Experimental Approaches To Constructs

Journal of Biomaterials Science, Polymer Edition
ISSN: 0920-5063 (Print) 1568-5624 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsp20
Experimental approaches to vascularisation within

tissue engineering constructs
Md. Sarker, X.B. Chen & D.J. Schreyer
To cite this article: Md. Sarker, X.B. Chen & D.J. Schreyer (2015) Experimental approaches to
vascularisation within tissue engineering constructs, Journal of Biomaterials Science, Polymer
Edition, 26:12, 683-734, DOI: 10.1080/09205063.2015.1059018
To link to this article: http://dx.doi.org/10.1080/09205063.2015.1059018
Accepted author version posted online: 08

Jun 2015.
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Date: 10 March 2016, At: 23:07
Journal of Biomaterials Science, Polymer Edition, 2015

Vol. 26, No. 12, 683734, http://dx.doi.org/10.1080/09205063.2015.1059018
REVIEW ARTICLE
Experimental approaches to vascularisation within tissue engineering
constructs
Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.
Md. Sarkera*, X.B. Chena,c and D.J. Schreyera,b

a
Division of Biomedical Engineering, College of Engineering, University of Saskatchewan, 1A26,
57 Campus Drive, Saskatoon, SK, S7N 5A9, Canada; bDepartment of Anatomy and Cell Biology,
College of Medicine, University of Saskatchewan, 5800 Saskatoon City Hospital, Saskatoon, SK,
S7K 0M7, Canada; cDepartment of Mechanical Engineering, College of Engineering, University
of Saskatchewan, 3B47, 57 Campus Drive, Saskatoon, SK, S7N 5A9, Canada
(Received 26 March 2015; accepted 2 June 2015)

Tissue engineering opens up a new area to restore the function of damaged tissue or
replace a defective organ. Common strategies in tissue engineering to repair and
form new tissue containing a functional vascular network include the use of cells,
growth factors, extracellular matrix proteins, and biophysical stimuli. Yet, formation
of well-distributed, interconnected, and stable vascular network still remains
challenging. In addition, anastomoses with host vasculature upon implantation and
long-time survival of the new blood vessel in vivo are other critical issues to be
addressed. This paper presents a brief review of recent advances in vascularization
in vitro as well as in vivo for tissue engineering, along with suggestions for future
research.
Keywords: vascularization; angiogenesis; vasculogenesis; biomaterial; growth
factors; scaffold
1. Introduction
A major challenge in the creation of new tissues by tissue engineering is the limitation
of diffusional mass transfer into new tissue having a thickness of more than
100200 m,[1] causing ischemia and cell death. Vascularization overcomes the limit
of diffusional mass transfer for delivery of various types of nutrients, oxygen, growth
factors, biochemical signaling factors, and removal of carbon dioxide, and metabolic
waste.[2] The 100200 m limit regulates the critical distance necessary between two
successive capillaries to prevent an ischemic condition.[3] This issue must be
considered in any experimental or therapeutic effort to regenerate functional tissue of
appreciable size using a tissue engineering approach.
Two mechanisms are responsible for the generation of a vascular network in vivo:
vasculogenesis and angiogenesis. Vasculogenesis generally takes place at an embryonic
stage, while angiogenesis occurs mostly in postnatal life.[4,5] The term vasculogenesis
denotes the assembly of endothelial progenitor cells (EPCs) that eventually form capillaries and a vascular network [6] in response to various growth factors, then undergoes
remodeling to form functional vasculature. Angiogenesis refers to the sprouting of new
blood vessels from existing ones due to specic angiogenic signals.[4] Engineered
*Corresponding author. Email: mas921@mail.usask.ca
2015 Taylor & Francis
684
Md. Sarker et al.
tissue can be vascularized by either process, but must ultimately result in anastomosis
with host vasculature.
2. Different types of blood vessels and their functions

The vascular system is a geometrically complex network consisting of arteries that
supply blood to tissues, and veins that remove it (Figure 1). Major arteries branch out
into successively smaller arterioles, metarterioles, and arterial capillaries. Capillaries
combine downstream to form venules, and then major veins.
Arterioles typically have diameters in the range of 10300 m and are composed of
concentric layers of smooth muscle cells (SMC), basement membrane and endothelial
cells (ECs).[7] A metarteriole is a short vessel which branches out from an arteriole
with a smaller diameter. Generally, a metarteriole consists of only two layers; SMCs
surrounding ECs. At the point where capillaries branch out from metarterioles, there
exists a loop of SMCs called the precapillary sphincter. The function of precapillary
sphincter is to control the blood ow to the capillary bed and to ensure enough time to
facilitate diffusion and other mass transfer mechanism between capillaries and nearby
cells. To keep the blood ow continuous, a bypass vessel called the thoroughfare channel connects the metarteriole with the venule. The capillaries consist only of ECs having an outer layer of protein-based basement membrane. However, pericyte cells, which
are responsible for paracrine signaling and capillary blood ow, can be found within
the capillary basement membrane. The capillaries are also known as microvessels, and
have a diameter of 410 m. The transition from capillary to venule takes place gradually and the approximate diameter of post-capillary venule is 1050 m. Post-capillary
venules then fuse with a connecting venule whose diameter is 50300 m.[8]
Capillaries are the principle site of mass transfer. The difference between the
pressure of blood within the capillaries and the interstitial uid pressure causes
the movement of uid into or out of capillaries. Generally, three types of capillaries are
Figure 1.
Blood circulatory system inside the body (Reproduced from [288]).
Figure 2.
685
Structure of different types of capillaries (Reproduced from [288]).
seen in the body: continuous, fenestrated and sinusoidal (Figure 2). Most capillaries
within the body are the continuous type. These have tight junctions as well as intercellular clefts between the ECs, and are covered with basement membrane. Water, gas
molecules, ions, and other water soluble molecules can pass in or out of continuous
capillaries through diffusion, vesicles, and intercellular clefts. However, high molecular
weight molecules are transported across ECs by pinocytosis. Fenestrated capillaries
have 6080 nm pores in their ECs and are covered with basement membrane.
Fenestrated capillaries carry out mass transfer through diffusion, vesicles, intercellular
clefts, and pores. This type of capillary is found within the endocrine glands, intestines,
pancreas, and kidneys. Sinusoidal capillaries are leaky in nature and have 3040 m
pores, much larger than those seen in fenestrated capillaries. Sinusoidal capillaries have
incomplete basement membrane covering and more intercellular clefts compare to other
capillaries. White and red blood cell and serum protein can pass through the walls of
sinusoidal capillaries. Generally, sinusoidal capillaries are found in bone marrow, liver,
spleen, and adrenal gland.[9]
3. Mechanisms of blood vessel formation in vivo
3.1. Vasculogenesis
EPCs from bone marrow or blood are mainly responsible for pre- and post-natal vasculogenesis through a complex series of steps (Figure 3). At the initial stage, EPCs move
from their source and enter unvascularized tissue in response to chemo-attractant factors, such as granulocytemonocyte colony stimulating factor, granulocyte colony
stimulating factor, vascular endothelial growth factor (VEGF), basic broblast growth
factor (bFGF), placental growth factor, erythropoietin and stromal cell-derived factor-1
(SDF-1).[10] Moreover, factors secreted from ischemic tissue cause nitric oxide (NO)
production that activates extracellular proteases, especially matrix metalloproteases-9
(MMP-9), and facilitates EPC migration.[11]
Homing of EPCs into ischemic tissue site requires chemotaxis, adhesion, and transendothelial migration. After moving out from bone marrow traveling through blood
vessels, EPCs undergo chemotaxis toward a particular ischemic region in response to
gradients of chemokines, such as SDF-1, interleukin-8 (IL-8), growth-regulated oncogene-, and CC chemokine.[12,13] The chemokines also stimulate EPCs to promote
adhesion with the inner layer of blood vessels. EPCs pass through the endothelial lining
686
Figure 3.
Md. Sarker et al.
Blood vessel formation by vasculogenesis (Reproduced from [289]).
of blood vessels by a specic transendothelial migration mechanism. Once at the basement membrane, EPCs initiate their invasion mechanism by rupturing it and remodeling
basement membrane proteins, as well as the extracellular matrix (ECM) of the interstitial space, using extracellular proteases, such as MMP-9, cathepsin L, urokinase-type
plasminogen activator, and tissue-type plasminogen activator, to reach the ischemic tissue site.[14,15] Then EPCs accumulate at the ischemic site; form a vascular pattern by
differentiating into ECs and interacting with existing ECs and ECM. EPCs attached to
the ECM proliferate under the inuence of VEGF, immunoglobulin, and epidermal
growth factor (EGF),[16,17] and differentiate due to monocyte chemoattractant protein1, insulin-like growth factor-1 (IGF-1), SDF-1, VEGF, and platelet-derived growth
factor (PDGF).[18,19]
3.2. Angiogenesis
During angiogenesis, new capillaries sprout from existing blood vessels in response to
various biochemical signals and expand into ischemic tissue following tracks, gradients,
or attractive or repulsive signals. Any unnecessary vascular plexus caused by random
sprouting undergoes remodeling according to the requirements of specic tissue.
687
Functional non-uniformity of ECs along the blood vessel wall caused by VEGF
gradients plays a critical role in angiogenic sprouting. Mechanistically, specic ECs
respond to a chemo attractant by extending as tip cell, then propagating in different
directions by forming stalk-like structure, which eventually acquire a lumen.[20,21] To
communicate with the surroundings, tip cells extend multiple lopoida which are sensitive to various guidance molecules and specic growth factors, such as VEGF-A, FGF,
angiopoietin-1 (Ang-1), EGF, and IL-8. The spatial as well as temporal distributions of
these factors are very crucial for guiding the growth of new capillaries within the
ischemic tissue.[22] For example, a VEGF-A gradient in tissue plays an important role
for lopodia extension from the tip cell, and the corresponding receptor for VEGF-A is
VEGFR-2, which is expressed by the tip cell.[23] Angiogenesis in ischemic or newly
formed tissue is a complicated process which can be demonstrated through some steps
(Figure 4).
At the beginning, ischemic tissue secretes VEGF which dilates the existing blood
vessel through the redistribution of intercellular adhesion molecules as well as modication of the cell membrane structure. It also increases vascular permeability with the
help of NO [24,25] and assists the relocation of plasma protein to form temporary
structures to facilitate EC migration. Besides VEGF, Ang-2 contributes to angiogenic
sprouting through the degradation of ECM protein and the removal of SMCs from the
outer surfaces of capillaries.[25,26] Degradation of basement membrane and ECM are
vital for EC migration, while microvascular ECs themselves secrete some matrix metalloproteases, such as MMP-2, MMP-3, MMP-9, along with protease inhibitor mainly
tissue inhibitor, of metalloproteinase-2, which coordinately regulate basement membrane and ECM degradation [27] by causing various spatial and temporal factors to
Figure 4.
Blood vessel formation by angiogenesis in brain cancer (Reproduced from [290]).
688
Md. Sarker et al.
generate non-uniformity along the wall of blood vessel.[28,29] In addition, another

important feature of extracellular proteolytic degradation is the release of some growth
factors, such as IGF-1, VEGF, and FGF-2, which also facilitate angiogenic
sprouting.[30]
The progression step is associated with the migration and proliferation of ECs due
to the inuence of VEGF, angiopoietins (Angs), FGFs, and PDGF. Among them, Ang1 regulates the interaction between ECs and periendothelial cells during migration,
[25,31] FGF facilitates EC growth via the recruitment of mesenchymal cells,[32] and
PDGF enhances EC growth through the recruitment of pericytes and SMCs.[33] In
addition, other factors involved in EC proliferation and migration include neuropeptides, IGF-1, erythropoietin, hepatocyte, and interleukins.[34,35]
After migration and proliferation of EC into ECM, ECs eventually differentiate to
form cord-like structure and a lumen starts developing due to the inuence of VEGF
and Ang. Interestingly, Ang-1, VEGF121, and VEGF165 help to increase lumen
diameter, while VEGF189 decreases lumen diameter.[36]
4. Tissue engineering approaches to blood vessel formation
Well-distributed and interconnected vascular network is required to maintain the viability of large cell population in engineered tissue. While tissue engineering is eventually
emerging as a potential approach to regenerate tissue and organ, formation of 3D
vascular network within the neotissue still remains challenging. Current strategies to
form complex vascular channel within engineering construct or growing tissue can be
categorized as scaffold-based and scaffold-free approaches (Figure 5). Strategies
(Table 1) associated with scaffold-based approaches are, construction of 3D matrix with
appropriate biomaterial by biofabrication technique or stacking multiple micropatterened thin planar surfaces or micro tissue modules, engineering new tissue with
decellularized matrix, functionalization of scaffolds with cells, angiogenic factors (AFs)
or proteins, transfection of seeded cells by gene therapy to obtain sustained release of
AFs and prevascularisation to maintain viability of the seeded cells. In contrast, in scaffold-free approach cell sheets are assembled to promote vascularized tissue.
4.1. Scaffold-based aproaches
4.1.1. Scaffold fabrication biomaterial
The choice of matrix biomaterial has profound impact on scaffold vascularization in tissue engineering. Like natural ECM, the biomaterial should facilitate vascular cell
biomaterial interactions, which would activate numerous signaling pathways to promote
vascular cell survival, proliferation, differentiation, and migration. A tissue scaffold
experiences different types of force upon implantation in vivo, such as compression,
shear, torsion, and tensile force. Therefore, the biomaterial should have sufcient
mechanical strength to sustain those forces to keep the scaffold architecture intact.
Moreover, controlled biodegradability is desired so that over time, the scaffold could
maintain its mechanical strength while the biomaterial is replaced with a vascular network and newly formed tissue. Furthermore, cytotoxicity has to be considered for its
impact on implanted or host cell survival in vivo or during preparation in vitro.
Generally, polymers are used as biomaterials for the fabrication of scaffolds.[37]
Biopolymers can be classied into three categories in terms of preparation or source:
synthetic, natural, and hybrid.
Figure 5.
689
Different approaches for vascular network formation.
4.1.1.1. Synthetic polymer. Synthetic polymers used for tissue engineering offer
scaffold fabrication exibility while maintaining desired mechanical properties of the
engineering construct. However, lack of cell adhesion peptides in the molecular structure of synthetic materials limits their application for vascular tissue engineering. A
number of biodegradable, biocompatible, and non-toxic synthetic polymers have shown
potential for tissue engineering applications. Among them, polyglycolic acid (PGA),
polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), poly-L-lactic acid (PLLA),
poly--caprolactone (PCL), polyethylene glycol (PEG), and polyhydroxyalkanoates
(PHAs) have frequently been explored in different studies of tissue-engineered graft
vascularization.[38]
When exposed to pulsatile conditions, PGA mesh tube scaffolds seeded with
porcine carotid SMCs were eventually replaced by blood vessel-like structures while
maintaining uniform SMCs distribution and collagen content close to that of native vessels.[39] In vivo experiments have also shown PGA scaffolds as promoters of vascularization. PGA scaffolds seeded with bovine artery cells degraded completely after
11 weeks of implantation in the pulmonary artery of sheep and were replaced by new
tissue having 73.9% collagen content with respect to native tissue.[40] Similarly, in an
in vivo study, PEG diacrylate hydrogel was implantated subfascially into SpragueDawley rats and kept in vivo for three weeks. Interestingly, even in the absence of
incorporated growth factors, vascularized tissue ingrowth was reported throughout the
entire PEG gels having pore size 50150 m.[41]
Derivatives of PLA (polymer of lactide) have also shown their angiogenic potential
in different studies. For example, in vivo implantation of tubular scaffold fabricated
690
Md. Sarker et al.
Table 1.
Tissue engineering approaches to blood vessel formation.
Approaches
Description
A.
Scaffold based approach
A.1. Scaffold fabrication
Synthetic polymers (e.g. PGA, PLA, PLGA, PLLA, PCL, PEG,
biomaterial
and PHAs); natural polymers (e.g. collagen, bronectin, brin,
elastin, silk broin, HA, alginate, chitosan); decellularized matrix
(e.g. allogenic or xenogenic tissue or organ graft)
A.2. 3D printing
3D Fabrication (e.g. 3D plotting, inkjet printing, laser-based
approaches
biofabrication and stereolithography) technique to fabricate
complex vascular network; microfabrication (e.g. microuidics,
micromolding, lithographic, direct laser write) technique to indent
micro pattern on polymer or cell seeded hydrogel to allow
complex network formation at micro level; modular assembly of
patterned micro modules generated from self-assembled
aggregation, microfabrication of cell seeded hydrogels, or direct
printing method to form macro tissue assembled by random
packing, stacking of layers or directed assembly
A.3. Addition of cells
Autologus mature ECs (e.g. human umbilical vein, human dermal
microvascular, human vascular), stem cells (e.g. embryonic stem
cell, MSC), EPCs (e.g. peripheral blood derived, umbilical cord
blood derived), vascular wall cells (e.g. pericytes, vascular SMCs,
pulmonary artery SMCs)
A.4. Addition of growth
VEGF, FGFs, PDGF, Angs, TGF-
factors
A.5. Gene therapy
Viral vectors (e.g. retrovirus, lentivirus, adeno-virus and adenoassociated virus) and nonviral vectors (e.g. plasmid DNA)
A.6. Prevascularisation
In vitro or in vivo culture prior to implant to form vasculature and
maintain cell viability
B.
B.1.
Scaffold free approach

Cell sheet technique
Temperature controlled cell attachment, culture and detachment
followed by stacking multiple layers
with porous lms of poly-D, L-lactic-co-glycolic acid facilitated the ingrowth of

brovascular tissue, which eventually formed a vascularized, tubular tissue.[42] To
investigate the combined effect of PLLA and collagen gel on vascularization, porous
PLLA scaffold was exposed to in vitro condition, where the pores were lled with the
mixture of human aortic SMCs and collagen gel, resulting in the rapid formation of a
smooth inner layer around the vascular graft.[43] Another composite polymer of lactide
PLGA was explored to study the release prole of VEGF. VEGF was directly incorporated into PLGA scaffolds and pre-encapsulated in PLGA microspheres. It was reported
that direct incorporation of VEGF resulted in 4060% release within ve days in vitro,
while encapsulation of VEGF within microspheres resulted in prolonged release. When
implanted into subcutaneous pockets of SCID mice, VEGF released from PLGA
scaffolds signicantly enhanced local angiogenesis.[44]
Like PLA, composites and derivatives of PCL have been identied as a vasculature
enhancer in several studies. In a comprehensive review, Woodruff et al. presented the
attractive features of PCL (e.g. inexpensive, FDA-approved, and superior rheological
and viscoelastic properties) to demonstrate its applicability for the fabrication of longer
term degradable implants suitable for a specic anatomical site.[45] In vivo implantation of PCL/PLA tubular scaffolds seeded with mixed cells obtained from femoral
691
veins of a mongrel dog showed no evidence of stenosis, thrombus, occlusion, or

aneurysmal formation within the tubular graft. Moreover, the vascular graft was
replaced by native tissue containing a large amount of ECM, where the ECs were
found to organize themselves in a linear way on the luminal surface of scaffold.[46]
Besides animal model study, in May 2000 a clinical trial was done on a four-year-old
girl to implant a pulmonary bypass graft made of PCL/PLA copolymer seeded with
autologous vein cells. This implantation was a milestone in pediatric cardiovascular surgery as the patient was doing well even after seven months of implantation and no
postoperative complications were reported.[47] Another derivative of PCL called poly
(lactide-co-caprolactone) (PLCL) was studied for tissue vascularization due to its biocompatibility, elasticity, and slow degradation property. In a study, PLCL vascular
grafts seeded with rabbit aortic SMCs were exposed to pulsatile ow in a perfusion
bioreactor for eight weeks. It was reported that the PLCL graft signicantly regulated
the proliferation of SMCs, increased collagen production, and promoted cell alignment
similar to that of native vascular smooth muscle tissues.[48]
The angiogenic potential of PHA derivatives and its composites were demonstrated
in different investigations. In a study, PHAPGA tubular scaffolds seeded with ovine
carotid artery cells were implanted into abdominal aortic segments of sheep for ve
months. No aneurysms were observed in the tubular graft; however, the percentage of
collagen, DNA content, as well as the mechanical strength of grown tissue, reached
close to that of native vessels.[49] Another research group maintained static/pulsatile
condition for PGA/PHA derivative-fabricated vascular grafts seeded with ovine vascular
myobroblasts and ECs. After 28 days of culture, it was reported that all samples
gained viable, dense, and conuent smooth tissue with high collagen content.[50]
4.1.1.2. Natural polymer. Biopolymer or naturally derived polymers can be classied
into two categories, such as; protein-based polymers and polysaccharidic polymers. In
tissue engineering, natural polymers are more advantageous over synthetic polymers in
many aspects, however, are not free from shortcomings.Some advantages and
drawbacks of natural polymers have been presented in Table 2.
Protein-based polymers, particularly collagen, brin, bronectin, elastin, and silk
broin, have frequently been explored in different studies for vascularization application. For example, it has been reported that freeze-dried collagen scaffold seeded with
dermal broblasts and bone marrow-derived stem cells enhance the proliferation of
EC.[51] Further, immobilized VEGF on collagen scaffolds promotes EC invasion and
proliferation.[52] Another group incorporated modied VEGF into collagen scaffolds
implanted subcutaneously into mice, resulting in an improved vascular network within
the implanted scaffold.[53] Besides, Fibrin tissue engineering scaffolds have also presented promising results for promoting blood vessel formation. Human microvascular
ECs were seeded into a 90% brin, 10% collagen matrix with combinations of added
growth factors; FGF-2 or VEGF, with TNF-.[54] This investigation suggests that brin matrix facilitates the formation of a vascular capillary network through matrix brinolysis by ECs, which occurs from the activities of plasmin and metalloproteinases. In
addition to providing specic cell adhesion sequences, a brin scaffold can store and
release angiogenic growth factors, such as VEGF-A165 and FGF-2, as well as plasmid
DNA to improve angiogenesis.[55] Another ECM protein bronectin has been considered as a vasculature promoter due to its enormous contribution in vascularization. In a
study, bronectin-coated collagen modules increased human vascular EC survival and
vessel formation when implanted in immunodecient mice compared to collagen
692
Table 2.
Md. Sarker et al.

Natural biomaterials for scaffold fabrication.
Biomaterial
Collagen
Advantages
(1)
(2)
Biocompatible,
biodegradable, ease
availability and modiability,
nonantigenic and controlled
protein release ability.[291]
Hydrophilic and enhance cell
interactions.[292]
Disadvanages
(1)
(2)
(3)
Hyaluronic
acid
(1)
(2)
Fibrin
(1)
(2)
(3)
Fibronectin
(1)
(2)
(3)
Alginate
(1)
Easily modiable,
hydrophilic, nonadhesive,
biodegradable,[297]
nonantigenic and noninammatory.[298]
Regulate cellular migration,
interaction, and
differentiation.[299]
Non toxic, non inammatory,
[302] non allergenic,[303]
non immunogenic[304],
economical,[305]
biodegradable [306] and
easily processable.[307]
Facilitates synthesis of
collagen [308] and promotes
angiogenesis.[309]
Enhances attachment,
migration and proliferation of
smooth muscle [310] and
ECs.[311]
(1)
(2)
(1)
(2)
(3)
Poor mechanical strength,

[293] deformability,
exibility and tensile
strength.[294]
High cost of purication,
handling complexity,[295]
rapid degradation, little
compression force
resistivity.[296]
Sterilization causes alteration
of protein structure.[291]
Requires purication to avoid
disease transmission.[300]
Rapid degradation rate, poor
mechanical strength,[301]
and forms scar tissue
in vivo.[294]
Inconsistency in
polymerization, fragile and
stress squeezable.
Structural weakness.[312]
Upon implantation, solubility
increases and structural
integrity decreases over
time.[313]
Critical role for angiogenesis

[314] and scaffold
vascularisation.[315]
Facilitates cell adhesion,[316]
migration,[317] proliferation
and alignment.[318]
Can be used to coat articial
biomaterials to support cell
adhesion, spreading [319] and
proliferation.[320]
(1)
(2)
High cost.[321]
Excessive mechanical stress
interrupts formation of
vasculature.[322]
Non inammatory,[321]
biocompatible, non toxic,
cheap and potential for cell
encapsulation and drug
delivery.[323,324]
(1)
Uncontrollable degradation
kinetics due to loss of
divalent ion and burst release
of protein drugs at higher
pH.[325]
(Continued)

Table 2.
(Continued).
Biomaterial
Advantages
(2)
pH sensitive behavior
facilitates drug delivery.[325]
Disadvanages
(2)
(3)
(4)
693
Poor mechanical
strength.[326]
Hydrophilic character
prevents it from adsorption
of cell adhesive protein.[327]
Lack of cell adhesion.[323]
modules without bronectin coating.[56] Similarly, implantation of a bronectin/

collagen I gel seeded with EC and mesenchymal cell facilitated the formation of wellperfused and long-lasting vascular network after implantation into mice.[57]
The role of structural protein elastin in vascularization is very signicant as genetically modied mice lacking elastin died within four days of birth from arterial obstruction resulting from subendothelial cell proliferation and reorganization of smooth
muscle into the lumen of artery.[58] Cross-linked soluble alpha-elastin discs were found
to facilitate vascular SMC adhesion,[59] whereas tropoelastin-coated surface was
reported as a promoter of EC attachment and proliferation.[60] Elastin composites have
also been investigated to vascularize tissue engineering grafts. For instance, in a study,
human coronary artery SMCs were cultured for seven days on aligned nanobrous
polyurethane scaffolds blended with the mixture of elastin and collagen. It was concluded that the growth of SMCs on elastin/collagen-blended scaffold increased by
224% compared to that of polyurethane.[61] Besides animal body-derived protein, silkworm-secreted protein silk broin showed promising results in several studies in tissue
vascularization. In a study, co-culture of human dermal microvascular endothelial cells
(HDMECs) and primary human osteoblast cells (HOCs) in 3D silk broin nets caused
ECs to form intertwined microcapillary-like structures.[62] In another study, 3D silk
broin scaffolds pre-cultured with HDMEC and HOC were implanted into
immunodecient mice. After 14 days, it was found that the microcapillary structures
pre-formed in vitro, perfused well with the host vasculature.[63]
Apart from protein-based polymers, polysaccharide polymers, mostly hyaluronic
acid (HA), alginate, and chitosan have been studied for tissue vascularization. For
example, subcutaneously injected HA combined with recombinant gelatin facilitates the
formation of vascular network as well as the deposition of ECM components in
rats.[64] Another group achieved controlled release of two angiogenic growth factors
(VEGF165 and PDGF-BB) from a hybrid mesh consisting of poly (3-caprolactone)collagen blend and HA hydrogel. They reported that the growth factor-loaded hybrid
mesh enhances cellular attachment and formation of vascular capillary network within
the tissue engineering construct during the culture of ECs and broblasts in a 3D
model.[65] However, HA has limitations in tissue engineering applications as it exhibits
poor cell attachment and mechanical properties.
Seaweed-derived polysaccharide alginate does not have EC adhesion properties, but
can be introduced through adsorption of proteins,[66] or by covalent incorporation of
different functional groups into the sugar backbone.[67] To facilitate vascularization
within alginate scaffolds, internal pores should be well interconnected and large enough
to allow ingrowth of blood vessels upon implantation in vivo.[68] For example, an
694
Md. Sarker et al.
alginate scaffold with 90% porosity and pore sizes ranging from 50 to 200 mm
facilitates human embryonic stem cell aggregation and the formation of void and tubelike structures in vivo.[69] Furthermore, growth factors can be incorporated in alginate
scaffold to enhance vascularization, for instance incorporation of VEGF in alginate
hydrogels enhance neovascularization into the matrix.[70,71]
Due to remarkable wound healing capability, chitosan and its composites were
explored for tissue engineering graft vascularization in different studies.[72] In a study,
porous chitosan, glycosaminoglycans (GAGs)-chitosan and dextran sulfate (DS)-chitosan scaffolds were implanted in dorsal subcutaneous pockets of male Sprague-Dawley
rats. The rats were sacriced after two weeks and it was noticed that chitosan, heparin
chitosan, and DS-chitosan scaffolds promoted cell proliferation, tissue ingrowth, and
formation of vascularized granulation tissue. Chitosan composites can also be used for
the controlled release of growth factors. To study the release prole of an AF from chitosan complexes, FGF-2 incorporated chitosan/heparin hydrogel was subcutaneously
injected into the back of male Sprague-Dawley rats. Controlled release of FGF-2 molecules for 20 days caused micro blood vessel and brous tissue formation around the
injected site.[73]
4.1.1.3. Decellularized matrix. Decellularization of tissue or whole organ causes the
formation of ECM protein-enriched three-dimensional bioscaffold. The acellular matrix
contains unique, tissue-specic, structural and functional molecules that regulate cellular phenotype and function, mechano-transduction, signaling, cellmatrix interactions as
well as tissue homeostasis.[74,75] Therefore, matrix decellularization followed by
recellularization with autologous cell could be a promising approach in tissue engineering. The technical difculty of removing all cellular remnants limits the application of
decellularized matrix due to the risk of a host immune response after transplantation.
Moreover, preserving the native properties of ECM, for example, three-dimensional
ultrastructure, surface topography, composition, bioactivity, and density of ligand distribution as well as internal network, such as nervous, vascular, and lymphatic networks, presents another technical challenge.[76] One of the important features of using
decellularized matrix is that it helps to rebuild the intricate vascular network within the
organ or tissue when the intact vascular spaces within the matrix are repopulated with
ECs.
Tissue decellularization can be accomplished by chemical, biologic, or physical
agents. Selection of an agent largely depends on tissue size, thickness, density, cellularity, and lipid content. Chemical agents, for example, acids and bases, hypotonic and
hypertonic solutions, ionic, non-ionic, and zwitterionic detergents, or solvents, can be
used to decellularize tissue based on the mechanism of solubilizing cytoplasmic components, denaturing proteins, dehydrating cells, disrupting nucleic acids, lipids, and proteins. However, chemical agents may cause some disruption of the ultrastructure of
ECM as well as damage of collagen, GAGs, and growth factors.[77,78] Biologic
agents, such as nucleases, trypsin, or dispase, are basically enzymes that work by catalyzing the hydrolysis of RNA and DNA chains or cleaving peptide bonds. The use of
biologic agents can also result in complications, for example, it can cause an immune
response, can disrupt ECM ultrastructure, or can remove collagen, laminin, bronectin,
elastin, and GAGs after prolonged exposure.[77,79] Physical techniques based on temperature, pressure, direct force, electroporation, perfusion, agitation, pressure gradient,
or super critical uid can also be used to remove cellular material from tissue. Physical
695
agents work by facilitating chemical exposure, bursting cells, or disrupting cell

membranes, but can cause disruption or damage of ECM due to physical force.[77]
Repopulation of a decullarized matrix with appropriate cells is a major engineering
challenge to be solved. The parenchymal cells, for example, hepatocytes, cardiomyocytes, epithelium, etc., are responsible for the specic functions of the organ, while
nonparenchymal cells, such as broblasts and ECs, help to maintain the functional
phenotype of the parenchymal cells and the cellular architecture of the tissue.[80,81]
Moreover, ECs eliminate the thrombotic barrier within decellularized matrix and protect
the parenchymal cells from the shear stress by maintaining blood ow within the
vascular network.[82,83] Therefore, the success of decellularized matrix as a scaffold
mostly depends on re-endothelialization of the intact vascular space. Autologus, allogenic, progenitor, or multipotent stem cells are frequently investigated for their ability
to repopulate decullarized tissues or organs. Non-immunogenic autologous cells are the
best choice because no immunosuppressive drug is required. However, harvesting complexity and inadequate proliferation capability limit the use of autologous cells to
repopulate acellular matrix. By some criteria, for example, required cell population,
urgency of implantation, simplicity of cell harvesting and expansion, and capability of
differentiation into desired cell types, allogenic cells could be more useful [75] if
immunogenic reaction and the use of immunosuppressive drugs can be tolerated.
Multipotent stem cells are a third option to repopulate decellularized matrix, although
challenges exist here in controlling or directing differentiation along organ-specic or
tissue-specic cell lineages.[76] Bioreactors with appropriate biophysical stimuli might
be used to assist in differentiation of stem cells.
A number of studies suggest a promising future for organ or tissue vascularization
using decellularized matrix. For example, rat hearts decellularized by coronary
perfusion and reseeded with cardiac or ECs were kept in a bioreactor, where a simulated cardiac physiology was maintained for 28 days. In this study, EC formed single
layers in both larger and smaller coronary vessels after day seven, and recellularized
heart exhibited pump function by day eight under physiological load with electrical
stimulation.[84] Similarly, lungs decellularized by detergent perfusion, reseeded with
epithelial or EC, and then perfused with blood and ventilated using physiologic pressures resulted in gas exchange similar to that of isolated native lungs by day ve.
Moreover, when these reseeded lung constructs were reimplanted into an orthotopic
position, they generated gas exchange for up to 6 h after extubation.[85]
4.1.2. 3D printing approaches to create vascular networks
Vascular cell survival, growth, signaling, gene expression, and phenotype during matrix
tissue culture is signicantly affected by the pore architecture, pore interconnectivity
and mechanical stiffness of the scaffold.[8688] The porous architecture of a scaffold
includes pore size, shape, porosity, and surface topography of the pores. Proper porosity allows cell migration, proliferation, and interaction that facilitate the formation of a
vascular network. In addition, porosity assists to transport nutrients and oxygen gas to
cells and remove metabolic waste from cell surroundings. However, excessive porous
structure might result in poor mechanical strength.
In bone tissue engineering, higher porosity and larger pore size of the scaffold
enhance vascularization and tissue growth. For example, a six-day in vitro investigation
on the proliferation of goat bone marrow stromal cells suggests that scaffolds having
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Md. Sarker et al.
70% porosity and 800 m average pore size enhance stromal cell proliferation
compared to scaffolds possessing 60% porosity and 700 m average pore size.[89]
Like porosity, the average pore size of a scaffold has a profound effect on tissue
growth and vascularization. A number of studies suggest that variation of pore size
affects cell functionality. For example, it was reported that implanted polytetrauoroethylene membrane having pore size 5 m under rat skin signicantly facilitates the
capillary blood vessel formation across the membranetissue interface.[90] When bronectin-coated porous silicon nitride substrates seeded with 3T3 broblasts and bovine
aortic ECs were kept in ex vivo condition for ve days, EC covered the scaffold pores
having diameter below 80 m. Interestingly, pore size under 30 m didnt display any
effect on EC coverage. Besides, broblast could cover pores of 50 m and less.[91]
Further, disc-shaped porous PLA scaffolds with pore size in the range of 63150 m
facilitated vascular SMC proliferation and matrix deposition during a four-weeks culture period.[92] Therefore, a potential strategy would be to fabricate scaffolds with targeted pore size that would promote the desired cellular activity, for example, tissue
ingrowth as well as blood vessel formation. The surface area of a scaffold is also an
important parameter to be considered as there exists a relationship between pore size
and available surface area for cell attachment where an RGD binding sequence motif
prevails. In general, small pore size increases specic surface area, which ensures minimal ligand density for the attachment of a critical number of cells.[93,94] On the other
hand, large pore size facilitates cell migration but reduces cell density.
Finally, pore interconnectivity within the scaffold can have a profound inuence on
neovascularization and tissue growth. The diffusional mass transfer of nutrient and oxygen gas and removal of waste metabolites can be interrupted due to improper pore
interconnectivity.[95] A -tricalcium phosphate scaffold fabricated by the casting technique was used in a rabbit model to investigate the effect of pore parameters on vascularized bone tissue formation. An increase in pore size increases the size of blood
vessels, but the increase in pore interconnectivity results in an increase of size as well
as the number of blood vessels.[96] As well, pore sizes greater than 400 m do not
support vascularization at all.[96] Scaffolds fabricated with PLGA biomaterial having a
small inter-pore distance along with minute pore size in the range of 520 m enhances
EC growth signicantly.[97]
Physical stiffness of a scaffold can enhance formation of focal adhesions and
cytoskeletal reorganization in ECs which regulate cell migration, proliferation, and differentiation, as well as cellcell and cellscaffold adhesion.[98,99] A number of studies
suggest that the spreading area of EC during focal adhesion increases with the increase
of biomaterials stiffness,[100,101] however, in a stiffer biomaterial, ECs like to make
cellbiomaterial interactions, rather than cellcell interaction that cause failure to form
vascular network. On the other hand, ECs take an elongated morphology in compliant
material whose Youngs modulus is around 1 kPa, augment cellcell interactions
instead of cellbiomaterial interaction [102], which result in the formation of
self-assembled vascular network.[100]
4.1.2.1. Rapid protyping technique and miscellaneous. To promote vascularization
within tissue engineering construct, two major factors are to be considered during scaffold fabrication, one is well-interconnected microchannels or micropores, and the other
is controlled positioning of vascular cells. Some studies suggest that incorporation of
channel or grooves into scaffold can promote the formation of vascular network as ECs
require aligned organization to change their function and phenotype to form
697
capillaries.[103,104] More specically, the depth and topography of microgrooves has

profound effect on cell alignment and functionality. Uttayarat et al. reported that maximum alignment of bovine aortic ECs can be obtained on bronectin-coated 1 m deep
microgrooved surface, while Hu and colleagues concluded that microgrooves having
wavy surface display better cell attachment, alignment, and survival compared to those
on the rectangularshaped microgrooved surface.[104,105]
To construct functional complex tissue with vasculature, different types of cells
should be seeded in an organized way as per their need and function, rather than in a
random fashion. Scaffolds fabricated with conventional techniques, such as gas foaming, electrospinning, salt-leaching, porogen melting, molding, ber deposition, and
freeze-drying, display uneven, uncontrolled, and undesired pore size, shape, wall thickness, pore interconnectivity, and morphology.[106] To fabricate the complex vascular
network layer by layer in a controlled and precise manner, CAD-based rapid prototyping (RP) techniques, such as 3D bioplotting, inkjet printing, laser-based biofabrication,
and stereolithography, can be used. However, in many cases, acellular structures were
seen to be fabricated by RP technique to keep cells free from the effect of unfavorable
fabrication conditions (e.g. heat, pressure, light, adhesives, cytotoxic solvents, or molding). One major aw of acellular approach is the inefcient and inhomogeneous postfabrication cell incorporation. Yet, a number of studies have been accomplished to
fabricate intricate microvascular pattern with RP technique. Of several approaches, one
promising approach was to incorporate sacricial laments into scaffold by 3D bioplotter
to get complex and well-interconnected vascular pattern (Figure 6). Lewis group
demonstrated the effectiveness of fugitive ink to create 3D microvascular network by
direct write assembly method.[107] In another study, they created vascular pattern by
direct write method using fugitive ink and encapsulated the whole 3D network into
gelatin methacrylate gel (GelMA). Then HUVECs were injected after evacuation of
fugitive ink. HUVECs were found to attach, proliferate, and differentiate to form vascular network.[108] To avoid cytotoxic organic solvents, and sacricial template-removal
associated extreme temperature or pressure, Wang et al. encapsulated sodium alginatebased sacricial vascular networks fabricated with 3D bioplotter into gelatin hydrogels
and then dissolved the sacricial pattern with EDTA solution. HUVECs were injected
into evacuated channel and found to form vascular lumens and network.[109] Another
research group, Chen and colleagues fabricated a patterned 3D network composed of
sacricial carbohydrate glass lament by bioplotter, dispensed ECM (e.g. brin gels,
matrigel) encapsulated cells and mixture of poly (ethylene glycol) diacrylate and
Figure 6.
3D approaches for vascular channel formation.
698
Md. Sarker et al.
acrylate-PEG-RGDS into a rectangular mold containing the lattice lament,

photo-crosslinked the PEG hydrogel to encapsulate the 3D network as well as ECM
embedded cells, and then dissolved the lattice with cell culture media to obtain the
interconnected empty vascular lumen. HUVECs were then seeded in the vascular
lumen, and perfused with blood under high-pressure pulsatile ow. Incorporated vascular pattern was able to sustain the metabolic function of primary rat hepatocytes in the
core of a thick, densely populated tissue constructs.[110] In another study, to access the
effect of complex vascular geometry on diffusion gradient, they prepared vascular channel structure in gelatin gel by photolithography, encapsulated the microuidic network
and therapeutic cells within collagen gel, ushed gelatin gel by external ow, and then
injected HUVECs into the vascular lumen. Injected HUVECs formed conuent monolayers within the open microuidic channels after attachment, alignment, and proliferation. In addition, the relationship between the geometry of vasculature and spatial
diffusive gradients which is responsible for angiogenic sprouting was also demonstrated.[111] To avoid the necrosis of large cell population in a thick tissue, vascular
pattern design parameter (e.g. vessel alignment, branching frequency and angles, and
tortuosity) is required to be optimized to ensure efcient convective diffusional mass
transfer (e.g. nutrients, metabolic wastes, gases, AFs, etc.) between cells and culture
medium or blood during in vitro or in vivo culture.[112] Therefore, several mathematical modeling and computational studies have been accomplished to optimize the architecture of vasculature considering the diffusional mass transfer, microuidic behavior,
and physiological data of human microvasculature.[113]
Controlled positioning of vascular cell into scaffold can be achieved through the
deposition of cell-laden hydrogels. Polymer hydrogels offer structural support and 3D
hydrated environment similar to in vivo condition for EC attachment, proliferation, and
differentiation as well as keep ECs free from fabrication-induced high shear
forces.[114] To maintain EC viability and function in culture, hydrogel crosslinking
and scaffold fabrication process should be biocompatible. Several studies frequently
applied stereolithography,[115] inkjet printing [116,117], and 3D bioplotting [118,119]
techniques to prepare cell (e.g. ECs, SMCs) laden hydrogel to fabricate vascular pattern. Besides, precise cell positioning, microchannel or interconnected porous structure
is required to avoid necrosis. To incorporate of EC-laden gel into microchannel, in a
study, liquid collagen containing EC suspension was distributed above micropatterned
poly (dimethylsiloxane) PDMS templates, the templates were then centrifuged to move
ECs into the channels, and then collagen was crosslinked to form gel. It was reported
that EC formed capillary tubes after culture with VEGF and bFGF over 2448 h.[120]
While microfabrication offers many promising features to pattern microcapillary network, time delay, use of nondegradable hydrogel, and handling complexity limits its
application. Therefore,an alternative way, such as mechanically removable spacer (e.g.
aligned array of wires), was investigated to create microchannel into hyrogel matrix.
Wray et al. used mechanical spacer to generate unbranched microchannel ranging from
152 to 787 m in diameter into silk scaffolds, and then they seeded human arterial
endothelial cells (hAECs) into the hollow channels along with bioactive agents. The
formation of a contiguous layer of hAECs around the channel wall was reported
seven days after cell seeding.[121] In addition, to ensure uniform cell seeding into
incorporated microchannels, spacers can also be used to transfer self-assembled cell
layer into hydrogel matrix. In a study, casted GelMA over EC-coated micrometric gold
rods positioned in a culture chamber was photo-crosslinked, an electrical potential was
applied to transfer the self-assembled layer, and then rods were removed to leave
699
behind the EC-assembled layer into the matrix. The same procedure was followed to
prepare and transfer double-layered cell and gel mixture to combine microvessel stabilizing 3T3 broblast cells with HUVECs.[122] Beyond non-sacricial laments,
microuidic/micromolding techniques were also investigated to fabricate sacricial pattern in a microscale. In a study, sacricial gelatin meshes were prepared by micromolding in patterned PDMS stamp. Hydrogels (e.g. brinogen, Type I collagen, matrigel)
were used to encapsulate the sacricial pattern, and then successive melting and ushing were done to empty the interconnected channels. The viability of injected HDMECs
into microchannel was excellent as HDMECs were seen to attach, spread, and proliferate.[123] Regardless of tremendous success with sacricial templates, dissolutionassociated cytotoxic reaction limits their applications.[124] In an effort to fabricate
sacricial templates-free scaffold, Bertassoni and colleagues bioprinted agarose bers,
casted PEGDA hydrogels over the bioprinted templates, and then photo-polymerized
them. Removal of bioprinted templates was easy, as agarose didnt adhere with casted
hydrogels. With this approach, a perfusable network could be incorporated into hydrogel matrix without producing any dissolution-related cytotoxicity.[125] Aside from
sacricial or removable laments, laser scanning lithography (LSL) was investigated to
create extremely precise micropattern on photo-sensitive hydrogel surface. In a study,
West group covalently incorporated RGDs and VEGF on LSL-generated micropatterned
poly (ethylene glycol) hydrogel surface. Tubules like formation in the micro-patterned
area were observed within two days, whereas no tubules formation was recognized by
day two for cells cultured on wide patterned lines.[126] As blood perfusion is very signicant to regulate vascular pattern, West and colleagues combined microfabrication
and self-assembly technique to study macro micro-scale transport to achieve biomimetic perfusion in vitro. They prepared PDMS housing with uid access ports by soft
lithographic techniques, injected PEG hydrogel containing the HUVEC-10T1/2 cell
mixture into the PDMS housing, photolithographically crosslinked the cell-laden hydrogel, and then initiated channel perfusion with normal physiologic ow rates. The perfusion between the fabricated microchannel and self-assembled vascular network
signicantly facilitated the convective diffusional mass transport.[127] Moreover,
signicant success for in vivo vascularization was reported while VEGF-mimetic peptide-bound PEGDA matrices were implanted into mice.[128] Besides microfabrication,
Garicia and colleagues studied functionalized hydrogel to promote vascularization
in vivo. They engineered photopolymerized PEG diacrylate hydrogel matrices containing protease-degradable sites, cell-adhesion motifs (e.g. arginineglycineaspartic acid),
and growth factors (e.g. VEGF) and implanted subcutaneously in male Lewis rats.
They reported that a signicant number of vessels formed into the matrices at two
weeks and the network became more densed at four weeks due to cell-demanded
sustained release of VEGF over two weeks.[129] As hydrogel density regulates the
mechanical strength, Putnam and colleagues investigated the effect of elevated brin
gel density on vasculature formation in vitro and in vivo. Their ndings suggest that
inhibition of vasculature formation in vivo due to elevated brin matrix density can be
partially lessened by co-delivery of mesenchymal stem cells (MSCs) with human
umbilical vein endothelial cells (HUVECs) into hydrogel.[130]
4.1.2.2. Microfabrication technique. Implanted, biodegradable tissue engineering scaffolds containing cells and growth factors successfully vascularize thin tissue (12 mm).
However, vascularization of thick tissue constructs (>2 mm) is still challenging as it
takes time for blood vessels to form and anastomose with the host blood supply. To
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Md. Sarker et al.
solve this issue, a microvascular network can be established by microfabrication within

the implantable scaffold in vitro.[131] Microfabrication and bio-microelectromechanical
techniques, are attracting attention due to their spatial resolutions of less than 10 m,
[132] a substantial improvement over conventional scaffold fabrication techniques such
as solvent casting and porogen leaching,[133] gas foaming [134], and three-dimensional
printing.[135] However, some parameters, such as selection of fabrication material,
oxygen concentrations in the microenvironment [136], and uid shear stresses within
the microuidic scaffold [137,138], signicantly affect the success and applicability of
microfabrication techniques.
Microfabrication generally involves preparation of lithographic mask to pattern the
blood vessel network according to a model,[113] formation of a master mold by thick
photoresist or plasma etching, casting the desired biopolymer into the master mold,
incorporation of a sacricial layer to facilitate lm removal from the master mold, and
curing the lms under vacuum (Figure 7). Single-layer microuidic networks are then
stacked together to form 3D scaffolds with complex vascular microchannels before
seeding with ECs in a bioreactor.[132] Real vascular networks consist of blood vessels
having various diameters. The direct-write laser technique can facilitate the precise
fabrication of multi-depth channels mimicking the in vivo structure.[139]
Synthetic nondegradable materials, such as poly dimethyl siloxane or silicon, are
often employed for microfabrication.[132] However, biodegradation and biocompatibility are important issues related to the integration of the vascular construct into the host
tissue as well as the potential for toxicity or inammatory responses. The biodegradable
polymers PLGA,[140] poly glycerol sebacate (PGS) [141], and silk broin [142] were
investigated for their suitability in constructing microuidic networks by microfabrication. PLGA exhibits rigid mechanical properties,[134] undesirable bulk degradation
kinetics,[143] limited biocompatibility [144], and cytotoxic degradation byproducts,
[145] all of which limit the applicability of PLGA to microuidic scaffolds. PGS offers
superior mechanical properties as well as better cellular response and morphology compared to PLGA.[143,144] PGS is inexpensive and attractive in terms of synthesizing
and processing simplicity to form layers up to 100 m.[146] A third possibility,
Figure 7.
Microfabrication technique for vascular network formation.
701
chemically functionalized silk broin, is a promising biopolymer for microfabrication

of vascular networks [142] because it offers improved cellular adhesion, controllable
degradation, and enhanced mechanical property compared to PGS lms.
Alternative to multiple-layer stacking techniques, lithographic processes and sacricial gel encapsulation methods can be applied to generate 3D microuidic networks
into hydrogels (alginate, collagen, brin, etc.) that can later be seeded with ECs for
vascular tissue engineering. A lithographic process has been used to form microuidic
channels within an alginate scaffold seeded with chondrocytes, and these channels
appear to support long-term cell survival and convective diffusion of soluble factors.[147] Examples of sacricial gel techniques include a study where Type I collagen
was used to encapsulate patterned Matrigel and dened shaped microcavities were
achieved within collagen after the digestion of matrigel by enzymes.[148] Similarly,
microuidic channels as narrow as 6 m were formed in collagen and brin gels using
embedded sacricial gelatin meshes. The channels generated in this way facilitate the
transport of macromolecules into the channels and from channels into the gel
matrix.[123]
4.1.2.3. Modular assembly. This sophisticated tissue engineering approach achieves
formation of macro tissue constructs through the assembly of smaller modules. Micro
modules can be prepared by self-assembled aggregation,[149] microfabrication of cellseeded hydrogels, [150]creation of cell sheets [151], or direct printing.[152] To promote
vascularization, micro modules containing target tissue cells have been coated with ECs
and perivascular cells prior to assembly.[153] The small size of the micro modules
facilitates the transport of oxygen and metabolites even if they are seeded with high
cell density.[154] The surface of the modules can also be modied by coating with
ECM proteins. For example, bronectin coating of collagen modules implanted in
immunodecient mice increases human vascular EC survival as well as blood vessel
formation.[56]
To form macro tissue, a number of methods can be adapted to assemble the micromodules, such as random packing,[155] stacking of layers,[156] or directed assembly.[157] In the random packing approach, EC-coated modules are packed together into
a larger chamber and perfused with blood or culture medium which facilitates the
formation of interconnected channels among the interstitial spaces between modules
(Figure 8). Antithrombogenic ECs assemble to form vascularized tissue, and promote
functional perfusion by delaying clotting time and inhibiting loss of platelets.[153]
Incorporation of stem and progenitor cells along with ECs can signicantly affect the
stabilization of blood vessels that develop when using the modular approach. For example, implanted collagen gel modules coated with rat aortic ECs into Sprague-Dawley
rats caused the formation of blood vessels within the rst seven days. The nascent
blood vessels eventually became mature and then lasted at least for 60 days. In these
simple constructs, the new blood vessels connected with the host vasculature, yet some
of them were leaky.[158] Interestingly, addition of bone marrow-derived stem cells to
the EC coated modules lead to formation of blood vessels having similar density, but
less leakiness.[159] However, the random-packed modular approach causes lack of
mechanical integrity in the resultant macro tissue and limits the use of secondary cells
other than ECs.
Sequential assembly is a more directed method used to construct large tissue from
modular tissues with a specic microarchitecture.[160] In this approach, microgels containing specic architectural designs are assembled in a controlled fashion to connect
702
Figure 8.
Md. Sarker et al.
Modular assembly technique for vascular network formation.
the micro channels of each microgel, resulting in the formation of a bifurcating and
interconnected network.[112] Thus, sequential assembly provides better control over
the relative spatial arrangement of the building blocks. For example, concentric PEG
microgel building blocks embedded with network of microchannels were assembled
sequentially by photo-crosslinking to achieve a natural blood vessel-like structure. The
inner layer was seeded with HUVECs, and the outer layer was coated with SMCs to
achieve a tube-like structure using a mineral oil immersion technique.[1] Formation of
vascular network by 3D fabrication technique has been summarized in Table 3.
4.1.2.4. Comparison among different fabrication techniques. It is well established that
oriented pores or microchannels are required to maintain cell viability in tissue engineering construct. In previous section, it was already stated that 3D fabrication techniques are associated with unfavorable conditions which led researchers to fabricate
acellular construct. However, post-fabrication cell seeding into scaffold demonstrates
many complexities in terms of efcient and homogeneous cell positioning. In contrast,
fabrication of cellular scaffold facilitates the controlled and homogeneous cell distribution. Compared to other fabrication material, hydrogels are more suitable to fabricate
cellular scaffold as it can ensure cell viability by providing in vivo like environment.
However, lack of mechanical strength limits the applications of hydrogel scaffold. In
addition, free radical photo polymerization or chemical crosslinking to polymerize
hydrogels can negatively affect the viability of encapsulated cells. Channel, which is
the key parameter to promote mass transport and directional growth of EC, can be
introduced into the 3D scaffold by using sacricial and non-sacricial laments.
Sacricial
laments/pluronic
F127
Mechanical spacer
3D Bioplotter
5% gelatin lament,
channel 400700 m
Micropatterned cells
3D Robotic
dispensing
Laser-guided direct
writing
Photolithography
and and
assembly of
structures
Multi-level
interconnected lumens
Chemical
Non sacricial agarose

laments
Hexagonal/woodpile
micropattern
3D Micromolding
Stereolithography
Photo,
thermal
Photo
Sacricial lattice
/sodium alginate
Bulk
polymerization
Photo
Thermal
Ionic,
photo,
thermal,
enzymatic
Thermal,
ionic
Sacricial
laments/carbohydrate
glass
Bulk
polymerization
Thermal
Agarose spacer
PEGDA
39 mg/mL
collagen
Matrigel
10 or 15%
GelMA
10% GelMA
Gelatin, agarose
and collagen
Multicellular
spheroids,
diameter 300
500 m
Alginate, brin,
pegda, matrigel
GelMA
GelMA
Photo
Photo
Scaffold material
Gelation
method
3D Bioplotter
Bulk
polymerization
Channel/pore/pattern/
spacer
Scaffold fabrication techniques for vascularisation.
Fabrication
technique
Table 3.
In vitro
HUVECs
HUVECs, SMCs/
encapsulation
HUVECs, VEGF
HUVECs/
immersion and
agitation
HUVECs/mixing
HUVECs
In vitro
In vitro
In vitro
In vitro
In vitro
In vitro
In vitro
CHO,
HUVSMCs,
HSFs
HUVECs
In vitro
In vitro
HUVECs
HUVECs
Additives/addition Model/target
technique
site
[1]
[331]
[330]
[329]
[125]
[109]
[110]
[328]
[122]
[108]
Ref
(Continued)
90% cell viability after two days,

in vivo like vessel formation
Conuent layer on 3 mg/ml

collagen after three days
Elongated structures after 24 h
Conuent layer by day four
Conuent layer on gelatin and

collagen, spheroids on agarose
after 34 days
Conuent layer by day seven
Conuent layer within one day
Conuent lumen of 10 mm long

and 618 15 m diameter,
stable geometry for 15 days
Spheroids fused within 57 days
to form vascular tubes of 0.9
2.5 mm diameter
Formed conuent layer, viability

>95% after 48 h
Channel conuence/lumen
formation

703
Photo,
thermal
Photo
Photo
Ionic
Micropatterned
Microchannels 50
1000 and 1550 m
200 m diameter
channels
LSL
Soft lithography
and
photolithography
CO2 laser
engraving system
Gelation
method
Photolithography
Micropatterned
(microfabrication)
Channel/pore/pattern/
spacer
(Continued).
Fabrication
technique
Table 3.
Alginate,
alginate-sulfate
PDMS, PEG
PEG, PEGDA
PDMS/2.4 mg/
mL collagen gel
Scaffold material
HUVECs, bFGF,
VEGF/immerse
and centrifuge
HUVECs, RGDS
and VEGF
RGDS, HUVEC
and 10T1/2,
injection
HUVECs, RGD/
HBP
Mice
In vitro
In vitro
In vitro
Additives/addition Model/target
technique
site
High vessel density after

eight weeks
lumens form in two days for

<35 m groove
Mature vessel by 4896 h
Conuent layer by 2448 h
Channel conuence/lumen
formation
[332]
[127]
[126]
[120]
Ref
704
Md. Sarker et al.
705
Recently, direct ink writing is considered to be an emerging technique to create 3D

vascular structures with sacricial laments.[161] While incorporation of sacricial laments (e.g. carbohydrate glass, fugitive inks, sodium alginate, Pluronic F127 etc.)
causes the formation of interconnected and branched channels, dissolution process
causes cytotoxicity, disruption of surrounding ECM, osmotic damage to encapsulated
cells.[124,125] On the other hand, non-sacricial laments (e.g. array of wires, agarose
lament, etc.) doesnt cause any toxic reaction, but cause uncontrolled channel size,
unbranched network, and structural damage to surrounding gels. Besides, direct write
approach, complex 3D vascular network can be formed by sequential stacking of thin
micro patterened surface. While enormous success has been achieved to create micro
vascular network by microfabrication technique, such as photolithography,[162] microcontact priniting,[163] micromolding [164]; material constrains (photo sensitivity),
layer stacking constrains, multiple-layer handling and assembling complexity, and timeconsuming compiling procedure are some of the hurdles that yet to be solved to form
complex 3D vascular network.[107,165] Besides stacking of microfabricated planar surfaces, compiling of micropatterned tiny modules has drawn the attention of researchers
to build larger biological tissues and organs with vascular network. Although modular
approach facilitates the formation of large tissue by assembling the functional microtissue through random packing,[166] stacking [156], or directed assembly,[157] technical
hurdles to form interconnected 3D macro vascular tree for larger tissue still remains
unsolved.[167] Besides, several limitations of modular approach, such as poor
Table 4.
Advantages and disadvantages of vasculature fabrication techniques.
Approaches
3D Fabrication
Advantages
Fabrication of microcapillary (diameter

410 m) network is possible with
stereolithography due to very high
printing resolution (1.3 m); however,
channels similar to arterioles (diameter
10300 m) and venules (diameter 10
300 m) can be fabricated with LIFT/
LGDW (resolution 10100 m), inkjet
(resolution 85300 m) and bioplotting
(resolution approx. 100 m).
Moreover, fabrication of highly dense
EC pattern is possible with LIFT/
LGDW, where as fabrication of macro
(in cm range) vascular network is
possible with bioplotting.[8,333]
Mirofabrication Precise control of microstructure to
form complex capillary vascular
network
Stereolithography can be applied for
photosensitive and biocompatible
biomaterial [334]
Modular
Facilitates desired geometry, superior
assembly
diffusion properties, high cell density
[335]
Cell sheet
Native mechanical property [160]
Disadvantages
Stereolithography exhibits material
(only photosensitive hydrogels)
constrains, LIFT/LGDW shows size
restrictions (e.g. not suitable in the
mm range), inkjet can damage EC
due to ow induced high shear
forces; and microcapillary (410 m)
network printiting is impossible with
3D bioplotter.[8,333]
Sacricial material may cause

swelling and nonidentical channel
[123]
Heat caused by molding or laser may
damage biomaterial as well as
incorporated ligand
Vascular network is not similar to
native tissue
Leakage,[151] requires cells that are
proliferative and can produce
sufcient ECM [160]
706
Md. Sarker et al.
mechanical integrity of fabricated tissue, limited cell types for incorporation, poor cell
viability due to time-consuming fabrication process, and random or uncontrolled structural pattern [160] of grown vasculature or tissue have to be optimized to promote
functional vascularization. Comparison of different vascular network fabrication
techniques has been summarized in Table 4.
4.1.3. Addition of cells

Blood vessels are composed of ECs, pericytes, or vascular SMCs.[168] ECs regulate
the activities of the inner layer of blood vessels, vascular SMCs maintain structural
integrity by producing structural proteins, such as elastin, collagen, and proteoglycans,
and pericytes regulate the formation of micro blood vessels and can differentiate into
SMCs, broblasts, and osteoblasts as required.[169] Fibroblast also provides structural
support to blood vessel by producing ECM.[170] So, to promote vascularization in a
tissue engineering construct, a promising strategy would be to incorporate ECs,
pericytes, or vascular SMCs and broblasts into an articial scaffold.
Angiogenic sprouting from the host vasculature into a tissue engineering scaffold
requires time, and a delay could result in ischemic cell death. But incorporation of
vascular cells into the scaffold ex vivo could result in the pre-formation of capillary-like
structures, which reduces the required time to anastomose with host vasculature and
achieve tissue perfusion. The potential sources for vascular cells are autologus, allogenic, or xenogenic.[171] However, the complications related with host immune
response limit the use of vascular cells that could be collected from allogenic or xenogenic sources. Therefore, autologous mature ECs, EPCs, and stem cells have been
investigated for use in tissue engineering applications. Inclusion of mature ECs facilitates the formation of vascular network within a tissue engineering scaffold.[172]
Among human body-derived ECs, HUVECs,[173] HDMECs,[174,175] and mature
human vascular ECs have been used for angiogenesis and vasculogenesis studies.[176,177] Unfortunately, ECs derived from different arteries, blood vessels, or
regions of the body can exhibit morphological and functional differences, demanding
attention for specic applications. For example, HDMECs are elongated, whereas
HUVECs are polygonal.[178,179] Artery-derived ECs are long and narrow, while
vein-derived ECs are short and wide.[180] Aortic-artery derived ECs are thicker than
pulmonary artery-derived ECs [178] and large vessel-derived ECs are inconsistent with
micro vessel-derived ECs in terms of functionality.[181]
Addition of cells to promote vascularization is compatible with a variety of scaffold
biomaterials. Biodegradable PLLA scaffolds loaded with HDMECs and implanted into
immunodecient mice resulted in the formation of functional microvessels that eventually anastomosed with the host vasculature.[172] Besides, brin matrix seeded with
ECs and preadipocytes, and implanted onto the chorioallantoic membrane of fertilized
chicken eggs became densely vascularized.[182] ECs from umbilical vein have also
been examined as potential cells for neovascularization by different groups. HUVECs
suspended in Matrigel were injected subcutaneously into immunodecient mice and
mature blood vessels were found after 30 days and remained stable up to
100 days.[183] In an attempt to investigate the effect of prevascularization on
anastomosis with the host vasculature, HUVECs and broblasts were co-cultured in brin gels for one week and then implanted subcutaneously into SCID mice. The rate of
vascularization was faster in the prevascularized compared to non-prevascularized
construct.[184] Combined effect of vascular cells and growth factors on tissue
707
vascularization was also explored, for instance, subcutaneous implantation of

matrigel-brin scaffold seeded with ECs and osteoblasts, and loaded with VEGF and
FGF-2 into SCID mice resulted in durable perfused vascular networks, which remained
functional for up to 60 days.[185] Further, the outcome of composite scaffold on neovascularization was investigated, for example, PLGA/PLLA scaffolds were seeded with
HUVECs, broblasts, and mouse myoblasts, and implanted subcutaneously into the
back of SCID mice. Formation of functional and perfused vascular network within the
implant was seen after 14 days of in vivo culture.[186]
Beside ECs, SMCs have also been investigated to promote vascularization for tissue
engineering applications. SMCs play a critical role in maintaining blood vessel integrity, diameter, blood pressure, and ow distribution.[187] When seeded into porous
polymer scaffolds, SMCs can facilitate growth of microvessels. For example, coculture
of EPCs with SMCs within porous polymer scaffolds resulted in the formation of
microvessels, while culture of EPCs alone failed.[188] SMCs, in a highly differentiated
and low proliferative state, can also enhance blood vessel formation in vitro in a proper
3D matrix environment.[189] Rat aortic SMCs were seen to differentiate into pericyte
cells during angiogenesis in vitro. These pericyte cells contribute much to form stable
vascular network by regulating the differentiation and maturation of microvessels.[190]
Inclusion of pulmonary artery-derived SMCs with microvascular ECs in PLLA scaffolds enriched with Matrigel also signicantly stabilizes the nascent blood vessel
in vivo.[191]
Although promising, challenges still exists for the use of mature ECs in tissue engineering scaffolds due to limited availability, proliferation capability, and poor functionality in the case of diseased or aged patients.[192,193] The alternative use of stem
cells obtained from embryos into tissue engineering constructs could be a satisfactory
solution as stem cells exhibit higher proliferation and differentiation ability to restore
tissue vascularization after ischemic events.[194] For example, ECs derived from
human embryonic stem cells and embedded in a bronectin/collagen gel could form
stable, perfused, and functional blood vessels in mice. Within these experiments, inclusion of bone marrow-derived mesenchymal precursor prevented rapid regression of the
new blood vessels and promoted the formation of stable and functional blood vessels
that lasted for more than 150 days.[195] Though stem cell implantation seems very
promising for vascular tissue engineering, there remains a technical challenge to harvest
cells with a desired phenotype through the expansion of multipotent stem cells.[171]
Moreover, post-implantation complications, such as formation of tumors, teratomas,
atheromas, or retinopathies, are also associated with stem cell implantation.[194]
Incorporation of EPCs into tissue engineering scaffold is perhaps more promising
compared to stem cells as EPCs are more committed to EC differentiation and still
exhibit satisfactory proliferation capability. EPCs can be derived from peripheral blood,
bone marrow, umbilical cord blood, liver tissue, or vascular walls for tissue engineering
application.[196,197] Inuences, such as growth factors, cytokines, and mechanical
shear stresses, regulate the differentiation of EPCs into mature ECs.[198] Evidence of
experimental investigation suggests that EPCs alone cant form stable and functional
vascular network after implantation in vivo, but require perivascular cells, such as
vascular SMCs or mesenchymal progenitor cells, for the stabilization of nascent blood
vessels.[199] Like mature ECs, the source of EPCs also affects the stability and longevity of neo-blood vessel. For example, new blood vessels developed in vivo from EPCs
derived from peripheral blood were unstable and regressed in three weeks, while blood
vessels developed from EPCs from umbilical cord blood lasted for more than four
708
Md. Sarker et al.
months.[200] The results of in vivo and in vitro culture of several vascular cells seeded
into scaffold have been summarized in Table 5.
4.1.3.1. Cell seeding with bioreactor. Perfusion bioreactor facilitates controlled and
homogeneous distribution of vascular cells, AFs and ECM protein throughout the
vascular graft. Cell seeding using a perfusion bioreactor can be advantageous, not only
to transport nutrients, oxygen, and remove metabolic waste throughout the bioengineered scaffold in a homogeneous fashion, but also to control other factors, such as
uid pH, temperature, ow velocity, and pressure. In particular, control of the mechanical environment can facilitate the formation vascular network within the scaffold. A
perfusion bioreactor was used to seed bovine aortic SMCs and ECs in a dynamic and
sequential manner onto tubular PGA scaffolds. Pulsatile ow conditions were maintained which facilitated cell proliferation, SMC differentiation, ECM deposition, and
conuent endothelial monolayer formation within the lumen.[201] Pulsatile ow also
signicantly regulates the structural organization of SMCs, a factor crucial for the
Table 5.
Cell types and behavior in tissue culture.

Required time after implantation
Cell types
Fibroblast,
HUVECs
Fibroblast,
HUVEC
HUVEC
Scaffold
CLS
biomaterial formation
HA
After
21 days
Fibrin gel
HUVEC,
VEGF, FGF2
HUVEC,
myoblast,
embryonic
broblast
HDMEC,
Preadipocyte
Matrigel
Fibrin
PLGA/
PLL
By
10 days
HDMEC
PLLA
sponges
Matrigel
enriched
PLLA
By
ve days
Collagen
gel
seven days By 11 days
Fibrin
Collagen
gel
Maturation
By ve days
By
10 days
By
four days
HDMEC,
Human
pulmonary
artery SMCs
Peripheral
blood
derived EPC,
10T1/2 cell
Umbilical cord
blood
derived EPC,
10T1/2 cell
Matrigel
Anastomosis
with host
vasculature
By 20 days
By 30 days
Regressed
after
60 days
By 30 days
four days
By 7
10 days
seven days
21 days
14 days
Implantation/
culture
Ref.
In vitro culture
[173]
Dorsal surface
of SCID mouse
Abdomen of
SCID mouse
Abdomen of
SCID mouse
[184]
[183]
[185]
Quadriceps
muscle of
SCID mouse
[186]
Chorioallantoic
membrane of
egg
Dorsal tissue of
SCID mice
Flank of SCID
mice
[182]
[172]
[191]
Cranial window [200]

Regressed
of SCID mice
by
three weeks
Regressed
after four
months
Cranial window [200]

of SCID mice
709
formation of stable vasculature.[202] Blood vessel ECs in vivo exposed to blood ow

experience shear stress and dynamic ow conditions that increase VEGF, PDGF-BB
expression by ECs compared to static condition.[203] Thus, dynamic culture parameters, such as pulsation, dynamic pressure, and ow rate, can be applied in a controlled
fashion using a perfusion bioreactor to mimic in vivo conditions during the formation
of a vascular network in vitro.
4.1.4. Addition of growth factors
In normal life, the capillary network differentiates and remodels itself in response to
extracellular signals to meet the physiological demand of the corresponding tissue and
eventually form larger arteries and veins. It is well established that up to twenty AFs
are involved in blood vessel formation and stabilization. Among them, VEGF, Angs,
PDGF, broblast growth factors (FGFs), and transforming growth factor- (TGF-)
have been repeatedly studied for tissue engineering scaffold vascularization.[204] The
angiogenic effect of different growth factors has been shown in Table 6.
VEGF is a key regulator for growth and survival of endothelium during vascularization.[205] Blocking VEGF with antibody hinders microvessel growth, cellular proliferation, and capillary tube formation.[206,207] In a study, human retinal pigment
epithelial cells exposed to insulin release VEGF in a sustained manner which causes
neovascularization in rat retina model.[208] As well, scaffolds containing VEGF-loaded
PLGA microspheres and hepatocyte cells and implanted into immunodecient mice
showed better cell survival and increased angiogenic sprouting, suggesting that VEGF
promotes capillary sprouting into the scaffold, which then facilitates hepatocyte cell survival.[209] VEGF dosing should be controlled properly as over expression of VEGF
might inhibit the function of vascular SMCs or pericytes, which could result in immature, leaky, and unstable blood vessel.[210,211] More usefully, VEGF dose for in vivo
application has been studied by incorporating it into implantable scaffolds through
physical bonding, or encapsulation in nanoparticles, microparticles, or hydrogels.[212]
VEGF loaded within heparinized chitosan-coated alginate hydrogels exhibited a prolonged release prole by delivering VEGF locally for 11 days in a linear fashion.[213]
Delivery strategy of multiple growth factors was also explored in several experiments,
for instance; it was concluded that sequential delivery of VEGF-A165 followed by
PDGF-BB with alginate hydrogels could induce an angiogenic effect after myocardial
infarction.[214] In a further renement, incorporation of alginate sulfate into alginate
hydrogel to bind VEGF, PDGF-BB, and transforming growth factor-1 (TGF-1)
resulted in sequential release of VEGF, PDGF-BB, and TGF-1 that enhanced vascular
density in a rat model.[215] Alternatively, VEGF immobilized in collagen scaffolds
promoted the penetration and proliferation of ECs, which enhanced vascularization.[52]
FGF-2 facilitates the angiogenic activity of VEGF as there exists a mutually dependent signaling pathway between FGF-2 and VEGF by which they promote angiogenesis.[216] Targeted delivery of FGF-2 appears to be a useful tissue engineering strategy.
Inclusion of EC covered cytodex-3 microcarriers and FGF-2 into brin gel increased
the average number of capillary-like formations per microcarrier.[217] Besides addition,
efforts were made in different studies to release AFs in a controlled way to enhance
vascularization. Controlled release of FGF-2 from PLGA microspheres incorporated
into an alginate scaffold promoted matrix vascularization after implantation on the
mesenteric membrane in rat peritoneum.[218] Similarly, controlled and localized release
of FGF-2 from gelatin microspheres at myocardial infarction sites in a rat model
Ang-1
FGF-2
PDGF
VEGF
Growth
factors
Table 6.
Acidic and basic gelatin

hydrogel
Collagen
Alginate
Covalent binding
In vitro
and in vivo
In vitro
and in vivo
In vitro
and in vivo
In vitro
Addition
Incorporation into
gel
Encapsulation into
microsphere
Absorption
In vivo
Addition
Microvascular fragments and

myobroblast (MF)
Collagen sponge
Chitosan/heparin hydrogel
In vitro
and in vivo
In vitro
In vitro
Mixing
Incorporation
Fibrin
In vitro
In vitro
In vivo
Analysis
type
Alginate hydrogel
Immobolization
Cross-linking
Growth factors
addition method
Collagen
Collagen
Biomaterials/scaffold
Angiogenic effect of different type of growth factors.
Myocardial infarction
of rat
Bovine capillary
endothelial cell
Dorsal skin excision
wound
Subcutaneously into
mice back
Mesenteric membrane
in rat peritoneum
Subcutaneously into
mouse back
Chorioallantoic
membrane assay
Mouse ECs
HUVEC
Chorioallantois
membrane of egg
HUVEC
Cell type/implantation
site
[52]
[336]
Ref.
Enhanced vascularization was seen for low

water content acidic gelatin
Endothelial cell proliferation, tube formation
and endothelial cell inltration
Enhanced MF proliferation, MF derived

VEGF and collagen Type I
Improved inltration of broblast and
capillary formation
Neovascularization and brous tissue
formation
Formation of large and matured blood vessels
[233]
[339]
[218]
[73]
[338]
[337]
Enhanced angiogenesis but destabilized

[3]
microvessels in longterm
Enhanced mature vessels and cardiac function [214]
Promoted inltration and proliferation of ECs

Promoted mitogenic activity
Enhanced microvessel and tissue ingrowth
Results
710
Md. Sarker et al.
711
enhanced vascularization, survival of transplanted myocytes, and cardiac function.[219]

However, FGF-2 alone cant form stable functional vascular network, instead combined
effect of multiple AFs are required. Combined delivery of FGF-2 and VEGF from
collagen-heparin sponges implanted subcutaneously in Wistar rats was more effective
in promoting the formation of dense, mature vasculature within the scaffolds than either
factor alone.[220]
PDGF stabilizes nascent blood vessels by regulating the proliferation and migration
of broblasts and SMCs [2] as well as promoting differentiation of mesenchymal cells
to mural cells.[221] Several studies suggest that the role of PDGF in the stabilization
of nascent blood vessel formed from angiogenic sprouting or vasculogenesis is very
signicant. In a study, PLGA scaffolds loaded with PDGF and VEGF were implanted
in both Lewis rats and non-obese diabetic mice. Sustained delivery of both growth factors resulted in large, dense, and mature blood vessels, whereas delivery of only VEGF
caused immature and unstable blood vessels.[222] Other types of growth factor Ang-1
and Ang-2 regulate vascularization by binding with Tie-2 receptor expressed on the EC
surface.[223] Mice lacking Ang-1 or Tie-2 have defects in the formation of a vascular
network, likely because Ang-1 signaling has a profound contribution in recruiting
SMCs to the nascent blood capillaries.[31] On the other hand, Ang-2 competes with
Ang-1 for Tie-2 receptor and destabilizes blood vessels by inhibiting the interaction
between ECs and the mural cells. Infact, Ang-2 facilitates angiogenesis by promoting
EC migration from the existing blood vessel to form new capillaries.
Besides, TGF- promotes angiogenesis via recruitment of AF secreting cells, such
as macrophages and broblasts. It also promotes differentiation of ECs and stabilizes
nascent blood vessels by recruiting mural cells.[224,225] It was reported that
genetically knocked out mice lacking TGF-1 died at midgestation due to defects in
yolk sac vasculogenesis and hematopoiesis.[226] During vascularization, concentration
dependent behavior of TGF regulates the formation of functional and stable vascular
network. At low concentration, TGF promotes EC migration and proliferation as well
as inhibits vessel maturation and mural cell differentiation. In contrast, at high concentrated state, TGF works opposite to the low concentrated state.[227]
4.1.4.1. AF loading and releasing approaches. A number of growth factors are
involved in neovascularization and some of them function in a sequential fashion.
Growth factors VEGF and FGF-2 regulate the initial stages of angiogenesis through the
formation of capillary-like structures, while PDGF-BB, Ang-1, TGF, sphingosine-1phosphate (S1P), hepatocyte growth factor (HGF) are involved in the later stages of
vascularization as they stabilize the CLS through the recruitment of SMCs and other
mural cells. Experimentally, sequential delivery of VEGF followed by S1P [228] or
FGF-2 followed by PDGF [229] signicantly enhance blood vessel density and stability
compared to simultaneous dual delivery. Interestingly, a reverse in the order of growth
factor delivery compels the factors to work against each other, resulting in decreased
vessel density and stability. Thus, uncontrolled early delivery of vessel maturation factors inhibits EC from forming capillaries, resulting in decreased vessel density and
diameter. Late delivery of AFs causes the regression of immature blood vessels through
the inhibition of mural cell recruitment.[228,229] Various AF loading and releasing
approaches have been shown in Figure 9.
Regulation of molecular binding affects the period and pattern of AFs delivery.
Basically, two types of bonds (e.g. non-covalent and covalent) are responsible for
attachment of AF on binding sites of scaffold biomaterial. Non-covalent bond is
712
Figure 9.
Md. Sarker et al.
AF loading approaches into scaffold.
established through hydrogen bonding, or hydrophobic, secondary, direct charge

charge, or indirect interactions. In different studies, several protein molecules, such as
heparin, bronectin, gelatin etc. have been used as excipient or coating agent to
immobilize the AFs on the specic binding sites. Besides, collagen, elastin, bronectin,
chondroitin sulphate, heparin sulphate, laminin, HA can be incorporated into polymer
gels to immobilize AFs as well as AF-inducing moieties.[230,231] The release kinetics
of immobilized AFs depends on the binding constant as well as environmental conditions, such as temperature, acidity, and hydrophobicity. While some bioproteins are
non-specic for AFs, short peptide sequences are more AF specic and can be incorporated into scaffold for AF binding and sustained release. For example, Maynard and
Hubbell identied a sulfated tetrapeptide which shows strong afnity for VEGF and
suggested to use the peptide for VEGF-binding applications.[232]
To get more prolonged release of AFs than that could be obtained by non-covalent
bonding, AFs can be incorporated into scaffold covalently by copolymerization or
chemical treatment. Chiu and Radisic covalently immobilized VEGF and Ang-1 onto
porous collagen scaffolds by carbodiimmide chemistry and reported increased EC proliferation compared to unmodied scaffolds and soluble growth factor controls.[233]
Similarly, Mann et al. covalently linked TGF-1 with PEG hydrogel and reported a signicant increase in ECM production of seeded vascular SMC compared to that
achieved by soluble TGF-1.[234] Besides, covalent conjugation of AFs with scaffold,
programmed release of AF is possible if AF could be physically attached with covalently linked AF binding proteins. Steffens et al. covalently incorporated heparin into
collagen matrices by carbodiimide chemistry and then physically immobilized VEGF to
the heparin. They exposed the modied matrices to the chorioallantoic membrane of
chicken embryos and to the subcutaneous tissue of rats, and reported that VEGF
physically bound to heparinized matrices display more potential to promote
vascularization compared to controls.[235] Beyond covalent cross-linking, potential
713
angiogenic activity can be obtained by modifying AF as a recombinant or fusion

protein. Kitajima et al. fused HGF to a collagen-binding domain (CBD) polypeptide
sequence derived from bronectin while keeping the function of the moieties of the
fusion protein intact. They reported that CBD-HGF protein caused better growth and
proliferation of ECs in vitro than that obtained by HGF dosing. Moreover, foursix-fold
more extensive blood vessel formation was reported in the scaffold after seven days of
subcutaneous implantation in rats compared to the controls.[236] Similarly, Zhao et al.
prepared another recombinant protein by fusing plasminogen-derived brin-binding
peptide Kringle1 (K1) to bFGF. The recombinant K1bFAF protein displayed high brin
afnity and promoted robust neovascularization as well as healed wound when
implanted into the wound sites of rats.[237]
A number of strategies have been implemented in different studies to load AFs into
tissue engineering construct. AF loading and release depends on scaffold fabrication
materials, way of entrapment, or types of bonding. Surface coating and encapsulation are
common strategies to directly load AF into fabrication biomaterial. AF can be encapsulated into a polymer matrix by simple mixing prior to solidication. Carriers can be used
to load AF by diffusion, impregnation or immersion mechanism as direct entrapment
could cause loss of biological activity of AF during processing steps.[238] Kaigler et al.
loaded VEGF into PLGA polymer matrices by mixing method and implanted the
scaffolds into Fisher rats. They retrieved the implants at 2, 6, and 12 weeks and reported
over two-fold increment in blood vessel number within the VEGF scaffolds compared to
PLGA scaffolds. Similarly, Elcin and coworker prepared VEGF-loaded alginate
microsphere by homogeneous mixing and noticed localized neovascularization at the
subcutaneous implantation site of the rat model due to controlled release of AF.[239]
Direct loading of AFs into the carrier couldnt control the relative release rates of
multiple AFs for sequential delivery. Therefore, alternative strategies, such as multilayered coatings with different release rates or covalent immobilization of AFs on carriers
can be applied to control relative timing and concentration of the AFs within the
developing tissue. In one study, multilayered alginate microbeads were used to encapsulate islets in the inner layer, while angiogenic morphogen FGF-1was incorporated on
the outer layer. To achieve faster release kinetics, the outer layer was prepared with 1%
alginate containing high manuronic acid, while 1.25% alginate having high guluronic
acid was used to prepare the inner core to get slower release kinetics.[240] Sequential
release of multiple growth factors can also be achieved through the use of specic
biomaterials that exhibit different equilibrium binding constant (KA) values for different
growth factors. For example, the KA sequence of VEGF, FGF-2, PDGF-BB, and
TGF1 for alginate sulfate can be presented as FGF-2 < VEGF < PDGF-BB~TGF1.
Thus, the higher the value of KA for a particular growth factor, the slower will be its
release.[241] Multi-phase scaffold loaded with AFs provides biomechanical and biochemical support to promote vascularization in a newly developed tissue by maintaining structural integrity as well as releasing multiple AFs in a controlled manner.
Richardson et al. mixed PLGA microspheres with particulate PLGA to make a porous
matrix, in which one growth factor was admixed with particulate PLGA polymer, while
another was encapsulated in PLGA microsphere to achieve controlled release of the
AFs. PLGA microspheres loaded with PDGF-BB exhibited slower release kinetics
compared to porous matrix loaded with VEGF.[222]
Beyond sustained and sequential release, spatial gradients of AF can be introduced
throughout the scaffold to get directional sprouting of blood vessels in the desired
direction. Dodla and Bellamkonda demonstrated a method to generate step and
714
Md. Sarker et al.
continuous gradient of growth factors along nerve regeneration conduit containing four
layers of gels, each with higher concentration of growth factor than the previous layer
in the direction of proximal to distal end. To generate step gradient, they increased the
concentration of growth factor in step-wise fashion for successive layers while one end
of layered scaffolds was dipped into the solution of growth factor to achieve continuous-gradient scaffold by controlled diffusion mechanism.[242] Similarly, Chen et al.
generated step gradient of VEGF into layered PLG scaffold containing PLG microspheres and implanted into ischemic hindlimbs of mice. They reported that gradient distribution of VEGF resulted in the formation of extensive directional sprout of vascular
networks, whereas, control conditions (scaffolds containing no VEGF, and scaffolds
containing uniform VEGF distribution) caused less-extensive vascular network formation in the ischemic limbs.[243] Besides, cell-demanded AF release is required for the
formation of stabilized and well organized vascular network. Insufcient amount of
VEGF causes EC apoptosis, while over expression results in leaky, immature, and
unstable vessels.[244] During vascularization, EC secretes MMPs to degrade surrounding matrix to allow EC migration. Cell-demanded AF release from scaffold can be
obtained by coupling AFs with MMP-degradable peptides or a specic chemical
linkage of AFs to a gel matrix.[231,245] While delivery of various AFs maintaining a
optimum ratio to promote stable vascularization is a critical issue yet to be solved,
platelet-rich plasma (PRP), which contains biologically determined ratios of multiple
AFs and concentrated platelets in a small volume of plasma, facilitates the delivery of
multiple AFs to form functional and stable vascular network.[246] Although several
attractive features (e.g. cheap, biologically safe, and ease availability) demonstrate the
viability of PRP as a potential agent of blood vessel promoter, short half-life period of
delivered AFs limits its application.[247,248] To achieve sustained and controlled
delivery of AFs, Kurita et al. designed an experiment to evaluate the effect of PRPincorporated gelatin microspheres on neovascularization. They prepared inbred Rat
blood-derived PRP and then injected PRP-incorporated gelatin hydrogel (PRP + Gel),
platelet-poor plasma (PPP), and PRP alone into the ischemic hind limb of rats.
Signicantly higher capillary density was reported for PRP + Gel group compared to
control, PPP, and PRP groups after four weeks of treatment.[249]
Besides direct delivery of AFs, indirect delivery strategy has also been explored
which involves the use of stimulus factors for specic cells that would release AFs at an
ischemic site. Some stimulus factors, such as bone morphogenic protein, hypoxia inducible factor-1, and sonic hedgehog homolog (SHH), can be used to stimulate cells to secrete
AFs as per the local requirement for the formation of a vascular network.[250252]
Afnity-based binding has been pursued in several studies to incorporate stimulus factors
into scaffold biomaterial. For example, Johnson and Wang incorporated SHH into submicron-sized emulsion-like spherical droplets called coacervate by high-afnity binding
with heparin to achieve a slow and sustained delivery for cardiac repair.[253] The special
features of this indirect approach are that it may be more convenient compared to direct
application, it facilitates the secretion of multiple growth factors which promote the maturation of nascent blood vessels, and it results in the natural formation of local gradients
as well as synchronization of various AFs at different stages of vascularization.
4.1.5. Gene therapy
While high production cost and short protein half-lives in vivo limit the application of
growth factors for vascular tissue engineering, gene therapy facilitates prolonged
715
overexpression of proteins.[254,255] Viral and non-viral vectors can be used to deliver

therapeutic genes. Nonpathogenic viral vectors are advantageous over non-viral ones
due to their high gene transfection efciency. Retrovirus, lentivirus, adenovirus, and
adeno-associated virus are four types of viral vectors that have been frequently
employed. Both retroviral and lentiviral vectors can be derived from viruses containing
a single-stranded RNA genome. Cells proliferating from infected cells contain therapeutic genes which exhibit stable and long-term expression. The advantages of retroviral
vectors are stable integration with host DNA, modiable tropism, ease of engineering,
and low immunogenicity. However, random integration with the host genome, potential
risk of mutagenesis, inability to infect non-dividing cells, and lack of specicity to
infect target cells are some of the disadvantages of retroviral vectors.[256] Lentiviral
vectors also offer some attractive features in gene therapy, for example, they are
capable of infecting both dividing and non-dividing cells, and their tropism can be
manipulated. However, the possibility of mutagenesis or oncogenesis and complications
of regulatory proteins in the packaging construct are the major drawbacks associated
with lentiviruses.[257] Overall, the size limitation of insertable genes limits the
application of retroviral and lentiviral vectors for gene therapy.
Adenoviral vectors (AV) have a double-stranded DNA genomeAdenoviral vectors
can infect both dividing and non-dividing cells, facilitate high level of transgene
expression, display better capacity for insertable genes, and have broad cell tropism.
However, short-term expression of the transgene, high immune response, and lack of
integration with host genome limits the use of AVs in gene therapy.
Adeno-associated viral vectors are derived from nonpathogenic viruses containing
single-stranded DNA. Adeno-associated viral vectors are infectious for both dividing
and non-dividing cells, nonmutagenic, and have low immunogenicity. They can promote wide-ranging cell tropism, gene integration, and long-term transgene expression.[258] However, the size restriction of insertable genes, slow kinetics as well as the
requirements of adenovirus for replication limits its use in gene therapy.[259]
In nonviral gene therapy, plasmid DNA is used to infect the target cell, where the
gene of interest is incorporated in a large double-stranded DNA sequence. The advantageous features of the nonviral vector approach are that it is inexpensive, nontoxic,
nonimmunogenic, non-cell-mutagenic, and there is no size restriction for the insertable
DNA sequence.[260] However, the low transfection efciency of nonviral vectors is a
major disadvantage which can be improved using physical and chemical approaches.
Physical methods, such as electroporation, sonoporation, magnetofection, laser irradiation, microinjection, electric eld-induced molecular vibration and ballistic gene guns,
can be applied to increase cell membrane permeability to effectively transfect primary
cells, progenitor cells, and stem cells.[261] Nonetheless, physical delivery methods are
not free from drawbacks, for example, they may trigger apoptosis, reduce cell viability
and can negatively affect cell phenotype.[262] Alternatively, in chemical methods,
cationic lipids, cationic polymers, cell-penetrating peptides, and inorganic nanoparticles
can be used to enhance DNA delivery efciency to target cells,[263] although large
doses of chemical vector to achieve high DNA transfection efciency can cause
cytotoxicity.[264]
A number of studies have used viral and nonviral vectors to evaluate the effect of
gene therapy in vascular tissue engineering. Adeno-associated viruses delivering VEGF
or ang-1 genes into ischemic mouse heart demonstrated that neovasculature caused by
single transfection of ang-1 gene or co-transfection of VEGF and ang-1 gene was less
leaky compared to transfection of VEGF alone. Besides, more capillaries were grown
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in co-transfected groups rather than only ang-1 transfected group.[265] Another study
suggests that plasmid encoding Ang-1 gene promotes collateral vessel formation in an
adult rabbit model having hindlimb ischemia.[266] When plasmids encoding FGF-2,
PDGF-BB, or their combination were injected into myocardial infarcted rat heart, the
plasmid expressing FGF-2 facilitated both capillary and arteriolar growth, and the plasmid encoding PDGF-BB mostly enhanced arteriolar development, whereas their combination increased the number of capillaries and arterioles, and enhanced capillary
stability compared to single-factor expressing plasmids.[267] The method of DNA
delivery can signicantly affect the success of neovasculature formation; PLGA
sponges containing plasmid DNA implanted into subcutaneous tissue of rats caused
sustained and widespread PDGF gene expression leading to enhanced matrix deposition
as well as blood vessel formation, while direct injection of the plasmid couldnt achieve
satisfactory results.[268]
Prior to in vivo transplantation, therapeutic cells are isolated from source, modied
exvivo with gene therapy, seeded into scaffolds and then the scaffolds are implanted
into targeted site. For bone tissue engineering applications, Jabbarzadeh et al. seeded
adipose-derived stromal cells (ADSCs), ECs, and transfected ADSCs with adenovirus
encoding the cDNA of VEGF into PLGA microsphere scaffolds and implanted subcutaneously in SCID mice. They reported that microspheres co-seeded with transfected
ADSCs and ECs showed the highest blood vessel density between 14 and 21 days after
implantation, compared to the blank scaffolds, or scaffolds coseeded with ADSCs and
ECs, or scaffolds seeded either with ECs or ADSCs alone.[269] In an attempt to reduce
brosis in muscle tissue, De Coppi et al. collected myoblasts from adult Lewis rat,
transfected the isolated cells with plasmid encoding VEGF and green uorescent
protein or with plasmid encoding a nonfunctional VEGF-alkaline phosphatase fusion
protein, suspended the transfected cells in collagen Type I gel, and then injected the gel
subcutaneously into nude mice and kept the scaffolds in vivo up to four weeks. Signicant vascular growth was reported for gels containing functional VEGF-transfected cells
compared to nonfunctional VEGF-transfected cells.[270] The method of DNA delivery
can signicantly affect the success of neovasculature formation; PLGA sponges containing plasmid DNA implanted into subcutaneous tissue of rats caused sustained and
widespread PDGF gene expression leading to enhanced matrix deposition as well as
blood vessel formation, while direct injection of the plasmid couldnt achieve satisfactory results.[268] To enhance hVEGF production and cell viability of transplanted stem
cells, Yang et al. utilized optimized poly(-amino esters)(PBAE)-DNA nanoparticles to
deliver hVEGF gene to human mesenchymal stem cells (hMSCs) and human embryonic stem cell-derived cells (hESdCs). Scaffolds seeded with PBAE/VEGF-modied
hMSCs and hESdCs were implanted into dorsal region of athymic mice. It was
reported that two- to four-fold higher blood vessel densities was seen in scaffolds
seeded with PBAE/VEGF-modied stem cells after two weeks of implantation
compared to control cells or Lipofectamine transfected cells.[271]
4.1.6. Scaffold prevascularization
A major challenge in scaffold vascularization is the time delay necessary to form new
functional vasculature following implantation. This delay could be reduced by prevascularization of the scaffold prior to implantation, as prevascularized tissue only
requires anastomosing with the host vasculature. Scaffolds can be prevascularized by
either an in vitro [186,272] or in vivo [273,274] approach. In vitro prevascularization
717
entails culture of different cell types at a controlled ratio in a bioreactor.[186,275] In

vivo prevascularisation is the vascularization of a scaffold implanted in a temporary
host site, prior to implantation at the targeted site.
4.1.6.1. In vitro prevascularization. During or after scaffold preparation, ECs can be
incorporated into a scaffold along with perivascular cells (SMCs, pericytes, etc.) and a
combination of growth factors (VEGF, TGF, FGF, etc.) that causes the formation of
capillary network within the scaffold ex vivo prior to implantation in vivo. To prevascularize the scaffold in vitro, controlled conditions and environment must be maintained.
Several studies demonstrated that in vitro prevascularization of scaffold reduces the
required time of inosculation with host vasculature after implantation in vivo. For
example, in a study, prevascularized and non-prevascularized collagen skin grafts
seeded with keratinocytes, broblasts, and ECs were transplantated into nude mice. It
was reported that prevascularized grafts took only four days to anastomose with host
capillaries, while 14 days were required to get a similar inosculation in the case of
non-prevascularized scaffolds.[276] A 3D multiculture system was used to generate prevascularized skeletal muscle tissue constructs containing myoblasts, embryonic broblasts, and HUVECs co-seeded into PLLA/PLGA polymer scaffolds. When implanted
into mice or rats, only the prevascularized tissue constructs showed functional vascularization, blood perfusion, and prolonged survival.[186] In bone tissue engineering,
in vitro co-culture of hMSCs and HUVECs generated a stable, 3D prevascular network
within 10 days, but anastomosis with host vasculature was limited after implantation.[277] In another study, rat adipose tissue containing microvessel fragments cultured
within collagen gels caused angiogenic sprouting by day ve and formed a collection
of elongated neovessels by day 11. The implanted fragments started interconnecting by
day three and remodeled itself into a mature vascular bed by day 28.[278] Prevascularization by reseeding appropriate cells into decellularized matrix has been used in
bladder tissue engineering. Decellularized porcine bowel was reseeded with SMCs and
urothelial cells, as well as EPCs, and connected to the host vasculature by
microsurgery. The prevascularized construct was effectively perfused with host blood
vessel after implantation, while blood clot was found in the non-prevascularized
construct.[279]
4.1.6.2. In vivo prevascularization. In this technique, scaffolds are vascularized at a
temporary site within the host body prior to transplantation into a targeted site. At the
beginning of prevascularization, the existing vein and artery of a selected arteriovenous
loop are enclosed into a polymer chamber containing matrix or empty space.[280] A
microvascular network is formed within the enclosed matrix, or vascularized granulation tissue is formed within an empty polymer chamber. Once the prevascularized tissue
is formed using this arteriovenous loop method, cells of the eventual target tissue are
seeded,[273,281] and the construct is transferred to the target site.[282] This approach
has been used to generate vascularization in different kinds of tissues, such as adipose,
[283] skeletal muscle,[284] pancreatic (islet),[281] and cardiac tissues.[285] For example, a Matrigel and FGF-2-enriched chamber was installed around the epigastric pedicle
of a diabetic mouse, resulting in the formation of highly vascularized adipose tissue
after 21 days.[273] Pancreatic islets were then transplanted into the prevascularized and
non-prevascularized chamber. Interestingly, signicant improvement in mouse blood
glucose regulation was achieved after three weeks due to islets transplanted into prevascularized chamber. This study suggests that prevascularized chamber promotes islets
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Md. Sarker et al.
survival and function, and can be used as a promising site for islets transplantation. To
repair infarcted myocardial tissue, cardiac patches were prepared by alginate scaffold
containing neonatal cardiac cells and mixture of AFs, placed on the omentum for prevascularization, and then relocated to the myocardial infarction site. Prevascularized
cardiac patches were structurally and electrically integrated with the host myocardium
after 28 days of transplantation.[274]
4.2. Scaffold-free approach

4.2.1. Cell sheet technique
Unlike techniques based on the construction of pre-formed scaffolds, the cell sheet
technique allows production of ECM material by cultured cells, which is then
implanted as a cell-ECM construct at the targeted site [151] (Figure 10). Cell-generated
ECM is likely to be more effective than articial scaffold matrix in regulating tissue
growth and maturation. For example, a ve-layer construct was prepared containing
alternative layers of myoblast and vascular EC sheets. This construct promoted the
formation of capillary-like network all over the structure within four days of in vitro
culture. When the ve-layered sheets were enclosed in brin gel and implanted into
nude rats, the HUVECs incorporated myoblast sheets resulted in the rapid formation of
blood vessels that were connected to the host vasculature.[286] Poly-N-isoproplyacrylamide has been reported as an advantageous culture surface for cell sheet preparation,
having a temperature-responsive property that facilitates cell attachment above 32 C
and detachment below 32 C. In addition, the surface promotes cell spread and proliferation at 37 C.[287] The cultured cells are separated from culture surface as a cell
Figure 10.
Cell sheet technique for vascular network formation.
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sheet upon conuence and multiple cell sheets are prepared following the same
methodology.
5. Summary and conclusion

Signicant improvement has been accomplished over the last decade in the area of
vascular tissue engineering. Challenges still exist to fabricate scaffolds using modern
fabrication techniques that mimic the 3D ultrastructure, surface topography, biophysical
and biochemical cues, and cell density and distribution of native tissue. Specic cells
are sensitive to the tissue-specic ultrastructure and biochemistry of ECM, and mismatch can cause problems. Another technical challenge of the scaffold-based approach
is to seed the scaffold with healthy and potent autologus vascular cells in sufcient
number as well as to maintain cell viability, functionality, and phenotype in vitro and
in vivo. Alternative opportunities using stem and progentitor cells exist instead of
autologous vascular ECs, but the risk of uncontrollable differentiation limits their
implementation at this time. Because of their potentially increased efciency and control of seeding vascular cells, perfusion bioreactors require more investigation. This
technology is still limited by the complexity of generating homogeneous cell distributions and providing appropriate biophysical stimuli to cultured cells. Growth factors
have been proven to be key regulators for vascularization, and more precise methods
are needed for controlling temporal, localized, sequential, or co-delivery of multiple
growth factors. Gene therapy in vascular tissue engineering offers an attractive
alternative due to the short half-life and high production cost of protein growth factors.
These relatively simple and direct approaches to revascularization are giving way to
more sophisticated technologies. The issues associated with the time delay of vascular
formation and anastomosis with host vasculature led researchers to the approach of
in vitro or in vivo prevascularization. The preparation of decellularized matrix promises
to produce the most bio-mimic scaffolds, but achieves limited success due to problems
regarding the decellularization and recellularization processes, and decellularizationassociated cytotoxic left over.
Scaffold-based approach to tissue vascularization may ultimately prove to be more
promising in terms of forming thick, complex tissue while achieving precise control
over the micro structure of blood vessels. Although capillaries and their interconnected
vascular network can now be formed by microfabrication technique, issues related to
tissue swelling and functional tissue formation still remain to be investigated. A modular approach facilitates better mass diffusion and cell seeding at high density, but
formation of dissimilar vascular structure in respect to native tissue can have negative
effects on tissue functionality. One possible way in forming functional vascularized tissue in vitro or in vivo could be the simultaneous formation of blood vessels in macro
and micro region. Combination of 3D and modular/bottom-up approaches could be able
to facilitate the fabrication of macro and complex 3D vascular network as well as the
positioning of cellladen, patterned mirogels around the vascular tree in a controlled
way so that those microtissues could modify themselves into vascularized tissue
through perfusion and self assembly. Another alternative, the cell sheet approach, holds
promise for highly proliferative cells while demonstrating poor tissue integrity, and
requires further investigation. Despite successes in many of the approaches described in
this review, no one technique is promising enough to ensure the generation of fully
stable and functional vascular network within all tissue engineering constructs. Tailored,
synergistic application of multiple different strategies to vascularize newly formed
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tissue in different situations may ultimately be required. Moreover, better methods are
also required to assess the fate of the newly formed blood vessel in terms of stability,
maturation, and remodeling after implantation, and their ability to remain functional for
long periods of time in vivo.
List of Abbreviations
VEGF
PDGF
FGF
TGF
IL-8
Ang
HGF
EGF
S1P
MCP-1
IGF-1
MMP
SDF-1
TIMP-2
GM-CSF
G-CSF
GRO-
PLLA
PLG
PGS
PLGA
PLL
CHO
HUVSMCs
HSFs
PEGDA
GelMA
RGDS
PEG
PDMS
HBP
LIFT
LGDW
SMC
vascular endothelial growth factor

platelet derived growth factor
broblast growth factor
transforming growth factor
interleukin-8
angiopoietins
hepatocyte growth factor
epidermal growth factor
sphingosine-1-phosphate
monocyte chemoattractant protein-1
insulin-like growth factor-1
matrix metalloproteases
stromal cell-derived factor-1
tissue inhibitor of metalloproteinase-2
granulocyte-monocyte colony stimulating factor
granulocyte-colony stimulating factor
growth regulated oncogene-
poly-L-lactic acid
poly (lactide-co-glycolide)
poly glycerol sebacate
poly lactic-co-glycolic acid
poly (L-lysine)
chinese hamster ovary cells
human umbilical vein smooth muscle cells
human skin broblasts
poly(ethylene glycol) diacrylate
gelatin methacrylate gel
arginine-glycine-aspartic acid-serine
poly(ethylene glycol)
polydimethylsiloxane
heparin-binding peptide
laser induced forward transfer
laser-guided direct writing
smooth muscle cells
Disclosure statement
No potential conict of interest was reported by the authors.
Funding
This work was nancially supported by the Saskatchewan Health Research Foundation [SHRF
Reference # 2784]; and the Natural Sciences and Engineering Research Council of Canada
[NSERC RGPIN-2014-05648].
721
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Date: 10 March 2016, At: 23:07

Journal of Biomaterials Science, Polymer Edition, 2015

Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.

Md. Sarkera*, X.B. Chena,c and D.J. Schreyera,b

(Received 26 March 2015; accepted 2 June 2015)

Md. Sarker et al.

Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.

2. Different types of blood vessels and their functions

Blood circulatory system inside the body (Reproduced from [288]).

Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.

Journal of Biomaterials Science, Polymer Edition

Structure of different types of capillaries (Reproduced from [288]).

Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.

Md. Sarker et al.

Blood vessel formation by vasculogenesis (Reproduced from [289]).

Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.

Journal of Biomaterials Science, Polymer Edition

Blood vessel formation by angiogenesis in brain cancer (Reproduced from [290]).

Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.

Md. Sarker et al.

generate non-uniformity along the wall of blood vessel.[28,29] In addition, another

Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.

Journal of Biomaterials Science, Polymer Edition

Different approaches for vascular network formation.

Md. Sarker et al.

Tissue engineering approaches to blood vessel formation.

Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.

Scaffold free approach

with porous lms of poly-D, L-lactic-co-glycolic acid facilitated the ingrowth of

Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.

Journal of Biomaterials Science, Polymer Edition

veins of a mongrel dog showed no evidence of stenosis, thrombus, occlusion, or

Md. Sarker et al.

Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.

Poor mechanical strength,

Critical role for angiogenesis

Journal of Biomaterials Science, Polymer Edition

Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.

modules without bronectin coating.[56] Similarly, implantation of a bronectin/

Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.

Md. Sarker et al.

Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.

Journal of Biomaterials Science, Polymer Edition

agents work by facilitating chemical exposure, bursting cells, or disrupting cell

Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.

Md. Sarker et al.

Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.

Journal of Biomaterials Science, Polymer Edition

capillaries.[103,104] More specically, the depth and topography of microgrooves has

3D approaches for vascular channel formation.

Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.

Md. Sarker et al.

acrylate-PEG-RGDS into a rectangular mold containing the lattice lament,

Journal of Biomaterials Science, Polymer Edition 2015.26:683-734.