IV. Incorporating Angiogenic Growth Factors Into The Tissue

A Systems Biology Approach to Promoting
Angiogenesis in a Tissue Engineered Scaffold
Mark Butler
Abstract—Vascularization of a tissue engineered construct is
critical to its long term viability in vivo. The formation of new
blood vessels, angiogenesis, is a key design parameter in the
development of a tissue engineered scaffold. The process of
angiogenesis results from a complex cascade of events involving
endothelial cell activation, migration and proliferation,
organization into immature vessels, association of mural cells
(pericytes and smooth muscle cells) with the immature vessels,
and matrix deposition as the vessels mature. Using systems
biology, the mechanisms and pathways required for each step in
the process of angiogenesis are being delineated. With this
understanding, more advanced polymeric scaffolds containing
angiogenic growth factors can be used to promote vascular
Index Terms—Tissue Engineering, Angiogenesis, Scaffold,
Systems Biology.
issue Engineering is an emerging field that involves the
development of a new generation of materials capable of
specific interactions with biological tissues. Combining
novel materials with living cells, the hope is to create a fully
functioning tissue. This is particularly useful in the realm of
organ tissue replacement where there is a limited availability
of donor organs or where, in some cases, (e.g., nerves) no
natural replacements are available. In engineering artificial
organs, a scaffold design is typically used. This involves
taking a small number of cells, perhaps from the patient, and
allowing them to grow on a three-dimensional porous scaffold
in vitro. With subsequent transplantation in vivo the tissue
would continue to grow, the scaffold would degrade, leaving
behind a fully functioning organ within the patient. The
scaffold design is thus key to the tissue engineered construct.
The scaffold must be biocompatible and degradable within the
body. It must be highly porous to allow the seeding of cells in
a three-dimensional space and allow the transport of oxygen
and nutrients to all cells through the development of a vascular
network. The lack of vascularization, however, has been the
pitfall of many tissue engineering designs resulting in the
failure of long-term survival of the implanted tissue [1-2].
When cells are first transplanted into a host, on a threedimensional porous scaffold, the area within the scaffold is
avascular and the transplanted cells are dependent on the
diffusion of nutrients and waste for survival [3]. This
diffusion limitation is adequate if the tissue is small (<1 mm
thick) or the cells’ metabolic needs are low. But in the case of
engineering an artificial organ, such as the liver, most cells
will die soon after transplantation because of mass transport
limitations [3]. Blood vessels are naturally brought into the
scaffold by the invasion of surrounding tissue, but this alone is
usually insufficient to provide necessary nutrients to cells
within the scaffold. It is thus necessary to induce the
formation of additional blood vessels in these porous matrices
to enhance the long-term survival of transplanted cells [3].
The hypothesis here is that using a systems biology
approach to understanding angiogenesis, the formation of new
blood vessels, the scaffold could be designed to encourage
blood vessel development. Angiogenic growth factors could
be incorporated into the scaffold chemistry, triggering
localized vessel recruitment from the surrounding tissue in the
host. Many research groups have shown the feasibility of
using growth factors to promote blood vessel growth and
growth factors have already been used successfully to promote
angiogenesis in cases of myocardial ischemia [4]. This is the
result of much progress made in the understanding of blood
vessel growth and development. A full systems understanding
of the process of angiogenesis, however, would provide more
tools for the researcher. A myriad of growth factors, released
at key time points in blood vessel development is likely
necessary for the sufficient vascularization of an artificial
Angiogenesis is defined as the growth of new blood vessels
from a preexisting microvascular bed [5]. Blood vessels can
be categorized into two classes: a) those lacking vascular
smooth muscle cells (eg. Capillaries) and b) larger vessels with
a muscular coat. Endothelial cells line the blood vessels and
are involved with the development of new vessels [5]. The
endothelial cell layer inside blood vessels remains dormant
until activated by a stimulus, such as hypoxic conditions or
growth factors. The process of angiogenesis occurs in the
following steps as outlined in Fig. 1. [5-6]:
1. Angiogenic growth factors are produced.
2. Growth factors bind to specific receptors located on the
endothelial cells (EC) of nearby preexisting blood vessels.
3. EC are activated once growth factors bind to their
receptors. Upon activation, EC produce enzymes.
4. Enzymes dissolve tiny holes in the basement membrane
surrounding all existing blood vessels.
5. EC proliferate and migrate out through the holes of the
existing vessel.
6. Specialized molecules called adhesion molecules, or
integrins (v3, v5) act like hooks to help pull the new blood
vessel sprout forward.
7. Enzymes such as matrix metalloproteinases (MMP) are
produced to dissolve the tissue in front of the sprouting vessel
tip in order to accommodate it. As the vessel extends, the
tissue is remolded around the vessel.
8. Sprouting EC roll up to form a blood vessel tube.
9. Individual blood vessel tubes connect to form blood vessel
loops that can circulate blood.
10. Finally, newly formed blood vessel tubes are stabilized by
specialized muscle cells (smooth muscle cells, pericytes) that
provide structural support.
migration and tube formation of endothelial cells and also
induces in these cells the expression of many other factors and
enzymes needed in the process of angiogenesis such as
urkinase-type plasminogen activator (uPA) and plasminogen
activator inhibitor-1 (PAI-1) [7]. In vivo, VEGF has been
shown to regulate vascular permeability, which is important
for the initiation of angiogenesis [7]. Hypoxic conditions, like
those found in ischemic tissues, have been found to trigger the
upregulation of VEGF from a variety of cell types to stimulate
the expression of VEGF receptors on endothelial cells [5].
Two VEGF receptors have been isolated and characterized on
endothelial cells. These are Flt-1 and Flk-1. It is thought that
the Flk-1 receptor bound with a VEGF molecule controls both
endothelial cell proliferation and the permeability of the vessel
[5]. The angiogenic actions of VEGF are depicted in Fig. 2.
Fig. 2. Actions of vascular endothelial growth factor VEGF
during angiogenesis [5]
6) pericyte recruitment
Fig. 1. Stages of Angiogenesis
Many growth factors have been found to initiate and control
the process of angiogenesis. A growth factor is defined as an
extracellular protein present in minute concentrations in vivo
that activates target cells through receptor binding [5]. In a
system’s biology sense these growth factors can be thought of
as “on/off” switches to control the mechanism of angiogenesis
discussed above. Although the complete complex mechanism
of angiogenesis has not been delineated, many of the growth
factors or “on” switches to promote angiogenesis have been
discovered. These include Vascular Endothelial Growth
Factor (VEGF), Fibroblast Growth Factor (FGF), PlateletDerived Growth Factor (PDGF) and Transforming Growth
Factor (TGF).
Vascular Endothelial Growth Factor (VEGF) is a major
regulator in the vascular system. In vitro, VEGF is involved in
the degradation of the extra-cellular matrix, proliferation,
The Fibroblast Growth Factor (FGF) family of proteins is
made up of at least 19 members. The two most important
members are acidic FGF (aFGF) and basic FGF (bFGF)
because these have the most angiogenic potential [5]. Acidic
and basic FGF are heparin binding monomers and are usually
found in association with the extracellular matrix. FGFs bind
to transmembrane tyrosine kinase receptors, named FGFR-1
through FGFR-4, which express overlapping binding patterns
of the FGFs. There are several pathways that FGFs act to
produce their mitogenic stimulus.
FGFs stimulate the
expression of proteases such as uPA and collagenase in
endothelial cells [5]. Basic FGF is known to stimulate
endothelial cell proliferation and migration and is also
produced by endothelial cells to recruit pericytes [5]. The
angiogenic actions of FGF are depicted in Fig. 3.
Fig. 3. Actions of acidic and basic fibroblast growth factors
during angiogenesis [5]
Platelet-derived growth factors (PDGF) are synthesized by
platelets, endothelial cells, and smooth muscle cells. When
PDGF is expressed by endothelial cells it acts as a mitogen for
the underlying smooth muscle cells which subsequently
promote smooth muscle cell expression of VEGF. PDGF
secreted from endothelial cells is also thought to be involved
in the recruitment of pericytes and the differentiation of
endothelial cell precursors [5].
factor into the scaffold with success. Perets et al (2002) found
a four-fold increase in the number of penetrating capillaries
into their bFGF-releasing scaffolds compared to the control
scaffold [8]. Using a VEGF secreting scaffold, Peters et al.
(2001) found a 160% increase in capillary density in their
scaffolds compared to the control [9]. These results suggest
that a polymer-based delivery system can be used engineer
more extensive vascular networks and to create a better
environment for the survival and proliferation of transplanted
cells. These single growth factor models are not, however,
perfect. It was observed by Perets et al. (2002) that there was
minimal capillary ingrowth into the pores in both the bFGF
loaded and control scaffolds [8]. This is not surprising as
angiogenesis is not governed by one single growth factor. A
large number of growth factors have been identified, each with
a particular role in the complex mechanism of angiogenesis.
Richardson et al. (2001) developed a novel system for dual
growth factor delivery in a polymer scaffold [10]. They
incorporated both VEGF and PDGF into their scaffolds by
simply mixing the VEGF and pre-encapsulating the PDGF
before mixing with the polymer (Fig. 4). VEGF is a well
known initiator of angiogenesis, but its presence is often not
sufficient for the formation of complex and mature
vasculature. PDGF promotes the maturation of blood vessels
by the recruitment of smooth muscle cells to the endothelial
lining of the growing vessels. The incorporation of both of
these growth factors coupled with the fact that the simple
mixing of VEGF gave a fast time release (to initiate
angiogenesis) while the pre-encapsulation gave a longer
release time of PDGF (to help maturation of vessels) resulted
in the rapid formation of a mature vascular network in the
scaffold [10].
D. TGF-β
Transforming Growth Factor β (TGF-β) acts as a stimulus
for angiogenesis through many pathways. Although the
complete mechanism is unknown, TGF-β induces the
expression of several angiogenic growth factors, acting
through paracrine signaling [5]. TGF-β has been shown to
upregulate the expression of PDGF in endothelial cells as well
as VEGF and bFGF expression by smooth muscle cells [5].
The most critical challenge that must be overcome to tissue
engineer scaffolds for artificial organs is how to induce
vascularization. One possible solution is the incorporation of
angiogenic growth factors, such as VEGF or FGF discussed
above, into the polymeric scaffold. Upon implantation, these
growth factors would be released from the polymeric scaffold
and trigger an angiogenic response.
Many studies have looked at incorporating a single growth
Fig. 4. Schematic of scaffold fabrication process used by Perets et al.
(2002) [10]. Growth factors were incorporated into polymer scaffolds by
either mixing with polymer particles before processing into scaffolds
(VEGF), or pre-encapsulating the factor (PDGF) into polymer
microspheres used to form scaffolds. The VEGF incorporation approach
results in the factor being largely associated with the surface of the
polymer, and subject to rapid release.
In contrast, the PDGF
incorporation approach is predicted to result in a more even distribution
of factor throughout the polymer, with release regulated by the
degradation of the polymer used to form microspheres [10].
An optimum scaffold would release many growth factors
with independent kinetics so that different factors could act at
distinct stages of vascular development. This could be
accomplished through the mode of incorporation of the growth
factor in the scaffold. Growth factors needed to initiate
angiogenesis could be simply mixed with the polymer to give
rapid release kinetics (eg. VEGF). Other growth factors
necessary at intermediate time points in the mechanism of
angiogenesis (eg. PDGF) could be encapsulated in polymers of
varying chemistries with different degradation times to give
the appropriate release kinetics. Researching polymeric
systems for release of multiple growth factors will therefore
become of high importance to the tissue engineer.
A polymeric system capable of releasing multiple factors
with distinct kinetics is useful only if key growth factors have
been identified. Although much research has been conducted
on understanding the mechanism of angiogenesis, a full
systems understanding would provide an exhaustive list of all
growth factors necessary and present at all time points during
vascular development.
Understanding the relationships
between growth factors and the pathways that they follow
during angiogenesis would allow the design of a scaffold that
closely mimics the signals and conditions found in vivo during
This would ultimately improve the
vascularization of the tissue engineered construct and render
feasible the possibility of engineering large artificial organs.
The systems biology approach to studying angiogenesis
would involve systematically perturbing the system. Perhaps,
the system could be taken as the vascularization of a
developing embryo, which could be genetically modified to
eliminate or replace the gene responsible for producing the
VEGF growth factor. How have the gene, protein, and
informational pathway responses changed due to this
perturbation? Taking this information, a mathematical model
that describes the structure of the VEGF pathway in
angiogenesis and how it responds to individual perturbations
could be established. Performing these types of experiments
would help to piece together the master plan that dictates the
overall process of angiogenesis. Hopefully, key nodes in the
network would be identified where perturbations have a
profound effect on the system. This knowledge would be
invaluable when designing a polymeric scaffold to help guide
the development of vasculature in a tissue engineered
The success of engineering large artificial organs is a dream
that has been limited by the lack of vascularization in porous
polymeric tissue engineered constructs. One solution to
promote angiogenesis is to incorporate angiogenic growth
factors into the polymeric scaffold that would be released as
the scaffold degrades. A systems biology approach to
angiogenesis would lead to a better understanding of the
mechanism of angiogenesis including all growth factors and
pathways involved. Many growth factors key to the process of
vascularization have been identified. These include VEGF,
FGF, PDGF and TGF. Incorporating a single growth factor
into a scaffold has been shown to have a successful angiogenic
response. This suggests that a polymer-based delivery system
can indeed be used engineer more extensive vascular networks
and to create a better environment for the survival and
proliferation of transplanted cells.
The process of
angiogenesis is governed not by a single growth factor,
however, but by a cascade of growth factors occurring in a
certain temporal sequence. A polymeric system capable of
releasing multiple growth factors with distinct kinetics is
needed to improve vascularization of the tissue engineered
construct and make the availability of large artificial organs a
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