restriction of blood flow, thus depriving heart muscles and other tissues of oxygen 4-6
.
Under such cases of severe atherosclerosis, endoprosthetic devices such as
cardiovascular stents are used for mechanically keeping the vessel open, thereby
restoring the blood flow 7-11
. Stents are elongated tubular metallic structures, with
either solid or lattice-like walls, deployed inside the artery by an angioplastic
procedure. Because of the need for the appropriate mechanical property compatible
with stent deployment to keep the vessels open, metals (including Ti, stainless steel,
Nitinol, and CoCr alloys) have been widely used as coronary stents 12
. A successful
vascular stent should support natural monolayer coverage of vascular endothelial cells
over the stent surface, so as to integrate it to the vascular wall 13-15
. The reappearance
of plaques at the stented site or in-stent restenosis (ISR) is a known problem
associated with using bare metal stents, eventually leading to thrombosis 16-19
. This
occurs mainly due to excessive vascular smooth muscle cell (VSMC) proliferation
and dysfunction of endothelial cells (ECs) as a result of the injury and inflammation
at the time of stent implantation. Numerous strategies have been adopted to overcome
this limitation pertaining to BMS, of which stent coatings using polymer, inorganic
materials, and drugs 12,20-23
are widely explored. Amongst them, the most promising
solution till date to address ISR is the use of drug eluting stents (DES) which relies on
the release of anti cell-proliferative, immunosuppressive or anti-thrombotic drugs
from a polymeric coating on the stent surface that inhibits the proliferation of smooth
muscle cells as well as reduces thrombus formation within the lumen 24
. However,
this strategy of drug induced inhibition of hyperplasia can also interfere with the re-
establishment of a healthy endothelium, leaving an unendothelialized bare metallic
surface or a dysfunctional endothelium. The polymeric degradation products from
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stent coatings can also further aggravate this situation. These can potentially enhance
the chance of thrombosis after the cessation of the drugs, forcing the patients to thrive
on treatments such as anti-platelet therapies for their lifetime 25-27
. Thus the problem
of thrombosis still remains unresolved. In this context, an approach, which is
essentially non-destructive to cells (unlike the case of DES), by specifically
promoting endothelialization process is desirable 28
. Establishment of a healthy
endothelial layer on stents could act as a natural anti-thrombotic barrier, preventing
thrombus formation and subsequently inhibiting smooth muscle proliferation and
platelet aggregation 29-31
. Immobilization of biological agents to promote
endothelialization and hemocompatibility such as fibrin, heparin sulfates, anti platelet
agents, antibodies, etc exhibited promising in-vivo results. However, the technical
complexities, processing cost, and their stability (activity) inside animal body are the
major associated challenges 12, 20-22
.
Moreover, permanent body implants such as stents which are in direct contact
with blood, requires a surface that is hemocompatible throughout its lifespan 21
. The
degree of thrombus formation subsequent to stent deployment is influenced primarily
by the choice of the stent material, site of implantation and blood flow. Despite
extensive anticoagulation approaches in stent design or adjuvant therapy during
angioplastic procedure, instances of stent thrombosis occurred as late angiographic
outcomes 32,33
. Hence, it is important that these device surfaces have minimal
adsorption of thrombogenic blood proteins and reduced interaction with coagulation
factors. Literature reports various strategies such as the use of heparin, antiplatelet
agents, inorganic coatings, polymers, etc for modifying biomedical prosthesis to
improve its hemocompatibility 34
. However, their extensive clinical uses on stents are
limited by the short plasma half life of antiplatelet agents/ anticoagulants and high
cost 34,35
. Herein, inorganic coatings are suggested as stent surface coatings to
improve their electro-mechanical properties and serve as a passive layer preventing its
exposure to blood 22,24
. However, ceramic coatings such as SiC, TiO2, diamond like
carbon (DLC) etc., on stent struts are brittle in nature, making it necessary to be
applied as a thin coating to make it crack-free and stable 36-54
. Conventional
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techniques such as dip coating, electrodeposition, etc make it extremely difficult to
achieve a reduced coating thickness, which remains their major challenge 55,56
.In this
context, nanosurface modification has been highlighted as a promising approach to
improve the cellular behavior and tissue integration of biomedical implants 57-63
.Such
surface modifications can be achieved by various methods which include
electrochemical processing, hydrothermal treatment, sandblasting/polishing etc.,
resulting in the formation of diverse ordered/disordered nanostructures on metallic
substrates 64-66
. The creation of nanoscale features on vascular metallic stent surfaces
can mimic the natural structure of the healthy vessel wall, making it more
cytocompatible 67
. Nanostructures could actively interact with the surrounding
biological environment and deliver specific signals to guide and control cell activities,
without the addition of exogenous chemical agents (i.e. growth factors, drugs) 64,65
.
Several studies have demonstrated the utility of nanostructured metallic titanium
surfaces in improving osteoblast response, osseointegration, and also in accelerating
endothelialization 65,67-69
. Titanium despite being well accepted for biomedical
implant applications is afflicted with inadequate mechanical properties, and hence is
not preferred for making stents 12,21
. Nevertheless, titanium in its oxide form, viz.,
titania (TiO2), owing to the excellent tissue response it renders, is proposed as a
suitable stent coating on bare metal stents, providing a hemocompatible surface; with
alterations in its surface chemistry and topography contributing to improved
biocompatibility 34,35,70-73
. The fact that nanoscale surface topography stimulates and
controls several molecular and cellular events at the tissue/implant interface has
prompted investigations of such topographies in the design of implantable metals 74,75
.
1.1.1 Scope of the Thesis
Stent surfaces that promote re-endothelialization would be a good option for
preventing stent thrombosis after long term implantation. Nanostructuring on vascular
metallic stent surfaces can mimic the natural structure of the healthy vessel wall,
making it more favorable for endothelial cells, promoting ECM secretion and
hemocompatibility, while subsequently minimizing VSMC growth, which is the
strategy adopted for this thesis work. This thesis work describes the generation of an
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array of different TiO2 nanostructures of distinct morphologies viz., Nanoleaves,
Nanopores, and Nanorods on metallic surfaces via a simple, scalable and cost-
effective technique and comparing their vascular cell response in-vitro. This strategy
of creating specific nanoscale topographies which are capable of naturally modulating
vascular cell behavior can be a cost-effective option for the regulation of the
fundamental factors responsible for in-stent restenosis (such as hyper smooth muscle
proliferation, endothelial dysfunction, platelet aggregation etc). Such biocompatible
metals would present a superior treatment modality without the use of costly drug
eluting stents that demand continual use of anti-platelet therapy for prolonged
durations and have long term toxicity effects. Hence such nanostructures are of great
translational value as a vital ‗polymer-less and drug-free‘ approach for surface
modifying clinically used bare metal stents based on stainless steel, and can be
proclaimed as a viable alternative to the drug eluting strategies.
1.2 Review of literature
1.2.1Blood vessel structure and function
Oxygenated blood is distributed to the heart via the coronary arteries which
are on the surface of the heart. The two primary coronary arteries are the left and right
main coronary arteries. These originate near the cusps of the aortic valve. Additional
arteries branch off the two main coronary arteries to supply the heart muscle with
blood. Since coronary arteries deliver blood to the heart muscle, any coronary artery
disorder or disease can have serious implications by reducing the flow of oxygen and
nutrients to the heart, which may lead to myocardial infarction and possibly death.
The vascular system is composed of arteries, capillaries and veins. The wall of an
artery consists of three layers which are represented in Fig. 1.3. The innermost layer,
which is in direct contact with the flow of blood, is the tunica intima. This layer is
lined with endothelial cells (ECs) which form flat pavement-like patterns on the
inside of the vessels and are surrounded by a connective tissue basement membrane.
The basement membrane separates endothelium from the underlying layers. ECs act
as a protective barrier and control the exchange of nutrients and fluid between blood
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and tissue, which is the basis for the maintenance of stability of the physiological
environment, essential to cell survival. The middle layer is the tunica media and it is
usually the thickest layer. It is composed of smooth muscle cells (SMC) and
extracellular matrix (ECM). It not only provides support for the vessel but also
changes the vessel diameter to regulate blood flow and blood pressure. The outermost
layer, which attaches the vessel to the surrounding tissue, is the tunica adventitia. It is
composed of connective tissue and contains primarily fibroblasts, elastic and
collagenous fibers. These fibers allow the vessel to stretch whilst preventing
overexpansion due to the pressure that is exerted on the walls by blood flow. Blood
passes through capillaries into venules, it then flows into progressively larger veins
until it reaches the heart. The walls of the veins have the same three layers as the
arteries, but develop thinner walls. The tunica media (smooth muscle) and tunica
externa (connective tissue) are much reduced and have the media composed primarily
of SMC, with relatively low amounts of elastic tissue 76
. Fig. 1.3 shows a clear picture
on comparison of the inner structure of arteries and veins.
Fig. 1.3 Schematic representation of the three layers of vascular vessel wall comparing that of
arteries and veins. Courtesy: http://apocketmerlin.tumblr.com
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1.2.2 Coronary heart disease
Cardiovascular disease broadly covers a range of conditions affecting both the
heart and the blood vessels. It is the leading cause of death in the aged western
population 77
. In America alone, almost 2,400 deaths occur each day as a result of the
disease, an average rate of one death every 37 seconds 78
. When atherosclerosis,
which is described as the hardening and loss of elasticity of arteries, occurs in the
coronary arteries it is referred to as coronary artery disease (CAD). This causes about
2100 deaths annually per million of the population in England and Wales (about
110,000 deaths in total). The disease is typically caused by the deposition of
atherosclerotic plaques (inflamed fatty deposits) on the inner wall of arteries 7, which
narrows the vessel lumen and obstructs the coronary arteries. This narrowing is
commonly referred to as a stenosis and leads to a consequential restriction of the
blood supply to the muscle cells surrounding the heart.
Coronary artery stenosis may be asymptomatic or may lead to shortness of
breath and angina, a chest pain. A critical reduction of the blood supply to the heart
may result in myocardial infarction or death. The atherosclerotic lesions that form
may also become fragile and rupture, resulting in thrombus formation and a further
restriction of blood flow. Fig. 1.1 depicts how atherosclerotic lesions gradually lead
to thrombosis.
Fig. 1.1 Diagram representing the atherosclerotic condition leading to thrombosis.
Image courtesy: http://www.thenewstribe.com
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The symptoms and treatment depend on which set of arteries are affected.
Peripheral artery disease mainly relates to vessels of the lower limbs 80
. However, the
symptomatic prevalence is mainly in vessels smaller than 6 mm internal diameter.
1.2.3 Stents and Coronary Disease
Stents are expandable meshed tubes used either to reinforce body vessels
possessing weak walls or to increase the internal diameter of an arterial vessel to
allow an improved flow of fluids such as blood. The use of arterial stents in particular
has grown significantly over the last 20 years due to an ageing population and the
changes in diet which has led to an increase in cardiovascular illness 81
. Estimates
vary, but it is predicted that coronary stents had a market value of $7.2 billion in 2012
and will continue to grow at a rate of 6% per annum thereafter. In 2009, over a
million US citizens received angioplasty/stent interventions. In the treatment of
coronary artery disease, stents offer a less invasive alternative to the Coronary Artery
Bypass Graft. It is estimated that for every bypass operation, there are four instances
of stents being employed as an alternative approach 82
.
Stents are fabricated by laser cutting shaped sections from a metal tube 83
.
For stenting, an opening is made in the patient (groin, arm or neck) and a catheter is
used to guide a deflated balloon inside a stent to the correct position in the artery 84-86
.
X-rays and dye flow are used to identify the area of the artery suffering from plaque
build-up and for associated stent positioning. Once positioned, the balloon is inflated,
causing the stent to expand and therefore the plaque to be pushed back against the
inner walls87,88
. Upon deflation and withdrawal of the balloon, the stent remains in
place. Fig. 1.2 depicts the steps involved in the intervention procedure of stent
deployment. In some instances, balloon inflation inside the artery is performed
without a stent (to assist initial widening) and then a stent is subsequently positioned
89,90.
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Fig. 1.2: Coronary stenting procedure (A) a catheter is fed into the femoral artery of the upper
leg. (B) The catheter is fed up to coronary arteries to an area of blockage. (C) A dye is released,
allowing visualization of the blockage. (D) A stent is placed on the balloon-tipped catheter. The
balloon is inflated, opening the artery. (E) The stent holds the artery open after the catheter is
removed. Courtesy: http://www.surgeryencyclopedia.com
1.2.4 History of coronary stenting
At the beginning of the 20th century, glass tubes that became a prototype of
stents were implanted into blood vessels of animals. In the next stage, with the origin
of percutaneous transluminal angioplasty (PTA), the expansion of blood vessels was
achieved by increasing the diameter of a catheter tube91
. This trial failed because of
migration and the development of thrombus. In 1985, Palmaz and his colleagues
developed the first balloon expanded stent 92
and just 1 year later, Gianturco
developed a balloon-expanded coil stent 93
made of stainless steel. The first self-
expandable stent, Wallstent, made of a cobalt (Co)–chromium (Cr) alloy (Elgiloy),
was clinically applied in 1986 94
. Another popular self-expandable stent, SMART,
consists of a superelastic nickel (Ni)– titanium (Ti) alloy 95
. Since the 1990s, stents
have been used in coronary arteries. Typical popular stents for this purpose are the
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Palmaz–Schatz stent and the Gianturco–Roubin stent. New designs and functions of
stents have also been developed. Today, for example, the majority of patients
undergoing percutaneous transluminal coronary angioplasty (PTCA) receive a stent.
However, restenosis follows PTCA in 30–40% of coronary lesions within 6 months
96,97. Although providing intra-arterial support with bare metal stents (BMS)
dramatically improves the angiographic and clinical outcome of patients to a
restenosis rate of 20–30% 96,97
, in-stent restenosis still remains a major limitation for
this approach with exaggerated intimal hyperplasia 98
. The advent of DES, which
release drugs such as sirolimus and paclitaxel for localized delivery, is a major
advancement in the evolution of stents. However, there is a risk of late stent
thrombosis (LST) associated with DES 99,100
.
1.2.5 Bare metallic stents (BMS)
Balloon expandable stents should have the ability to undergo plastic
deformation and then maintain the required size once deployed 101
. Self-expanding
stents, on the other hand, should have sufficient elasticity to be compressed for
delivery and then expanded in the target area101
.
The characteristics of an ideal stent have been described in numerous reviews 102-104
.
In general, it should have
(1) low profile—ability to be crimped on the balloon catheter supported by a guide
wire;
(2) good expandability ratio—once the stent is inserted at the target area and the
balloon is inflated, the stent should undergo sufficient expansion and conform to the
vessel wall;
(3) sufficient radial hoop strength and negligible recoil—once implanted, the stent
should be able to overcome the forces imposed by the atherosclerotic arterial wall and
should not collapse;
(4) sufficient flexibility—it should be flexible enough to travel through even the
smaller diameter atherosclerotic arteries;
(5) adequate radiopacity/magnetic resonance imaging (MRI) compatibility—to assist
clinicians in assessing the in-vivo location of the stent;
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(6) thromboresistivity—the material should be blood compatible and not encourages
platelet adhesion and deposition
(7) drug delivery capacity—this has become one of the indispensable requirements
for stents of the modern era to prevent restenosis.
1.2.6 Materials for BMS
Generally, the metals commonly used for manufacturing stents are 316L
stainless steel (316L SS), platinum–iridium (Pt–Ir) alloy, tantalum (Ta), nitinol (Ni–
Ti), cobalt– chromium (Co–Cr) alloy, titanium (Ti), pure iron (Fe), and magnesium
(Mg) alloys owing to their adequate mechanical properties which are compared in
Table1.1. A list of stent materials that are recommended for coronary stenting
applications based on their characteristics, with a rationale for its choice has been
depicted in Table 1.2.
i) Stainless steel (SS)
Stents fabricated from stainless steel, which is an iron (Fe)-based alloy,
contain over 18 % Cr, 8 % Ni73
. Stainless steel does not corrode in an oxygen-
containing atmosphere, but they corrode locally and sometimes form pits in chloride
solutions as found in some body fluids. Elements such as Ni, molybdenum (Mo),
copper (Cu), Ti, niobium (Nb), and nitrogen (N) are added to stainless steel to
improve its corrosion resistance, heat resistance, strength, and workability. The
metallurgical structure, strength, and corrosion resistance of stainless steel depend on
the concentrations of Ni and Cr, and stainless steels are categorized as ferritic (Fe–Cr
system), martensitic (Fe–Cr system), or austenitic (Fe–Cr–Ni system) types according
to the crystal phase. Amongst the wide variety of stainless steel available, 316L SS is
the commonly used one for biomedical applications, possessing low carbon content
(below 0.03%). Type 316L stainless steel has Fe, <0.03% C, 16-18.5% Cr, 10-14%
Ni, 2-3% Mo, <2% Mn, <1% Si, <0.045% P, <0.03% S. 2- 3% Molybdenum added
enhance the corrosion resistance steel as the stability of the passive film increases.
The main reason for the use of stainless steel for coronary applications is the good
balance of strength and elongation, which facilitates the manufacture of the stent, the
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plasticity for balloon expansion, and the maintenance of morphology to resist the
elastic recoil of blood vessels.
ii) Tantalum
Ta has excellent corrosion resistance because of its highly stable surface oxide
layer, which prevents electron exchange between the metal and the adsorbed
biological species 105,106
. It has been coated on a 316L SS surfaces to improve
corrosion properties, thereby enhancing the biocompatibility of 316L SS107
. It has
excellent fluoroscopic visibility because of its high density. It is an MRI compatible
material as it produces no significant artifacts because of its non-ferromagnetic
properties108,109
. Ta is also known for its good biocompatibility 110,111
. Enhanced
hemocompatibility was achieved by adding Ta to Ti oxide and the films showed
improved endothelialization rate as the percentage concentration of Ta increased 112-
113. Though the biocompatibility and visibility properties of Ta are superior to 316L
SS, the commercial availability of Ta stents is lower than 316L SS stents. This is
mainly because of its poor mechanical properties. Since the yield strength of Ta is
closer to its tensile strength, these stents have a higher possibility of breaking during
deployment.
iii) Titanium and alloys
Ti and its alloys have been extensively used in orthopedic and dental
applications because of their excellent biocompatibility114,115
. Its highly stable surface
oxide layer provides excellent corrosion resistance115, 116
.However, Ti is not
commonly used for making stents. Although Ti and Co–Cr both have high yield
strength in approximately the same range, Ti has a significantly lower tensile
strength. Thus, there is a higher probability of tensile failure of the Ti stents when
expanded to stresses beyond their yield strengths, which is the norm in balloon
expandable stent deployment. Alloying Ti with materials that reduce its yield strength
while retaining tensile properties might prove to be optimum. Because of its low
ductility, Ti stents are more prone to fracture. Because of these inadequate
mechanical properties, commercially pure Ti failed to make an impact as the sole
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stent material. However, the applications of Ti are not limited to coronary stent
applications. Ti-nitride oxide coating on 316L SS was found to be biologically inert
with reduced platelet and fibrinogen deposition, thereby reducing neointimal
hyperplasia117
. The Titan stent (Hexacath, France), which has implemented this
coating technique, has shown promising results in human clinical trials 118,119
. Also,
Ti-based Ta and niobium alloys, which have potential applications for stents, showed
excellent hemocompatibility 120
. One of the Ti alloys which is extensively used for
making stents is Ni–Ti.
iv) Co–Cr alloy
Co–Cr alloys, which conform to ASTM standards F562 and F90, have been
used in dental and orthopedic applications for decades 121
and recently have been used
for making stents. These alloys have excellent radial strength because of their high
elastic modulus (Table 1.1).
Table 1.1 Mechanical properties of the metals that are used for making stents; G. Mani et al.
Biomaterials 28 (2007)
The thickness of the struts is a critical issue in designing a stent 122,123
, hence,
the ability to make ultra-thin struts with increased strength using these alloys is one of
their main attractions124
. In addition to this, they are radio-opaque 125
and MRI-
compatible 126
. The cobalt alloy platform DRIVER stents (Medtronic Inc, USA) are
commercially available in Europe. Recently, FDA approved the L-605Co–Cr alloy
Guidant Multi-Link Vision stent for clinical use127
. Co–Cr alloys show high strength,
toughness, castability, corrosion resistance, and wear resistance. The corrosion
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resistance of Co–Cr alloys is better than that of stainless steel. The surface oxide film
of a Co–Cr–Mo alloy contains oxides of cobalt and chromium which impart corrosion
resistance to the alloy. Table 1.2 provides a list of materials that are recommended for
coronary stenting applications based on their characteristics, with a rationale for its
choice.
Table 1.2 Materials with ideal characteristics for coronary stent applications; G. Mani et al.
Biomaterials 28 (2007)
1.2.7 In-stent restenosis - mechanism, pathogenesis
Restenosis is the re-narrowing of a blood vessel causing a reduction of the
luminal size, consequently restricting blood flow after intravascular procedure.
Restenosis is mainly characterized by intimal hyperplasia and vessel remodeling128
.
The hyperplasia is an abnormal or unusual increase in the cells composing the intima.
Restenosis is a combined result of a biological response and mechanical reaction to
percutaneous coronary intervention (PCI). At the early phase of restenosis, elastic
recoil takes place due to the mechanical response of the elastic fibers of vascular wall
to overstretching by balloon catheter. The recoil occurs within minutes following
balloon deflation; the recoil may cause up to a 40% lumen loss 129
. However, this
phase may be totally eliminated by introducing a stent. The biological response to the
procedure is more complicated to eliminate, and it may consist of the following four
phases, 129-133
as described in Fig. 1.4.
i) Platelet Aggregation: Immediately after stent placement, endothelial denudation
and medial dissection results due to the mechanical injury of PCI [Fig. 1.4a]. The
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injury causes platelet aggregation and activation, producing a countless number of
various cell-signaling factors, initiating an inflammatory cascade and releasing
adhesion molecules that cause a thrombus formation129,130
.
ii) Inflammatory Phase: Over the next few days to weeks, a variety of white cells
will gather at the injury site, secrete their own factors, and exert their own influence
on the healing tissue. The inflammatory response can persist for months [Fig. 1.4b].
iii) Proliferation Phase: The inflammatory phase stimulates smooth muscle cells
(SMCs) migration and proliferation, in an attempt to repair the wound. This process is
enabled by leukocytes cells releasing and activating tissue-digesting enzymes,
forming a path for the SMCs to move. SMCs migrate to the thrombus that acts as a
scaffold, providing the substrate for neointimal formation. The migrating SMCs form
an overgrown, obstructing scar [Fig. 1.4c].
iv) Late Remodeling Phase: The final mechanism of restenosis response is the late
remodeling of the vessel. This produces a neointimal layer, which is mainly formed
by proliferating SMC and extracellular matrix (ECM). Inflammatory mediators and
cellular elements contribute to trigger a complex array of events that modulates
matrix production and cellular proliferation. As the amount of scar develops [Fig. 1.4
d], blood flow is gradually reduced. Additionally, there is evidential re-
endothelialization of partial segments of the injured vessel surface.
Fig. 1.4. A schematic representation of the restenosis process. (a) Platelet Aggregation:
Immediate result of stent placement with endothelial denudation and platelet/fibrinogen (not
shown) deposition. (b) Inflammatory Phase: A variety of white cells will gather at the injury site.
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(c) Proliferation Phase: Smooth muscle cells migrate and proliferate, creating the neointima in
an attempt to repair the wound. (d) Late Remodeling Phase: The neointima is changed from
predominantly cellular to a less cellular and more extracellular matrix-rich plaque. Kraitzer A, J
Biomed Mater Res B Appl Biomater. 2008
1.2.8 Strategies for combating in-stent restenosis
A considerable amount of research was invested in the prevention of in-stent
restenosis due to the substantial rate of the phenomenon. This interest prompted
various strategies that have been investigated and employed for the treatment of
restenosis. The stent strategy reduced restenosis caused by balloon angioplasty from
~50% to ~15% 129
. Early attempts at the prevention of restenosis focused on the
administration of antithrombotic agents, but there was limited success in trials134
.
With technologic advances and greater understanding of vascular pathobiology, novel
therapeutic strategies, such as local delivery of ionizing radiation, pharmacological
agents, and gene therapy, have been utilized to prevent coronary restenosis134,135
. It
became clear that although coronary stenting combined with antithrombotic therapies
had essentially eliminated the problem of elastic recoil, thrombus formation, and
vessel remodeling, in-stent restenosis remained a major problem129
. Today, it is
obvious that vascular SMC (VSMC) growth and migration trigger intimal
hyperplasia, which is the main cause of in-stent restenosis135
. Researchers have
adopted several strategies as below to address these issues.
i) Polymer coatings
Polymeric coatings on stents serve as a surface layer preventing its exposure to blood
and also to coat drugs. The most widely used materials for developing resorbable
stents or stent coatings on bare metal stents are the aliphatic polyesters including
poly-L-lactic acid, polyglycolic acid, poly-DL-lactic acid and poly-ε -caprolactone.
These polymers have been used as a coating material on stents to improve the
antithrombogenic properties of Ta137
, corrosion resistance of Ni–Ti138
and
biocompatibility of SS139
. A porcine model using poly-L-lactic acid demonstrated
minimal inflammatory and thrombotic response with good initial radial strength 140
.
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Phosphorylcholine is a naturally occurring neutrally charged phospholipid, a
component of the plasma membrane. It causes less platelet activation than stainless
steel. Stents coated with phosphorylcholine have been shown to be non-inflammatory
and non-thrombogenic in-vitro and in-vivo for six months or longer136
.
Phosphorylcholine also provides a basis for a drug eluting coating. Clinical trials with
this and other polymer-based stents to date have yielded highly variable results.
ii) Inorganic/Ceramic coatings
Inorganic strategies may also have potential. Silicon carbide has been
investigated for its ability to alter the electrochemical properties of the stent surface.
It has been suggested that the initiation of thrombosis is at least partly due to the
degeneration of blood proteins by electron transfer to the metal. The ideal surface,
from this point of view, is a semi-conductor such as silicon carbide. But, being brittle,
silicon carbide can only be applied as a thin layer. Systematic testing of the effect of
the silicon-carbide coated Tensum (Biotronik) stent upon cytotoxicity, hemolysis,
mutagenicity and hemocompatibility produced favourable results when compared
with Palmaz–Schatz (Cordis) and HepaMed (heparin) coated Wiktor (Medtronic)
stents 141
. Tantalum stents coated with silicon carbide deployed in rabbit iliac arteries
demonstrated complete endothelialization with minimal intimal proliferation142
.
Placement of eight silicon carbide-coated Palmaz–Schatz stents into patients suffering
from abrupt closure post-PTCA showed, at coronary angiography the next day,
patency of all the stents with no visible thrombus 143
. A series of 165 patients with
215 stents carried out using the Tensum (Biotronik) tantalum, balloon expandable,
silicon carbide-coated stent deployed in a group at high risk of restenosis and
thrombosis demonstrated 2% stent thrombosis. At six months, 32% of patients (24%
of stents) had a cardiac event144
. Likewise, other inorganic coatings have been tested
to demonstrate useful properties. A ‗diamond-like‘ carbon-coated stent exposed to
flowing platelet-rich plasma, produced less platelet activation and deposition and ion
release than uncoated stents 145,146
. Gold was suggested to be the ultimate inert stent
coating. A 5 µm thick gold coating applied to a stainless steel stent indeed showed
more than a halving of adherent thrombus mass compared with an uncoated stent 147
.
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However, disappointingly, a randomized study of 730 patients receiving a gold-
coated or bare stent revealed an excess of clinical events in the gold-coated group at
one year (24% vs 13%) 148
.
Biocompatible titanium-based coating was yet another material tested to coat
coronary stent surfaces. Previous examinations of the biocompatibility of various
metals have indicated that in addition to their excellent mechanical performance,
stainless steel and gold have an increased electrochemical surface potential, allowing
the transfer of electrons to proteins, such as fibrinogen. This promotes thrombus
formation and neointimal hyperplasia. In contrast, titanium, with its low
electrochemical surface potential, is biologically inert and has excellent
biocompatibility and inflammatory response149
. Titanium-nitride-oxide (TiNOX) is
proposed as a novel titanium alloy that renders metallic stents biologically inert and
prevents metallic foreign body reaction. Several preclinical and clinical studies were
taken up on this biocompatible coating offered by TiNOX on metallic stents of SS
and Cobalt Chromium. Porcine studies compared TiNOX-1 (ceramic) or TiNOX-2
(metallic) with uncoated stainless steel stents. Initial findings demonstrated reduced
neointimal formation and platelet adhesion 150
. A reduced neointimal hyperplasia up
to 50% at 6 week follow-up was observed. Clinical studies also demonstrated the
non-inferiority of TiNOX -coated stent in comparison with the first generation drug
eluting stents. A 3-year follow up report from the TITAX AMI trial demonstrated
better clinical outcome for TITANOX-coated stent compared with paclitaxel eluting
stents in terms of lower rate of Myocardial infarction (MI), cardiac death and stent
thrombosis in patients presented with acute MI151
.A 6-month follow-up study (TIBET
registry)showed excellent immediate clinical and angiographic outcome, with a low
incidence of major adverse cardiac events (MACE) at mid-term follow-up of Titan
stent implantation in 156 consecutive diabetic patients admitted to undergo
percutaneous intervention for at least one significant (50%) coronary lesion152
. A
similar follow up study conducted by the French Ministry of Health prospective
multicentre sought to explore the immediate outcome of the titanium-nitride-oxide-
coated bioactive stent, Titan2, in real-world practice and the incidence of major
Amrita Centre for Nanosciences and Molecular Medicine Page 19
cardiac events(EVIDENCE registry).In real-world practice, Titan2® stent
implantation revealed an excellent immediate outcome, with a low incidence of major
adverse cardiac events at 12-month follow-up153.
iii) Heparin-coated stents
Several anti-thrombogenic stent coatings 154,155
have been investigated,
with heparin being the most well known and extensively tested. Heparin has been
studied while covalently bound to the stent, as a ―passive‖ coating, and also as an
eluted drug. Heparin coated stents were associated with reduced platelet and
endothelial activation when compared to bare metal controls by plasma P-selectin and
E-selectin assessment in a small human study 156
.White cell and platelet activation in-
vitro have been shown to be reduced when heparin coated stents are compared to
gold, 157
SiC, 158,159
or bare metal stents, with prolongation of thrombosis time 160,161
.As
with some non-randomized series evaluating heparin coated stents, the clinical
incidence of sub acute stent thrombosis (SAT) was low in BENESTENT II
(randomized comparison of implantation of heparin coated stents with balloon
angioplasty in selected patients with coronary artery disease) although there was no
control bare stent group compared to the heparin coated Palmaz-Schatz group 162
.
Other randomized (COAST, heparin-coated stent placement for the treatment of
stenoses in small coronary arteries of symptomatic patients) 163
and non-randomized
comparisons to bare metal stents suggest similar SAT and ISR rates 164,165
.
iv) Drug eluting stents
Early difficulties with coronary stents included a risk of
early thrombosis (clotting) (Fig. 1.6A) resulting in occlusion of the stent166
. Coating
stainless steel stents with other substances such as platinum or gold did not eliminate
this problem167
. Drug eluting stents (DES) were developed and approved first in
2003, with significant initial success 168
. DES is capable of releasing single or
multiple bioactive agents from a polymeric coating on its surface into the bloodstream
and surrounding tissues. By adding a drug eluting coating, the rate of restenosis has
been reduced to about 5% or less169
. The drugs that may be useful in preventing ISR
Amrita Centre for Nanosciences and Molecular Medicine Page 20
fall into four major categories: anti-neoplastics, immunosupressives, migration
inhibitors, and enhanced healing factors24
. Heparin has been effective in reducing
both thrombosis and neointimal proliferation while sirolimus and paclitaxel were
mainly used for their anti-proliferative effects in blocking neointimal hyperplasia170
.
The anti-proliferative compounds commonly used in developing drug eluting stents
include paclitaxel, QP-2, actinomycin, statins, and many others. Paclitaxel was
originally used to inhibit tumor growth by assembling microtubules that prevent cells
from dividing. It has recently been observed to attenuate neointimal growth as well24
.
Paclitaxel stents, when placed in a culture of smooth muscle cells obtained from
human coronary atherosclerotic plaque, produced severe destruction of cytoskeletal
components of the cells, suggesting a possible strategy for in-vivo use, assuming the
problems of inflammation and radial strength can be overcome171
.
Immunosuppressive such as limus drugs are generally used to prevent the immune
rejection of allogenic organ transplants. The general mechanism of action of most of
these drugs is to stop cell cycle progression by inhibiting DNA synthesis. Everolimus,
sirolimus, tacrolimus (FK-506), ABT-578, interferon, dexamethasone, and
cyclosporine belong to this category. The sirolimus derived compounds appear to be
promising in their ability to reduce intimal thickening 24
.The first successful trials
were of sirolimus-eluting stents. A clinical trial in 2002 led to the approval of
sirolimus-eluting Cypher stent in Europe in 2002 172
.Soon thereafter, a series of trials
of paclitaxel-eluting stents (PES) led to FDA approval of the Taxus stent in 2004 173
.
The Xience V everolimus eluting stent was approved by the FDA in July 2008
and has been available in Europe and other international markets since late 2006.
However, late stent thrombosis has been a major concern with DES platforms due to
delayed endothelialization as seen clearly from Fig. 1.6B. The newer generation of
DES stents which utilize strategies such as biodegradable polymeric coatings,
biodegradable stents and polymer-free approaches can be a possible solution to this
problem. To date, the most successfully tested drug eluting stents have been coated
with synthetic polymers: poly-n-butyl methacrylate and polyethylene–vinyl acetate
Amrita Centre for Nanosciences and Molecular Medicine Page 21
with sirolimus, and a poly(lactide-co-caprolactone) copolymer with paclitaxel eluting
platforms 24
.
Fig. 1.6 (A) Schematic representation of late stent thrombosis associated with DES. (B) Partially
endothelialized DES in comparison to BMS ; EES- everolimus-eluting stent; PES- paclitaxel-
eluting stent; SES- sirolimus-eluting stent; ZES- zotarolimus-eluting stent (C) Different
generations of DES. Image courtesy: medimoon.com/2013; M. Joner et al., J Am Coll Cardiol.
2008; M.J. Patel et al, Acta Pharm, 2012
In addition to coated metallic stents, a new category of completely
biodegradable stents appeared in the early 2000. Bioabsorbable DES helps to achieve
excellent acute and long-term results, but disappear completely within months,
thereby avoiding the need for prolonged dual antiplatelet therapy. In the late 1990s, a
bioabsorbable (Igaki–Tamai, Japan) stent, made of a high-molecular-mass poly-L-
lactic acid (PLLA), was implanted in 15 patients (25 stents) to evaluate the feasibility,
safety, and efficacy of the PLLA stent174
. No major cardiac events, except for repeat
A B
C
Amrita Centre for Nanosciences and Molecular Medicine Page 22
angioplasty, developed within 6 months. A still larger study of 50 elective patients
(63 lesions, 84 stents) also showed promising results175
.
The IDEAL biodegradable stent (Bioabsorbable Therapeutics, Inc, Menlo
Park, CA) consisting of a backbone of poly-anhydride ester based on salicylic acid
and adipic acid anhydride and a coating of sirolimus, potentially rendered the stent
with both anti-inflammatory and antiproliferative properties176
. Most notably, the
polymer was associated with reduced inflammation compared with a standard BMS
and Cypher stent, which was likely due to the anti-inflammatory properties of
salicylic acid following absorption by the vessel wall after its release. Drug elution
was found to be complete after 30 days, and complete stent degradation occurred over
a 9- to 12-month period. In July 2009, the results from 11 patients enrolled in the
multicenter first-in-humans Whisper trial showed stent safety and confirmed
structural integrity of the stent, with no evidence of acute or chronic recoil.
Unfortunately, insufficient neointimal suppression was demonstrated, which could be
possibly due to the consequence of inadequate drug dosing, particularly considering
that the surface area dose of sirolimus is only a quarter of that found on the Cypher
stent177
. ABSORB (Abbott Vascular, Santa Clara, CA, USA)is a Bioresorbable
Vascular Scaffold (BVS) system that elutes everolimus in a similar way to XIENCE
V and then resorbs naturally into the body leaving no permanent scaffold. The safety
and performance of the Absorb BVS system was previously established in 131
patients from the first-in-man ABSORB trial. Further, clinical outcomes after twelve-
month follow up trials in 512 patients (ABSORB EXTEND) showed low rates of
MACE and scaffold thrombosis 178
.
The polymer-free approaches of developing DES include carbon based
films, micro structured reservoirs, ceramic coatings, bioabsorbable coatings, drug
coatings etc 179
. Limited evidence exists regarding the long-term performance of
polymer-free (PF) DES in comparison to permanent polymer DES. Recent studies
report the results of the long term performance and clinical outcomes of different
polymer-free and polymeric DES after randomized trials. The LIPSIA Yukon trial
randomized 240 patients with diabetes mellitus to a polymer-free sirolimus eluting
Amrita Centre for Nanosciences and Molecular Medicine Page 23
stent (Yukon Choice, Translumina) versus a polymer-based paclitaxel-eluting stent
(Taxus Liberté, Boston Scientific)180
.In another randomized trial, the 5-year efficacy
and safety of a PF sirolimus-eluting stent versus a permanent polymer paclitaxel-
eluting stent (PES) in the setting of the Intracoronary Stenting and Angiographic
Restenosis-Test Equivalence Between Two Drug-Eluting Stents (ISAR-TEST) was
investigated in a total of 450 patients undergoing percutaneous coronary intervention
181. Overall, both trials demonstrated no significant differences in the rates of
myocardial infarction, definite stent thrombosis, target lesion revascularization, non-
target vessel revascularization, and stroke between patients assigned to the polymer-
free and polymeric DES platforms. The polymer-free platforms were unsuccessful in
establishing any significant advantage over the polymer-based DES in terms of the
primary end point late lumen loss, while the 5 years follow up showed similar clinical
for both181
.
An updated meta-analysis of bioabsorbable (BP-DES) versus durable
polymer drug-eluting stents (DP-DES) was performed in 20,005 patients with
coronary artery disease by Lupi et al. in 2014. The study concluded significantly
reduced late lumen loss and late stent thrombosis rates for BP-DES in comparison to
DP-DES, without clear benefits on harder endpoints. The efficacy of BP-DES in
preserving lumen patency seemed larger for sirolimus and novolimus DES182
.
Likewise, bioabsorbable polymer-based biolimus-eluting stents established superior
clinical outcomes as against BMS and first-generation DES, and similar rates of
cardiac death/MI, and Target Vessel Revascularization (TVR) compared to second-
generation DP-DES, but higher rates of definite ST than cobalt-chromium
everolimus-eluting stents183
.
1.2.9 Limitations of polymer-based drug eluting stents
Drug eluting stents with durable polymer coatings have by now replaced the
bare metallic stents by significantly reducing late lumen loss and have revolutionized
the practice of percutaneous coronary intervention. However, there are still
unresolved issues pertaining to the use of polymer based drug eluting stents.
Amrita Centre for Nanosciences and Molecular Medicine Page 24
i) Polymeric degradation
The first generation DES such as, Cypher and Taxus employed thick durable
polymeric layer to incorporate the antiproliferative drugs and to render their
controlled release. The presence of these durable polymers has been linked to
inflammatory responses and local toxicity in preclinical analysis184, 185
. Furthermore,
durable polymers used in first-generation DES were associated with mechanical
complications such as polymer delamination, ―webbed‖ polymer surface etc.,
leading to stent expansion issues(Fig. 1.5A) as well as non-uniform coating, resulting
in erratic drug distribution(Fig. 1.5B) 140
.
Fig. 1.5 Limitations of stent polymeric coatings (A) Degradation/ delamination (B) non uniform
coating Image courtesy: Waksman, Interventional Cardiology, 2010
In recent years, considerable trials have been made in clinical research on
the development of novel drug carrier systems including absorbable (or
biodegradable) polymers and non-polymeric stent surfaces. These new platforms
provide better deliverability, radio-opacity, flexibility, and radial strength and also
facilitate reduced dosage of currently used antiproliferative drugs. However, such
polymeric coatings are also afflicted by certain issues as discussed below.
ii) Delayed endothelialization
The long-term safety of polymer-based DES has been a major concern due to
impaired arterial healing, which in turn leads to late stent thrombosis. The anti-
proliferative drugs from DES would hamper the growth of endothelial cells apart
from inhibiting smooth muscles and platelets [Ref].This affects the re-
A B
Amrita Centre for Nanosciences and Molecular Medicine Page 25
endothelialization of stent struts, leaving the bare stent surface to contact with blood
provoking inflammatory cell infiltration and/or cause long-term drug sequestration
within the arterial wall186
. However new research on using more endothelial cell
friendly drugs to coat stents such as everolimus and zotarolimus have resulted in
improved endothelial coverage after implantation. In addition, literature on the use of
polymer-besides for interventions involving multiple stents (such as bifurcations or
overlapping stents) indicate that, there are chances for local arterial toxicity
aggravation that occurs when drug and polymer concentrations are substantially
increased187
.
iii) Stent thrombosis
Polymer-based drug eluting stents have been associated with an increased
risk of very late stent thrombosis compared with bare metal stents. This could be
endorsed fairly to hypersensitivity reactions towards the polymeric coatings, resulting
in inflammation, late stent malapposition, and stent thrombosis 24, 186
. Moreover,
delayed stent endothelialization results in platelet aggregation and stent thrombosis at
a later stage, mainly after the exhaustion of the drugs (late stent thrombosis) 188
. This
makes the patients depend on prolonged anti-platelet therapy after coronary stent
implantation.
1.2.10 Role of regenerative medicine and nanotechnology for
cardiovascular implants
The endothelial monolayer that lines the normal blood vessel serves as a
bioregulator of cardiovascular physiology. The vascular endothelium is versatile and
multifunctional with many synthetic and metabolic properties. These include the
regulation of thrombus and platelet activation, adhesion and aggregation 189
as well as
modulation of vascular tone and blood flow 190
. It also controls SMC migration and
proliferation 191
. ECs secrete and express numerous growth factors, extracellular
matrix products, anti-thrombotic and pro-coagulant factors. ECs are intimately
involved in maintaining a non-thrombogenic blood-tissue interface. Re-
endothelialized segments of artery are often associated with less neointimal
Amrita Centre for Nanosciences and Molecular Medicine Page 26
thickening and the absence of an intact EC lining therefore predisposes
cardiovascular implants; including bypass graft and stents; to platelet deposition,
thrombus and implant failure. As a result, investigators have developed methods to
promote the endothelialization of vascular grafts prior to implantation by
transplantation of ECs in-vitro, a process called EC seeding. The cellular engineering
approach called ‗seeding‘ involves lining the lumen of the graft in-vitro with EC 192,
193. Recent advances in science and technology, especially progressive development
in nanoscience and nanotechnology, offer novel nano-structured materials with
enhanced characteristics which make them the material of choice for various
applications 194, 195
.
Nanotechnology is concerned with manipulation at the molecular or atomic
level to provide useful applications 196,197
. The main unifying theme is the control of
matter on a scale smaller than 1 micrometer, normally between 0.1-100 nanometers,
as well as the fabrication of devices on this same length scale. Materials reduced to
the nanoscale can show very different properties compared to what they exhibit on a
macroscale, enabling unique applications197
. To cite a typical example, a material
such as gold, which is chemically inert at normal scales, can serve as a potent
chemical catalyst at nanoscales. Much of the fascination with nanotechnology stems
from these unique quantum and surface phenomena that matter exhibits at the
nanoscale. The difficulties involved in fulfilling the numerous ideal characteristics
using traditional synthetic materials have lead biomaterials research towards the fields
of nanotechnology and tissue engineering.
In cardiovascular arena, researchers have utilized nanotechnology to create
stent surfaces that express novel advantageous properties such as anti-
thrombogenicity and biostability, either through surface texturing or via the
incorporation of functionalities on material surfaces198
. Surface characteristics of the
biomaterial used for stent fabrication is well known to influence stent-biology
interactions. The primary surface characteristics of a stent material, which influence
thrombosis and neointimal hyperplasia, include surface energy, surface texture,
surface potential, and the stability of the surface oxide layer 199,200
. An in-vitro study
Amrita Centre for Nanosciences and Molecular Medicine Page 27
showed that the adherence, growth and proliferation of endothelial cells on Ta films
were much better than on 316L SS and Ti films 201
. In another study, the sputter
coating of a Ti–Ta target produced a surface that showed better endothelialization
because of the changes in the microstructure of the natural Ti-oxide film produced 202
.
The above cases imply that changes in surface energies or surface texturing of a
biomaterial can induce changes in their biological behavior. However, the nature of
the coating although biocompatible, should not lose its integrity during the stent
placement and expansion, causing adverse effects. Also, similar to surface charge, an
optimal range of surface energy and surface roughness can promote better
endothelialization on the metallic biomaterials.
It is well established in literature that nanoscale surface topography has
significant effects on cellular behavior. This has been corroborated by the recent
studies by our research group on nanostructuring of metallic titanium showing that
osteoblast/endothelial cells do respond to nanoscale surface differently203,204,70
.
Literature reports also provide ample evidences for the effects of nanostructuring in
promoting enhanced cellular adhesion in-vitro and in-vivo. A nanoscale rough surface
of poly(lactic-co-glycolic acid) (PLGA) produced by NaOH treatment when
compared with smooth PLGA substrate, enhanced rat aortic SMC adhesion and
proliferation, and decreased rat aortic EC adhesion and proliferation 205,206
. Similar
effect was observed on nanostructured Ti versus conventional Ti 207
. Human coronary
artery SMCs were found to adhere and proliferate significantly more on aligned
electrospun poly (l-lactid-co-ε-caprolactone) or P(LLA-CL) nanofibers than on
randomly oriented nanofibers and the P(LLA-CL) film as well as tissue culture PS
(TCPS) control 208
. These altered cell responses to nanoscale topography stems from
several factors such as (i) changes in protein adsorption at the nanoscale surface, (ii)
the dimension of the nanoscale structures which regulates cell adhesion and
spreading, subsequently gene expression, proliferation and differentiation and (iii) the
nanostructure-induced alteration of the elasticity of substrate surface 209,210
. These
effects are beautifully illustrated in Fig. 1.7 wherein surface roughness and stiffness
are found to influence the attachment and proliferation of cardiomyocytes.
Amrita Centre for Nanosciences and Molecular Medicine Page 28
Fig. 1.7 Schematic representation of the effect of nanorough surface on cell behavior compared
to a flatter surface. (A) Shows the adsorption of ECM proteins immediately when substrates
implanted or soaked in media. (B) Indiactes the cardiomyocytes adhered to the substrates and
begin to grow. (C) Due to mimicking native myocardium ECM in surface features, more
cardiomyocytes on nanorough stiff substrates were adhered and grown than on conventional and
plain nanorough substrates.Stout et. al Int J Nanomedicine. 2012
1.2.11 Titania nanosurface modification
Current efforts are directed towards maximizing biocompatibility of
the implant material, which means to optimize cell adhesiveness and to support
physiological cell reactions such as spreading, proliferation, migration and
differentiation. Major strategies to improve cell adhesiveness of implant biomaterial
Amrita Centre for Nanosciences and Molecular Medicine Page 29
include surface roughening, etching or modifying by physical and chemical methods,
and/or coating with adhesive proteins of the extracellular matrix such as collagens,
fibronectin, and laminins. In addition, evidence is accumulating that not only the
surface chemistry of the biomaterial, but also the surface topography at nanoscale is a
critical parameter for cellular recognition of biomaterials. It is well known that the
native oxide layer on titanium (Ti) implants is responsible for its superior
biocompatibility and tissue integration. Recent efforts have targeted titanium dioxide
(TiO2 or titania) as a good candidate for surface modification at the nanoscale,
leading to improved nanotextures for enhancing host integration properties. TiO2
nanotubes on a Ti implant surface with different diameters (30, 50, 70, and 100 nm)
created by a simple electrochemical anodization process showed minimal
inflammatory response of macrophages in-vitro, clearly demonstrating the nanosize
effect on immune cells 211
. Studies also demonstrated that bone marrow stem cells
(MSC) respond to titanium chips covered with TiO2 nanotubes in a size-dependent
manner, with a maximum of cell adhesion, proliferation, and migration rates on 15
nm diameter nanotubes indicating the influence of nanosructural dimensions on cell
behavior 212,213
. It was demonstrated that fabrication of such surface nanofeatures by
compressing Ti nanoparticles 207
or by creating rationally designed nanopatterns 214
on Ti resulted in a substantial increase in endothelial adherence compared to bare Ti.
Surface modification of Ti by the creation of ordered TiO2 nanotubes has resulted in
improved osteoblast attachment, function, and proliferation215,216
.
In a previous study conducted by our own group, osteoblast cell response
was found to be greatly improved on hydrothermally modified TiO2
nanostructures203,204
. Nanomodification of Ti generating TiO2 surfaces has proved
beneficial in promoting endothelial proliferation as well as migration onto stent
surfaces214, 216, 217
Recent studies also suggest that TiO2 nanotubes are beneficial for
endothelial cells, encouraging more ECM secretion and functions, while inhibiting
VSMC growth 101,102
.Nanostructures developed by coating the Ti surface with rosette
nanotubes of DNA base analogues have been reported to influence endothelial
attachment and spreading, 216
implying in general that nanostructuring of metallic
Amrita Centre for Nanosciences and Molecular Medicine Page 30
surfaces might be a promising approach to faster endothelialization. Brammer et al
also reported that, TiO2 nanotubular structures have influence on endothelial
functionality with increased Nitric oxide/endothelin-1 ratio pointing to their
antithrombogenicity 217
. A recent report on stainless steel 316L coated with nano-
structured TiO2 layer exhibited improved blood compatibility, in terms of both blood
platelet activity and coagulation cascade, which can decrease the thrombogenicity of
blood contacting devices which were made from stainless steel 218
.
1.2.12 Micro and nanomodifications on coronary stents
Surface topography as mentioned earlier, is known to modulate cell response
significantly219
. The technology of creating nano and micro features on coronary
stents to modulate vascular cell behavior has been employed by various stent
companies. Recent examples of stents that contain a microporous coating include the
Corel-C™ stent that is fabricated from CoCr with a carbon nanoparticle coating
(Relysis Medical, India) 220
. Microporous surfaces have also been incorporated into
polymer based bioabsorbable stents 221
. Sandblasting has been used to create pores
between 1 to 100 μm on the surface of stainless steel stents, an example of which is
the Yukon™ stent platform (Translumina, Hechingen, Germany) 222
. The
BioFreedom (Biosensors Inc.) is a 316L stainless steel stent platform without polymer
and coated with biolimus A9 (Fig. 1.8A).
Fig. 1.8 Micro and nanomodifications on coronary stents. (A) Without polymer. The image of
scanning electron microscope showing the surface of the BioFreedom stent with biolimus A9
A
B
C Ci
Cii
Ciii
Civ
Amrita Centre for Nanosciences and Molecular Medicine Page 31
impregnated micropores in the abluminal side of the strut. (B): SEM view of a Setagon stent (x
20,000) showing alumina nanoporous stent surface (Ci) Scanning electron micrographs of
microporous Hap Coating, (Cii) cross section of the Hap coating (Ciii) final coating including the
Hap filled with sirolimus formulation, (Civ) cross section of the final coating. (Image courtesy:
Interven Cardiol, 2011, Future Medicine Ltd; Informa Healthcare 2007; A. Abizaid, Circulation:
Cardiovascular Interventions, 2010)
Preclinical studies have reported lower lesion scores, fewer struts with fibrin,
granulomas and giant cells, a significantly lower percentage diameter stenosis and
greater endothelialization than the sirolimus eluting Cypher stent223
. However, the
rapamycin eluting Yukon™ stent was found to be clinically inferior to the Cypher
stent, as well as a version of the Yukon™ stent coated in a drug containing
bioabsorbable polymer222
. At least two examples exist of nanoporous stent surfaces
are in the market. The Jomed™ coating consists of a thin aluminum base layer, which
is then subjected to an acidic solution that converts the aluminum into a thin ceramic
nanoporous aluminum oxide 224
(Fig. 1.8B, Setagon stents). A recent report suggests
that particle debris may be released from the stent surface 225
. MIV Therapeutics, Inc.
has developed a stent with a hydroxyapatite (Hap) coating that is 0.30 to 1 μm in
thickness with a porosity of 40-60% in volume 226
(Fig. 1.8C). This stent has shown
promising responses in both animal studies and in an initial clinical study after
adsorption of sirolimus 227
.
Amrita Centre for Nanosciences and Molecular Medicine Page 32
Summarizing, various strategies have been adopted by researchers to address the
limitations of bare metallic stents as tabulated in Fig 1.9.
Fig. 1.9 Strategies to combat in-stent restenosis
Drug coated stents
• Anti-neoplastic– Paclitaxel, ABT 578 • Immunosuppressant– Sirolimus– Tacrolimus
Inorganic coatings HAp, Iridium oxide, TiN & TiO
2 coatings, carbon-based films such as
diamond-like carbon (DLC) and carbon nitride (CN), Al2O
3, silicone
carbide
Endothelialization promoting modifications
• Antibody immobilization (CD31) for EPC capture • Bioactive peptide, Estradiol– VEGF
Bio-chemical modifications
• Heparin coating • Biomimetic polymers – Phosphorylcholine,
Hyaluronic acid (HA)
Amrita Centre for Nanosciences and Molecular Medicine Page 33
1.3 Aim and overall hypothesis of the thesis:
This thesis work focuses on the development of inorganic (ceramic) coatings
of bio/hemocompatible, nanotextured titania on Stainless Steel substrates and
thereafter on stents as a cell-friendly approach to realize three-fold benefits:
provide an anti-thrombotic layer
inhibit platelet aggregation
concurrently improve endothelialization and inhibit SMC over-proliferation.
AIM:
The aim of the study is to improve the current limitations of existing bare
metallic stents through a simple surface nanostructuring approach, without the use of
any anti-proliferative drugs or polymers, to combat in-stent restenosis.
HYPOTHESIS:
Surface modification of bare metal stents by creating hemocompatible
titania nanostructural features can present a more cell-friendly
environment for vascular cells, aiding in rapid functional
endothelialization, without inducing hyperproliferation of smooth muscle
cells or platelet adhesion, thereby regulating the causative factors for in-
stent re-stenosis and thrombosis.
1.4 Specific objectives of the thesis
The following are the specific objectives of this thesis work:
Fabrication of an array of uniform nanofeatures of TiO2 on metallic titanium
substrates/stent prototypes via hydrothermal processing by varying different
processing parameters such as time, temperature and concentration of the
reaction medium.
Amrita Centre for Nanosciences and Molecular Medicine Page 34
Comparative in-vitro evaluation of endothelial and smooth muscle cell response
(proliferation, apoptosis and cytoskeleton organization, etc.) on various
nanosurfaces, functionality of the formed endothelium and time for complete
endothelialization.
Checking for in-vitro hemocompatibility of nanomodified Ti plates/stents under
static and dynamic flow conditions, and assessment of the same on
endothelialized surfaces.
Optimization of hydrothermal process for nanotexturing of TiO2 onto Ti coated
stainless steel substrates for better biological response, corrosion resistance and
mechanical integrity.
Assessment of ion leaching profiles, corrosion resistance and mechanical
properties of the titania coating on stainless steel substrates as per ISO standards.
Translation of TiO2 nanostructured coating on to stainless steel coronary stents.
Crimping and expansion tests to prove the adhesion and stability of TiO2 coating
on SS stent and evaluation of the coating durability in-vitro under high shear
stress dynamic flow.
Amrita Centre for Nanosciences and Molecular Medicine Page 35
1.5 References
1. Global status report on non-communicable disaeses 2010. Geneva, World
Health Organization, 2011
2. Global atlas on cardiovascular disease prevention and control. Geneva, World
Health Organization, 2011
3. C.D. Mathers and D. Loncar, PLoS Med, 2006, 3, e442
4. M. Anthea, L. Roshan. J. Hopkins, C.W. McLaughlin, S. Johnson, M.Q.
Warner, D. LaHart and J.D. Wright, Human Biology and Health, 1993, NJ:
Prentice Hall, Englewood Cliffs, ISBN 0-13-981176-1
5. R. Ross, Nature, 1993,362, 801–809
6. A.V. Finn, M. Nakano, J. Narula, F.D. Kolodgie and R. Virmani. Arterioscler
Thromb Vasc Biol 2010, 30, 1282–1292
7. G. Dangas, F. Kuepper, Circulation, 2002, 105, 2586-2587
8. R.F.J. Shepherd and R.E. Vlietstra, The history of balloon angioplasty, ed. R.E.
Vlietstra, and D.R. Holmes, F.A. Davis Company, Philadelphia, 1987, p. 1–17
9. B. Doyle, C.S. Rihal, C.J. O'Sullivan, R.J. Lennon, H.J. Wiste, M. Bell, et al,
Circulation, 2007, 116, 2391–2398
10. R.K. Myler and S.H. Stertzer. Coronary and peripheral angioplasty: historical
perspective. ed. E.J. Topol, W.B. Saunders Company, Philadelphia, 1994, p.
171–185
11. P.W. Serruys, P.D. Jaegere, F. Kiemeneij, C. Macaya, W. Rutsch, G.
Heyndrickx, et al., New Engl J Med, 1994, 331, 489–495
12. G. Mani, M.D. Feldman, D. Patel and C. Mauli, Biomaterials, 2007, 28, 1689–
1710
13. P. Schuler, D. Assefa, J. Ylanne, N. Basler, M. Olschewski, I. Ahrens, T. Nordt,
C. Bode and K Peter, Cell Commun Adhes, 2003, 10, 17-26
14. J.Y. Chen, Y.X. Leng, X.B. Tian, L.P. Wang, N. Huang, P.K. Chu and P. Yang,
Biomaterials, 2002, 23, 2545-2552
15. S. Choudhary, M. Berhe, K.M. Haberstroh and T.J. Webster, Int J
Nanomedicine, 2006, 1, 41-49
Amrita Centre for Nanosciences and Molecular Medicine Page 36
16. M.G. Wolf, D. Moliterno, A. Lincoff and E. Topol, Clin Cardiol 1996, 19, 347–
56
17. A.C. Newby and A.B. Zaltsman. J Pathol, 2000, 190, 300–309
18. K.H. Mak, G. Belli, S.G. Ellis and D.J. Moliterno. JACC ,1996, 27, 494–503
19. N. Kipshidze, G. Dangas, M. Tsapenko, J. Moses, M.B. Leon, M. Kutryk, et al.,
J Am Coll Cardiol, 2004, 44, 733–739
20. O.F. Bertrand, R. Sipehia, R. Mongrain, J. Rodes, J.C. Tardif, L. Bilodeau, G.
Cote and M.G. Bourassa, JACC, 1998, 32, 562–571
21. M.M.H. van Beusekom, P.W. Serruys and W.J. van der Giessen. Coronary
Artery Disease, 1994, 5, 590-596
22. A. Kraitzer, Y. Kloog and M. Zilberman, J Biomed Mater Res B Appl Biomater,
2008, 85, 583-603.
23. B. Bhargava, G. Karthikeyan, A.S Abizaid and R. Mehran, BMJ, 2003, 327,
274–279
24. M.J. Patel, S.S. Patel, N.S. Patel and N.M. Patel, Acta Pharm, 2012, 62, 473–
496
25. M.K. Reddy, J.K. Vasir, S.K. Sahoo, T.K. Jain, M.M. Yallapu, et al.,
CircCardiovasc Interv 2008, 1, 209–216
26. J. Daemen, N. Kukreja, P.H. van Twisk, Y. Onuma, P.P. de Jaegere, et al., Am J
Cardiol, 2008, 101, 1105–1111
27. D.A. Siqueira, A.A. Abizaid, R. Costa J de, F. Feres, L.A. Mattos, et al., Eur
Heart J 2007, 28, 1304–1309
28. A. Abizaid, J.R. Costa, Circ Cardiovasc Interv 20103: 384–393
29. Inoue T, Croce K, Morooka T, Sakuma M, Node K and Simon DI, JACC
Cardiovasc Interv, 2011, 4, 1057–1066
30. E.S. Pollak, C.A. Buck, J.Loscalzo, G.A. Zimmerman, R.P. McEver, D.B.
Cines, Blood, 1998, 91, 3527-3561
31. M. Feletou. The Endothelium: Part 1: Multiple Functions of the Endothelial
Cells—Focus on Endothelium-Derived Vasoactive Mediators, ed F.M. San
Amrita Centre for Nanosciences and Molecular Medicine Page 37
Rafael, Morgan & Claypool Life Sciences, 2011,Chapter 2Multiple Functions
of the Endothelial Cells
32. J.C. Palmaz, J Endovsc Threr, 2004, 11, II-200–II-206.
33. T. Rau, J. Schofer, M. Schluter, A. Seidensticker, J. Berger, D.G. Mathey, J Am
Coll Cardiol, 1998, 31, 275–280
34. A. deMel, B.G. Cousins,. A.M. Seifalian, Int J Biomater, 2012, 2012, 8
35. C. Werner, M.F. Maitz, C. Sperling, J Mater Chem, 2007, 17, 1–10
36. H.D. Zheng, A.Z. Sadek, M. Breedon, D. Yao, K. Latham, J.D. Plessis, K.
Kalantar- Zadeh, Electrochem Commun, 2009, 11, 1308
37. O.K. Varghese, M. Paulose, C.A. Grimes, Nature Nanotechnology, 2009, 4, 592
38. R.A. Herman, A. Rybnikar, A. Resch, B. Marki, E. Alt, A. Stemberger, JACC,
1998,413A
39. E. Edelman, P. Seifert, A. Groothuis, A. Morss, D. Bornstein, C. Rogers,
Circulation 2001, 103, 429–434
40. Dahl JRV, Haager PK, Grube E, Gross M, Beythien C, Kromer EP, et al. Am J
Cardiol 2002, 89, 801–805
41. G. Danzi, C. Capuano, M. Sesana, A.D. Blasi, S. Predolini, D. Antoniucci.
Cardiovasc Interv 2002, 55, 157–62
42. C. Seliger, K. Schwennicke, C. Schaffar, Eur Heart J, 2000, 21, 286
43. F. Sgura, C.D. Mario, F. Liistro, M. Montorfano, A. Colombo, G.E.Grube,Herz,
2002, 27, 514–517
44. Z.H. Zhao, Y. Sakagami, T. Osaka. Can J Microbiol, 1998, 44, 441–447
45. C.D. Mario, E. Grube, Y. Nisanci, N. Reifart, A. Colombo, J. Rodermann, et al.
Int J Cardiol 2004, 95, 329–331
46. A. Bolz, M. Schaldach, Artif Organs, 1990,14, 260–269
47. M. Unverdorben, B. Sippel, R. Degenhardt, K. Sattler, R. Fries, B. Abt, et al.
Am Heart J 2003, 145, E17
48. P. Schuler, D. Assefa, J. Ylanne, N. Basler, M. Olschewski, I. Ahrens, et al. Cell
Commun Adhes, 2003, 10, 17–26
Amrita Centre for Nanosciences and Molecular Medicine Page 38
49. S. Monnink, A.J. van Boven, H.O. Peels, I. Tigchelaar, P.J. de Kam, H.J. Crijns,
et al. J Investig Med, 1999, 47, 304–310
50. U. Kalnins, A. Erglis, I. Dinne, I. Kumsars, S. Jegere, Med Sci Monit, 2002, 8,
116–120
51. L.F. Tanajura, A.A. Abizaid, F. Feres, I. Pinto, L. Mattos, R. Staico, et al.
JACC, 2003, 58A
52. M. Unverdorben, K. Sattler, R. Degenhardt, R. Fries, B. Abt, E. Wagner, et al. J
Interv Cardiol, 2003, 16, 325–33
53. C. Bickel, H. Rupprecht, H. Darius, C. Binz, B. Hauroder, F. Krummenauer, et
al. J Interv Cardiol, 2001, 14, 407–413
54. K. Gutensohn, C. Beythien, J. Bau, T. Fenner, P. Grewe, R. Koester, et al.
Thromb Res 2000, 99, 577–585
55. N. Barati, M.A. Faghihi Sani, Color Colorants Coat, 2009, 2,71-78
56. P. Fauchais and A. Vardelle, Materials Science-Advanced Plasma Spray
Applications, ed. H.S. Jazi, ISBN 978-953-51-0349-3, 2012, Chapter 1 Thermal
Sprayed Coatings Used Against Corrosion and Corrosive Wear
57. H. Liu and T. J. Webster, Biomaterials, 2006, 28, 354
58. K. von der Mark, S. Bauer and P. Schmuki, Cell Tissue Res, 2010, 339,131
59. G.A. Horley, Small, 2006, 2, 3
60. G.M. Whitesides, Nat Biotechnol, 2003, 21, 1161
61. J.Y. Rho, L.K. Spearing and P. Zioupos,Med Eng Phys, 1998, 20,92
62. 19 N. J. Sniadecki, R.A. Desai, S. A. Ruiz and C.S. Chen, Ann Biomed Eng,
2006, 34, 59
63. M. M. Stevens and J. H. George, Science, 2005, 310, 1135
64. S. Lavenus, J.C. Ricquier, G. Louarn and P. Layrolle, Nanomedicine,2010, 5,
937–994
65. V.V. Divyarani, K. Manzoor, M. Deepthy, N. Selvamurugan and S. V. Nair,
Nanotechnology, 2009, 20, 195101
66. C. Yao, E.B. Slamovich and T.J. Webster, J. Biomed. Mater. Res., Part A, 2007,
85, 157–166
Amrita Centre for Nanosciences and Molecular Medicine Page 39
67. E. Fine, L. Zhang, H. Fenniri, T.J. Webster, Int J Nanomedicine, 2009, 4, 91–97
68. K. Popat, L. Leoni, C. Grimes and T. Desai, Biomaterials, 2007, 28,3188–3197
69. L. Zhang, T.J. Webster, Nano Today, 2009, 4, 66—80
70. C.C. Mohan, P.R. Sreerekha, V.V. Divyarani, S.V. Nair, K.P. Chennazhi, D.
Menon, J Mater Chem, 2012, 22, 1326-1340
71. C.C.Mohan, K.P. Chennazhi, D. Menon, Acta Biomater, 2013, 9, 9568-77
72. Simpson JP, Geret V, Brown SA, Merritt K,NBS Spec. Publ, 601 1981, 395-422
73. Takao Hanawa, J Artif Organs, 2009, 12, 73–79
74. F. Variola, J.B. Brunski, G. Orsini, P. T. de Oliveira, R. Wazen, A. Nanci,
Nanoscale, 2011, 3, 335–353
75. G. Manivasagam, D. Dhinasekaran and A. Rajamanickam, Recent Pat Corros
Sci, 2010, 2, 40-54
76. M.J. Mulvany, Resistance arteries, structure and function: proceedings of the
Third International Symposium on Resistance Arteries, Denmark, 1991
77. J. D. Hooi, A. D.Kester, H. E. Stoffers, P. E. Rinkens, J. A. Knottnerus, and J.
W. van Ree, 2004, J Clin Epidemiol, 57, 294-300
78. W. Rosamond, K. Flegal, K. Furie, A. Go, K. Greenlund, N. Haase, et al.,
Circulation,2008, 117, e25-146
79. B. Mayer, J. Erdmann, and H. Schunkert, Clin.Res.Cardiol, 2007, 96, 1-7
80. J. E. Kirkpatrick, J Insur Med, 1999, 31, 37-38
81. J. Fajadet, A. Chieffo, European Heart Journal, 33, 36-50
82. Report #C245, Worldwide Coronary Stents Market, 2008-2017, MedMarket
Diligence 2009.
83. J. Butany, K. Carmichael, S.W. Leong, and M.J. Collins, J Clin Pathol, 2005,
58, 795–804
84. E. Nikolsky, L. Gruberg, S. Pechersky, M. Kapeliovich, E. Grenadier, S.
Amikam, et al., Catheter Cardiovasc Interv, 2003, 59, 324-328
85. S.E. Nissen, J.C. Gurley, C.L. Grines, et al., Circulation, 1991, 84, 1087–1099
86. S.E. Nissen, J.C. Gurley, Int J Card Imaging, 1991, 6, 165–177
87. B.F. Waller, C.A. Pinkerton and J.D. Slack, Circulation, 1992, 85, 2305–2310
Amrita Centre for Nanosciences and Molecular Medicine Page 40
88. F. Schiele, N. Meneveau, M.F. Seronde, et al. Int J Cardiovasc Intervent, 2000,
3, 207–213
89. S.C. Smith, T.E Feldman, J.W. Hirshfeld, et al., Circulation, 2006, 113, 156–
175
90. F.J.H Gijsen, F. Migliavacca, S. Schievano, L. Socci, L. Petrini, A. Thury, J.J.
Wentzel, A.F.W van der Steen, P.W.S Serruys, G. Dubini,BioMedical
Engineering OnLine, 2008, 7, 23
91. C.T. Dotter, M.P. Judkins, Circulation, 1964, 30, 654–670
92. J.C. Palmaz, R.R. Sibbitt, F.O. Tio, S.R. Reuter, J.E. Peters, F. Garcia, Surgery
1986, 99, 199–205
93. G.S. Roubin, K.A. Robinson, S.B. King, C. Gianturco, A.J. Black, J.E. Brown,
R.J. Siegel, J.S. Douglas, Circulation, 1987, 76, 891–897
94. U. Sigwart, J. Puel, V. Mirkovitch, F. Joffre, L. Kappenberger, N Eng J Med,
1987,316, 701–706
95. C.C. Phatouros, R.T. Higashida, A.M. Malek. AJNR, 2000, 21, 732–738
96. P.W. Serruys, P.D. Jaegere, F. Kiemeneij, C. Macaya, W. Rutsch, G.
Heyndrickx, et al. New Engl J Med, 1994, 331,489–495
97. D.L. Fischman, M.B. Leon, D.S. Baim, R.A. Schatz, M.P. Savage, I. Penn, et al.
New Engl J Med 1994, 331, 496–501
98. J. Holmes, Am J Cardiol, 2003, 91, 50A–53A
99. A.T.L Ong, E.P. McFadden, E. Regar, P.P.T. deJaegere, R.T. vanDomburg, P.W.
Serruys, J Am Coll Cardiol, 2005, 45, 2088–2092
100. I. Iakovou, T. Schmidt, E. Bonizzoni, L. Ge, G. Sangiorgi, G. Stankovic, et al. J
Am Med Assoc, 2005, 293, 2126–2130
101. A.Taylor,Metals. ed. U. Sigwart,: W.B. Saunders Company Ltd; London,1996.
p. 28–33
102. R.A. Schatz, Circulation, 1989, 79, 445–457
103. S.C. Wong, R.A. Schatz. New York: Futura Publishing Company, Inc., 1993, p.
3–19
Amrita Centre for Nanosciences and Molecular Medicine Page 41
104. J.H. Rundback, R .Leonardo, G.N. Rozenbilt. New York: Thieme; 2000, p. 1–
22
105. H. Zitter, H.Plenk, J Biomed Mater Res, 1987, 21, 881–896
106. P. Johnson, J. Bernstein, G. Hunter, W. Dawson, L. Hench, J Biomed Mater Res
1977, 11, 637–656
107. F. Macionczyk, B. Gerold, R. Thull, Surf Coat Technol, 2001, 142, 1084–1087
108. G. Teitelbaum, M. Raney, M. Carvlin, A. Matsumoto, K. Barth, Cardiovasc
Intervent Radiol, 1989, 12, 125–127.
109. A. Matsumoto, G. Teitelbaum, K. Barth, M. Carvlin, M. Savin, E. Strecker.
Radiology, 1989, 170, 753–755
110. H. Matsuno, A. Yokoyama, F. Watari, M. Uo, T. Kawasaki, Biomaterials 2001,
22, 1253–1262
111. Y.X. Leng, J.Y. Chen, P. Yang, H. Sun, J. Wang, N. Huang. Nucl Instrum
Methods Phys Res Sec B: Beam Interact Mater Atoms, 2006, 242, 30–32
112. J.Y. Chen, Y.X. Leng, X.B. Tian, L.P. Wang, N. Huang, P.K. Chu, et al.,
Biomaterials 2002, 23, 2545–2552
113. J.Y. Chen, Y.X. Leng, X. Zhang, P. Yang, H. Sun, J. Wang, et al. Nucl Instrum
Methods Phys Res Sec B: Beam Interact Mater Atoms 2006, 242, 26–29
114. J.B. Park, In: Biomater Sci Eng. New York: Plenum Press, 1987, p. 193–233
115. J.R. Davis. Metallic materials. In: Handbook of Medical Devices, Materials
Park: ASM International, 2003, p 21–50.
116. H.A. Farzad, M.T. Peivandi, S.M.R.Y. Sani, Eng Fail Anal, 2007, 14, 1205-
1217
117. S. Windecker, I. Mayer, G.D. Pasquale, W. Maier, O. Dirsch, P.D. Groot, et al.,
Circulation, 2001, 104, 928–933
118. M. Mosseri, I.Tamari, M. Plich, Y. Hasin, M. Brizines, A. Frimerman, et al.
Cardiovasc Revasc Med, 2005, 6, 2–6
119. S. Windecker, R. Simon, M. Lins, V. Klauss, F. Eberli, M. Roffi, et al.
Circulation, 2005,111, 2617–22
Amrita Centre for Nanosciences and Molecular Medicine Page 42
120. V. Biehl, T. Wack, S. Winter, U. Seyfert, J. Breme, Biomol Eng, 2002,19, 97–
101
121. J.B. Brunski, Metals. ed. B.D. Ratner, A.S. Hoffman, F.J. Schoen, J.E. Lemons,
materials in medicine, San Diego, 2004, p. 137–153
122. C. Briguori, C. Sarais, P. Pagnotta, F. Liistro, M. Montorfano, A. Chieffo, et al.,
J Am Coll Cardiol, 2002, 40, 403–409
123. A. Kastrati, J. Mehilli, J. Dirschinger, F. Dotzer, H. Schuhlen, F.J. Neumann, et
al., Circulation, 2001, 103, 2816–2821
124. S. Rittersma, Rd. Winter, K Koch, M. Bax, C. Schotborgh, K. Mulder, et al.,
AmJ Cardiol, 2004, 93, 77–80
125. D. Kereiakes, D. Cox, J. Hermiller, M. Midei, Bachinsky W, Nukta E, et al. Am
J Cardiol, 2003, 92, 463–466
126. A. Klocke, J. Kemper, D. Schulze, G. Adam, B.K. Nieke. J Orofac Orthop,
2005, 66, 279–287
127. US Food and Drug Administration, Center for Devices and Radiological Health,
‗‗MULTI-LINK VISIONTM RX & OTW coronary stent system: P020047,
2003
128. M.N. Babapulle, M.J. Eisenberg, Circulation, 2002,106, 2859–2866
129. TC. Woods, A.R. Marks, Annu Rev Med, 2004, 55, 169–178
130. A.L. Lewis, L.A. Tolhurst, P.W. Stratford, Biomaterials, 2002, 23, 1697–1706
131. S.H. Hofma, H.M. van Beusekom, P.W. Serruys, W.J. van Der Giessen, Curr
Interv Cardiol Rep, 2001,3, 28–36
132. F.G. Welt, C. Rogers, Arterioscler Thromb Vasc Biol, 2002, 22, 1769–1776
133. S.H. Duda, T.C. Poerner, B. Wiesinger, J.H. Rundback, G. Tepe, J. Wiskirchen,
K.K. Haase, J Vasc Interv Radiol, 2003, 14, 291–301
134. J.E. Sousa, P.W. Serruys, M.A. Costa, Circulation, 2003, 107, 2274–2279
135. L. Garza, Y.W. Aude, J.F. Saucedo, Curr Opin Cardiol, 2002, 17, 518–525
136. A.L Lewis, J.D Furze, S. Small, J.D. Robertson, B.J. Higgins, S. Taylor, and
D.R. Ricci, 2002, J Biomed Mater Res, 63, 699-705
Amrita Centre for Nanosciences and Molecular Medicine Page 43
137. D.M. Whelan, W.J.van der Giessen, S.C, Krabbendam, E.A.van Vliet, P.D
Verdouw, P.W. Serruys, H.M. van Beusekom, 2000, Heart, 83, 338-345
138. M. Galli, A. Bartorelli, F. Bedogni, N. DeCesare, S. Klugmann, L. Maiello, F.
Miccoli, T. Moccetti, M. Onofri, V. Paolillo, R. Pirisi, P. Presbitero, P.
Sganzerla, M.Viecca, S. Zerboni, and G. Lanteri, J Invasive Cardiol, 2000, 12,
452-458
139. M. Galli, L. Sommariva, F. Prati, S. Zerboni, A. Politi, R. Bonatti, S. Mameli,
E. Butti, A. Pagano, and G. Ferrari, 2001, Catheter Cardiovasc Interv, 53, 182-
187
140. S. Hossainy, S. Prabhu,J Biomed Mater Res A, 2008, 87, 487–493
141. S.H.J. Monnink, I. Tigchelaar, A.J. van Boven et al., Circulation, 1998, 17, 856
142. W. van der Giessen, H.M. van Beusekom, S.H. Hofma et al. Eur Heart J, 1996,
17, 178
143. A. Bolz, M. Amon, C. Ozbek et al., Tex Heart Inst J, 1996, 23, 162–166
144. B. Heublein, K. Pethig, A.M. Elsayed, J Invas Cardiol, 1998, 10, 255–262
145. C. Beythien, K. Gutensohn, T. Kenner et al, Eur Heart J, 1998, 19, 497
146. C. Beythien, K. Gutensohn, P. Kuhnl, et al., J Am Coll Cardiol, 1998, 31, 413A
147. R.A. Herrmann, A. Rybnikar, A. Resch et al., J Am Coll Cardiol 1998; 31:
413A
148. A. Schomig, A. Kastrati, F.J. Neumann et al., Circulation, 1998, 17, 855
149. D.E. Kandzari, J.E. Tcheng, and J.P. Zidar, Cardiovasc Interv, 2002, 56, 562-
576
150. S. Windecker, I. Mayer, P.G. De, W. Maier, O. Dirsch, G.P De, Y.P. Wu, G
Noll, B. Leskosek, B. Meier, and O.M Hess, Circulation, 2001, 104, 928-933
151. P. Karjalainen, Eurointervention, 2010, 6, Supplement H
152. M.1.Valdes Chavarri, A. Bethencourt, E. Pinar, A. Gomez, J.F. Portales, F.
Pomar, et al., Heart Vessels, 2012, 27, 151-8
153. M. Angioi, P. Barragan, S. Cattan, F. Collet, P. Dupouy, P. Durand, Archives of
Cardiovascular Diseases, 2012, 105, 60-67
Amrita Centre for Nanosciences and Molecular Medicine Page 44
154. R. Herrmann, G. Schmidmaier, B. Markl, et al., Thromb Haemost, 1999, 82,
51–57
155. E. Alt, I. Haehnel, C. Beilharz, et al., Circulation, 2000, 101, 1453–1458
156. Y. Beaudry, S. Sze, B. Fagih, et al., J Invasive Cardiol, 2001,13, 628–31
157. K. Christensen, R. Larsson, H. Emanuelsson, et al, Biomaterials, 2001, 22,
349–355
158. A. Tarnok, A. Mahnke, M. Muller, et al., Cytometry, 1999, 38, 30–39
159. C. Bickel, H.J.Rupprecht, Darius H, et al. J Interv Cardiol, 2001, 14, 407–413
160. C. Beythien, K. Gutensohn, J. Bau, et al. Thromb Res, 1999; 94, 79–86
161. R. Blezer, L. Cahalan, P.T. Cahalan, et al. Blood Coagul Fibrinolysis 1998, 9,
435–440
162. P.W. Serruys, B. van Hout, H, Bonnier, et al, Lancet 1998, 352, 673–81
163. M. Haude, T.F. Konorza, U. Kalnins, et al, Circulation 2003,107, 1265–1270
164. J. Wohrle, E. Al Khayer, U. Grotzinger, et al., Eur Heart J, 2001, 22, 1808–
1816
165. M.C. Vrolix, V.M. Legrand, J.H. Reiber, et al., Am J Cardiol, 2000, 86, 385–
389
166. S.B. Donald, Harrison's Principles of Internal Medicine, ed: D.L. Kasper, A.S.
Fauci, D.L. Longo, E. Braunwald, S.L. Hauser, & J. L. Jameson. McGraw-Hill,
New York: 2005, p. 1459–1462
167. A. Kastrati, A. Schomig, J. Dirschinger, et al. Circulation, 2000,101,2478–
2483
168. M. Valgimigli, C.A.G. van Mieghem, A.T.L. Ong, J. Aoki, G.A. Rodriguez
Granillo et al., Circulation, 2005, 111, 1383-1389
169. E.P. McFadden, E.E.Stabile, E. Regar, E. Cheneau, A.T Ong. et al., Lancet,
2004, 364, 1519-1521
170. R.Virmani, A. Farb, G. Guagliumi, and F.D Kolodgie, 2004, Coron Artery Dis,
15, 313-318
171. R. Voisard, E. Alt, R. Baur et al. Eur Heart J, 1998, 1, 376
172. P.W. Serruys, M.J. Kutryk, A.T. Ong, N. Engl. J. Med, 2006, 354, 483–495
Amrita Centre for Nanosciences and Molecular Medicine Page 45
173. "New Device Approval - P030025 - TAXUS Express2 Paclitaxel-Eluting
Coronary Stent System". Archived from the original on 2008-02-03. Retrieved
2008-02-25
174. I. Autio, U. Malo-Ranta, O.P. Kallioniemi, and T. Nikkari, 1989, Artery, 16,
72-83
175. T. Tsuji, H. Tamai, K. Igaki, Y.S Hsu, K. Kosuga, T. Hata, Circ J, 2004, 68:
135
176. Y. Onuma, P.W. Serruys, Circulation, 2011, 123, 779-797
177. R. Jabara, L. Pendyala, S. Geva, J. Chen, N. Chronos, K. Robinson,
EuroIntervention, 2009, 5, F58– F64
178. A Abizaid, J Costa, A Bartorelli, R Whitbourn, R van Geuns, B Chevalier, et
al., EuroIntervention, 2014, article in press.
179. A. Abizaid and J. R. Costa, Circ Cardiovasc Interv. 2010;3:384-393.
180. T Stiermaier et al. CathCardiovascInterv, 2014, 83, 418-2
181. King L etal. Catheter Cardiovasc Interv. 2013 81, E23-8
182. Lupi etal, Catheter Cardiovasc Interv, 2014, , 83, E193-206
183. Palmerini, J Am Coll Cardiol, 2014, 63, 299-307
184. T.F. Luscher, J. Steffel, F.R. Eberli, M. Joner, G. Nakazawa, Tanner FC,
Virmani R et al., Circulation, 2007, 115, 1051–1058
185. G.V. Silva, M.R. Fernandes, R. Madonna, F. Clubb, E. Oliveira, P. Jimenez-
Quevedo, Catheter Cardiovasc Interv, 2009,73, 801–808
186. Virmani R, Guagliumi G, Farb A, et al.,Circulation, 2004, 109, 701–705
187. A. Hoye, I. Iakovou, L. Ge, et al., J Am Coll Cardiol. 2006, 47, 1949–1958
188. DR. Holmes, D.J. Kereiakes, S. Garg, et al., J Am Coll Cardiol, 2010, 56, 1357-
65
189. M. Herring and A. Gardner, J. Glover, Surgery, 1978, 84, 498-504
190. P. Zilla, R. Fasol, M. Deutsch, T. Fischlein, E. Minar, A. Hammerle, O.
Krupicka, and M. Kadletz, J.Vasc.Surg. 1987, 6, 535-541
191. M.A Wahab, K.Y Mya and C.B He, J Pol Sci Part A –PolChem 2008, 46,
5887-5896
Amrita Centre for Nanosciences and Molecular Medicine Page 46
192. K.Zeng, L. Wang, S.X Zheng and X. F Qian, Polymer, 2009, 685-695
193. Z.Ghalanbor, S. A Marashi, and B. Ranjbar, Med.Hypotheses, 2005, 198-199
194. T. Kubik, K. Bogunia-Kubik, and M. Sugisaka, Curr.Pharm.Biotechnol,
2005, 17-33
195. M.B.Habal, Arch.Surg, 1984,119, 843-848
196. J.Palmaz, Tex Heart Inst J, 1997, 24,156–159
197. Y. Huang, E. Verbeken, E. Schacht and I.D. Scheerder. Local drug delivery
using drug-eluting stents. ed: P.W. Serruys, M.B. Leon, A. Colombo, M.J.
Kutryk, London Martin Dunitz Ltd; 2002. p. 319–335
198. Y.X. Leng, J.Y. Chen, P. Yang, H. Sun, J. Wang and N. Huang, Nucl Instrum
Methods Phys Res Sec B: Beam Interact Mater Atoms, 2006, 242, 30–32
199. Chen JY, Leng YX, Zhang X, Yang P, Sun H, Wang J, et al. Nucl Instrum
Methods Phys Res Sec B: Beam Interact Mater Atoms, 2006, 242, 26–29
200. V.V Divya Rani, K. Manzoor, D.Menon, N. Selvamurugan and S.V Nair,
Nanotechnology, 2009, 20, 195101
201. V .V. Divya Rani, V.K. Lakshmanan, V.C Anitha, K. Manzoor, D. Menon and
S.V Nair, Acta Biomater, 2012, 8,1976-1989
202. J. Gao, L. Nikalson and R. Langer, J Biomed Mater Res, 1998, 42, 417–424
203. D.C. Miller, A. Thapa, K.M. Haberstroh and T.J. Webster. Biomaterials 2004,
25, 53–61
204. S. Choudhary, K.M. Haberstroh and T.J. Webster, Tissue Eng, 2007, 13, 1421–
1430
205. Kwon IK, Kidoaki S and Matsuda T. Biomaterials 2005, 26, 3929–3939
206. Y. Yang and K.W. Leong, Nanomed Nanobiotech, 2010, 2, 478-495
207. K. Kuraishi, H. Iwata, S. Nakano, S. Kubota, H. Tonami, M. Toda, N. Toma,
et.al, J Biomed Mater Res Part B: Appl Biomater, 2009, 230–239
208. M.L.Chamberlain, K.S. Brammer, G.W. Johnston, S. Chien, S. Jin, J Biomater
Nanobiotech, 2011, 2, 293-300
209. S. Bauer, J. Park, K. von der Mark, P. Schmuki, Acta Biomater, 2008, 4, 1576-
1582
Amrita Centre for Nanosciences and Molecular Medicine Page 47
210. J. Park, S. Bauer, P. Schmuki, K. von der Mark, Nano Lett, 2009, 9, 3157-3164
211. J. Lu, M. P. Rao, N. C. MacDonald, D. Khang and T.J. Webster, Acta
Biomater, 2008, 4, 192–201
212. K. Popat, L. Leoni, C. Grimes and T. Desai, Biomaterials, 2007, 28, 3188–
3197
213. C. Yao, E. B. Slamovich and T. J. Webster, J Biomed Mater Res, Part A, 2007,
85, 157–166
214. T. W. Chung, D. Z. Liu, S. Y. Wang and S. S. Wang, Biomaterials, 2003, 24,
4655–4661
215. E. Fine, L. Zhang, H. Fenniri and T. J. Webster, Int J Nanomed, 2009, 4, 91–97
216. L. Peng, M.L. Eltgroth, T.J. LaTempa, C.A. Grimes and T.A. Desai,
Biomaterials, 2009, 30, 1268–1272
217. K.S. Brammer, S.H. Oh, J.O. Gallagher and S. Jin, Nano Lett., 2008,8, 786–
793.
218. J.H. Yi, C. Bernard, F. Variola, S.F. Zalzal, J.D. Wuest, F. Roseiand A. Nanci,
Surf. Sci., 2006, 600, 4613–4621
219. B. Bhargava, N.K. Reddy, G. Karthikeyan, R. Raju, S. Mishra, S. Singh, et al.
Catheter Cardiovasc Interv, 2006, 67, 698–702
220. Y.W. Ye, C. Landau, J.E. Willard, G. Rajasubramanian, A. Moskowitz, S.
Aziz, et al. Ann Biomed Eng. 1998, 26, 398–408
221. R. Wessely, J. Hausleiter, C. Michaelis, B. Jaschke, M. Vogeser, S. Milz, et al.
Arterioscler Thromb Vasc Biol, 2005, 25, 748–753
222. J. Hausleiter, A. Kastrati, R. Wessely, A. Dibra, J. Mehilli, T.
Schratzenstaller, et al. Eur Heart J, 2005, 26, 1475–1481
223. J. Mehilli, R.A. Byrne, A. Wieczorek, R. Iijima, S. Schulz, O. Bruskina, et al.
Eur Heart J. 2008, 29, 1975–1982
224. H. Wieneke, O. Dirsch, T. Sawitowski, Y.L. Gu, H. Brauer, U. Dahmen, et al.
Catheter and Cardiovasc Interv, 2003, 60, 399–407
225. M. Kollum, A. Farb, R. Schreiber, K. Terfera, A. Arab, A. Geist, et al.
Catheter Cardiovasc Interv, 2005, 64, 85–90
Amrita Centre for Nanosciences and Molecular Medicine Page 48
226. A Rajtar, G. L. Kaluza, Q. Yang, D. Hakimi, D. Liu, M. Tsui, M. Lien, D.
Smith, F. J. Clubb, and T. Troczynski, EuroIntervention, 2006, 2,113–115
227. J.R. Costa, A. Abizaid, R. Costa, F. Feres, L.F. Tanajura, A. Abizaid, et al.
JACC Cardiovasc Interv, 2009, 2, 422–427