Biofunctionalization of Biomaterials for Accelerated in Situ
Endothelialization: A Review
Achala de Mel,†Gavin Jell,‡,§Molly M. Stevens,‡,§and Alexander M. Seifalian*,†,⊥
Centre of Nanotechnology, Biomaterials and Tissue Engineering, UCL Division of Surgery & Interventional
Science, University College London, London, United Kingdom, Department of Materials and Institute of
Biomedical Engineering, Imperial College London, London, United Kingdom, and University Department of
Surgery, Royal Free Hampstead NHS Trust Hospital, London, United Kingdom
Received June 24, 2008; Revised Manuscript Received August 7, 2008
The higher patency rates of cardiovascular implants, including vascular bypass grafts, stents, and heart valves are
related to their ability to inhibit thrombosis, intimal hyperplasia, and calcification. In native tissue, the endothelium
plays a major role in inhibiting these processes. Various bioengineering research strategies thereby aspire to
induce endothelialization of graft surfaces either prior to implantation or by accelerating in situ graft
endothelialization. This article reviews potential bioresponsive molecular components that can be incorporated
into (and/or released from) biomaterial surfaces to obtain accelerated in situ endothelialization of vascular grafts.
These molecules could promote in situ endothelialization by the mobilization of endothelial progenitor cells (EPC)
from the bone marrow, encouraging cell-specific adhesion (endothelial cells (EC) and/or EPC) to the graft and,
once attached, by controlling the proliferation and differentiation of these cells. EC and EPC interactions with the
extracellular matrix continue to be a principal source of inspiration for material biofunctionalization, and therefore,
the latest developments in understanding these interactions will be discussed.
Coronary artery disease is a major cause of mortality and
morbidity1with two main surgical options available. Percuta-
neous transluminal coronary angioplasty/stenting is used in cases
of less than 70% occlusion, and bypass surgery is performed in
cases with a greater degree of occlusion. Drug eluting stents
have within the last 10 years received considerable research
attention and some notable successes in clinical trials.2-5
However, issues relating to the drug elution lifespan and the
consequences once this elapses remain, including incidences of
late thrombosis and restenosis.6Furthermore, there is evidence
to suggest that drug elution may inhibit endothelial cell (EC)
proliferation.6For patients requiring bypass surgery, saphenous
vein, internal mammary artery, internal thoracic, or radial artery
are the grafts of choice, however, 5-30% of patients have no
suitable veins/arteries available due to previous use or diseased
vein wall. A thriving synthetic graft industry therefore exists,
largely concerning expanded polytetrafluroethylene (ePTFE) or
polyethylene terephalate (Dacron). However, despite their
prevalence, these grafts offer low patency due to noncompliance,
thrombogenic surface, and the tendency to form intimal hyper-
Existing synthetic materials are also not suitable for coronary
bypass or for small caliber (<5 mm) arteries where the occlusion
rates are highest. The engineering of novel cardiovascular grafts
is therefore urgently required and two main approaches exist.
The first approach involves the development of a permanent
vascular replacement, which has a nonadhesive, inert, nonbio-
fouling surface. The second approach aims to biofunctionalize
a degradable or a nonbiodegradable graft in such a way that it
can activate a cascade of biological processes that eventually
regenerates or replaces a functioning tissue (Figures 1 and 2).8
IH and thrombosis are the principal mechanisms causing
cardiovascular graft failure and various strategies exist to inhibit
* To whom correspondence should be addressed. Tel.: 0044 20 7830
2901. E-mail: firstname.lastname@example.org.
†University College London.
‡Department of Materials, Imperial College London.
§Institute of Biomedical Engineering, Imperial College London.
⊥Royal Free Hampstead NHS Trust Hospital.
November 2008Published by the American Chemical SocietyVolume 9, Number 11
Copyright 2008 by the American Chemical Society
10.1021/bm800681k CCC: $40.75
2008 American Chemical Society
Published on Web 10/03/2008
these processes and prolong graft patency,9including coating
of grafts with various anticoagulants (heparin), antiplatelets
factors (glycoprotein IIb/IIIa inhibitors), and antiproliferating
The endothelium is an active organ that maintains vessel
integrity with dynamic mechanisms that prevent thrombosis and
IH.12,13Studies involving in vitro endothelialization of grafts
with cultured EC prior to implantation have shown that a
confluent endothelium prevents thrombogenic complications and
improves long-term patency.14-17In vitro endothelialization
procedures are however labor intensive and require a consider-
able amount of time and expertise to extract and culture the
cells, making the process costly and limited to specialty centers.
Hence, materials that promote in situ endothelialization of
cardiovascular implants (without IH or thrombus formation
during endothelium development) would be highly desirable.18
In vivo circulating EC, endothelial progenitor cells (EPC),19and
the less well-defined CD133+CD34+CD45-progenitor cells20,21
could potentially be recruited onto a modified graft for in situ
endothelialization. A subset of CD14+monocytic cells have
also been shown to be able to differentiate into EC.22Interest-
ingly, these bone marrow derived monocyte lineage cells that
can differentiate into mature EC have been reported to inhibit
IH to a greater degree than EPC.23
Protein adsorption and subsequent cell attachment/behavior
in response to an implanted foreign material is determined by
a variety of material properties including surface chemistry,
topography, dissolution rate, and the micro/macro mechanical
elasticity. Material surface properties can therefore be modified
by physicochemical modification and biofunctionalization to
promote desirable protein and cellular interactions (Figure 1).
In the emerging and dynamic research field of in situ endothe-
lialization, a multitude of biomaterial biofunctionalization
approaches can be found. This article will review research
involved in accelerating spontaneous in situ endothelialization
(including the EPC mobilization and, promoting the adhesion,
proliferation, and differentiation of these cells) on biomaterials
for cardiovascular applications (Figure 2). The interactions
between cell receptors and extracellular matrix (ECM), which
have inspired (and will continue to inspire) biomaterial bio-
functionalization, will be discussed, as will methods to provide
protection from thrombosis and IH during endothelium formation.
2. Promoting Endothelialization on Biomaterials
2.1. Mimicking the Extracellular Matrix. Cells are inher-
ently sensitive to physical, biochemical, and chemical stimuli
from their surroundings.24-26In vivo cells are in intimate contact
with the extracellular matrix (ECM), which is formed from a
complex connection of proteins, glycoproteins, and proteogly-
cans. The ECM provides not only structural support but also
contains a reservoir of cell signaling motifs and growth factors
that guide cellular anchorage and behavior. The local cell
environment or “niche” provides defined environmental cues
that determine cell-specific behavior, including selective recruit-
ment, proliferation, differentiation, and the production of the
numerous proteins needed for hierarchical tissue organization.27
A plethora of ECM compositions exist, each containing various
concentrations of insoluble macromolecules (fibrillar proteins,
e.g., collagen and glycoproteins), which interact with proteins
on cell surfaces and soluble macromolecules such as growth
factors.28The organization, density, spatial geometry, and
biochemistry of these ECM components determine mechanical
strength, cell response, and ultimately, hierarchical tissue
Features of the ECM such as nanoscale topography, optimized
mechanical properties, and presentation of bioresponsive motifs
have inspired multiple examples of biomaterials design for tissue
engineering scaffolds.29-31One strategy in vascular research
is to present endothelium derived macromolecules or their cell
interacting domains onto vascular grafts to mimic features of
the ECM and thereby assist specific cell adhesion.32
Figure 1. Examples of various physical, chemical, and biofunctionalization techniques to enhance in situ endothelialization. The topography
(e.g., random roughening (a) or geometrically arranged features (b)) and chemistry (e.g., plasma etching or UV irradiation (c)) of the biomaterial
influences protein adsorption and subsequent cell behavior. Different topographical features are invariably accompanied by chemical
heterogeneities. Cellular response to topographical features is, therefore, influenced both by the differential adsorption of various extracellular
macromolecules onto the various topographical structures and the physical adjustment of cell morphology and cytoskeletal organization caused
by feature shape. Increased biofunctionality can be achieved by attaching specific peptide motifs (Table 1), which can bind to cell receptors
(e.g., integrins such as Rv?3) and induce “firm” cell anchorage. Various techniques to attach these peptides exist including passive coating (d),
covalently linking (e), and presenting peptide linkers to sequence specific peptides from the environment (f). Presentation of the peptides at the
correct concentration, formation, and spatial arrangement determines biological response. The biofunctionality of the graft can also be further
enhanced by including biologically recognized and responsive peptide sequences (e.g., protease sensitive degradation sites) that control scaffold
degradation. Topographical, chemical, and biofunctional surface structuring can enable dynamic interactions with the surrounding ECM/blood
and enable the rapid establishment of a functional endothelium.
Biomacromolecules, Vol. 9, No. 11, 2008 de Mel et al.
Table 1. Summary of Potential Peptides Derived from the ECM Proteins or other Moieties Intended To Enhance Vascular Graft Endothelializationa,b
application/polymer suface modification
Guidant Tetra stentsspassive coating stents
deployed in porcine coronary arteries
v EC coverage V neointimal and stenosis areas
RGD cross-linked fibrin gel
ePTFE vascular graftssprecoating and shear stress
exposure in a pulsatile circulation (12 dyn/cm2)
v EC retention
RGD and WQPPRARI146
PTFE film surface-ammonia plasma treated; covalent
conjugation with printing
v adhesion, spreading, and migration of EC
CS-1 binding motif147(EILDVPST)
and the RGD motif (GRGDSPC)
covalently conjugated to PET film PET reacted with
ethylene diamine, succinic anhydride, DCC, DMF,
NHS, and peptide with DMF
CD34+cells umbilical cord blood
v cell expansion
CRGD, CREDV, and cyclic peptide
peptides with N-terminal cysteine, chemisorbed on
v cell adhesion greatest on CCRRGDWLC
RGD, YIGSR, and IKVAV149
covalently attached to an aminated polycaprolactone
polymer surface using carbodiimide chemistry
adipose-derived stem cells
v adhesion and proliferation
YIGSR as chain extenders and PEG as a soft
segment (PUUYIGSR-PEG) in the polyurethane
BAEC blood platelets
no platelet adhesion v EC adhesion, spreading, and
migration and superior mechanical properties
attached to collagen as cross-linkers
corneal epithelial cell
v adhesion and proliferation
PEGspolyurethane prepolymer extended with
SGG[K[N(O)NO]-]4GGS and GGYIGSRGGK the
diazeniumdiolate peptide, SGG[K[N(O)NO]-]4GGS
f SGGKKKKGGS reacted with NO
BAEC and SMC and platelets
v EC adhesion, migration, and NO encouraged
increased EC proliferation V platelet adhesion to
PEG-containing polyurethane and significant V with
covalent immobilization on ePTFE grafts by an
atmospheric plasma four P15-treated grafts were
implanted as arteriovenous grafts between the
femoral artery and vein or the carotid artery and
jugular vein in two sheep
HUVEC and HUASMC
v adhesion, proliferation of EC, and endothelialization
on the lumen V adhesion and proliferation of SMC
(3× thicker neointimal hyperplasia in controls)
REDV and VAPG152
peptides coated on microfluidic devices subjected to
EC and SMC
v adhesion of EC with REDV and SMCs with VAPG V
shear stress v adhesion
KREDVY and REDVY153
covalently bound peptides on gold patterned SiO2
v adhesion, V apoptosis, and necrosis of adhered
AG73 peptide RKRLQVQLSIRT154
cubcutaneously injected Cultrex BME supplemented
with AG73, also in vitro
induced EC tube formation and v sprouting of aortic
rings and angiogenic response
A13: RQVFQVAYIIIKA, A99:
covalent coupling to MB-chitosan coated tissue
HT-1080 human fibrosarcoma cells
and human foreskin fibroblasts
v cell adhesion
passive surface coating on polystyrene, ethyl vinyl
acetate, ePTFE, polycarbonate, titanium, and
EC, SMC, epithelial cells,
myoblasts, and osteoblast
v cell adhesion on polystyrene, EVA, ePTFE, and
titanium v tissue integration into ePTFE in vivo
fibronectin and laminin, and the
laminin peptide biotin-IKVAV157
soft protein lithography (microcontact printing) to
transfer biotinylated ECM proteins on
LRM55 astroglioma and primary rat
selective adhesion to patterned areas with
biotin-conjugated proteins, significant neuriteextension, viability, and development into
functionally active synapses
peptide amphiphile aqueous solution mixed with
suspensions of NPCs f a transparent gel-like solid
the density of bioactive epitope in the cell
environment was varied
v selective differentiation epitope density affects
biomimetic construct peptide fluorosurfactant
polymer, adsorbed on ePTFE.
v EC selective attachment, growth, shear stability V
affinity for platelets
avidin-biotin binding systems159
avidin receptors through anadsorbed film of biotinylated bovine serum albumin (b-BSA)160
avidin adsorbed on flat, 2D, and highly porous 3D
poly L-lactic acid surfaces glass substrates coupled
with avidin receptors through an adsorbed film of
b-BSA; avidin-treated slides were then seeded with biotinylated BAEC
biotinylated Hep G2 cells also
efficient, rapid attachment of Hep G2 cells v initial
BAEC adhesion, spreading rates, and strength of
coatingsautologous and/or xenologous vascular
prostheses implanted in the bilateral carotid arteries of rabbits
aKey: AHSVEC, adult human saphenous vein EC; BAEC, bovine aortic EC; BME, basement membrane extract; ECM, extracellular matrix; EC, endothelial cells; ePTFE, exp&ed polytetrafluoroethylene; (HPA)EC,
human pulmonary artery EC; HUVEC, human umbilical vein EC; NPC, neural progenitor cells; PEG, polyethylene glycol; PET, poly(ethylene terephthalate); SMC, smooth muscle cells; v, increased; V, reduced.bRGD
has received considerable research interest (and reviewed in depth elsewhere161). The table only presents a few applications where RGD is used in combination with another peptide and in novel approaches.
Accelerated in Situ EndothelializationBiomacromolecules, Vol. 9, No. 11, 2008
2.2. Peptides: Functional Domains of ECM Compo-
nents. Various ECM peptide sequences, which have been
determined to influence cell behavior, have been isolated and
grafted on materials to enhance biological properties, for
example, REDV,33PHSRN,34RGD, and GRGDSP from fi-
bronectin,35laminin-derived recognition sequences, IKLLI,
IKVAV,36LRE, PDSGR, RGD, and YIGSR, and collagen type
I derived sequence, DGEA.37These peptide ligands can directly
interact with cell receptors. Table 1 details the various ECM
proteins or derived peptides that have been incorporated into
modified polymers for vascular graft engineering and to promote
graft patency. Of the peptides investigated, the RGD peptide
has perhaps featured in the largest number of biomaterials
studies (Table 1). In cardiovascular bioengineering, biofunc-
tionalization of polycarbonate polyurethane urea, and nano-
composite polymers with RGD/modified RGD and heparin, has
been reported to increase EC and EPC adhesion.38-43
In addition to direct incorporation of peptides into biomate-
rials, the engineered peptides can themselves be used as building
blocks. Elastin engineered from its constituent polypeptides and
cross-linked to exhibit properties of native elastin is one such
example.44The bottom-up approach used by nature to create
the biopolymers of the ECM (e.g., collagen self-assembly) is
increasingly being imitated by biomaterial scientists in the
fabrication of new synthetic materials.45Various approaches in
using proteins as scaffolds for creating vascular grafts have been
2.3. Natural Polymers versus Artificial Extracellular
Matrix Proteins. ECM-derived collagen, fibronectin, laminin,
and vitronectin have been shown to facilitate cell attachment
as they possess intrinsic biological recognition sites for cells
via integrin receptors.47These ECM components or their
functional components are obtained as naturally extracted and
purified adhesive proteins, synthesized oligopeptides, or geneti-
cally engineered recombinant proteins.
There are certain undesirable effects involved with naturally
extracted and purified adhesive proteins, including immune
response, risk of infection, purity, and the structural complexity
(which minimizes any potential manipulation). Synthesized
oligopeptides are considered as a solution for many of the issues
involved with natural proteins.
Peptides may have some advantages over larger natural
proteins. For example, fibronectin (a natural RGD-containing
protein), despite its EC binding affinity, has been shown to
induce platelet activation, whereas a recombinant RGD-fusion
protein, cellulose-binding domain (CBD)-RGD has shown
enhanced cell adhesion and an inhibition of platelet activation
on polyurethane (PU).48,49Similarly, GFOGER,50a collagen-
mimetic peptide has been shown to selectively promote R2?1
integrin binding leading to osteoblast differentiation compared
to full-length type I collagen, thus, indicating that specific
biofunctional domains act differently when within the native
ligand.51This effect has also been shown with a galactose
carrying synthetic ECM polymer with enhanced attachment and
liver specific functions of hepatocytes.52It has also been
observed that when natural (extracted, isolated, and purified)
basement membrane proteins such as collagen and fibronectin
were compared with synthesized RGD and heparin bonded
polymers, natural proteins showed significantly reduced EC
However, despite their wide applications and benefits, it has
also been shown that some oligopeptides have a lower activity
compared to the native ligand due to the absence of comple-
mentary or modulatory domains and amino acids at sites distant
from the cell-binding domain, which can affect cell behavior.53
Genetic engineering of recombinant proteins eliminates many
of the issues involved with natural proteins and provide better
options for incorporating full-length cell binding domain proteins
and other significant amino acid sequences which may act as
cell interactive components.53,54The synthesis of more complex
components of a particular ECM protein by recombinant
technology, thereby permits more accurate mimicking of ECM
components and functions. For instance, the engineered CS5,
RGD binding domains derived from naturally occurring ECM
elastin, fibronectin-like domains provided enhanced endothelial
adhesion (via REDV33in this instance) and increased the
elasticity of small diameter vascular grafts.55
3. Cell-Material Interactions
3.1. Ligands on Biomaterials. Cell-material interaction can
be divided into receptor mediated and nonreceptor mediated
interactions. Nonreceptor mediated interactions are discussed
later and this section will focus on ligand-receptor interactions.
Ligands, are defined herewith as biomolecules that interact with
receptors such as integrins. Relevant research on integrins is
discussed below. The geometric spatial arrangement of
ligands,34,56,57their density,58,59orientation, conformation,60as
well as stereochemistry of the sequence61can all affect the
specificity of cell behavior and therefore require consideration
Figure 2. In situ graft endothelialization. Bioresponsive vascular grafts can target several biological processes to promote in situ endothelialization,
including (1) promoting the mobilization of EPC from the bone marrow, (2) encouraging cell-specific (circulating EC, EPC, and stem cells)
homing to the vascular graft site, (3) providing cell-specific adhesion motifs on the vascular grafts (of a predetermined spatial concentration),
and (4) directing the behavior of the cells post-adhesion to rapidly form a mature fully functioning endothelium capable of self-repair.
Biomacromolecules, Vol. 9, No. 11, 2008 de Mel et al.
for the biofunctionalization of vascular implants. For example,
studies have shown that EC have a greater binding affinity for
immobilized cyclic RGD compared to linear RGD and particu-
larly with amino acids of stereochemistry D.62The cyclic peptide
is believed to more closely mimic the conformation of the native
ligand. This strong and rapid cell attachment with cyclic RGD
would be beneficial for in situ endothelialization. Interestingly,
in addition to enhancing EC adhesion, a series of cyclic RGD
peptides have also been shown to act as potent, selective
antagonists for the platelet integrin R2?3(GPIIb/IIIa) and inhibit
platelet-mediated thrombus formation.63
Ligand solubility may also be a critically relevant factor for
efficient biofunctionalization of biomaterials. Water-soluble
RGD-containing peptides with low molecular weight (a few
amino acid units) strongly associate with integrins and inhibit
the attachment of cells.48Furthermore, RGD spatial clustering
is also known to be important in determining EC adhesion and
the strength of the adhesion.64
3.2. Integrins. Integrins are a large family of heterodimeric,
transmembrane, cell surface proteins that govern the adhesive
interactions between cells and macromolecular components of
the ECM via binding motifs such as the RGD sequence (Figure
3;65which binds to Rv?3integrin).66,67Binding to integrin
receptors activates various signaling paths that, among other
functions, mediate cell attachment, proliferation, organization
of the Actin cytoskeleton, and formation of focal adhesions.62,63
Integrins are recognized to have a critical role in cell-biomaterial
interactions and therefore protein/peptide ligands that interact
with integrins are considered as targets to control receptor
mediated cell adhesion. However, most integrin receptors are
not specific to a particular ligand (e.g., ?3) and have affinity
toward a variety of ligands (or ligand combinations) and thus
their reputation as “promiscuous receptors”.68
Currently, EC have been shown to express at least 13 different
integrins, depending on their state of development, differentia-
tion, and function.69The migration of EPC to ischemic tissues
is believed to involve R4?1integrins, while ?1, Rv?3, and Rv?5
are involved in mediating the adhesion of progenitor cells during
endothelialization.20Furthermore, ?2has shown a significant
role in EPC homing to sites of ischemia and in neovascular-
ization.70Interestingly, R4integrins are absent in platelets, and
ligands aimed at interacting with these receptors may be of
promise for in situ endothelialization of vascular grafts.71
Apart from integrins, c-kit is another receptor expressed by
EPC that may be important for in situ endothelialization. Stem
cell factor (SCF) is a transmembrane protein that is a ligand
for c-kit and has been shown to be involved in EPC adhesion
to the endothelium.72,73CXCR2 is another receptor expressed
in EPC, which plays a key role in homing of EPC to sites of
injury and neovascularisation.74
4. EPC Mobilization
Circulating EPC are relatively low in abundance and,
therefore, for accelerated in situ endothelialization, it may be
possible to mobilize progenitor cells from the bone marrow and
increase circulating numbers.75HMG-CoA reductase inhibitors,
estradiol, peroxisome proliferator activated receptor gamma
(PPAR-γ) agonists, CXCR4 antagonists (T140 and AMD3100),
VEGF, erythropoietin, angiopoietin-1, granulocyte colony-
stimulating factor (G-CSF); GM-CSF, stromal cell-derived
factor-1,76-78hepatocyte growth factor,79leukemia inhibitory
factor, and interleukin-8 are some factors recognized to increase
the number of circulating EPC.80Factors that are involved in
the EPC proliferation and differentiation are equally significant.
Some recent experiments proving the effectiveness of certain
biochemical factors involved in mobilization, proliferation, and
differentiation of EPC and EC are summarized in Table 2. These
recognized, soluble, signaling proteins can be incorporated into
biomaterials for their controlled release or injected prior to graft
insertion to increase EPC numbers.
The action of these signaling factors depends upon their local
concentration, interaction with other signaling molecules (growth
factors, cytokines, chemokines etc), cell type, and activation
state of the cell. For example, VEGF is recognized for a rather
positive role in endothelialization81due to its role in promoting
EC proliferation and migration, but it is now emerging that
VEGF also induces undesirable IH, as shown when released
from ePTFE grafts.82,83
The soluble form of SCF, which is also expressed by EC is
known to be involved in EPC mobilization from the bone
marrow,72,73,84thereby suggesting SCF as a potential candidate
to be incorporated into vascular grafts or alternatively injected
in vivo to mobilize EPC to promote endothelialization (as shown
with G-CSF).85However, SCF is involved in inducing IH86and
SCF deficiency has been suggested to offer vascular protective
effects.87Precise control of SCF release may therefore be critical
for accelerated endothelialization, and if SCF proves to con-
tribute to rapid EPC mobilization and rapid endothelium
formation with its binding interactions with the c-kit receptor,
it may have an overall beneficial effect.
Integrins, as discussed previously, play a major role in
biomaterial-cell interactions. The R4 integrins are expressed
in colony-forming EPC, in bone marrow mononuclear cells.
Administration of anti-R4 integrin antibodies to downregulate
R4 integrin function has also been shown to increase EPC
mobilization from the bone marrow into peripheral circulation.88
Clearly, many of the peptides/factors identified above (CSF,
EPO, soluble SCF, and those listed in Table 2) for the
mobilization of progenitor cells from the bone marrow are also
involved in other physiological pathways such as inflammation75
and adverse effects have been reported in clinical trials (e.g.,
restenosis).89,90Precise control of release or a combinational
drug release approach and new EPC specific targets may greatly
enhance the clinical impact of this area of research.
Figure 3. Integrin receptors; reprinted from Hynes, R. O. Integrins:
Versatility, Modulation, and Signaling in Cell Adhesion. Cell 1992,
69, pp 11-25, with permission from Elsevier; http://www.sciencedirect.
com/science/journal/00928674.65Integrins are heterodimeric, trans-
membrane, and cell surface receptors. Cell specificity is determined
by variation of R- and ?-subunits. The outer extracellular domain is
involved in interacting with the molecules of the ECM ligands.
Accelerated in Situ EndothelializationBiomacromolecules, Vol. 9, No. 11, 2008
5. Techniques for Presenting and Delivering
Various approaches to immobilize, correctly present and
control the release of peptides/proteins (as detailed in Tables 1
and 2) from vascular grafts exist (Figure 2). The identification
of successful techniques to functionalize a particular material
is a challenge and is of great significance in designing grafts to
enhance endothelialization. Each protein/peptide attachment
technique needs to be tailor-made, considering the material,
protein/peptide of interest and also the anticipated interaction/
delivery of the attached moiety via appropriate spatiotemporal
kinetics. It is crucial to select suitable modification techniques,
as unoptimized material surfaces can promote a number of
undesirable effects, (e.g., free radical formation, inflammatory
response, fibrous encapsulation, etc.) and lead to graft failure.
Free radicals are linked to the material surface type and to
loosely adhered cells on a material. Thus, an unoptimally seeded
or formed endothelial layer on a synthetic polymer also has the
potential to induce free radicals.91
Peptides and proteins can be used for solid or soluble state
signaling and biomaterials modified accordingly.92Solid state
signaling often requires chemical modifications. Covalent
coupling enables peptides/proteins to be distributed uniformly
throughout the material or restricted to the surface. Table 3
details methods for covalently attaching proteins to materials
depending on available functional groups. Biomolecule distribu-
Table 2. Summary of Some Factors Involved in Controlling Cell Mobilization, Proliferation, and Differentiationa
factor/ applications applicationcell type/modelmodeloutcome
recombinant human G-CSF
injected before balloon
injury in the rat artery a
BM replaced with
also in wire-mediated
vascular injury produced in
the femoral artery of mice
darbe protein therapy in
patients and assessment
of CD34+circulating stem
stimulation with CCN1 in
vitro and in mouse model
to detect effect on EPC
BM-derived EPCin vitro
v circulating c-Kit+/Flk-1+
cells v re-endothelialization
also, v circulating EPC:
re-endothelialization and V
CD34, cSCs EPC from
patients with renal anemia
clinical and in vitro
v number of cSC and v EPC
in vitro and also C57BL/6
recruitment of CD34+
progenitor cells endothelial
v circulating rat EPC and
adhesiveness of cultured
v migration, and v
differentiation to mature
EC and also colonization
treated with simvastatin
BM-derived EPCin vitro
chemotaxis and migration
evaluated with a trans-well
culture system, adhesion
tested in dynamic and
EPO added to cultured EPC
from healthy subjects and
subjects with congestive
heart failure treated with
EPO also, recombinant
human EPO injected after
wire injury of the femoral
artery of mice and BM
replaced by GFP or
balloon-injured arteries of
athymic nude rats and
activated with MCP-1
mouse embryonic EPC in vitro
EPC in vitro
v in adhesion and
proliferation of EPC also, V
neointimal formation and v
v adherence of BM-MLCs
onto injured endothelium,
differentiation into EC-like
cells V neointimal
hyperplasia, no adhesion
of PB-derived CD34-/
v EC proliferation V cell
v cell adhesion, proliferation,
resisted shear stress
thrombo-resistant cell layer
v epithelial cell coverage
heparin and nonheparin-like
EC on cross-linked gels of
derivatized dextran, plain
HUVEC seeded on the FG
matrix with ECGF
ECGF (immobilized with
HUVEC in vitro
covalently attached to
VEGF variant, coupled to
fibrin via a
cleavage to release VEGF
ePTFE grafts implanted at
the level of the abdominal
human corneal epithelial
EPC (human umbilical cord
v EC proliferation, EPC
maturation into EC
male Lewis rat model
v endothelialization and
aKey: BM, bone marrow; BM-MLCs, bone marrow derived CD34-/CD14+monocyte lineage cells; ECGF, endothelial cell growth factor; EGF,172
epidermal growth factor; EPO, erythropoietin; FG, fibrin glue; GFP, green fluorescent protein; G-CSF, granulocyte colony-stimulating factor; HUVEC,
human umbilical vein EC; MCP-1, monocyte chemoattractant protein-1; VEGF, vascular endothelial growth factor. v ) increased, V ) decreased.
Biomacromolecules, Vol. 9, No. 11, 2008de Mel et al.
tion throughout the bulk of the engineered material would have
beneficial effects in cardiovascular grafts where these proteins/
peptides induce neovascularisation/angiogenesis throughout the
graft wall and, thus, support endothelium development. Endo-
thelialization on a vascular graft requires EC recruitment from
sites other than the anastomotic border and angiogenesis may
facilitates transinterstitial migration of EC.93
Surface adsorption of peptides/proteins is widely employed
for surface modification and has been shown to mediate cell
interactions with biomaterials.94Passive adsorption offers limited
control over the orientation of the ligands. Simple coating of
surfaces with ECM proteins, such as fibronectin or collagen,
proves to be less efficient compared with covalent grafting, and
a poly(carbonate-urea)urethane graft conduit covalently modified
with RGD and heparin showed better retention of EC when
compared to simple coating of the same proteins.41
6. Spatial Arrangements of Peptides
6.1. Microarchitecture and Nanofabrication. It is recog-
nized that cells are sensitive to microscale and nanoscale patterns
of chemistry and topography, and much recent attention has
been focused on nanoscale surface engineering to control the
density, orientation, and overall efficiency of peptides or protein
presentation.25,95Micro- and nanoscale grooves and patterns can
be used as a means of orientating cells, guiding cell migration,
and differentiation.96Topographically patterned poly(D,L-lactic
acid (PDLLA) surfaces have shown enhanced adhesion of
vascular cells compared to conventional, nonpatterned PLGA.97
Cell behavior is directly influenced by the surface structures
such as grooves, pits, or ridges and indirectly through the
differences in protein adhesion/conformation partly caused by
the chemical differences (e.g., surface charges) resulting from
the topographical structures.98The order (as opposed to
randomness) of nanoscale patterned surfaces has also recently
been shown to influence cell behavior.99The use of molecular
self-assembly to form nanometer-thick coatings offers a precise
method of surface engineering and has been reviewed.100In
our group, we use a nanocomposite polymer for small diameter
graft design, which has siloxane groups in the form of polyhedral
oligomeric silsequioxane (POSS). POSS rises to the surface of
the polymer101to cause nanotopographical changes that in turn
may be partly responsible for the antithrombogenic effects
including inhibition of factor X activity, increased clot strength,
clot lysis, and overall lower platelet adsorption.102
Many naturally derived biomaterials form nanoscale fibers
and can mimic natural self-assembly processes to form func-
tional polymers103in which assemblies are driven by weak
noncovalent bonds. When considering biological systems, which
are in contact with water, hydrophobic interactions are particu-
larly significant in driving the self-assembly of peptides,
proteins, or lipids.100Self-assembling peptides have shown
promise in nerve104,105and myocardial tissue engineering.106,107
Some osteogenic peptides such as ALK (osteogenic growth
peptide), DGR (osteopontin cell adhesion), and PGR (RGD
binding) have shown significantly enhanced proliferation and
osteogenic differentiation of MC3T3-E1 (mouse osteoblastic
cells).108Similar self-assembling biomaterials could possibly
be applied to promote endothelialization.
6.2. Other Techniques for Promoting Desirable Cell-
Vascular Graft Interactions. UV (γ ) 254 nm) irradiation is
a widely used surface modification technique to increase
polymer surface hydrophilicity. The technique has been shown
to increase EC adhesion and proliferation on a nanocomposite
polymer used in vascular graft fabrication.109On cross-linked
hyaluronan, in addition to endothelialization, UV irradiation has
been shown to decrease platelet activation with a corresponding
reduction of p-selectin expression, which is a marker of platelet
Certain shear stresses have also been shown (when applied
on seeded vascular grafts or as a preconditioning procedure) to
promote EC retention and endothelialization.111-113Shear stress
activates EC mechanotransduction signaling pathways which
cause changes in EC phenotype.114
7.1. Inhibiting Thrombogenicity (Prior to Endothelium
Formation). For long-term patency of vascular grafts, it is vital
to prevent undesirable cell and protein adhesion. To this end,
several functional moieties, PEG (polyethylene glycol),115-118
dextran,119and PEO (polyethylene oxide),120,121have been
incorporated onto polymers to achieve nonadhesive and non-
biofouling surfaces. This is particularly significant as the
adsorption of undesirable plasma proteins and cells on graft
surfaces can lead to inflammatory reactions and thrombus
formation. In addition to preventing inflammatory reactions,
these functional groups play a role in preventing thrombotic
Furthermore, nonadhesive molecules can be incorporated not
only to create a nonadhesive surface, but also applied in
combination with other ligands to promote cell-type specific
adhesion.123,124For example, dextrans are hydrophilic polysac-
charides which show a similar effect to PEG in regard to their
ability to decrease cell adhesion on biomaterial surfaces.
Dextrans, PEG, and PEO can also be chemically modified along
the polymer backbone with cell-selective peptides to promote
specific cell adhesion.125,126
To prevent thrombus formation and IH prior to neo-
endothelialization, various antithrombogenic agents have been
incorporated into graft surfaces.9,11,127Nitric oxide (NO),
Table 3. Chemical Modifications for Covalent Coupling of
Proteins/Peptides to Polymers (Modified from Hirano and
reactive groups on
hydrazine and nitrous acid
aKey: Single letter codes for amino acids, which comprise the peptides
are G, Gly; A, Ala; L, Leu; M, Met; F, Phe; W, Trp; K, Lys; Q, Gln; E, Glu;
S, Ser; P, Pro; V, Val; I, Ile; C, Cys; Y, Tyr; H, His; R, Arg; N, Asn; D,
Asp; T, Thr; DCC, dicyclohexylcarbodiimide; DMT-MM, 4(4,6-dimethoxy-
1,3,5-triazin-2-yl)-4-methylmorpholinium chloride; EDC, 1-ethyl-3-(3-di-
methylaminopropyl) carbodiimide hydrochloride.
Accelerated in Situ EndothelializationBiomacromolecules, Vol. 9, No. 11, 2008
which is produced by the EC, is found to be effective in
preventing the activation and adhesion of leucocytes, limiting
platelet aggregation and thrombus formation and preventing
vascular smooth muscle cell proliferation,128thus improving
graft patency. Various polyurethane grafts have been modified
to release NO, with some promising in vitro results in terms
of preventing platelet adhesion and activation.129L-Arginine,
a NO precursor, has been used to coat external jugular veins
and grafted onto contralateral carotid artery of rabbits,130
while spermine/NO, which was applied on balloon injured
arteries of rats131and also in a rabbit model132has been
reported to reduce neo IH. Among various NO donors,
diazeniumdiolates133-135and s-nitrosothiols136,137are two
common groups that have been incorporated into biomaterials
for controlled release of NO.
7.2. Platelets: Friend or Foe. In vascular graft engineering
the prevention of platelet adhesion, platelet activation, and
thrombosis are driving forces for in situ endothelialization.
Among many other detrimental roles, it has also been reported
that platelets enhance the differentiation of CD34+cells to
become foam cells and, therefore, potentially induce vascular
disease.138However, somewhat surprisingly, platelet activa-
tion is necessary for EPC homing and the differentiation of
CD34+cells into EC and therefore promote endothelializa-
tion.139The peptide SFLLRN has been found to be important
in expanding EPC ex vivo by activating the thrombin receptor
PAR-1,140suggesting that SFLLRN may also promote EPC
mobilization in vivo if released from vascular implants.
Platelets therefore appear to have a dual role and manipulation
of the positive effects could potentially aid accelerated in
8. Future Challenges
Various approaches to promote endothelialization on graft
surfaces have been explored with particular emphasis on the
incorporation and presentation of cell ligands (peptides/proteins)
on the graft surface to promote cell adhesion and direct
differentiation. However, when considering the induction of in
situ endothelialization of vascular grafts, it is also important to
consider factors involved in EC/EPC mobilization, the number
of circulating EC/EPC and homing of these cells to the vascular
If recognized proteins/cytokines, such as EPO and SCF, are
to be used with grafts, there needs to be optimization of methods
to enable the presentation of relatively large proteins in such a
manner as to retain the requisite biological functionality in shear
stress environments over a time frame that can induce endot-
helialization while avoiding other undesirable effects. The
orientation, geometric arrangement, and density of peptide
presentation are vital for determining the desired cellular
While there is rapid progress in the understanding of vascular
progenitor cell biology, there is still much to be learned.141,142
These gaps in understanding of biology, active pathways, and
functioning of vascular progenitor cells also contribute to the
challenge of designing grafts, which could selectively bind cells
to promote rapid endothelialization without thrombotic com-
plications. Furthermore, there is currently a relative lack of
suitable in vivo models to prove the efficacy of biofunctionalized
biomaterials on endothelialization. Indeed, animal models have
shown spontaneous and rapid endothelialization on graft materi-
als, which is not a true reflection of endothelialization that occurs
in humans with cardiovascular disorders. Use of older animals
has been considered a solution.14Flow circuit studies that
simulate hemodynamic blood flow have also proved to offer a
good representation of vascular graft-circulating cell interac-
The controlled release and presentation of biologically
relevant motifs or signals should be such as to maximize
endothelialization, while minimizing thrombogenicity and other
systemic complications, while any detrimental effects of surface
modification procedures on mechanical properties of the polymer
graft such as in burst strength and overall compliance must also
be carefully considered.143
EPC adhesion and the endothelialization process to some
extent resemble an injury response. For example, the mech-
anisms associated with platelets and c-kit and, thus, endot-
helialization promoted by certain proteins appears to be
coupled with thrombosis and IH. A major challenge is to
manipulate the delicate balance between promoting in situ
endothelialization, while inhibiting thrombosis and IH. In situ
endothelializable grafts need to be protected during the time
of implantation and complete endothelialization, probably up
to 6 weeks, and, among such protective mechanisms, NO
elution is of keen interest.
Vascular graft in situ endothelialization has gained con-
siderable recent attention as a means of increasing graft
patency and to improve the biological function of grafts by
inhibiting thrombosis and IH. Current research has identified
many peptides/proteins and various factors and numerous
techniques that could be used for biofunctionalization of
biomaterials. This is a dynamic and rapidly evolving field,
where there is a critical clinical need for “off the shelf” grafts,
which promote accelerated, spontaneous, in situ endothelial-
Amino Acid Abbreviations
G ) glycine
A ) alanine
L ) leucine
M ) methionine
F ) phenylalanine
W ) tryptophan
K ) lysine
Q ) glutamine
E ) glutamic acid
S ) serine
P ) proline
V ) valine
I ) isoleucine
C ) cysteine
Y ) tyrosine
H ) histidine
R ) arginine
N ) asparagine
D ) aspartic acid
T ) threonine
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