(Reference Series in Biomedical Engineering) Wolfgang Holnthoner, Andrea Banfi, James Kirkpatrick, Heinz Redl (Eds.) - Vascularization For Tissue Engineering and Regenerative Medicine-Springer Interna

Angiogenesis: Basics of Vascular Biology
Victor W. M. van Hinsbergh
Abstract
Angiogenesis occurs by two mechanisms sprouting angiogenesis and intussus-
ceptive angiogenesis. Intussusceptive angiogenesis starts with the formation of an
intravascular pillar, which can be extended, so that a vessel tube becomes
separated in two parallel branches. Sprouting angiogenesis regards the outward
formation of small new vascular branches that starts via invasion of endothelial
sprouts into the extracellular matrix. These sprouts are led by a tip cell that
strongly responds to exogenous angiogenic factors, of which VEGF-A is the
most prominent. This chapter describes molecular steps and metabolic responses
that occur within the tip cell and the subsequent signaling that alters the behavior
of the adjacent stalk cells. Subsequently, lumen formation, anastomosis, and
restoration of perfusion occur, as well as selective removal of excess vascular
branches by pruning. The process of angiogenesis is enforced by postnatal
vasculogenesis, which represents the recruitment of circulating true endothelial
progenitor cells (late outgrowth EPCs or endothelial colony forming cells) to an
area in need of blood supply, and is further supported by recruitment of myeloid
early-outgrowth EPCs that have an auxiliary function. The detailed studies on the
sprouting process itself have to be placed into a (patho)physiological context to
be able to generate functional microvascular networks. From combined compu-
tational modeling and experimental studies it has become clear that formation of a
new microvascular network requires the mutual interplay between sprouting,
redistribution (remodeling), and pruning of endothelial tubules into a functional
vascular bed.
V.W.M. van Hinsbergh (*)

Department of Physiology, Institute for Cardiovascular Research, VU University medical center,
Amsterdam, The Netherlands
e-mail: v.vanhinsbergh@vumc.nl
# Springer International Publishing AG 2016 1

W. Holnthoner et al. (eds.), Vascularization for Tissue Engineering and Regenerative
Medicine, Reference Series in Biomedical Engineering,
DOI 10.1007/978-3-319-21056-8_1-1
2 V.W.M. van Hinsbergh
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Neovascularization in Early Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3 Different Forms of Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
4 Intussusceptive Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
5 Dominant Growth Factors in Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
6 Balancing Angiogenesis: Endogenous Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
7 Sprouting Angiogenesis: Initiation and Invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
8 Extension and Stabilization of Sprouts: Dll4/Notch Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
9 Guidance of Tip Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
10 Metabolic Control of Sprouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
11 Lumen Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
12 Anastomosis and Vessel Stabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
13 Pruning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
14 Lymphangiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
15 Postnatal Vasculogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
16 Generation of a Functional Microvascular Bed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
17 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
1 Introduction
Functions of the various organs in the body are supported, monitored, and connected
by three major systems: the circulatory system with blood vessel ramifications
throughout the whole body, which distributes oxygen, nutrients, and hormones;
the neuronal system; and the immune/inflammation system of leukocytes that patrol
throughout the body and monitor infection, inflammation, and healing, which in part
occur via blood and lymphatic vessels. While arteries and veins are mainly involved
in the transport of the blood propelled forward by the heart, the distribution and
exchange of oxygen and nutrients occurs by and in the microcirculation. The
microcirculation consists essentially of a large number of network loops that connect
the arteries and veins. Their perfusion in the adult organ is controlled by the arterial-
venous blood pressure difference generated by the cardiac output and the vascular
resistance that is largely controlled by arteriolar vasoregulation, capillary recruit-
ment, and shear forces exerted by the blood on the endothelial surface. Exchange
occurs mainly in the capillaries where the ratio between endothelial surface and
blood volume is much higher than in the other vessels. As the diffusion of oxygen
usually spans only a few layers of cells, the capillaries of the microvascular network
must be distributed in an adequate way to fulfill the metabolic demand of all tissue
cells. In a healthy tissue increases in the metabolic demand is compensated by
vasodilation of resistance arterioles and recruitment of underperfused capillaries.
However, if the overall microvascular bed cannot cope with the demand, e.g.,
because of growth, exercise, tissue injury and repair, or inflammation, ischemic
damage has to be prevented by expanding the microvascular bed by angiogenesis
(and collateralization if the proximal arterial blood supply has been interrupted).
Angiogenesis is also needed when a graft with tissue-engineered cells is implanted.
Angiogenesis: Basics of Vascular Biology 3
Without connection to the circulatory system the implanted cells will largely die,
even if they contain prefabricated endothelial tubules.
In this chapter we shall discuss basic aspects of angiogenesis. Three main types of
angiogenesis are recognized: sprouting angiogenesis, intussusceptive angiogenesis,
and vasculogenesis. Most mechanistic information comes from animal models, such
as vascularization of the chicken embryo and developing zebrafish and development
of the mouse retina vasculature, in which sprouting angiogenesis has been investi-
gated extensively. These insights may help to induce sprouting and to generate
perfused connections between grafts and the host vasculature. In addition, we shall
discuss postnatal vasculogenesis as a mean to obtain patient-specific endothelial
cells and to enforce neovascularization. However, the body needs more than endo-
thelial tubes only. Understanding how to generate microvascular networks by using
the body’s own ability to differentiate a functional microvascular bed is the next
challenge that is needed to master neovascularization in disease and tissue repair.
2 Neovascularization in Early Development
During early embryonic development, progenitors of vascular endothelial cells,

so-called angioblasts, have become differentiated at two different locations. In the
blood islands of the yolk sac and extraembryonic membranes they surround hemo-
poietic cells, while within the embryo itself, angioblasts orientate as cords of cells
that fuse together and form a tubular structure, the dorsal aorta. The angioblasts of
the blood islands subsequently expand and form a capillary plexus, a lumen
containing network of endothelial cells, throughout the extraembryonic tissue
(Risau and Flamme 1995). This plexus further expands by intussusceptive and
sprouting angiogenesis (see below) thus approaching vascular structures within the
embryo itself (Eichmann et al. 2005b; Risau 1997). There, sprouting angiogenesis
from the dorsal aorta results in the branching of intercostal vessels and early veins.
Part of the dorsal aorta develops into a primitive heart. As soon as the heart starts
contracting blood starts moving; first partially back and forward, but soon as a
circulation in one direction. It also connects to the preexisting plexus tubes in an
ordered fashion. The blood flow itself becomes a determining component in the
spread of these connections, in the further differentiation of arteries and veins, and in
the functional properties of the whole vascular system (Jones et al. 2006).
It is important to stress that while the initial vasculogenesis and formation of the
primitive capillary plexus occur in the absence of blood flow, thereafter the circu-
lating blood and particularly the accompanying shear forces on the endothelium
become an important determinant of the subsequent shape and functionality of
various vessels of the circulation. It controls functions of the arterial and microvas-
cular endothelium, among which is the production of the vasodilator nitric oxide
(Busse and Fleming 1998; Song and Munn 2011). Together with the metabolic
demand of the surrounding tissue it determines the pattern of the local microcircu-
lation of healthy tissues.
3 Different Forms of Angiogenesis
The expansion of the primitive vasculature occurs by sprouting and intussuscep-

tive angiogenesis (Fig. 1). Intussusceptive angiogenesis leads to the splitting of
microvessels along its length axis. To that end, invaginations from the inner
surface of the vessel form a bridge within the vessel lumen. This bridge can expand
and divide the luminal compartment along the vessel length, which results in two
parallel or branching vessels. Sprouting angiogenesis refers to the formation of
new vasculature by branching off of sprouts and anastomosis of sprouts forming a
new perfused vessel. In sprouting angiogenesis, which is also observed in wound
healing and pathologies of the adult, an existing vessel starts by branching off one
or more of its endothelial cells. It requires that the endothelial cells degrade the
original basement membrane, migrate through it into the interstitium, and form –
by migration and cell division – a chain of endothelial cells with a lumen at the side
of the original vessel and a so-called tip cell at the invading tip of the chain. The tip
of the sprout induces another vessel also to sprout, after which both sprouts
anastomose and perfusion becomes established (Isogai et al. 2003; Lenard et al.
2013).
Intussusceptive and sprouting angiogenesis can be enforced by incorporation of
circulating endothelial progenitor cells, in particular endothelial colony-forming
cells, which insert in the newly forming endothelial lining and have a high
Intussusceptive angiogenesis Sprouting Angiogenesis
Fig. 1 Intussusceptive (splitting) angiogenesis (a) and sprouting angiogenesis (b)

proliferative potential. These cells may harbor an important regenerative capacity

and will be discussed separately as postnatal vasculogenesis.
The need for intussusceptive and sprouting angiogenesis is large during devel-
opment, tissue healing, or prolonged inflammation, when the metabolic demands are
very high and cannot be matched by existing vasodilation mechanisms. Once the
tissue stabilizes and the metabolic demand decreases, the number of branches in the
vascular bed can become reduced again by selective absorption of excess micro-
vessels, a process called pruning, which adapts the vasculature to the new metabolic
need (Benjamin et al. 1999; Korn and Augustin 2015).
4 Intussusceptive Angiogenesis
While sprouting angiogenesis refers to the expansion of microvessels that radiates

from the basolateral side of the endothelial lining into the interstitium, intussus-
ceptive angiogenesis occurs within the vascular lumen (Burri and Tarek 1990;
Makanya et al. 2009; Risau 1997). A hallmark of intussusceptive angiogenesis is
the formation of the so-called intussusceptive pillar, a structure that starts as
protrusive extensions of the endothelium (usually junction protrusions) within
the vessel lumen that bridge both inner sides of the vessel lumen (Burri and
Tarek 1990; Paku et al. 2011). Subsequently, the simultaneous occurrence of
local detachment of the basal membrane from the underlying interstitium and
contractile shape changes of the endothelial cell causes the movement of the cell
body into the intraluminal bridge together with its adherent basement membrane
(Paku et al. 2011). Endothelial cells connected from both sides of the vessel finally
form a pillar with a collagenous center, into which pericytes and fibroblasts
migrate. Intussusceptive angiogenesis proceeds rapidly by using existing endothe-
lial cells and does not require endothelial proliferation. While originally cylindrical
in shape, intussusceptive pillars expand and induce vessel duplication as well as
vessel branching (Mentzer and Konerding 2014). Furthermore, flow can induce
multiple pillars, thus facilitating the formation of bifurcations in the embryo.
Intussusceptive angiogenesis is not limited to embryogenesis but has an important
contribution to angiogenesis in juvenile and adult organs, in particular in the lung
(Ackermann et al. 2014).
Because intussusceptive angiogenesis occurs within the vessel lumen, it is more
difficult to study it than sprouting angiogenesis. While considerable progress has
been made in elucidating the mechanisms of sprouting angiogenesis, the mecha-
nisms involved in intussusceptive angiogenesis are largely still unknown. On the
basis of gene deletion experiments in mice and subsequent mechanistic studies it has
become evident that VEGF receptors, which are well known for their effects on
sprouting angiogenesis, and angiopoietin receptors, are required for intussusceptive
angiogenesis (Baum et al. 2010; Risau 1997; Winnik et al. 2009) Exogenous
addition of erythropoietin, which is induced by hypoxia, also stimulated intussus-
ceptive angiogenesis (Crivellato et al. 2004).
5 Dominant Growth Factors in Angiogenesis
The mechanisms of sprouting angiogenesis have become better understood and

involve a series of events that are stimulated and controlled by angiogenic growth
factors, receptors, and inhibitors, of which VEGF-A and its receptors play a pivotal
role. In search of factors that controlled the growth tumor vasculature many different
pro- an antiangiogenic factors have been recognized (see Carmeliet and Jain 2000;
Folkman and Klagsbrun 1987 for review), including fibroblast growth factors (FGF)
FGF-2 and FGF-1 (originally called ECGF), which are potent stimulators of endo-
thelial proliferation when exogenously administrated and commonly used for the
culture and propagation of endothelial cells (Gospodarowicz et al. 1976; Maciag
et al. 1981). However, only after the discovery and cloning of VEGF-A (Carmeliet
et al. 1996; Ferrara et al. 1996), its family members VEGF-C and D (Joukov et al.
1996; Yamada et al. 1997) and the VEGF receptors (Dumont et al. 1998; Fong et al.
1995; Shalaby et al. 1995) insight in the molecular regulation of developmental and
regenerative angiogenesis became available. Experimental data from the last two
decades have put specific emphasis on the dominant role of four major receptor-
mediated regulatory factor families that display a dominant control of angiogenesis
and vascular modeling. They regard the VEGF/VEGF receptor family (Adams and
Alitalo 2007; Ferrara et al. 2003), the Notch receptors and their ligands, in particular
Delta-like ligand-4 (Dll4) (Hellstrom et al. 2007; Liu et al. 2003; Noguera-Troise
et al. 2006; Ridgway et al. 2006), endothelial members of Wnt signaling as recently
recognized (Dufourcq et al. 2002, 2008; Guillabert-Gourgues et al. 2016; Korn et al.
2014), and finally the angiopoietin/angiopoietin receptor system, which influences
remodeling of angiogenic vessels in a context-dependent manner (Augustin et al.
2009; Suri et al. 1996). These factors and receptors are strictly regulated. Full
deletion, and in many cases also haplodeficiency (lack of one allele), causes severe
disturbances in vascular development and usually lethality of the embryo (Carmeliet
et al. 1996; Ferrara et al. 1996; Hellstrom et al. 2007).
6 Balancing Angiogenesis: Endogenous Inhibitors
Once a vessel is established it becomes less responsive to angiogenic factors.

Antiangiogenic factors become dominating. They switch off angiogenesis and
support endothelial quiescence. Many antiangiogenic factors are proteins that inter-
fere with the signaling of angiogenic growth factors and many of them are deposited
in the extracellular matrix of maturing vessels. These latter ones include matrix
proteins like thrombospondins 1 and 2 (TSP-1, TSP-2) and the proteoglycans
decorin and osteoglycin. TSP-1 can interact with many proteins and contains two
angiogenesis inhibiting TSP- domains (Iruela-Arispe et al. 1999). Inhibition occurs
in particular through interaction with integrins and CD36 and CD47 antigens.
Interaction of TSP-1/TSP-2 with CD36 causes recruitment of the intracellular Src
homology 2 domain-containing protein tyrosine phosphatase (SHP-1) to the
VEGFR-2, which interferes with VEGF signaling (Chu et al. 2013). Binding of
CD47 to TSP-1 withdraws CD47 from interaction with VEGFR-2 and also limits
VEGFR-2 signaling (Kaur et al. 2010). However, other interactions have also
reported affecting FGF-2 signaling (Pagano et al. 2012) and endothelial cell migra-
tion, survival, and apoptosis. Decorin inhibits angiogenesis by multiple mechanisms
(Neill et al. 2012), while osteoglycin prevents binding of VEGF to the extracellular
domain of VEGFR-2 (Wu et al. 2017).
Another important physiological regulator of angiogenesis is (soluble) VEGFR-1
(Fong et al. 1995). VEGFR-1 is required for normal vascularization, but angiogen-
esis and development are normal when its tyrosine kinase domain is lacking
(Hiratsuka et al. 1998). VEGFR-1 acts as a decoy receptor of VEGF-A and thus
limits the extent of sprouting. The importance of VEGFR-1 in limiting angiogenesis
was underlined by a study showing that the cornea requires soluble VEGFR-1 to
prevent its vascularization (Ambati et al. 2006). Furthermore, pigment epithelium
derived factor (PEDF) contains a peptide region in its N-terminal part (residues
24–57), which balances VEGF-induced angiogenesis in the eye and other tissues
(Dawson et al. 1999; Doll et al. 2003). Its mechanisms of action is still unclear, but
among several suggestions inhibition of Wnt signaling has been put forward (Park
et al. 2011). Finally, as pericellular proteases are required for invasion of the
angiogenic sprouts into the interstitial matrix, proteases inhibitors and proteolyti-
cally generated peptides also inhibit angiogenesis (see next section).
7 Sprouting Angiogenesis: Initiation and Invasion
The endothelium of a quiescent vessel is enwrapped on its basolateral side by a

firm basement membrane and usually is covered by mural cells. To escape from
the inner vascular lining, mural cells have to give way and endothelial cells have
to degrade their basement membrane to allow radial outgrowth of vessel sprouts.
Detachment of adherent pericytes can be the consequence of degradation of the
basement membrane, in which pericytes are embedded, or can be induced by
interference with essential growth factors for mural cells such as platelet-derived
growth factor (PDGF) and angiopoietin-1 (Ribatti et al. 2011; Wilkinson-Berka
et al. 2004). Degradation of the basement membrane is achieved by highly
controlled proteolysis that occurs at the surface of the endothelial cell and
which is comparable to that of other invading cells. The main proteases that
have been recognized in this process are (membrane-type) matrix meta-
lloproteinases (MMPs), in particular MT1-MMP (also called MMP-14),
MMP-2, and MMP-9, and the urokinase-type plasminogen activator/plasmin
system (Chun et al. 2004; Hotary et al. 2002; Koolwijk et al. 1996; Pepper et al.
1990; Stratman et al. 2009; van Hinsbergh et al. 2006; van Hinsbergh and
Koolwijk 2008). These proteases evoke site-specific pericellular proteolysis by
either being a membrane-anchored protein or via receptor binding to the cell
membrane. This pericellular proteolysis is under the additional control of specific
inhibitors, such as tissue inhibitors of metalloproteinases (TIMPs), the
MMP-inhibitor RECK (reversion-inducing cysteine-rich protein with Kazal
motifs), and plasminogen activator inhibitor-1 (PAI-1) (Handsley and Edwards

2005; Noda et al. 2003; Oh et al. 2001; Pepper et al. 1990). Important inducers of
this pericellular proteolytic activity are angiogenic factors, such as VEGF-A and
FGF-2, and inflammatory mediators, in particular tumor necrosis factor-α (TNFα).
The proteolytic activity also cleaves many proteins in the basement membrane and
interstitium, thus liberating additional angiogenic growth factors as well as angio-
genesis inhibiting peptides such as endostatin (O’Reilly et al. 1997) and tumstatin
(Maeshima et al. 2002) (see also Egeblad and Werb 2002; van Hinsbergh et al.
2006). As a result, the endothelial cell projects extrusions through the gap in the
basement membrane, which on guidance of angiogenic factors activate the cell to
acquire new properties, namely that of the so-called tip cell (Gerhardt et al. 2003).
The tip cell is the prime invading cell of an angiogenic sprout and is characterized
by multiple filopodia, which harbor many VEGFR-2 molecules, which display a
high activity and turnover (Sawamiphak et al. 2010). They also contain neuropilin-1,
which supports VEGFR-2 activation (Soker et al. 2002; Fantin et al. 2013), and the
cytokine receptor CXCR4, which enables the cell to sense gradients of the strong
chemokine stromal derived factor (SDF-1) (Strasser et al. 2010). In response to these
factors, the tip cell starts invading the surrounding matrix while maintaining contact
with its neighboring endothelial cells. This leads to a string of endothelial cells that
invade the interstitial matrix led by the tip cell. The cells that follow the tip cell are
called stalk cells and are able to proliferate.
Many conditions which create high VEGF-A concentrations, such as acute
hypoxia and tumors, induce sprouting. While it is generally accepted that a
VEGF-A gradient is an important inducer and activator of tip cells and that filopodia
of tip cells respond to VEGF by their receptor VEGFR-2, the VEGF gradient is not
directly translated to a higher content of VEGFR-2 in filopodia or tip cells. However,
not only the number of receptors exposed at a certain moment is important, but
endocytosis of ligand-binding VEGFR-2 and VEGFR-3 is necessary for down-
stream signaling by these receptors (Nakayama et al. 2013; Sawamiphak et al.
2010; Wang et al. 2010; Zhang and Simons 2014).
In addition to VEGFR-2, VEGFR-3 is also encountered in angiogenic sprouts.
During early development, VEGFR-3, which binds VEGF-C and VEGF-D, is
present in endothelial cells, in particular in veins and the microvasculature and is
essential for proper development of blood vessels (Dumont et al. 1998). However,
when development proceeds, VEGFR-3 becomes restricted to the lymphatic endo-
thelium (Kaipainen et al. 1995) and some fenestrated endothelia (Partanen et al.
2010, FASEB J). After birth, VEGFR-3 becomes highly reexpressed in the tip cells
of angiogenic sprouts, e.g., in the neonatal mouse retina and in tumor angiogenesis
suggesting a controlling role in the tip cell (Tammela et al. 2008). Indeed, stimulation
of VEGFR-3 augmented VEGF/VEGFR-2 mediated angiogenesis. However, the
contribution of VEGFR-3 may be complicated as VEGFR-2/VEGFR-3 hetero-
dimers have also been recognized in angiogenic sprouts (Nilsson et al. 2010).
Genetic experiments recently indicated that, while VEGFR-3 signaling is pivotal
in lymphangiogenesis, VEGFR-3 required VEGF/VEGFR-2 signaling to act on
angiogenesis (Zarkada et al. 2015). This indicates that VEGFR-3 was unable to
induce angiogenesis without VEGF/VEGFR-2 signaling as had been suggested

earlier (Benedito et al. 2012).
Internalization of ligand-binding VEGFR-2 – as well as VEGFR-3 – required the
presence of ephrinB2 without need for interaction with its Eph receptor (Bochenek
et al. 2010). Deletion of ephrinB2 abrogated VEGF-induced sprouting, while over-
expression of ephrinB2 enhanced sprouting in a disorganized way. In the developing
mouse retina, Nakayama et al. (2013) demonstrated that endocytosis of ligand-
binding VEGFR-2 and VEGFR-3 was facilitated by a complex of the proteins
Disabled-2 (Dab2), ephrinB2, and Par-3. Dab2, a clathrin-associated sorting protein,
coupled VEGFR-2 to the cell polarity protein Par-3 and ephrinB2. Endothelium-
specific deletion of Dab2 prevented endothelial sprouting, similar as ephrinB2
deletion did. Intracellularly VEGFR-2 activity causes multiple phosphorylations,
depending on the VEGF species and isoform (Fearnley et al. 2016; Olsson et al.
2006). This not only causes gene induction of Dll4 and other proteins but also the
phosphorylation of the atypical protein kinase PKCλ and probably PKCζ, which can
inactivate and dissociate Dab2 by phosphorylation at Ser24 (Nakayama et al. 2013),
thus reducing VEGF receptor internalization and signaling.
8 Extension and Stabilization of Sprouts: Dll4/Notch

Signaling
Interestingly, activation of VEGFR-2 and VEGFR-3 induces the expression of

Delta-like ligand-4 (Dll4), a ligand of the receptor Notch. One might expect that
all endothelial cells start tipping, but secretion of Dll4 by the tip cell counteracts this
by activating Notch-1 on adjacent endothelial cells. Activation of Notch-1 causes
receptor shedding and subsequent liberation and translocation of the intracellular
part of the receptor to the nucleus, which activates transcription of specific genes.
This leads to multiple effects within the adjacent endothelial cells (stalk cell)
including a shift in the balance between VEGFR-1 and VEGFR-2 towards the
inhibitory VEGFR-1, which suppresses a tip cell response (Harrington et al. 2008;
Potente et al. 2011; Fig. 2). The importance of Notch/Dll4 regulation is underpinned
by the observations that (haplo)deficiency of Dll4 or Notch-1 causes massive
disorganized sprouting in the developing murine retina but few properly perfused
vessels (Hellstrom et al. 2007; Krebs et al. 2000).
The high (intra)cellular VEGFR-2 activation that stimulates the invasive proper-
ties of tip cells is not accompanied by proliferation of the tip cell, which rarely
divides (Gerhardt et al. 2003; Ruhrberg et al. 2002). On the other hand, the reduced
VEGFR-2/VEGFR-1 ratio in stalk cells limits tip cell formation but still allows
proliferation of the stalk cells that are in the sub-sprouting area and which encounter
a lower VEGF-A concentration than the tip cells. Although the VEGF-A concentra-
tion is lower, it is still sufficient to induce endothelial cell proliferation, as in vitro
experiments showed that the concentration of VEGF-A required to induce prolifer-
ation is far less than that that is needed for migration and invasion of endothelial cells
(Koolwijk et al. 1996). Apparently other factors determine the lack of proliferative
Surface-bound proteolytic activity
10
VEGFR-2 and VEGFR-3 on Filopodia

respond to VEGF gradient
Internalization of VEGF:VEGFR-2 and -R3

complexes
required: Dab2, ephrin, Par-3;
inhibited by atypical PKC
Dll4 Notch1
VEGF/VEGFR-2 and -3 signaling Notch1 activation and nuclear signaling via its NICD
Induction of Dll4
Transcription: Induction PTEN Increase in cell polarity
↑VEGFR1 decoy receptor (VE-cadherin, β-catenin,
Top cell Notch blocked by ↓VEGFR2, ↓VEGFR3 pals, Par-3)
Jag1 interaction ↑Jag1
Nuclear FOXO1 Repulsion
Growth factors, inhibits Myc and (podocalyxin, CD34)
insulin proliferation Lumen formation
Jag1 IP3K
Notch1
AKT
↑Glycolysis FOXO1 FOXO1-P
- - - - Cell division
- - c
- -
Dll4 Notch1
VE-cadherin
Tip cell
Sprouting
Stalk cells
Fig. 2 Responses of and interplay between tip and stalk cells during sprouting angiogenesis. The sprouting tip cell responds to stimuli such as a gradient of VEGF-A
(green dots). VEGF binding to VEGFR-2 causes internalization and signaling which induces Dll4. Tip cells display glycolysis. Dll4 activates the transmembrane
V.W.M. van Hinsbergh
receptor Notch1, of which the Notch intracellular domain (NICD) is liberated and induces many effects (box) in the adjacent stalk cells (see text for details)
response of the tip cell, such as the overall organization of the cytoskeleton, the high
metabolic demand of the sprouting cell, and possibly the location and protein
interactions of the activated VEGF receptor-2 itself. However, the differences
between tip and stalk cells are relative and not absolute. Tip and stalk cells are
interchangeable phenotypes (Jakobsson et al. 2010). Once the tip cell becomes
exhausted, one of the adjacent stalk cell can take over the tip function, after which
the original tip cell returns in a stalk cell role and phenotype. In addition to increased
VEGFR-1 transcription, Notch activation causes the transcription of Jagged-1,
another Notch ligand, which limits Dll4 binding indirectly, and which is thought
to couple back to the tip cell, thus facilitating endured sprouting (Potente et al. 2011;
Yoon et al. 2016).
An interesting addition came from the work of Serra et al. (2015), who demon-
strated that Dll4-Notch1 activation exerts its antiproliferative effect on endothelial
stalk cells in the sprouting front of the murine retinal vasculature via activation of
PTEN (phosphatase and tensin homolog). It had been observed earlier that
endothelium-specific deletion of PTEN in mice caused increased mitotic and migra-
tory responses to angiogenic growth factors and abnormal angiogenesis (Hamada
et al. 2005; Huang and Kontos 2002). Furthermore, PTEN was also indicated as a
strong candidate to explain the antiangiogenic phenotype observed in various
angiogenesis models after deletion of the miR-17-92 cluster. This indicates that the
miR-17-92 cluster, which is upregulated via a VEGF/ERK/Elk-1 pathway, reduces
PTEN and facilitates endothelial cell proliferation and angiogenesis (Chamorro-
Jorganes et al. 2016). In the murine retina, Serra et al. (2015) found that PTEN
had no effect on the Notch-dependent phenotypic determination of stalk vs. tip cells.
However, endothelium-specific deletion of PTEN caused increased endothelial cell
division in particular in the sprouting front and resulted in excessive branching,
reduced sprout length, and substantially increased vessel width, thus causing vascu-
lar hypertrophy. Mice with a twofold overexpression of PTEN exhibited the reverse
phenotype, i.e., decreased vessel width and increased sprout length, with no changes
in the number of branches and sprouts. Indeed, PTEN activation, which appeared
driven by Dll4-activated Notch signaling, inhibited endothelial cell proliferation in
stalk cells located at the sprouting front (Serra et al. 2015). Cell proliferation was not
affected in the sub-frontal area of the developing retinal vasculature, in which no
PTEN activation was encountered. This is in line with the finding that endothelial
cell division, which is needed for the expansion of the new vessels, occurs largely in
this sub-sprouting area.
9 Guidance of Tip Cells
It has been anticipated that the VEGF-induced response of sprouting endothelial

cells is fine-tuned in a similar way as axonal sprouts (growth cones) by guidance
receptor molecules that attract and repulse tip cells (Eichmann et al. 2005a). This
would explain how the sprouting vessel finds its way and creates together with other
sprouts an orderly vascular network that covers the whole tissue. Among the
guidance receptors that have been recognized in sprouting endothelial cells are
neuropilins (interact with VEGF, placenta growth factor, and semaphorins),
UNC5B (a receptor for netrins that is induced by hypoxia) (Dakouane-Giudicelli
et al. 2011; Navankasattusas et al. 2008), plexins that recognize various semaphorins
(Cora et al. 2014; Klagsbrun and Eichmann 2005), Eph ephrin receptors (Adams
et al. 1999; Barquilla and Pasquale 2015), and the receptor ROBO4 (involved in
maintaining vessel integrity) (Koch et al. 2011). They interact with defined families
of ligands or counter-receptors. They modulate the direction of sprouting but can
also be involved in other processes, such as the phenotypic determination of the new
vessel, such as arterial-venous differentiation (ephrin-Eph) (Adams et al. 1999; Swift
and Weinstein 2009) or enforcement of endothelial junctions (UNC5B-netrin-1;
ROBO-4-UNCB5) (Kim et al. 2011; Koch et al. 2011). Several reviews provide
details of these interactions (Klagsbrun and Eichmann 2005; Potente et al. 2011).
The challenge is to integrate the myriad of these interactions in a general model, in
which effects are separated in tissue-specific effects and general effects that deter-
mine the formation of an all-tissue-cells-reaching functional vascular network.
Indeed, many of these guidance receptors have a direct or indirect effect on
VEGFR-2 signaling or provide information on the distance between vessels
(in various directions), presence of other tissue cells, and metabolic demand of
nearby cells.
10 Metabolic Control of Sprouting
Important regulators of perfusion control in a microvascular bed are the shear

stresses of blood flow, the oxygen tension, and the metabolic demand of the tissue.
Perfusion of the newly formed microvessel and the accompanying shear stress are
important for its stabilization. In the absence of blood perfusion, the newly formed
tube will finally collapse (unless continuously sustained by hypoxia-induced VEGF-
A). Stimulation of neovascularization is required in areas with a deficit in oxygen
supply, while an excess of oxygen can induce vascular pruning, the selective
abrogation of vessel branches, particularly in an immature vascular bed. The lack
of oxygen induces several metabolic adaptations in tissue cells, in which the
stabilization of the α-subunits of hypoxia inducible factors (HIFs) play an important
role. Upon stabilization HIFα subunits are translocated to the nucleus where they
form a complex with HIF-1β and change the expression of many hundreds of genes,
among which VEGF-A (Manalo et al. 2005; Ning et al. 2004; Semenza 2003;
Takeda et al. 2004). All tissue cells express HIF-1α, but the expression of HIF-2α
(indicated by the gene name EPAS1: endothelial PAS domain-containing protein-1)
is much more cell specific and abundant in endothelial cells. While activation of
HIF-1α causes the induction of many genes including a strong increase in VEGF-A
transcription and hypersprouting (Shweiki et al. 1992), activation of the in endothe-
lial cells abundant HIF-2α causes limitation of the number of tip cells and stabiliza-
tion and elongation of new sprouts (Skuli et al. 2012). In other words, tissue cells in
the hypoxic environment produce massive amounts of HIF-1-induced VEGF-A,
while endothelial cells respond to it and have their own hypoxic program, in which
the additional expression and stabilization of HIF2α is necessary to obtain stable
new vasculature. Skuli et al. (2012) showed an important role for HIF-2-mediated
induction of Dll4 and angiopoietin-2 in the limitation of tip cell formation in mic.
However, it cannot be excluded that HIF-2 has multiple effects on endothelial
sprouting, at least in human endothelial cells (Nauta et al. 2016; Takeda et al.
2004). Studies in mice that were haplodeficient for prolyl dehydrogenase-2
(Mazzone et al. 2009) also pointed to the stabilization of the endothelial monolayer
by HIF-2α, which was accompanied by increased levels of VE-cadherin and the
decoy receptor soluble VEGFR-1.
Hypoxia induces increased transcription of all enzymes in the glycolysis pathway,
which enhances the capacity of endothelial cells to generate ATP. Several studies
have pointed to the importance of glycolysis for endothelial ATP generation
(>85%), while mitochondrial oxidation have only a minor contribution (De Bock
et al. 2013; Quintero et al. 2006). Instead, mitochondria may have other important
regulatory functions in endothelial cells, including facilitating de novo nucleotide
synthesis via fatty acid oxidation for sustained endothelial growth (Schoors et al.
2015) and modulating endothelial function by reactive oxygen radicals (Quintero
et al. 2006). Endothelial cell migration and invasion is mainly under the control of
glycolytic ATP production, while interference with mitochondrial activities has little
effect. De Bock et al. (2013) demonstrated that important regulatory enzymes of the
glycolysis pathway associate with f-actin fibers in lamellipodia and filopodia thus
providing extra ATP to these for sprouting essential structures. Furthermore, inhibi-
tion of the glycolysis pathway markedly attenuated sprouting in vitro and in vivo
(Cruys et al. 2016; Schoors et al. 2014).
The induction of angiogenesis – as well as its later shutdown when vessels
stabilize – shifts the phenotype of the endothelial cells from a stable cell to a
sprouting cell, and later on from a proliferating cell to a stable cell that has many
functions including controlling hemostasis, vasodilation, and influx of nutrients,
hormones, and leukocytes. These shifts also require metabolic adaptation of the
cell. A recent study by Wilhelm et al. (Wilhelm et al. 2016) identifies the protein
FOXO1- via c-Myc signaling – as an important regulator of endothelial proliferation
and ATP generation. While mitogenic factors activate c-Myc signaling and tran-
scription regulation, c-Myc activity becomes inhibited by the nuclear presence of
FOXO-1, which thus suppresses cell proliferation. Endothelium-specific deletion of
FOXO-1 in the mouse retina causes increased proliferation and causes hyperplastic
sprouting, while overexpression of FOXO-1 results in thinning and reduced
branching of the outgrowing vasculature and decreased expression of glycolytic
enzymes. Physiologically, inhibition of FOXO-1 activity occurs via the phosphory-
lation of FOXO-1 by the kinase AKT (protein kinase B) (Nakae et al. 2001), which
evokes rapid translocation of FOXO1 out of the nucleus, thus leaving nuclear c-Myc
active. The regulation of FOXO-1 may be well connected to the effects of PTEN
(Serra et al. 2015) described above. A major effect of PTEN is the dephosphorylation
of inositol trisphosphate (IP3) by which activation of IP3 kinase and its substrate
AKT is abrogated. This leads to dephosphorylation of FOXO-1 and restores nuclear
FOXO-1 accumulation, which makes FOXO-1 available for inhibition of nuclear

c-Myc and thus suspends cell proliferation.
11 Lumen Formation
As soon as the sprout consists of several endothelial cells, a vascular lumen is

formed. Initial studies on zebrafish indicated that the lumen was formed by the
fusion and exocytosis of endothelial vesicles (Kamei et al. 2006). Subsequent studies
showed that in mammalian sprouting models a lumen is usually formed by separa-
tion of adjacent endothelial stalk cells after the formation of stable VE-cadherin
junctions (Zeeb et al. 2010). In 3D cultures, endothelial sprouts with VE-cadherin
mutations that cannot interact with one of the polarity proteins pals1 or Par-3 failed
to form a lumen (Brinkmann et al. 2016). This underscores that acquiring endothelial
junctions and cell polarity is needed for lumen formation. The generation of stable
homotypic VE-cadherin interactions also recruits the strongly negative charged
sialomucins CD34 and podocalyxin, which facilitate lumen formation as they
cause electrostatic repulsion of the cells (Strilic et al. 2009).
Involvement of PAR-3 in lumen and tube formation of endothelial cells in a
collagen matrix in vitro was also observed (Davis et al. 2011; Sacharidou et al.
2010). In these experiments PAR-3 acted as a central player in a large complex that
on one side involved Par-6 and CDC42 and on the other side the junctional adhesion
molecules JamB and JamC that interacted with MMP14 (MT1-MMP). These exper-
iments underline the importance for cell polarity in lumen formation, but specific
effects on lumen formation versus canalization within the collagen gel allowing
tubule formation are difficult to discriminate.
12 Anastomosis and Vessel Stabilization
Time-lapse movies of zebrafish with green-fluorescent labeled endothelium illustrate

that, when expanding venous sprouts appear in the vicinity of arteries, they induce or
encounter short branches from the arterial side to which they spontaneously merge
(Lawson and Weinstein 2002). Once merged, the tubular connection allows the
perfusion by the blood, after which the vessels becomes stabilized and further
mature. When mixtures of endothelial cells and mesenchymal stem cells were
transplanted, newly formed endothelial tubes connected to the existing circulation
and survived for extended periods, demonstrating that anastomoses between graft
and host vessels do form and are functional (Koike et al. 2004; Levenberg et al.
2005). In a similar way, when two perfused plexuses of differently labeled endothe-
lial cells are placed in each other vicinity in a microfluidic system, the two plexuses
merge, a process facilitated by VEGF (Diaz-Santana et al. 2015; Song et al. 2012). It
shows that endothelial cells in principle are able to anastomose by themselves
without involvement of other cells. However, this does not exclude the support of
additional cells in facilitating anastomosis formation and further stabilization of the
connected vessel. Indeed, in vivo coverage by pericytes, which represent or derive

from mesenchymal stem cells, was needed to maintain tubules that were formed by
exogenously added human endothelial cells in mice in vivo (Au et al. 2008b).
There is ample evidence that specific types of leukocytes can contribute to the
patterning of vascular beds. Chalothorn et al. (2007; Chalothorn and Faber 2010)
demonstrated marked differences in vascular collaterals and anastomoses in various
tissues of the mouse that depended on the genetic immunological background of these
mice. Furthermore, cytotoxic T cells have been observed in newly vascularized retina
being involved in removing excess neovessels, called vascular pruning (Ishida et al.
2003). A specific role in angiogenesis is anticipated for a subgroup of macrophages
that express the angiopoietin receptor Tie-2 (Nucera et al. 2011). Tie-2 containing
macrophages have been reported to chaperone anastomosis formation in the mouse
hindbrain (Fantin et al. 2010). Similarly, macrophages were encountered at the tips of
sprouts in the mouse retina (Outtz et al. 2011). In those macrophages that were in
close contact with endothelial cells at sites of developing or just completed anasto-
moses, active Notch signaling was observed. In favor of a proposed role of macro-
phage Notch in anastomosis formation, Outtz et al. (2011) observed that macrophage-
specific deletion of Notch1 was accompanied by the occurrence of long unbranched
tip cells, which may reflect incomplete anastomosis formation.
In the context of tissue engineering, it is of interest to note that formation of
anastomoses leading to a tubular network of implanted human endothelial cells in a
mouse host may proceed by a specific mechanism. Using fluorescent markers for the
various cells and a window technique to follow the vascularization process, Cheng
et al. (2011) describe in detail how endothelial cells partly dissociate from the tubular
network, cause detachment of pericytes from the host microvessels, and subse-
quently form a sheet of human endothelial cells around the host vessel. This is
prevented by inhibition of matrix metalloproteinases (MMPs) and accompanied by
upregulation of MMP-14 (MT1-MMP) and MMP-9. In a couple of weeks, the
human endothelial cells replaced the host endothelial cells and blood perfusion
gradually propagated through the human endothelial tubules. Cheng et al. (2011)
called this process “wrapping and tapping” and demonstrated its occurrence both in
brain and dermal skin host vasculature.
The newly formed endothelial tubes need to be stabilized and perfused. Stabili-
zation occurs by flow-mediated shear forces, development of a basement membrane,
and the recruitment of pericytes. The order of sequence is not always clear and can
vary in different conditions. The stalk cells of the new sprouts form rapidly a
continuous basement membrane and subsequently acquire contact with pericytes.
In mature vessels pericytes are embedded within the endothelial basement mem-
brane (Bruns and Palade 1968). Pericytes are encountered in all blood vessels but are
very sparse in postcapillary venules and normally absent in lymphatic capillaries.
Their density is highest at the arteriolar side of capillaries and least at the venular side
(Armulik et al. 2011; Nees et al. 2013). At the arterioles several pericytes are densely
wrapped around one endothelial cell, while at the venular side of capillaries one
pericyte can be in contact with several microvascular endothelial cells by long
protrusions.
13 Pruning
Not only development and growth but also inflammation and repair ask for a high
delivery of oxygen and nutrients. This high metabolic demand induces expansion of
the vascular bed. Once these processes cease the metabolic demand decreases, an
excess of vessels is present for normal tissue functioning. Hence, an additional type
of adaptation is required, namely limiting the number of vascular branches, also
called vascular pruning. This process is an intrinsic property of the angiogenesis
process itself and is regulated by vessel contraction and/or closure of vessel
branches, which is followed by disintegration of the endothelial contacts and
apoptosis. Alternatively, endothelial cells can migrate into the tissue and subse-
quently incorporate in an adjacent expanding vessel, which may be energetically
more efficient rather than being lost by detachment or apoptosis. Indeed, in zebrafish
Kochhan et al. (2013) observed each of these three mechanisms in vascular
remodeling, i.e., endothelial apoptosis, migration, and reusage in adjacent vessels.
Pruning can also occur as part of metabolic disease in particular diabetes, where it is
called vascular rarefaction, and contributes to underperfusion of the tissue. Although
the regulating players may be different in vascular development and disease, the
principal mechanisms are probably comparable.
The forces that drive vascular pruning are still poorly understood. It is unclear
whether withdrawal of factors plays an initiating role, such as suggested for VEGF
withdrawal (Benjamin et al. 1999; Mourad et al. 2008) or whether actively secreted
pruning inducing factors are involved (Korn and Augustin 2015). As blood flow
helps stabilizing new vessels, limited blood flow, e.g., after partial contraction of the
vessel, will facilitate the pruning process. Adjacent cells can also contribute. While
pericytes are well known to stabilize the endothelial lining of a newly formed vessel,
it is still uncertain if their detachment is required first or whether endothelial
apoptosis also occurs independently. However, given their location within the
endothelial basement membrane it is likely that their mutual communication will
be affected during the pruning process. Furthermore, macrophages are often encoun-
tered at areas of vascular pruning and might well contribute to the vascular
remodeling process. In the mouse retina the involvement of cytotoxic T cells has
been implicated in endothelial apoptosis (Ishida et al. 2003), further pointing to the
importance of immune and inflammatory cell surveillance in vascular pruning.
14 Lymphangiogenesis
Once circulation is established, lymphatic vessels start developing, initially by

sprouting from venous vessels (Adams and Alitalo 2007). Lymphangiogenesis has
many similarities with angiogenesis, but specific players are involved, in which
VEGFR-3 and its ligands VEGF-C and VEGF-D play a specific role, as well other
factors that were recognized in the last decade (Aspelund et al. 2016; Karaman and
Detmar 2014). The pathophysiology of lymphangiogenesis is extensively discussed
Late outgrowth EPC = ECFC
Bone marrow
Early outgrowth
derived EPC (myeloid)
?Blood vessel
derived
Hypoxic
area
Bone
marrow
Postnatal vasculogenesis
Return to
(ECFCs and myeloid EPCs)
bone marrow
Fig. 3 Postnatal vasculogenesis is characterized by the incorporation of circulating endothelial

colony forming cells (ECFCs, late outgrowth endothelial progenitor cells (EPCs)) and is stimulated
by monocytic cells (early outgrowth EPCs) that produce angiogenic growth factors and may
contribute to anastomosis formation and branching. The myeloid (monocytic) cells are part of a
surveillance system and return to the bone marrow when angiogenesis support is no longer required
in the chapter on lymphangiogenesis in this book (Holnthoner), to which the reader

is referred.
15 Postnatal Vasculogenesis
During angiogenesis the division rate of endothelial cells increases several orders of
magnitude from once in 1–3 years to almost daily (Hobson and Denekamp 1984).
The huge increase in proliferation puts forward the question whether there exists a
population of endothelial cells with a very high proliferation potential. In search of
circulating endothelial cell progenitors Asahara et al. (1997) and other investigators
(see Hirschi et al. 2008 for review) recognized that a very small population of
circulating cells can be induced to form colonies of rapidly proliferating cells that
express endothelial markers. These cells were able to support angiogenesis either by
paracrine stimulation or by incorporation within the endothelial lining (Basile and
Yoder 2014; Fig. 3). It has become clear that vasculogenesis not only occurs during
embryonal development but also postnatally and even in adult life. Recruitment,
differentiation, and vessel incorporation of endothelial progenitor cells (EPCs)
provide additional endothelial cells for expanding the new vasculature. Under
influence of VEGF, SDF-1, GM-CSF, or tissue hypoxia, endothelial progenitor
cells are mobilized from the bone marrow or other tissues into circulation and
subsequently home to an area in need of increased oxygen supply. Once recruited
into ischemic or inflamed sites, true endothelial progenitor cells differentiate under
influence of pro-angiogenic factors into mature endothelial cells, which subse-
quently incorporate into the existing vascular endothelial lining and proliferate, or
initiate assemblage of new vascular structures (Asahara et al. 2011; Au et al. 2008a;
Hirschi et al. 2008; Melero-Martin et al. 2007).
The progenitor cells involved in postnatal vasculogenesis are most studied in man,
and some differences in their origin and characteristics may occur in rodents. At
present, the scientific literature recognizes two distinct types of endothelial progenitor
cells (EPCs) each of them participating during vascularization in different manner.
Depending on the time of their clonal outgrowth in vitro after isolation from peripheral
blood EPCs colonies have been clustered as early and late outgrowth EPCs. The
so-called early outgrowth EPCs belong to myeloid lineage (Piaggio et al. 2009). They
display several endothelial markers but are not true endothelial cells. They participate
in angiogenesis in a paracrine fashion by releasing pro-angiogenic factors (Pula et al.
2009; Rehman et al. 2003; Sahoo et al. 2011). On the other hand, the late outgrowth
EPCs also referred as endothelial colony forming cells (ECFCs) or blood outgrowth
endothelial cells do not belong to the hematopoietic cell lineage. They exhibit a
pronounced vascularization (vasculogenic) ability in vivo by physically incorporating
into newly formed blood vessels (Melero-Martin et al. 2007; Yoder et al. 2007).
While the myeloid early outgrowth EPCs are recruited from the bone marrow, the
origin of circulating ECFCs remains still unanswered. Estimations of the incorpo-
ration of bone marrow-derived cells in heterologous transplantation patients revealed
less than 0.5% contribution of bone marrow-derived cells in the vascular lining of
growing blood vessels (Peters et al. 2005; Wickersheim et al. 2009), suggesting
another source. In addition to peripheral and cord blood (Asahara et al. 1997; Ingram
et al. 2004), other human tissues such as white adipose tissue (Lin et al. 2013), lung
(Asosingh et al. 2009) and the intima of large blood vessels (Ingram et al. 2005) have
been used to isolate human ECFCs. The large variability in tissues may point to a
vascular origin of the ECFCs. Indeed, the existence of a complete hierarchy of
endothelial progenitor cells in cultures of umbilical vein, aorta, and pulmonary artery
endothelial cells derived from the vessel wall indicates that the intimal space of
blood vessels might act as a reservoir of cells with potent vascularization and
reparative potential (Alvarez et al. 2008; Ingram et al. 2005; Naito et al. 2012).
As ECFCs are derived from individual patients and can be propagated in large
numbers, they can be used for studying endothelial cells from individual patients in
various types of disease. Furthermore, they bear the potential to act as an excellent
source of patient-specific endothelial cells in tissue engineering applications
(Asahara et al. 2011; Basile and Yoder 2014; Silvestre et al. 2013; Tasev et al. 2015).
16 Generation of a Functional Microvascular Bed
The efficient transport of fluid requires a hierarchy of vessels throughout the

circulation. They decrease steadily from large muscular arteries to a fine space-
filling meshwork of thin exchange capillaries and subsequent merge into venules and
a Optimal O2 exchange
c Actual microvascular
structure
Art
Cap
Ven
PO2
high
Optimal fluid low

distribution (convection)
Fig. 4 Optimal exchange of oxygen and nutrients require a dense network as schematically
depicted in a, while the convective flow asks for a gradual distribution in afferent and efferent
vessels (b). In the living tissues these two extremes are combined in a complex network (c), in
which not only the topological distribution is important for blood supply and exchange to all tissue
cells according to metabolic demand of these cells. But also communication of local capillaries with
the proximal arterioles and distal veins is needed for ensuring optimal blood flow. Messages to the
venous part go by the flow of blood, but upstream messages ask for another signaling system along
the vasculature itself. Art arteriole, Cap capillary, Ven venule, PO2 oxygen tension (From Pries and
Secomb (2014), www.physiologyonline.org)
veins that conduct the blood to the heart. After initial formation of the circulatory
system, angiogenesis controls the local expansion of microvessels upon metabolic
demand or injury. The blood flow to the expanding vasculature can be increased
rapidly by vasodilation and in time by outward remodeling of arteries and arterioles
(and, in case of vessel obstruction, by collateralization). However, the generation of
a functional microvascular network is a dynamic process that must obey both
efficient convective transport of the blood over relatively large distances and simul-
taneously reach all tissue cells via a dense space-filling network, in order to assure
that all tissue cells receive sufficient oxygen and nutrients (Fig. 4). Oxygen supply is
the most critical because of its limited diffusion distance. In a landmark study,
Secomb et al. (2013) used computational modeling to generate a microvascular
bed that fulfils the two requirements of convective transport and limited distance
to all tissue cells. In this model Secomb used 33 parameters regarding blood
properties (4), tissue oxygen handling (3), VEGF release and consumption (3),
structural vessel adaptation (11), sprouting angiogenesis (10), and vascular migra-
tion (2).
Its physiological implications were further explained in a subsequent review by
Pries and Secomb (2014). While inadequate oxygen supply induces angiogenesis
according the model input, the model predicted that contemporary interplay between
sprouting angiogenesis, vascular remodeling, and pruning was required for
obtaining a full coverage of oxygen delivering vessels in the tissue. Furthermore,
Secomb’s model provided a mechanism for selection of vessels that contributed to
convective transport and oxygen delivery. Redundant vessels will shrink and finally
be pruned unless or until it appears that shear stress increases (limitation of convec-
tive transport) or oxygenation becomes insufficient (exchange deficit). Finally, as
pointed out by Pries and colleagues (Pries and Reglin 2016; Pries and Secomb 2014)
the message of adequate perfusion must be delivered to proximal and distal parts of
the vascular network for proper adaptation of these vessels and to make certain that
no functional shortcut between arterial and venous circulation occurs. While the
flowing blood can easily transport message from the capillaries to the venular part,
reaching the arteriolar part requires upstream delivery of that message. Pries and
Secomb (2014) suggested electrical coupling of endothelial cells, in which gap
junctions are involved. While such coupling has been demonstrated reaching several
endothelial cells (Kameritsch et al. 2012), its action over relatively long distances
has still to be demonstrated. As an alternative one may consider whether neuronal
sprouts extending from the adrenergic nerves that closely follow arteries and arteri-
oles may help and pick up signals that translate into an adaptive vessel response
upstream.
While Secomb’s model reported the formation of a microvascular network in
preset conditions that closely reflect healthy tissue, it allows the introduction of
many variables that reflect disease conditions. Its predictions can be used to improve
our mechanistic insight in the regulation of angiogenesis, while computational
models also provide a potential platform for evaluating minimal requirements for
anastomosis and survival of endothelial tubules in a tissue-engineered grafts.
17 Conclusion
Much stimulated by the initial studies on the vascularization of tumors, research on

sprouting angiogenesis has developed to a level at which (patho)physiological
angiogenesis becomes understood at a molecular mechanistic level. The behavior
of tip and stalk cells at the initiation of sprouting angiogenesis received much
attention, while in the last years also the formation of anastomoses and initial
perfusion, two pivotal processes in angiogenesis, became more clear. Additional
mechanisms, in particular intussusceptive angiogenesis and postnatal vasculogenesis
are acting, of which still more has to be learned. They bear additional opportunities
for improving neovascularization. It has become clear that formation of a new
microvascular network requires the mutual interplay between sprouting,
redistribution (remodeling), and pruning of endothelial tubules into a functional

vascular bed. Control of generating such network will be the next step to achieve.
Using both computational modeling and dedicated 3D-culture and in vivo experi-
ments their mutual interactions result in a fine-tuning of understanding the normal
and conditional requirements for network formation for optimal neovascularization.
Here it becomes important to also use the body’s own ability to fine-tune its
vascularization and the functions of the new vasculature. This will help to recover
specific functions that are attributed to distribution and exchange regulation in
microvessels, such as vasoregulation, glycocalyx expression and functioning, con-
trolled exchange of nutrients and hormones, hemostasis regulation, and adaptive
recruitment of leukocytes. After several decades of angiogenesis research there is
still much to do, but steadily we come nearer mastering neovascularization in disease
and tissue repair.
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Stimulation by Light
S. Chaudary, S. Rieger, H. Redl, and P. Dungel
Abstract
Tissue engineering techniques, to replace wounded or missing tissue, are advanc-
ing rapidly to ensure the speedy recovery of patients. However, this field faces
limitations of cells and biomaterials which prevents the acceleration of regener-
ation. Low level light therapy, a physical therapy, shows potential in enhancing
and supporting the existing medicinal treatments. Visible light in the red and near-
infrared range has shown to have positive stimulatory effects on various types of
cells involved in wound healing and tissue regeneration. As angiogenesis is an
essential part of this process, light therapy was investigated in multiple studies to
see its beneficial effect on vessel formation. In vitro, in vivo, and in a clinical
setup, LLLT therapy proved that it is capable of stimulating not only endothelial
cells but other cells such as fibroblasts, smooth muscle cells, and lymphocytes
which are involved in the vessel formation process. It triggers the activation of
cytochrome c oxidase, which leads to the production of NO, ROS, and ATP in the
mitochondria. These molecules appear to act as secondary messengers initiating
ERK/Sp1 and PI3K signaling pathway, which in turn leads to proliferation,
migration, and the synthesis of proangiogenic factors. This data indicates that
LLLT could be a promising adjuvant treatment in the future.
S. Chaudary • S. Rieger • H. Redl • P. Dungel (*)

Austrian Cluster for Tissue Regeneration, Ludwig Boltzmann Institute of Experimental and Clinical
Traumatology, Vienna, Austria
e-mail: sidrah.chaudary@trauma.lbg.ac.at; stefan.rieger@trauma.lbg.ac.at; office@trauma.lbg.ac.
at; peter.dungel@trauma.lbg.ac.at

DOI 10.1007/978-3-319-21056-8_4-2
2 S. Chaudary et al.
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 History of Low-Level Light Therapy (LLLT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3 Coherent Light (Laser) Versus Incoherent Light (LED) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
4 Biphasic Dose Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
5 Interaction of Light with Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
6 General Mechanism of LLLT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
7 In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
7.1 Secondary Angiogenic Effects of Supporting Cells After Exposure to LLLT . . . . . . . . 15
8 In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
1 Introduction
One of the biggest challenges for engineered organs in the field of tissue engineering
and regenerative medicine to date is the development of tissue, organs, and scaffolds,
which are perfused adequately, to ensure the proper supply of gases, nutrient
metabolites, and signaling molecules to the tissue, once it is transplanted (Phelps
and García 2010). The maximum diffusion distance of nutrients and oxygen from the
blood vessels to the tissue is 200 μm (Carmeliet and Jain 2000); therefore, either the
complex three-dimensional engineered structure needs an integrated vascular net-
work to support cell survival or extrinsic stimulation which would activate angio-
genesis in vivo (Lovett et al. 2009). Insufficient blood delivery leads to functional
limitations of bioengineered constructs, failure to perform accordingly, and ulti-
mately to rejection by the recipient’s body.
Tissue engineering aims not only to mimic the physiological wound healing
process but also to accelerate and improve it. To be able to simulate regeneration,
one needs to comprehend the complex progression, development, balance, and
interactions of each molecule that plays a role in wound healing. Only through this
understanding can a tissue engineer know which cells and mediators need to be
present at what time and in which concentration, to be able to get the best outcome.
Due to trauma, the physiological balance becomes instable, which the body tries
to recover from again as soon as possible. In case the stress is too much, external help
is needed to repair the imbalance. Several strategies, such as cell and scaffold
transplantation, insertion of multicellular spheroid to develop capillary-like sprouts,
biomaterials supplemented with growth factors to induce angiogenesis, macro-
tissues consisting of cell-embedded hydrogels or even ex vivo formed vessel con-
structs, have been employed so that faster and successful vascularization and
consequently wound healing can be achieved (Rouwkema et al. 2008; Lovett et al.
2009). However, these therapies have their limitations, as the constructed tissue
samples are only a few millimeter thick, it is questionable if the metabolic demand of
the cellular components is being met, the transferring of the construct is problematic,
and the efficient integration of the construct into the systemic vasculature also is not
ideal (Lovett et al. 2009; Chong 2015; Laschke and Menger 2015).
Stimulation by Light 3
As the research to enhance and improve tissue-engineered products is ongoing,

alternative therapies to already existing procedures are being sought. Physical thera-
pies, such as low-level light therapy, extracorporeal shockwave therapy, or electro-
magnetic therapy, have shown to facilitate the process of healing and enhancing
physiological functions (Chung et al. 2012; Hamblin and Huang 2013). Low-level
light therapy (LLLT) is a form of phototherapy, which induces a biomodulative
response in the tissue that is exposed to it, to reduce inflammation, edema, and pain
(Aimbire et al. 2006; Castano et al. 2007; Chow et al. 2007), alleviate neurogenic
issues (Christie et al. 2007; Chow et al. 2009), and enhance tissue repair and regener-
ation, which also includes the healing of deeper tissues, nerves, and vasculature (Bisht
et al. 1994; Gigo-Benato et al. 2005; Demidova-Rice et al. 2007; Fushimi et al. 2012).
One of the first applications of LLLT was in healing skin ulcers (Mester et al. 1971,
1972, 1976). Light therapy in the red and near-infrared range (NIR) (600–1100 nm)
has generated beneficial biological effects in various injury models. It not only
influences cellular dysfunction, cell proliferation, migration, and adhesion and pre-
vents apoptosis but also improves collagen and cytokine synthesis and increases
mediator expression (Huang et al. 2009). This could be due to the ability of red
wavelength to penetrate deep into injured tissue, allowing noninvasive therapy to
augment would healing. Multiple in vitro (Zhang et al. 2003; Hawkins and Abrahamse
2006) and in vivo studies (Bisht et al. 1994; Gigo-Benato et al. 2005; Demidova-Rice
et al. 2007; Barolet 2008; Chung et al. 2012) have shown that LLLT affects all three
phases of wound healing (Chung et al. 2012). It stimulates the secretion of inflamma-
tory mediators, such as prostaglandin (Chow et al. 2009); modulates growth factor and
cytokine production (Kipshidze et al. 2001; Mvula et al. 2010; Saygun et al. 2012;
Gupta et al. 2013); induces the proliferation, migration, and even differentiation of
various cells involved in wound healing (Hawkins and Abrahamse 2007; Fushimi
et al. 2012; Saygun et al. 2012); triggers angiogenesis (Chen et al. 2008); accelerates
collagen synthesis; increases tensile strength (da Silva et al. 2010; Prabhu et al. 2012);
and prevents the formation of fibrosis (Mamalis et al. 2016), leading to the quicker
closure of wounds (Demidova-Rice et al. 2007; Fushimi et al. 2012). It elicits these
effects by increasing mitochondrial metabolism (Hu et al. 2007) and ATP synthesis
(Karu 1999; Hamblin and Demidova 2006; Karu and Pyatibrat 2011) and stimulating
expression of numerous genes involved in cellular proliferation, migration, and
inhibiting apoptosis among others (Chen et al. 2011).
Prior to further indulging in the role of LLLT in wound healing, more precisely
angiogenesis, it is important to enhance our understanding of the origin, ideal
parameters, stimulation power, and influential mechanism by getting a short over-
view about low-level light therapy.
2 History of Low-Level Light Therapy (LLLT)
Light therapy from various sources has been one of the oldest modes of therapy for
different pathological conditions by the ancient Egyptians, Indians, and Chinese
(Daniell and Hill 1991). Niels Finsen received the Nobel Prize in 1903 for
developing a “chemical rays” lamp to treat tuberculosis. He became famous for

using artificial irradiation sources and thereby initiating the era of modern photo-
therapy (Roelandts 2005; Hönigsmann 2013). After Finsen came a dip in the field of
phototherapy, which was however uplifted with the coming of the laser. Soon after
the invention of the ruby laser in the 1960s, the biostimulatory function of the
helium-neon (He-Ne) laser was discovered, rather by accident. In 1967, Professor
Mester conducted experiments on mice at the Semmelweis University in Hungary to
investigate the carcinogenic effect of the laser. However, he was pleasantly surprised
when he realized that the hair on the shaven mice back exposed to the low-level laser
grew back much quicker compared to the non-illuminated mice (Mester et al. 1967).
Thereupon, he coined the term “laser biostimulation.” While further studying the
functions of the He-Ne laser, he detected a rapid wound healing effect in mice
(Mester et al. 1972). Upon confirming his results, he soon applied his findings on
human patients, who suffered from nonhealing skin ulcers (Mester et al. 1968).
From that point onwards, over the past five decades, multiple researchers around
the world have been working on figuring out the physical, biological, and molecular
background of the effects of low-level light therapy, as it is known in modern times.
This therapeutic technique is referred to as “low level” as the energy density used is
much lower compared to other lasers used for ablating, cutting, and thermally
coagulating tissue. Apart from that, it is also known as “cold laser” or “soft laser,”
because its usage produces far less heat and therefore less damage to the tissue in
comparison to halogen lights, solar, and UV light sources.
Since the discovery of the photobiomodulatory influence of lasers upon cells, it
was thought that merely lasers, which emit coherent light, could only have positive
effects on cells. However, over the years, a source of noncoherent light, the light-
emitting diodes (LEDs) have been tested and proposed as a better alternative, due to
its multiple advantages over lasers (Barolet 2008). The National Aeronautics and
Space Administration (NASA) mainly developed the research field of LED photo-
biomodulation. Initially, it was tested in space for the growth of plants after they
were exposed to a specific wavelength. Astronauts faced the problem of reduced
wound healing in space due to zero gravity, which was also an issue for Navy Seals
in submarines under conditions of high atmospheric pressure. Based on the positive
results by NASA, LED therapy became a renowned therapy option in the field of
tissue regeneration. There are still divergences between investigators, who promote
or reject the LED as a low-level light therapy source, based upon the limited research
data that is currently available (Barolet 2008; Sobanko and Alster 2008).
3 Coherent Light (Laser) Versus Incoherent Light (LED)
Coherence is the measure of the ability of the photons in a wave to interfere with one
another. Therefore, coherent light is defined to have an emission of photons in a
beam form. The beams have the same frequency and travel in unbroken wave chains
(constant relative phase). This gives them the characteristic of being parallel. In
contrast, noncoherent light has a much lower degree of coherence and photons
oscillate in different directions. Also, a frequent and random change in phases

between the photons is observed, which vary with time and position.
Light amplification by stimulated emitting electromagnetic radiation (LASER), a
source of coherent light, is the conventional light source used for low-level therapy.
It has been seen as the ideal supply of light due to its spatial coherence, narrow
low-divergence beam, and because it can be manipulated with different kinds of
lenses. However, the disadvantage of these properties that many scientists forget is
the inability to spread and diffuse deep into the tissue.
On the other hand, incoherent light sources, such as light-emitting diodes (LEDs),
can spread and diffuse into tissue specifically due to its characteristics. They were
introduced, in 1962, as a “practical semiconducting electronic component” due to its
petite and robust structure. Initially, only low-intensity red light-emitting diodes
exited; however, this has changed over the years, and a variety of different wave-
length diodes are available now. LEDs release energy in the form of photons by
merging the present electrons with the device’s electron holes, creating an electro-
luminescent effect and high power. The color of the light depends on the energy gap
of the semiconductor. Apart from their pea-size structure, they produce far less heat
compared to lasers, are available in multiple wavelength ranges, have a narrow band
of electromagnetic radiation, can illuminate large areas, and, most importantly, are
very cost-effective (Moreno and Sun 2008).
Laser-favoring scientists have partially attributed the positive biological results
of the cold laser to its coherence (Mester et al. 1985). Conversely, modern-day
scientists have carried out laser and LED comparative studies to determine if there
is any difference in their effectivity. Among the first to test the two light sources,
Dall Agnol et al. (2009) separately illuminated diabetic wound tissue of type I
diabetic rats with a laser and LEDs and examined the tissue histomorphologically.
His group observed that the wounds exposed to LEDs had more efficiently reduced
the wounds’ diameters; however, 168 h post-illumination, similar effects could be
seen. Another group investigated the proliferative and differentiative influence of
both light sources on pre-osteoblasts. Illumination with different intensities (3 and
5 J/cm2) and duration (50 and 83 s) showed that proliferation after 24 h had
increased in the cell group illuminated by the 5 J/cm2 laser; however, after
48, 72, and 96 h, there was no difference between any of the illuminated groups.
On the other hand, illumination could not trigger differentiative effect in the
pre-osteoblasts (Pagin et al. 2012). Histological staining of illuminated dorsal
cutaneous wounds showed that angiogenesis was increased in all LED and laser
groups (of different wavelengths) and that coherence was not the decisive factor
for the outcome (de Sousa et al. 2013). Interestingly, during the analysis of the
impact of light on cutaneous wounds in iron-deficient rodents, the laser light had a
better result on healthy animals, whereas the LED had a significant higher wound
closure rate in anemic subjects (Oliveira Sampaio et al. 2013). It should be taken
into account that due to the width of the light beam of LED, the power (W) needs to
be adjusted to a higher value compared to the laser, so as to achieve the same final
dose (J/cm2) for both light sources and thus be able to compare their therapeutic
effect (Corazza et al. 2007).
These sets of data support the theory that the beneficiary consequences of laser
and LEDs are independent of light coherence. Karu goes as far as to say that the
coherence of the light is lost in the superficial layers of the skin prior to being
absorbed by the biological chromophores and therefore plays no role and is not a
prerequisite for the photomodulatory effects (Karu 1999).
4 Biphasic Dose Response
For any medication or therapy to work, there has to be an active ingredient, and a
certain dose of that active ingredient needs to be taken so that the health can improve.
In the case of LLLT, the active ingredients are the illumination parameters, and the
dose is the irradiation time (Huang et al. 2009). There are multiple parameters that
need to be considered to elicit a positive influence on the tissue, such as wavelength,
illumination intensity, pulse rate, repetition frequency, and of course the final dose
being received. To make the determination and evaluation easier and more compa-
rable across the field, researchers working with cold light therapy have boiled down
the various aspects down to one factor: the final dose. The final dose is expressed as
the energy density (J/cm2), which can be calculated by multiplying the power (Watts)
used with the time (seconds) the tissue was exposed to the illumination. Although
this simplistic equation is used to calculate the final dose, it does not necessarily
correspond to the actual dose received or elicit the same response in the tissue. For
example, the effect of double the power and half the time would not be the same if
the power was halved and the time was doubled.
This kind of response to the exposed illumination parameters is called biphasic
response. This biphasic response directly correlates with the properties of the
wavelength and energy density absorbed by the chromophores in the tissue. It has
been proven that insufficient illumination energy or time results in no response. On
the other hand, too much of the final dose can lead to an inhibition of cellular
functions (Lanzafame et al. 2007). Just as there is an optical window for the
wavelengths which the cells are exposed to, in the same way there is an optical
combination of energy and duration which stimulates the therapeutic response
(Chung et al. 2012).
In 1887, Hugo Schulz demonstrated how low doses of different poisons stimu-
lated the yeast metabolism. In collaboration with Rudolph Arndt, they coined the
“Arndt-Schulz law” (Mester et al. 1985) which proved that weak stimuli tend to
trigger an acceleration of the metabolic activity. An increase in dose stimulates it
further; however, a rise in stimuli concentration will reach a peak that will ultimately
inhibit it.
This law has been summarized in a 3D model graph (Arndt-Schulz law diagram)
to depict the relationship between irradiance and time or fluence in a possible
biphasic response, again concluding that a deficient amount of power density or
time will have no influence on the pathological tissue, whereas overpower would
hinder the stimulatory effect.
To comprehend and reconstruct the “Arndt-Schulz law” in the field of photobi-

ology, Ginsbach (1979) illuminated rats with an He-Ne laser at different irradiances
(12.4 mW/cm2 and 45 mW/cm2) and found that that the higher irradiance showed an
effect whereas the lower did not (Reddy 2003). Shefer and his colleagues discovered
that illumination of infarct hearts in rats with three different irradiances (2.5,
5, 25 mW/cm2) showed varying degrees of recovery, the maximal beneficial effect
being at an irradiation of 5 mW/cm2 (Shefer et al. 2001). Huang et al. (2009) stated
that “fluences of red or NIR as low as 3 or 5 J/cm2 will be beneficial in vivo, but a
large dose like 50 or 100 J/cm2 will lose the beneficial effect and may even become
detrimental” (Huang et al. 2009).
An attempt has been made to explain the reason behind the inhibitory effect of the
biphasic response. Whereas, it is true that LLLT triggers the production of ROS,
which are beneficial in a certain amount. However, if that amount is exceeded due to
the increased exposure of the cells to illumination, they can have harmful effects,
even leading to the death of the cell (Huang and Zheng 2006). In the same way, the
excessive release of NO through photolysis of nitrosylated proteins can be unfavor-
able for the cell (AlGhamdi et al. 2012). Activation of unwanted secondary messen-
gers can trigger the activation of pathways which have a damaging effect. Gao and
Xing (2009) demonstrated that a “healthy” dose prompted signaling of the prolifer-
ative pathway (Gao and Xing 2009), whereas Wu et al. (2009) observed that an
overdose of illumination led to the activation of the mitochondrial caspase-3 path-
way, which led to the apoptosis of the cells (Wu et al. 2009). Kipshidze et al. (2001)
found that long exposures of smooth muscle cells, myocytes, and fibroblasts led to a
decreased production of VEGF, and it has been demonstrated that higher doses of
LLLT were cytotoxic for vascular cells (Deckelbaum et al. 1993; Kipshidze et al.
1998). Therefore, it is crucial to determine the optimum dose and illumination
duration, in order to prevent inhibitory effects.
5 Interaction of Light with Tissue
For light to be able to have an effect on cells in human tissue, it has to conform to two
laws of photobiology. According to the first law, a molecular chromophore or
photoacceptor needs to be present in the tissue that interacts with the photons
according to their electronic absorption bands (Sutherland 2002). The second law
of photobiology states that the absorbing tissue has to have certain optical properties
to take up light of certain wavelengths to initiate its effects (Hamblin and Demidova
2006). In any mammalian tissue, cells may contain one or more chromophores, such
as hemoglobin, cytochrome c, flavin, NADH-dehydrogenase, and melanin, to name
a few (Aziz-Jalali et al. 2012; de Sousa et al. 2013; Chaves et al. 2014).
In photomedicine, the ultraviolet (UV) region is generally divided into three
regions: the UV-C region (100–280 nm), the UV-B region (280–320 nm), and the
UV-A region (320–400 nm). The visible light region is generally defined between
400 and 760 nm, and the infrared region lies above 760 nm. Only radiation of a
certain wavelength lying between 400 and 800 nm coming from the sun reaches the
earth; the rest is absorbed by the stratospheric ozone. Therefore, it is reasonable that
wavelengths in the abovementioned range will induce a biological effect that is
desired in the pathological tissue.
Because the optical window lies between 600 and 1500 nm, the use of LLLT in
animals and patients almost exclusively involves red and near-infrared light
(600–1100 nm) (Karu and Afanas’eva 1995). This could be due to the fact that
light in this spectrum range can penetrate the deepest, through the skin, muscle, and
vasculature, in addition to lacking carcinogenic and mutagenic properties of UV
light. Multiple studies provide evidence that wavelength in the red and near-infrared
range are optimal for the use in low-level light therapy. Taniguchi et al. (2009)
showed its proliferative effect in fibroblasts and Chen et al. (2008) in endothelial
cells. Apart from that, it not only promotes recovery from ischemia in
cardiomyocytes (Zhang et al. 2009), but also enables muscle regeneration (Weiss
and Oron 1992) and aids wound and retinal healing (Conlan et al. 1996; Eells et al.
2004). In neuronal cells, red/NIR light prevents neurotoxic effect of cyanide and
azide (Desmet et al. 2006; Wong-Riley et al. 2005), restores axonal transport in
Parkinson’s disease (Trimmer et al. 2009), and exerts a neuroprotective effect against
optic neuropathies (Rojas et al. 2008).
6 General Mechanism of LLLT
Mitochondria, which initially were known to be the powerhouse of the cell, also
have other functions attributed to them. In 1989 it was proposed that monochromatic
visible and NIR illumination, used in LLLT, was absorbed by components of the
mitochondria respiratory chain, which triggered the cellular response on a molecular
level (Karu 1989).
The respiratory chain is embedded in the inner membrane of the mitochondria
and consists of several complexes: NADH dehydrogenase (complex I), succinate
dehydrogenase (complex II), cytochrome bc1 (complex III), cytochrome c oxidase
(complex IV), ATP synthase, and two unattached diffusible components – ubiqui-
none and cytochrome c. The end products of the citric acid cycle, NADH and
succinate, highly charged with electrons are reduced by complexes I and II, respec-
tively. The released energy promotes the passage of electrons through the other
complexes and is coupled with the transport of protons across the inner membrane
into the intermembrane space, creating a proton concentration gradient. This gradi-
ent upholds the electrical and pH membrane potential and also facilitates the
phosphorylation of ADP to ATP in the ATP synthase.
To elucidate which component of the cell responds to low-level light therapy and
triggers the cellular response, absorption spectra were measured for the
abovementioned biological complexes in oxidized and reduced states. Among
them cytochrome c oxidase was recorded to have an action spectra closest to that
of NIR spectra (Hamblin and Demidova 2006). Due to the conformational change of
the cytochrome c oxidase in the oxidized and reduced form (Szundi et al. 2001), the
wavelength observed ranged in different parts of the red to near-infrared spectrum

(613.6–846 nm) (Karu et al. 2005a).
To further confirm the role of cytochrome c oxidase (Pastore et al. 2000), it was
isolated and illuminated with an He-Ne laser, which resulted in an increased
oxidation and electron transfer, which is a consequence of absorbed photons.
Amplification of the electron transfer reaction results in an increased production of
ATP (Yu et al. 1997; Pastore et al. 2000), which facilitates other molecular mecha-
nisms in the cell, such as opening of ion channels or activation of secondary
messengers.
It was formally believed that cytochrome c oxidase only reduced water to oxygen,
which was designated as Cco/H2O (Poyton et al. 2009). During the reaction, water is
reduced by a series of one-electron transfers, which has the potential to generate
superoxides (O2•-), hydrogen peroxide (H2O2) and hydroxyl ions (OH-), which are
collectively known as reactive oxygen species (ROS). The activation of the mito-
chondrial respiratory chain by illumination leads to the increased production of these
ROS compared to non-illuminated cells (Eichler et al. 2007). However, it is still
unclear what concentration has a proliferative effect and is not harmful for the cell.
Apart from that, the “singlet-oxygen hypothesis” states that other molecules, such as
porphyrins and flavoproteins, can be stimulated by certain wavelengths as well to
react with oxygen, which leads to a transfer of energy and produces reactive species,
such as singlet oxygen (Hamblin and Demidova 2006).
Cytochrome c oxidase was also found to have another enzymatic function – the
reduction of nitrite to nitric oxide during the Cco/NO reaction. A directly propor-
tional increase in NO production could be observed with a raising nitrite concentra-
tion and decreasing pH level (Poyton et al. 2009). Zhang et al. (2009) confirmed this
in a hypoxia/reoxygenation cardiomyocyte model, where the condition of hypoxia/
reoxygenation damaged cells improved after exposure to NIR illumination. They
reported that this protective effect of NIR was litigated through a nitric oxide-
dependent mechanism, which was not totally produced by nitric oxide synthases
(NOS) (Zhang et al. 2009).
Nitric oxide (NO) has been established as a signaling molecule; however, it also
reversibly binds to cytochrome c oxidase and consequently inhibits its functions.
Implementing the “NO hypothesis” (Karu et al. 2005b) has shown that the illumi-
nation of the inhibited cytochrome c oxidase releases NO through which on the one
side cytochrome c can resume its function and NO potentially diffuses into the
surrounding to act as a second messenger molecule setting off other reactions.
Following this train of thought, performed experiments have shown a release of
NO into the blood (Mitchell and Mack 2013) and the associated increase in the blood
flow after illumination, which was a result of the vasodilatory effects of NO in the
microcirculation of the skin. It was also shown to increase lymphatic drainage in
lymphedemas and reduced swelling after trauma (Donadee et al. 2011).
Overall there is an agreement that certain molecules in the cell such as nicotin-
amide adenine dinucleotide (NAD/NADH), nicotinamide adenine dinucleotide
phosphate (NADP/NADPH), and glutathione/glutathione disulfide couple
(GSH/GSSG) have the potential to be reduced and thereby elicit a cascade effect.
In fact, multiple essential regulation pathways are mediated via the cellular redox
state, and their conformation change activates intracellular signaling molecules and
enzymes, regulates RNA/DNA/protein synthesis, and influences the cell cycle
(Honda et al. 2005).
As the established concept of photoactivation of mitochondrial chromophores
sinks in, another theory is slowly finding acceptance in this field. The traditionally
known pathway transduces signals from the nucleus, through the cytoplasm to the
organelles. However, this is not the only existing mechanism of communication
within the cell. As the influence of low-level light therapy has become more and
more apparent, it has been affiliated with the term mitochondrial retrograde signal-
ing, which is the transmission of information to the nucleus related to the change that
occurs in the mitochondria. This signaling pathway has been established in the
budding yeasts Saccharomyces cerevisiae (Liu and Butow 2006), plant cells
(Rhoads and Subbaiah 2007), as well as myocytes (Biswas et al. 1999) and cancer
cells (Erol 2005).
This signaling pathway makes it comprehendible how the response of the
snowball effect triggered in the mitochondria by low-level light can be detected
in the cytosol, nucleus, and even extracellularly. The biostimulation not only has
an effect on the organization of the cytoskeleton in the cytosol but also influences
the distribution of endoplasmic reticulum and protein synthesis (Oliveira et al.
2009). Furthermore, as mentioned earlier, a cascade of secondary messenger
activation is sparked along the pathway toward the nucleus, such as the c-Met, a
member of the tyrosine protein kinase receptors, via phosphorylation (Shefer et al.
2001), which has the potential of further setting off the Ras/Raf/MEK/ERK, PI3K/
Akt/eIF4E (Shefer et al. 2003), PI3K/Akt/eNOS (Uruno et al. 2004), and
PLC-gamma/PKC pathways. This is followed by transcriptional factor activation,
such as Fos, Jun, NF-kB, and p53, among others (Yang et al. 1996; Kirlin et al.
1999; Alaluf et al. 2000).
7 In Vitro
Light therapy has been used to accelerate would healing for the past 50 years (Mester
et al. 1971), especially promoting the formation of improved microvasculature
(Schindl et al. 2003; Chen et al. 2008; Feng et al. 2012; Moore et al. 2005) despite
the fact that mammalian adult endothelial cells are one of the least proliferative cell
types under physiological condition (Hobson and Denekamp 1984). Angiogenesis is
an essential biological response in the healing process, as the high metabolic activity at
the injury site demands more oxygen and nutrients (Diegelmann and Evans 2004).
Signals from surrounding fibroblasts, macrophages, and other endothelial cells trigger
vascular formation, and stressed endothelial themselves secrete required factors which
function in an autocrine manner. Neovascularization is stimulated via VEGF, bFGF,
and TGF-β in combination with cofactors (Tonnesen et al. 2000). The VEGF family
members are key regulators of angiogenesis and consist of VEGF-A, VEGF-B,
VEGF-C, VEGF-D, VEGF-E, and PIGF, and each subtype further has multiple
isoforms (Crawford et al. 2009). VEGF-A is the main proangiogenic factor and binds
to two different VEGF receptors, VEGFR-1 and VEGFR-2. Binding of VEGF-A to
VEGFR-1 triggers the formation of capillary structures, whereas binding to VEGFR-2
initiates endothelial cell proliferation; the inhibition of either factor or receptor leads to
decreased diameters, density, vascular permeability, and ultimately to apoptosis of the
cells (Roeckl et al. 1998; Qazi et al. 2009). Góralczyk et al. in two separate studies
investigated the gene expression of VEGF, secretion of VEGF-A, the presence of
VEGF receptors (sVEGFR-1 and sVEGFR-2) in the supernatant, and the proliferation
of endothelial cells after exposing them to low-level light therapy at a wavelength of
635 nm (red) and an energy density of 0 J/cm2, 2 J/cm2, 4 J/cm2, and 8 J/cm2
(Góralczyk et al. 2015). They observed an increase in proliferation in all the groups
apart from the control; however, the highest level of cell proliferation was found to be
in the groups with the energy density of 2 and 4 J/cm2. These results were to those of
Schindl et al. (2003) using the same energy density (Schindl et al. 2003). Furthermore,
they saw a reduction of VEGF-A, sVEGFR-1, and sVEGFR-2 in the supernatant, the
least concentration being in the group illuminated with 2 and 4 J/cm2. The low
concentration of VEGF-A could be explained by its binding to the cell membrane
receptor which leads to the activation of proliferation of the cells, as could be seen
from the results. Soluble sVEGFR-1 and sVEGFR-2 receptors are important to bind to
excess VEGF in the supernatant to prevent an overstimulation of proliferation and
angiogenesis of endothelial cells acting as a negative regulator (Jain et al. 2012; Ebos
et al. 2004).
Szymanska’s group (2013) showed that low-level light therapy in the 635 nm and
830 nm range and also at doses of 2, 4, and 8 J/cm2 influences the proliferation of
endothelial cells (Szymanska et al. 2013). Interestingly, VEGF concentration
decreased inversely proportional to the proliferation of the cells, indicating its
binding to the receptors and thereby eliciting its response. VEGF initiates and
regulates the initial stages of angiogenesis. It is 50,000 times more potent than
histamine in permeabilizing vessel membranes, causing an efflux of plasma into
the extracellular space, thereby initiating the coagulation of fibrinogen and organi-
zation of extracellular matrix. This leads to the migration of endothelial cells and is a
trigger of vessel germination (Shibuya 2001). Application of an 830 nm laser led to a
decrease in TGF-β secretion, whereas 635 nm increased the production of TGF-β in
a dose-dependent manner. Interestingly, different wavelengths had varying influ-
ences on the proliferation and secretion of growth factors; this could be explained by
the different levels of photoreception by the cell. Szymanska et al. (2013) hypoth-
esized that TGF-β acted as a proliferation inhibitor, as its diminished secretion at a
dose of 8 J/cm2 led to an increased endothelial cells proliferation. TGF-β is an
important proangiogenic factor, which is synthesized by multiple cell types and
organs. It influences cells differently during growth and differentiation. TGF-β
mainly stimulates the increased production of collagen I and protease inhibitor
secretion, promoting angiogenesis; whereas it suppresses the expression of degra-
dation proteases and inhibits endothelial cell proliferation (Werner and Grose 2003;
Bierie and Moses 2010), with a cofactor, it will stimulate angiogenesis (Lawrence
and Diegelmann n.d.). TGF-β binds to TβRI or TβRII activating the Smad pathway,
which then conducts the signal from the cell membrane to the nucleus. There they
bind to the transcription factors, regulating the gene expression (Pepper 1997).
Feng et al. (2012) demonstrated that LLLT promotes VEGF expression and
vascular endothelial cell proliferation through the activation of the ERK/Sp1 path-
way. Using a 632.8 nm laser, they showed that a significant increase in proliferation
was observed at a dose of 1.8 J/cm2 (Feng et al. 2012). They attributed this effect to
the activation of the ERK pathway by its gradual dissociation from MEK and its
translocation to the nucleus already 30 min after illumination, where it stayed active
for almost 90 min. In the nucleus, the concentration of ERK and phosphor-ERK
binding to Specificity protein 1 (Sp1) increased significantly and was inhibited in the
presence of PD98059. The phosphorylation of Sp1 led to its activation and enhanced
its binding activity to the VEGF promoter, which was hindered in the presence of
mithramycin-A. The phosphorylation of the Thr453 and Thr739 on the Sp1 enabled
the recruitment of more Sp1, which increased the transcription efficacy of VEGF.
VEGF expression augmented following LLLT compared to the control group and
was remarkably decreased when cells were pretreated with inhibitors such as
PD98059 or mithramycin-A. This led to a transition of the cells from G1-to-S
phase and an increased proliferation of the endothelial cells. A change in the
phosphorylation level of Sp1 is regulated by ERK, Akt, and PKC-ζ, which is a
special transcription factor for the G1 cell cycle phase in epithelial cells (Grinstein
et al. 2002), and it also activates the cyclin D1 promoter in vascular endothelial cells
mediated by Ras-dependent pathway (Nagata et al. 2001). Sp1 is known to regulate
the expression of multiple genes implicated in cell growth, proliferation, and angio-
genesis in response to physiological or pathological stimuli (Santiago et al. 2007;
Gong et al. 2012; Li et al. 2011). It regulates VEGF expression by directly binding
with its prototypic Cys2/His2-type zinc finger motif to the high GC-rich motif in the
proximal regions of the VEGF promoter (Briggs et al. 1986; Kadonaga et al. 1987;
Wu et al. 2007).
MAP kinase pathway components are evolutionarily conserved signaling mole-
cules which enable the transduction of extracellular commands to intracellular
responses. The Ras-dependent ERK pathway is a classical MAP kinase pathway
which is activated by mitogenic factors, differentiation stimuli, and cytokines. This
pathway is essential for the G1-to-S phase progression and is known for its stimu-
lation of proliferative regulators of the cell cycle and interference with anti-
proliferative genes. Mainly anchored to MAPK/ERK kinases in the cytoplasm, it
translocates to the nucleus upon activation, to phosphorylate and thereby activate
numerous substrates (MacKenzie et al. 2000; Cui et al. 2008; Meloche and
Pouysségur 2007). It has been shown that LLLT mediates its activating and prolif-
erative effect of quiescent satellite cells via the MAPK/ERK pathway (Shefer et al.
2001).
The group of Chen et al. (2008) hypothesized that the endothelial proliferation
and migration was stimulated by low-level light therapy through the activation of the
PI3K signaling pathway (Chen et al. 2008). After exposing endothelial cells to a
632.5 nm laser at a final dose of 0.26 J/cm2, they observed the upregulation of eNOS
on gene and protein level, which led to the proliferation of the HUVECs. The
observed formation of capillaries and neovascularization effect was diminished after

a PI3K pathway inhibitor, LY294002, as added, confirming that the eNOS expres-
sion was upregulated via the PI3K pathway. As mentioned earlier, Pore et al. (2004)
showed that upregulation of VEGF could also be possible through the Akt pathway
mediated by Sp1 (Pore et al. 2004). Inhibition of Sp1 by siRNA prevented the
induction of VEGF mRNA. Other groups have shown that the PI3K/Akt pathway
can increase VEGF expression via the hypoxia-inducible factor 1 (HIF-1), which in
turn activated eNOS and leads to the synthesis of NO and thereby to endothelial cells
activation (Zundel et al. 2000; Laughner et al. 2001; Brugarolas et al. 2003).
Another hypothesis is that during hypoxia, VEGF gene activation occurs under
the influence of hypoxia-inducible factor HIF-1a. Importins a and b actively trans-
port HIF-1a to the nucleus, where it combines with HIF-1b to form an active HIF-1
complex. This complex binds to the transcriptional region of the gene, and with the
recruitment of transcription factors, P-CREB and P-STAT2 among others, the
transcription of the VEGF gene is initiated (Ahluwalia et al. 2010; Ke and Costa
2006; Levy et al. 1995).
Apart from that, Chen et al. (2008) also confirmed that migration occurred upon
illumination, which was due to an increase in vinculin protein expression, a con-
served cytoskeletal protein localized in both cell-cell and cell-extracellular matrix
junctions (Jockusch and Rüdiger 1996). Staining of vinculin showed that it was
localized at the two edges of the cell and formed focal adhesion contacts. For
recruited cells to be able to move to the wound site, they need to migrate, which
involves the extension of their lamellopodia at the leading edge, adhesion to the
surrounding extracellular matrix, and contraction at the rear of the cell. In this
process, the constant formation and dissolving of adhesion sites need to be repeated
multiple times till the cell reaches its destination (Bailly et al. 1998; Heath and
Holifield 1991; Lauffenburger and Horwitz 1996; Ridley et al. 2003). Vinculin, a
cytoskeletal protein, is involved in cell to cell and cell to extracellular matrix
interaction by linking cytoskeletal actin with transmembrane receptors, integrins
and cadherins (DePasquale and Izzard 1987). Furthermore, it takes part in the
formation of the cell morphology and also in the migration of the cell (Zachary
2003). Recent studies have shown that signals generated by VEGFR-2 in endothelial
cells promoted the recruitment of vinculin to the leading edge of the cell and oriented
with their lamellopodia protrusion in the direction of migration during angiogenesis
and wound healing (Li et al. 2001).
Ankri et al. (2010) illuminated endothelial cells in “stressed condition” with
visible light in the 400–800 nm range with a final dose of 39 J/cm2 and observed
that blue light stimulated more nitric oxide (NO) production compared to red light,
offering an alternative wavelength to release intracellular NO bioavailability (Ankri
et al. 2010). In the study of Ankri et al. (2010), they showed that NO release was
triggered in “stressed” cells compared to no release in normal cells, which confirmed
the findings of Karu et al. that photobiostimulation occurs in cells which are in a
pathophysiological condition and the healthy cells remain unaffected. They also
proved that the concentration of NO formed after illumination in stressed cells was
not higher than cells in normal medium. It is very important that NO concentrations
remain at physiological level, as excess levels would lead to the reaction of NO with
O2 to form ONOO , a harmful oxidant, causing damage to the mitochondria and
suppression cytochrome oxidase activity (Halliwell and Gutteridge 1986).
Nitric oxide is a semi-stable free radical which functions as an important biolog-
ical messenger. NO is known to mediate vascular smooth muscle relaxation, causing
dilation of blood vessels; it also stimulates mitochondrial biogenesis and apoptosis
and is involved in energy production (Brown 2003). Additionally, it modulates
cytokine cascades and thereby participates in wound healing processes (Schwentker
et al. 2002). There are non-enzymatic (nitrosothiols) and enzymatic (nitric oxide
synthase) sources for NO. Nitrosothiols, such as nitrosoglutathione, react to light in
the UV range, as well as wavelengths longer than 500 nm, releasing NO during the
process of photolysis (Singh et al. 1995; Sexton et al. 1994). On the other hand,
when light interacts with enzymes such as nitric oxide synthase (NOS), they also
produce NO; however, nothing is decomposed (Zhu et al. 1997). NOS contains four
major molecules that interact with light, namely, heme, FMN, FAD, and tetra-
hydrobiopterin to produce NO. Apart from that, NOS can also take up free electron
released by photoactivated NADPH oxidase complex in the cytosolic membrane to
synthesize NO (Lubart et al. 2005). An increase of calcium levels in the cell induced
by visible light also triggers NOS to form NO (Lavi et al. 2003). Furthermore, NOS
has two constitutive forms, neuronal NOS and endothelial NOS, and an inducible
form, inducible NOS. nNOS and eNOS activation is calcium dependent; however,
hypoxia is stronger stimuli for eNOS. iNOS is also triggered during cellular stress
but is calcium independent. Endothelial cells express eNOS and iNOS, their activity
being augmented by cellular ROS, which are also produced following visible light
illumination (Lubart et al. 2005).
Induction of the enzymatic activity of NOS can take hours; however, studies have
shown that ROS, such as H2O2, is able to trigger iNOS synthesis in endothelial cells
within minutes via a calcium-dependent eNOS phosphorylation (Thomas et al.
2002), which stays activated over several hours (López-Ongil et al. 1998). Illumi-
nation has also been shown to trigger the release of H2O2 in various cells, which lead
to iNOS activation that can last up to 1 day (Zadeh et al. 2000). Blue light seems to
be more effective than red light in the activation of NOS, which can be explained on
the one hand by the presence of heme and flavins in the NOS protein and on the other
hand by the increased production of ROS, which activate NOS as mentioned above
(Banan et al. 2001; Ikeda et al. 1999; Eichler et al. 2005).
Recently it has been discovered that cytochrome c oxidase, part of the mitochon-
drial respiratory chain, not only reduces oxygen to water but is also able to reduce
nitrite to NO (Cco/NO) in multiple cells, including endothelial cells (Poyton et al.
2009). A decrease in cellular pH and increased nitrite concentrations augment the
Cco/NO reaction rate. Although Cco/NO releases NO under normal oxygen condi-
tions, its activity enhances during hypoxia (Poyton and Ball 2011). Ball et al. (2011)
concluded from their study while illuminating cells with low-intensity broad spec-
trum light that 590 14 nm light stimulated Cco/NO activity under normoxic and
hypoxic conditions, thereby not influencing the cytochrome c oxidase reduction of
oxygen to water (Ball et al. 2011). In another case, the disassociation of NO from
cytochrome c oxidase after exposure to 595–597 nm light was confirmed, increasing

its bioavailability (Hayashi et al. 2007; Sarti et al. 2000). This enables the use of NO
intracellularly for signaling and extracellularly for vasodilation and other signaling
cascades (Poyton et al. 2009). Illumination led to delayed NO production in rat
hearts (Borutaite et al. 2000), cardiomyocytes (Yang et al. 2011), and arteries (Zhu
et al. 1997). While studying NO synthesis in cardiomyocytes after illumination with
near-infrared light, Zhang et al. (2009) noticed that not all of the NO produced came
from iNOS (Zhang et al. 2009). Apart from Karu et al. (2005b) observed a stronger
attachment of HeLa cells to glass after low lower laser illumination at 904 nm, which
was attributed to NO (Karu et al. 2005). These findings provide a substitute to the
enzymatic source of cellular NO.
Hsu et al. (2010) showed the improvement in the adhesion power of endothelial
cells to poly(carbonate)urethane (PU) biomaterial vascular grafts after illumination
with 632.8 nm laser at a final dose of 1.18 J/cm2 (Hsu et al. 2010). The cells were
illuminated prior to harvesting and subsequently seeded on the PU graft. The
illuminated endothelial cells showed changed morphology was more resilient
against shear stress and increased extracellular calcium and nitric oxide. The authors
hypothesized that the increase in calcium due to illumination triggers the Ca2+/
calmodulin-dependent eNOS which catalysis NO synthesis. Increased calcium
efflux acts as a signal transducer influencing the adhesion of integrins by bringing
about a conformation change in their matrix-binding site or by their attachment to
actin filaments. The illuminated cells also secreted more sGAG after being seeded on
the biomaterial graft for 72 h, which assists in their attachment. An improved
attachment of the cells is important for anchoring-dependent cells during the prolif-
eration process, as without it, the cells would go into apoptosis (Kipshidze et al.
2001), as was shown by Karu et al. (1996). Biomaterials are often porous, have an
uneven surface or ridges, and therefore would make it difficult to illuminate cells
directly on the graft, as the light would not be able to penetrate through the material
to reach the cells; therefore, it is advisable to preexpose the cells to LLLT for better
results. Furthermore, it has been suggested that prospective NO-releasing biopoly-
mers upon light stimulation could enable a long-term use of vascular grafts without
causing negative effects such as systemic vasodilation (Fleser et al. 2004).
7.1 Secondary Angiogenic Effects of Supporting Cells After

Exposure to LLLT
As mentioned earlier, wound healing is a dynamic process, where multiple cells

secrete cytokines and mediators, thereby interacting and influencing one another.
Fibroblasts are essential during the proliferation and remodeling phase, especially as
they secret components of the extracellular matrix needed to support the vessels
formed during angiogenesis. Apart from that, secretion of KGF-1, KGF-2, and IL-6
from fibroblasts cause keratinocytes to migrate, proliferate, and differentiate into
epidermis cells (Smola et al. 1993; Xia et al. 1999) and can continue the process by
self-expressing IL-6 and NO. Furthermore, keratinocytes are prompted to express
VEGF, which guides angiogenesis in the injured tissue through upregulation by NO

in endothelial cells. During this phase, fibroblasts and endothelial cells proliferate
the most. VEGF is secreted not only by keratinocytes but also by fibroblasts,
macrophages, and platelets and even endothelial cells present at the edge of intact
capillaries. The lumen formation of endothelial cells is stabilized by the enveloping
of smooth muscle cells; the presence of different components of the extracellular
matrix (ECM), such as elastin and collagen secreted by fibroblasts; the assembly of
basement membrane; and the adhesion of pericytes (Costa et al. 2007; Conway et al.
2001). Interferon-inducible protein (IP-10) seems to give the signal to turn off most
of the components of the proliferation phase. It reduces the recruitment of fibroblasts
by inhibiting EGF-induced fibroblast migration and inhibits interferons.
Kipshidze et al. (2001) showed in their study that myocytes, vascular smooth
muscle cells, and fibroblasts responded to low-level light therapy at a wavelength of
632 nm and doses between 0.10 and 6.3 J/cm2 with secretion of VEGF, the
fibroblasts responding least sensitive toward LLLT (Kipshidze et al. 2001). Apart
from that, endothelial cells were exposed to the secretome of illuminated smooth
muscle cells, after which they showed an increased proliferation compared to VEGF-
conditioned medium, indicating that SMC secrete a more potent form of VEGF or
other factors that also increase proliferation in endothelial cells.
Light therapy activates multiple genes in fibroblasts, as shown by Zhang et al.
(2003). One hundred eleven of the investigated genes were regulated by red light
irradiation, which belonged to ten functional categories. Eight genes which play a
stimulatory role in cell proliferation and three genes involved in the energy metab-
olism and respiration chain were upregulated, whereas genes related to apoptosis or
stress response were downregulated. In another study conducted by Martignago
et al. (2015), they showed that illumination led to the activation of the genes for
collagen (COL1 α1) and VEGF in cultured fibroblasts (Martignago et al. 2014).
Houreld et al. (2014) profiled 84 genes in fibroblasts in response to light therapy in
the 660 nm range and found that 43 genes were upregulated whereas 33 were
downregulated (Houreld et al. 2014). Of the upregulated genes, some belonged to
the ECM components, remodeling enzymes, and adhesion molecules or were
involved in signal transduction. Fibroblasts, keratinocytes, and macrophages secrete
matrix metalloproteinase (MMPs) to clear the extracellular matrix from inflamma-
tory debris and enabling cells to migrate deeper into the matrix. Their inhibition
prevents the formation of new capillaries. Chen et al. (2008) showed that near
infrared could upregulate the production of matrix metalloproteinase 2 (MMP2) in
fibroblasts at both the protein and transcriptional levels (Chen et al. 2008).
Smooth muscle cells support and strengthen the blood vessel structure and also
synthesize connective tissue components of the artery such as collagen and elastin.
To find out if light therapy could improve the function of smooth muscle cells,
Gavish et al. (2006) investigated its effect on porcine primary aortic smooth muscle
cells (Gavish et al. 2006). They irradiated the cells at a wavelength of 780 nm and
found that it not only increased proliferation but also augmented collagen synthesis
by twofold, modulated the equilibrium between regulatory matrix remodeling
enzymes, and inhibited pro-inflammatory IL-1-b gene expression.
Platelets are activated after injury to vessels by the presence of exposed ECM
components, such as integrins, fibrinogen, von Willebrand factor and thrombin; this
in turn stimulates the platelets to produce prostaglandin and leukotriene. These
mediators lead to vasoconstriction and acts as a chemoattractant to neutrophils and
fibroblasts. Apart from that, the clotted platelets release fibrin, complement, seroto-
nin, platelet factor IV for coagulation, antimicrobial activity, induction of vascular
permeability, and chemoattraction of fibroblasts and monocytes, respectively.
Low-level light therapy is able to modulate the secretion of angiogenic factors by
T-lymphocytes as shown by Agaiby et al. (2000). Their group was able to show that
endothelial cells proliferated after being incubated in the secretome of
T-lymphocytes that were illuminated at 820 nm.
Stem cells also contribute to wound healing by differentiating into multiple cell
lineages after paracrine growth factor stimulation. They also impact the wound
milieu by stimulating recruitment, migration, proliferation of endogenous cells,
and angiogenesis (Brunt and Klausner 1988). Martignago et al. (2015) showed
that illumination with a 660 nm laser increased proliferation in adipose-derived
mesenchymal stem cells and elevated VEGF and VEGF receptor levels needed for
vessel formation (Martignago et al. 2015).
8 In Vivo
With these in vitro data, it was not far to seek to investigate the effects of LLLT
in vivo, and despite the fact that the underlying mechanisms of low-level light
therapy have not yet been unraveled, it has been applied to patients in various
clinical areas, with demonstrated positive effects for the patients (Mester et al.
1971; Boschi et al. 2008; de Sousa et al. 2013). LLLT finds applications in a variety
of clinical fields such as in dermatology, oncology, surgery, dentistry, and veterinary
medicine with beneficial therapeutic outcome for treatment of nerve and muscle
disorders, joint and back pain management, and wound healing (Tchanque-Fossuo
et al. 2016; Dungel et al. 2014; Bensadoun and Nair 2012; Walsh 1997).
In various in vivo studies, primarily in rodent models, LLLT has been shown to
accelerate tissue regeneration, increase neovascularization, and promote angiogen-
esis in wound healing. In 2014 Dungel et al. could show that illumination of a flap
wound in mice significantly reduced necrosis 7 days post-surgery (Dungel et al.
2014). These results support the findings of a study from Cury et al. who in 2013
showed in a similar setting that illumination of skin flap in rats leads to increased
angiogenesis, HIF-1α and VEGF expression, and decreased MMP-2 activity (Cury
et al. 2013). These findings were furthermore supported by a recent study from
Wagner et al. in which the effects of photobiomodulation during oral wound healing
was investigated. In this study it could be shown that illumination of oral ulcers at
two different intensities increased angiogenesis during oral wound healing and
significantly increased IL-1β expression and decreased TNF-α expression (Wagner
et al. 2016). In another study performed in 2015, Park et al. could show in a mouse
model, that LLLT significantly improved adipose-derived mesenchymal stromal cell
(ASC) transplantation efficacy by diminishing apoptosis of transplanted ASCs and

by increasing neovascularization and tissue regeneration (Park et al. 2015).
Aside from traumatic wounds, LLLT finds application in diabetes-related impair-
ment and tissue regeneration. Patients with diabetes mellitus often face complica-
tions in wound healing, like the clinical manifestation of diabetic foot ulcers (DFU).
Due to the chronic state of hyperglycemia in diabetic patients, an unbalanced level of
matrix metalloproteases (MMP) establishes, which leads to excessive degradation of
the extracellular matrix, ultimately leading to reduced tensile strength of the skin and
defective wound healing (Guo and Dipietro 2010). This chronic impairment of
wound healing predisposes the affected patients to severe infections, leading to the
fact that one out of six DFU patients will require a limb amputation, with a following
5-year mortality rate of up to 77% (Vuorisalo et al. 2009). A recent paper from
March 2016 reviewed four randomized control trials (RCT) involving 131 partici-
pants, with the main aim to investigate the use of LLLT for treatment of DFU. All
studies demonstrated positive healing outcomes with LLLT compared to placebo or
control groups, and there were no adverse events associated with LLLT treatment
(Tchanque-Fossuo et al. 2016).
It is being presumed that the capability of LLLT to alleviate the symptoms of
inflammation is due to immune cells responding to irradiation with light in the
visible spectrum, and this may justify its beneficial effect in clinical therapy dem-
onstrated in treatment of various chronic inflammatory diseases. LLLT is able to
stimulate the degranulation of mast cells, and as a result pro-inflammatory cytokines
like TNF-α are released, and this degranulation of cytokines leads to an increase of
leukocyte infiltration into surrounding tissue (Bayat et al. 2008). Moreover, LLLT
facilitates an enhanced proliferation, activation, and motility of lymphocytes and
also increased phagocytic properties of macrophages (Young et al. 1989; Inoue et al.
1989; Hawkins et al. 2005). It has also been shown, that upon LLLT in models of
inflammation, like osteoarthritis, a decrease in the infiltration of neutrophils and the
gene expression of pro-inflammatory IL-1β, IL-6, and TNF-α could be achieved
(Aimbire et al. 2008; Bortone et al. 2008; Alves et al. 2013). Furthermore, Wu et al.
found that the treatment is also able to inhibit the inflammatory response in adipose-
derived stem cells (ADSCs) after stimulation with LPS. They concluded that this
inhibitory effect was mediated by the regulation of cyclic AMP and NF-κB
(Wu et al. 2013).
Another very important area of application, where LLLT can have significant
impact on the immune system, is the field of tissue engineering, where autologous or
allogeneic cells are used to improve or replace biological tissues. It might be possible
to increase the wound healing and evade the resulting immune reaction of the host to
the new or foreign cells through LLLT. Due to the new era of tissue engineering and
the emerging role of stem cells in this field, the question arose whether LLLT could
have beneficial effects on transplanted stem cells, as these cells have a high proba-
bility of being rejected by the recipient tissue. For example, transplanted spheroids of
human adipose-derived stem cells (hASCs) in skin flaps of mice which were treated
with LLLT showed increased wound healing, including the neovascularization and
regeneration of skin appendages. Higher secretion levels of growth factors could be
measured in the LLLT-treated group, as well as higher survival of hASCs (Park et al.
2016). Due to the superior accessibility of superficial wounds, they seem to be best
suited for LLLT, and a variety of studies already demonstrated efficacy of light
therapy in suitable models (Dungel et al. 2014; Hawkins et al. 2005; Fekrazad et al.
2015).
Another branch in tissue engineering is the repair of bone defects and concluding
autogenous bone engraftment. A study by Barbosa et al. tried to evaluate the effects
of LLLT on an autogenous bone graft, stabilized with fibrin sealant derived from
snake venom. Altogether, the results showed that LLLT exhibited positive results in
bone repair via its effects on bone metabolism and consolidation of fractures via
osteoblastic stimulation and increased bone mineral density. They were also able to
find increased angiogenesis upon the laser treatment (Barbosa et al. 2016).
Studies have suggested that the anti-inflammatory, anti-edema, and
pro-angiogenic property of LLLT can act transcranially, as an effective treatment
modality for stroke (Huang et al. 2012). It is suggested that the mechanism of action
of LLLT on neurons in culture is through the stimulation of cytochrome c oxidase, a
process called photobiomodulation. This stimulation results in increased cellular
respiration, including increased ATP levels and ROS generation which leads to
activation of redox-sensitive genes and related transcription factors including
NF-κβ (Rojas and Gonzalez-lima 2013; Migliario et al. 2014). Further, data from
for traumatic brain injury (TBI) animal models which were treated with transcranial
LLLT have shown improvements in neurological functioning, reduction of the brain
lesion size, reduction of neuro-inflammation, and stimulation of the formation of
new neurons. Brain-derived neurotrophic factor (BDNF) and Synapsin-1 were
significantly upregulated upon LLLT in these lesions, and they concluded that
these improvements are mediated by this upregulation of BDNF which in fact
encourage synaptogenesis (Xuan et al. 2014; Rojas and Gonzalez-lima 2013).
The beneficial effects attributed to LLLT, such as acceleration of wound healing,
promotion of analgesia, modulation of inflammatory processes, and its antimicrobial
activity, when combined with photosensitizers, are also well-received in the field of
dentistry (Bjordal et al. 2006; Chow et al. 2007; Demidova-Rice et al. 2007; Koshi
et al. 2011). In dentistry, LLLT finds application in a broad range of fields, including
oral wound and nerve tissue healing, control of pain and edema, and bone
biostimulation (Angelov et al. 2009; Rochkind et al. 2007).
9 Conclusion
As organ regeneration techniques in the field of tissue engineering are advancing,

supportive physical therapies are also being sought to enhance and facilitate the
processes involved in tissue recovery. Among other therapies, low-level light ther-
apy is promising, as it has shown to reduce inflammation, accelerate proliferation,
and positively stimulate cells. Initially low-intensity lasers were used for their
therapeutic effects; however, with the development of LEDs, slowly and steadily,
the lasers were supplanted, as LEDs can spread and diffuse more widely,
illuminating more area compared to the coherent light of lasers and are more cost-
effective. Multiple wavelengths have shown to elicit stimulatory effects; however,
light therapy in the red and near-infrared range penetrates deeper into the tissue
compared to other wavelengths and is therefore more effective as shown in the
literature. It is important to note that the light therapy has a biphasic effect, as seen in
the “Arndt-Schulz law,” because of which the correct dose and intensity need to be
determined prior to application.
LLLT, as can be seen from the examples mentioned earlier, influences different
types of cells involved in the regenerative process and wound healing. As angio-
genesis is a critical step in both prior mentioned process, it was of utmost importance
to critical examine the role LLLT could potentially play in its propagation. Endo-
thelial cells, the main cells involved in angiogenesis, are one of the least proliferative
cells under physiological conditions, which makes it even more urgent to find a form
of therapy that could stimulate their proliferation and activation. VEGF is an initiator
and key player in angiogenesis secreted by endothelial cells. LLLT has been able to
stimulate its expression on gene and protein level, as well as promote the prolifer-
ation of endothelial cells in multiple studies. It has been shown that the proliferative
effect of LLLT is triggered through the activation of the ERK/Sp1 pathway which
leads to a transition of the cells from G1-to-S phase. Another study demonstrates that
proliferation and migration of endothelial cells occurred through the triggering of the
PI3K signaling pathway, by activation of eNOS. Red light therapy also leads to an
increase in proangiogenic factors, such as TGF-β and NO. These positive effects are
initialized in the mitochondria, where the interaction of red light with cytochrome c
oxidase in the electron transport chain occurs. This leads to an increase in mito-
chondrial respiration and elevated production of ROS, NO, and ATP, which act as
secondary messengers to activate the pathways mentioned above.
Wound healing and tissue regeneration are a dynamic process involving multiple
cells. The interplay and communication between different cells promote angiogen-
esis. Fibroblasts, myocytes, lymphocytes, and vascular smooth muscle cells secreted
VEGF upon light stimulation which supports vessel formation and proliferation of
endothelial cells. Light-stimulated fibroblasts also produced collagen and other ECM
components giving support to the formed vessels. MMPs secreted by fibroblasts,
keratinocytes, and macrophages enabled the propagation of new capillaries.
The secretion of proangiogenic factors such as VEGF, HIF-1α, and IL-1β has also
been observed in vivo. Light therapy increases neovascularization and therefore
accelerates wound healing, reduces necrosis, and prevents apoptosis. Translating
these results clinically, it has been shown with success that patients suffering from
diabetic foot ulcers and inflammation for multiple years experienced positive healing
outcomes with LLLT.
Summarizing all results, it is conclusive that LLLT has stimulatory effects on
numerous cells involved in tissue regeneration. This is of benefit, as it has the
potential to act as a supportive therapy and therefore facilitate the healing process.
In the foreseeable future, it is expected that the research on the applications of LLLT
will increase and that novel approaches to treatment of a variety of diseases will be
developed using this noninvasive, cost-effective approach.
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27 Oct 2016
Targeting the Cellular “Oxygen Sensors”:
Hypoxia Pre-Conditioning and Stabilization
of Hypoxia-Inducible Factors
Hermann Agis
Abstract
The development of novel strategies in tissue engineering and regenerative
medicine is inspired by the knowledge on the cell biological processes underlying
regeneration. A clear key element in the early phase of healing is the cellular
response to hypoxia. Novel therapeutic approaches target the cellular “oxygen
sensors” by applying hypoxic pre-conditioning and pharmacologically simulated
hypoxia. The cellular response to hypoxia is highly conserved, is well-
orchestrated, and relies on hypoxia-inducible factors, which require labile tran-
scription factor subunits and initiate among other pathways the cellular adaption
to hypoxia and increase the production of pro-angiogenic factors. Hence,
targeting cellular oxygen sensors is considered to be a promising strategy for
tissue engineering and regenerative medicine. In this chapter, an overview of the
current knowledge on the biology of hypoxia is given. Furthermore, we will
review current research in the application of hypoxia-based strategies such as
hypoxia pre-conditioning and prolyl hydroxylase (PHD) inhibitors for tissue
engineering and regenerative medicine.
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 The Biology of Hypoxia: Hypoxia-Inducible Factors as Key Players . . . . . . . . . . . . . . . . . . . . . . 3
3 Pharmacologically and Genetically Simulated Hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4 Potential Applications for Hypoxia-Based Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
H. Agis (*)
Department of Conservative Dentistry and Periodontology, School of Dentistry, Medical University
of Vienna, Vienna, Austria
Austrian Cluster for Tissue Regeneration, Vienna, Austria
e-mail: hermann.agis@meduniwien.ac.at

DOI 10.1007/978-3-319-21056-8_5-1
2 H. Agis
4.1 Ischemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
4.2 Inflammatory Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
4.3 Tissue Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5 Ex Vivo Hypoxia-Based Pre-conditioning Strategies in Tissue Engineering and
Regenerative Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
6 In Vivo Hypoxia-Based Strategies in Tissue Engineering and Regenerative
Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
7 Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
1 Introduction
The development of novel strategies in tissue engineering and regenerative medicine

needs to be guided by the knowledge on the cell biological processes underlying
regeneration. As a biotechnologist working in the field of regenerative dentistry,
applying this philosophy opened a door to the field of hypoxia’s role in tissue
regeneration, in particular healing of the bone, periodontium, and dental pulp. I am
grateful for the privilege to share this fascination by writing this chapter.
The aim of tissue engineering is to replace damaged tissue using constructs
composed of scaffolds, cells, and signaling factors, employing the principles of
life science, medicine, and engineering (Chen and Jin 2010). However, a key
challenge in the clinical application of these bioengineered constructs is poor
diffusion of oxygen into avascular defect sites, in particular in large critical size
defects (Liu et al. 2015). To overcome this challenge in regenerative medicine, we
need strategies which are inspired by the biology underlying the healing process.
The cellular response to hypoxia is highly conserved, is well-orchestrated, and relies
on the interplay of cellular “oxygen sensors” and a complex “cell biological machin-
ery” that among other processes induces the production of pro-angiogenic factors
(Fraisl et al. 2009). These factors are key players in development and regeneration as
they initiate the formation of blood vessels and the ingrowth of repair cells including
mesenchymal progenitor cells (Wang et al. 2007; Thangarajah et al. 2009). Further-
more, their function extends this role as links to the cellular metabolism and
erythropoiesis have been shown (Fraisl et al. 2009; Maes et al. 2012; Yousaf and
Spinowitz 2016). The sometimes limited efficiency of regenerative approaches
which rely on the time delivery of single recombinant growth factors has highlighted
in clinical trials that the complexity of an angiogenic response is tough to mimic
artificially (Hadjipanayi and Schilling 2013). Hence, targeting the “cell biological
machinery” which drives the pro-angiogenic response in regeneration via the “oxy-
gen sensors” to induce a well-balanced pro-angiogenic response is considered a
promising approach for tissue engineering and regenerative medicine (Fraisl et al.
2009; Hadjipanayi and Schilling 2013).
In particular for patients with compromised healing, e.g., by aging and diabetes,
the impaired responsiveness to hypoxia involving the cellular “oxygen sensors”
suggests that strategies targeting this signaling pathway have high potential to
Targeting the Cellular “Oxygen Sensors”: Hypoxia Pre-Conditioning. . . 3
Fig. 1 Schematic diagrams illustrating the various hypoxia-based approaches. (a) Fields for
therapeutic application are ischemic diseases, inflammatory diseases, and tissue injury. (b)
Hypoxia-based approaches can be applied in vivo and ex vivo using cells or the supernatant of
cells (secretome). Hypoxia signaling can be induced physically, genetically, and pharmacologically.
Adopted with modifications form (Müller et al. 2017 in press)
support healing and thus improve their life quality (Duscher et al. 2015). Based on
the knowledge on the key role of hypoxia in regeneration, hypoxia-based approaches
can be easily and efficiently applied, even if we do not understand the full complex-
ity of the pro-angiogenic response yet. These approaches include in vivo and ex vivo
application of prolyl hydroxylase (PHD) inhibitors, hypoxia pre-conditioning for
regenerative medicine, and gene therapy (Fig. 1).
The aim of this chapter is to provide an overview of the biology of hypoxia and to
review the current research on hypoxia-based approaches with the focus on PHD
inhibitors and hypoxia pre-conditioning for tissue regeneration and regenerative
medicine.
2 The Biology of Hypoxia: Hypoxia-Inducible Factors as Key

Players
Regeneration begins with the formation of a blood clot (Araujo et al. 2010). The
coagulation cascade is initiated, leading to the conversion of fibrinogen into fibrin
(Mosesson 2005). A fibrin clot forms and acts as temporary matrix in the defect. The
accumulated platelets within the blood clot release the content of their granules: a
plethora of signaling molecules, including platelet-derived growth factors and trans-
forming growth factor-β, which attract mesenchymal progenitor cells and endothelial
cells (Gruber et al. 2004; Kandler et al. 2004). A pro-angiogenic microenvironment
is established and the granulation tissue organizes. However, the early phase of
healing is characterized by catabolic and hypoxic conditions due to poor diffusion of
4
Fig. 2 The complex “cell biological machinery” of hypoxia-inducible transcription factors. (a) Hypoxia and angiogenesis are coupled by hypoxia-inducible
transcription factor (HIF)-1α involving the release pro-angiogenic factors such as vascular endothelial growth factor (VEGF). (b) Similar to HIF-1α, also HIF-2α
H. Agis
oxygen into the avascular defect area. The immigrating cells have to withstand and
respond to these harmful conditions. Also cell transplantation strategies have to
overcome these conditions, in particular in critical size defects where prolonged
hypoxia compromises the survival of the transplanted cells (Hadjipanayi and Schil-
ling 2013). Furthermore, cellular responsiveness to growth factors and differentia-
tion factors such as bone morphogenetic protein (BMP)-2 and BMP-6 is reduced
under hypoxia suggesting that the effectiveness of approaches that rely on single
recombinant growth factors or differentiation factors is reduced under these condi-
tions (Gruber et al. 2008). Thus, the development of a functional blood vessel
network to bring physiological levels of oxygen into larger critical size defects is
crucial for the success of healing. When oxygen levels are below ~6%, hypoxia-
induced signaling is initiated in a broad spectrum of tissues to induce angiogenesis
and reach physiologic oxygen levels in the tissue.
Temporary cells can withstand hypoxia (Werle et al. 2016). They have a complex
“cell biological machinery” to respond to these hypoxic conditions. They can adapt
to hypoxia which is relevant during development and the early phase of healing. The
physiologic minimal oxygen level varies in the different tissues. In bone, physiologic
oxygen levels can be 1.3% (Spencer et al. 2014; Werle et al. 2016). The ambient
oxygen levels of about 21% that we breathe are much higher than these oxygen
levels. This further suggests that the current cell culture models might not totally
recapitulate the in vivo situation with regard to the oxygen levels. Moreover,
activation of the hypoxia signaling pathway is known to increase exponentially
when oxygen levels decrease below 8% (Ehrismann et al. 2007; Werle et al.
2016). Based on the knowledge on the mechanism underlying the cellular response
to hypoxia, novel approaches for tissue engineering and regenerative medicine are in
development.
The key mediator of cellular response to hypoxia is the heterodimeric transcrip-
tion factor hypoxia-inducible factor (HIF) (Fig. 2). The two HIF subunits α and β are
constitutively expressed (Fraisl et al. 2009). The α subunit comes in several isoforms
such as HIF-1α, HIF-2α, and HIF-3α. The labile transcription factor subunit HIF-1α
which is degraded under normoxia links angiogenesis to oxygen levels and has
therefore become a promising target in several fields of medicine including tissue
regeneration and cancer treatment (Fraisl et al. 2009; Duscher et al. 2015, 2015).
Similar to HIF-1α, HIF-2α is degraded under normoxic conditions and stabilized
under hypoxic conditions. HIF-1α and HIF-2α share some functions including their
Fig. 2 (continued) is degraded under normoxic conditions involving PHD and FIH-1 activity. The
two isoforms HIF-1α and HIF-2α have overlapping and distinct function such as in angiogenesis.
However, they have also unique functions. The glycolytic pathway is controlled by HIF-1α,
whereas erythropoiesis is a unique function of HIF-2α. (c) For HIF-3α, different modes of actions
were described. Full-length HIF-3α is an oxygen-regulated transcription activator. The HIF-3α is
induced by hypoxia at the mRNA and protein stability level. Some HIF-3α variants can inhibit
HIF-1α and HIF-2α function by binding to HIF-1β. (d) Other HIF-3α variants have constitutative
activity (Adopted with modifications from Maes et al. 2012 and Duan 2016)
6 H. Agis
role in controlling pro-angiogenic signaling. HIF-3α of which some variants are

degraded under normoxia has a more complex functional spectrum. In addition to
having distinct target genes, some HIF-3α variants share similar target genes as
HIF-1, while other variants of the subunit have inhibitory effects on HIF-1α- and
HIF-2α-related activity (Yang et al. 2015; Duan 2016).
The availability of the hypoxia sensible HIFα is controlled by enzymes which
require oxygen as cofactor. Under normoxic conditions (Fig. 2), prolyl hydroxylase
domain-containing enzymes (PHD) and factor-inhibiting HIF (FIH)-1 hydroxylate
HIFα and thereby initiate their degradation. These PHD1, 2, and 3 hydroxylate two
specific proline residues of HIF-1α. (Fraisl et al. 2009) The von Hippel–Lindau
(VHL) binds to HIF-1α and ubiquitinates the transcription factor which is then
degraded in the proteosome. Under normoxic conditions, FIH-1 also hydroxylates
HIF-1α and HIF-2α at a specific asparagine residue. This hydroxylation prevents
binding to cofactors and thereby inhibits HIF-1 signaling. (Fraisl et al. 2009) Similar
to HIF-1α also HIF-2α is degraded under normoxic conditions involving PHD and
FIH-1 activity (Fig. 2).
In hypoxic conditions (Fig. 2), the oxygen-dependent PHD1, 2, 3, and FIH-1 are
inhibited and degraded by seven in absentia homolog E3 ubiquitin ligases (Fraisl
et al. 2009). Hence, HIF-1α and HIF-2α accumulate intracellularly and bind the β
subunit. This complex of α and β subunit can then induce genes which contain
hypoxia response elements in their promotor region (Fraisl et al. 2009). The two
isoforms HIF-1α and HIF-2α have overlapping and distinct function (Elvidge et al.
2006; Maes et al. 2012).
For HIF-3α differential modes of actions for the various variants were described
(Zhang et al. 2014a; Duan 2016; Yang et al. 2015). Full-length HIF-3α is an oxygen-
regulated transcription activator (Fig. 2). The HIF-3α is induced by hypoxia at the
mRNA and protein stability level. Similar to HIF-1α, the oxygen depending variants
of HIF-3α are hydroxylated and subjected to proteosomal degradation. Overlapping
target genes of HIF-3α and HIF-1α are involved in apoptosis, proteolysis, metabo-
lism of glucose and amino acid, and PPAR signaling (Zhang et al. 2014a; Duan
2016). Distinct functions of HIF-3α involve genes responsible for NOD-like recep-
tor signaling and Jak–STAT signaling. Some variants of HIF-3α can inhibit HIF-1α
and HIF-2α function by competing for the binding to HIF-1β. Other HIF-3α variants
are dominant negative regulators of HIF-1α and HIF-2α. Other HIF-3α variants have
constitutive activity (Zhang et al. 2014a; Duan 2016) (Fig. 2).
To date, a wide spectrum of hypoxia-induced target genes have been identified,
highlighting the key role of HIF-1α and HIF-2α in angiogenesis, cell survival,
energy metabolism, inflammation, and erythropoiesis (Rabinowitz 2013). Control-
ling angiogenesis is a function which both, HIF-1α and HIF-2α, have in common.
However, the glycolytic pathway is governed by HIF-1α, whereas erythropoiesis is a
unique function of HIF-2α. Among the target genes are vascular endothelial growth
factor (VEGF), platelet-derived growth factor-BB, placental growth factor,
angiogenin, angiopoietin-2, angiopoietin-like 4, stromal-derived factor 1 (SDF 1),
stem cell factor, and interleukin (IL)-8, which are key molecules in angiogenesis and
tissue regeneration. Also, cellular proteolytic activity is modulated by hypoxia
Fig. 3 The balance in the

secretome. Hypoxia and
hypoxia-based approaches
shift the balance in the cellular
secretome to an enhanced
pro-angiogenic activity by
increasing the pro-angiogenic
factors and decreasing
anti-angiogenic factors
(Rabinowitz 2013). Hypoxia can induce a pro-angiogenic shift in the cellular

secretome via stabilizing the labile oxygen-sensitive HIF subunits and by increasing
pro-angiogenic factors and decreasing anti-angiogenic factors (Fraisl et al. 2009)
(Figs. 2 and 3) (Elvidge et al. 2006; Maes et al. 2012).
VEGF has been considered a key factor to stimulate angiogenesis and healing in
regenerative medicine (Fraisl et al. 2009). These strategies include gene therapy
approaches and local application of the recombinant protein. Angiogenin, also
known as ribonuclease 5, is a small 123 amino acid protein involved in angiogenesis
(Fujio et al. 2015). Angiopoietin-2 is an angiogenic growth factor and a member of
the angiopoietin family (Isidori et al. 2016). Angiopoietin-like 4 has been reported to
have pro- and anti-angiogenic activity (Hato et al. 2008; Zhu et al. 2012) and is
involved in hard tissue resorption (Knowles et al. 2010). IL-8, also known as
neutrophil chemotactic factor, induces chemotaxis and migration in granulocytes
and phagocytosis. IL-8 is expressed in cell constructs in the early phase of healing,
stimulates proliferation and survival of endothelial cell, and is considered a potent
promoter of angiogenesis (Li et al. 2003; Schraufstatter et al. 2003). IL-8 is involved
in wound repair, rheumatoid arthritis, and tumor growth (Koch et al. 1992; Morelli
et al. 2011). SDF-1 is a chemokine that is essential for mobilization and recruitment
of mesenchymal stem cells (Yellowley 2013). SDF-1 initiates this process by
binding to its receptor CXCR4 (Yellowley 2013). Recombinant SDF-1 and over-
expression of SDF-1 can increase bone regeneration (Jin and Giannobile 2014).
Under conditions which are associated with compromised tissue repair, HIF-1α can
be compromised, and levels of VEGF and SDF-1 can be below the critical threshold
level (Thangarajah et al. n.d., 2009). The concept to target HIF to stimulate the
production of pro-angiogenic growth factors can help to support regeneration.
Support comes from genetically modified mice (Rankin et al. 2012; Weng et al.
2014).
Overexpression of HIF-1α in mature osteoblasts leads to dense and highly
vascularized long bones due to increased VEGF production. In line with this,
also blockage of HIF degradation in mice lacking VHL in osteoprogenitors and
osteochondral progenitors showed enhanced vascularization and increased bone
8 H. Agis
quality (Rankin et al. 2012; Weng et al. 2014). Also knockout of VHL and mice
overexpressing HIF-1α showed increased angiogenesis and bone formation in a
distraction osteogenesis model and in a critical-sized bone defect, respectively (Wan
et al. 2008; Zou et al. 2011, 2011, 2012). In line with this, the knockout of HIF-1α in
osteoblasts highlights the importance for compromised bone regeneration and angio-
genesis (Wan et al. 2008). Similar knockout of HIF-1α in fibroblasts decreased wound
healing and neovascularization (Duscher et al. 2015). Together, these findings high-
light the importance of HIF-1α in the cellular response under hypoxia and its high
potential as target for strategies for tissue healing in regenerative medicine based on
pharmaceutical stabilization of HIF-1α and gene therapy.
Among the PHD responsible for degradation of the labile HIF subunits, PHD2 is
crucial for the regulation of intracellular HIF-1α and HIF-2α levels and thus
hypoxia-induced signaling (Vogel et al. 2010; Kim and Yang 2015; Lee et al.
2015). However, the role of PHD is not limited to controlling the cellular response
to hypoxic conditions but must be seen in a much broader perspective. PHD1 is
involved in cell proliferation via degradation of cyclin D1 (Zhang et al. 2009).
Furthermore, PHD1 controls the levels of the nuclear factor kappa-light-chain-
enhancer of activated B cells and is involved in degradation of the large subunit of
RNA polymerase II Rbp1 (Oliver et al. 2009; Kim and Yang 2015). Also PHD3 has a
broad spectrum of functions which include involvement in cell migration, apoptosis,
the immune system, as well as neural development (Place and Domann 2013). PHD3
can function as scaffolding protein thereby regulating numerous pathways (Luo et al.
2011; Garvalov et al. 2014; Kim and Yang 2015). Overall PHD are involved in a
broad spectrum of cell biological functions. These roles of PHD need to be consid-
ered when designing strategies that target PHD, in particular when applying phar-
macological inhibitors which unspecifically impede PHD (Fig. 4).
3 Pharmacologically and Genetically Simulated Hypoxia
An innovative therapeutic approach to enhance healing, in particular in situations

where healing capacity is compromised, is pharmacologically simulated hypoxia
(Mace et al. 2007; Botusan et al. 2008; Thangarajah et al. 2009; Duscher et al. 2015).
This strategy aims to increase the production of pro-angiogenic factors in the defect
by pharmacological stabilization of the transcription factor HIF-1α and HIF-2α by
inhibition of hydroxylases that initiate degradation of HIF subunits with prolyl
hydroxylase (PHD) inhibitors also known as hypoxia mimetic agents (Fig. 4)
(Mace et al. 2007; Botusan et al. 2008; Thangarajah et al. 2009; Duscher et al.
2015). In addition to oxygen, the three PHD and FIH-1 require 2-oxoglutarate and
Fe2+ which serve as cofactors (Fraisl et al. 2009). PHD inhibitors can inhibit binding
of 2-oxoglutarate or Fe2+ to the PHD, thereby inhibiting their activity and preventing
the degradation of the HIFα subunits (Fraisl et al. 2009). Several PHD inhibitors
have been shown to induce a hypoxia-like response including unspecific and specific
pharmacological inhibitors of PHD. These inhibitors include dimethyloxallyl gly-
cine (DMOG), deferoxamine (DFO), L-mimosine, and Co2+ (Fraisl et al. 2009).
Fig. 4 Prolyl hydroxylase (PHD) inhibitors (Inh.) to stabilize the labile hypoxiainducible tran-
scription factor (HIF) subunits. PHD inhibitors stabilize HIF and thereby induce a hypoxia-like
cellular response (Adopted with modifications from Maes et al. 2012)
DMOG is a nonspecific inhibitor of 2-OG for all PHD and FIH-1. DFO is an iron
chelator that strongly inhibits FIH-1 and with decreasing intensity also PHD3 and
PHD1 but not PHD2. L-Mimosine is also an inhibitor of 2-OG activity. By blocking
of PHD and FIH-1, the PHD inhibitors cause enhanced intracellular levels of HIF-1
and a sustained activation of hypoxia-responsive genes (Fraisl et al. 2009) (Table 1).
The therapeutic potential of PHD inhibitors lies in their potential to provoke the
release of pro-angiogenic factors, independent of the local oxygen tension
(Ben-Shoshan et al. 2008; Shen et al. 2009) While gene expression profiles upon
hypoxia and PHD inhibitors show similar patterns, they are not identical and may be
induced via HIF-1- and HIF-2-independent pathways (Cummins et al. 2006; Elvidge
et al. 2006). For the PHD inhibitor DMOG, these genes include the serine–threonine
protein kinase oncogene PIM1 (Bachmann and Moroy 2005), an aspartate hydrox-
ylase (Dinchuk et al. 2002), and the growth differentiation factor GDF15 (Bootcov
et al. 1997). GDF15 has been previously shown to be induced by anoxia indepen-
dently of HIF (Albertoni et al. 2002) Furthermore, DMOG can inhibit procollagen
prolyl hydroxylases (Baader et al. 1994) and may inhibit other family members
(Aravind and Koonin 2001; Elkins et al. 2003). Also other transcription factors than
HIF are responsive to hypoxia including NFkB. Hypoxia can increase the sensitivity
of the NFkB pathway to pro-inflammatory cytokines. The regulator of NFkB IKK
contains a prolyl hydroxylation site like in the oxygen-dependent degradation
domain of HIF-1α (Cummins et al. 2006). Furthermore, the redox balance affects
the Wnt signaling pathway which is involved in cell survival and regeneration
showing another link between hypoxia and regeneration which has not been
shown for PHD inhibitors. Taken together, the cellular response to hypoxia is
10 H. Agis
Table 1 An assembly of prolyl hydroxylase inhibitors sorted by the underlying principle

Principle Inhibitor Specificity
Mimics 2-OG Dimethyloxallyl glycine Inhibits PHDs, FIH, CPH
N-Oxalyl-D-phenylalanine Inhibits FIH over PHD2
Blocks active site FG-4497 Inhibits PHDs, FIH
TM 6089
Compound A
Dealanylalahocin analogues
8-Hydroxyquinolines
Pyrazolopyridines
Blocks active site, TM 6008 Inhibits enzymes that require Fe2+
iron (Fe2þ) chelator Ethyl 3,4-dimethylbenzoate
FG-0041 Iron (Fe2+) chelator and
blocksactive site; inhibits
enzymes that require Fe2+
FG-2229 (ciclopirox olamine) Inhibits enzymes that require Fe2+
L-Mimosine Fe2þ
Iron (Fe2+) chelator Deferoxamine Inhibits enzymes that require Fe2+
Hydralazine
Replace Fe2+ Metal ions such as Co2+ and Cu2+ Inhibits enzymes that require Fe2+
as co-factor
Adopted with modifications from Fraisl et al. (2009)
complex and might not be totally recapitulated by PHD inhibitors, and hypoxia
mimetic agents might not be the perfect terminology (Cummins et al. 2006; Elvidge
et al. 2006). This has to be considered when designing therapeutic strategies.
Therefore, it is not surprising that also controversial effects of PHD inhibitors
have been described. PHD inhibitors can interfere with the cellular processes
involved in tissue regeneration including proliferation via cell cycle arrest and the
modulation of the plasminogen activation system (Agis et al. 2012; Müller et al.
2012; Wehner et al. 2014). Furthermore, it is unknown how the unspecific inhibition
of prolyl hydroxylase activity modulates the production and quality of extracellular
matrix. Interestingly, the collagen cross-linker lysyl oxidase is increased by HIF-1
suggesting potential impact of PHD inhibitor treatment (Levental et al. 2009;
Schietke et al. 2010). A strategy to overcome potential unwanted effects such as
inhibition of proliferation via cell cycle arrest for regenerative approaches is the
application of the secretome of stem cells that are ex vivo incubated in the presence of
PHD inhibitors. Also the application of stem cells that were pretreated with PHD
inhibitors or genetically modified cells has been shown to be a viable alternative.
Overall, pharmacologically and genetically simulated hypoxia represents a promis-
ing tool for tissue engineering and cell-based therapies.
Several strategies have been applied to exploit hypoxia signaling including
hypoxia conditioning, the application of conditioned medium of hypoxia-treated
cells and repeated injection of PHD inhibitors into the defect site. These approaches
stimulate angiogenesis and wound healing in vitro and wound healing and bone
regeneration in animal models (Donneys et al. 2013; Hou et al. 2013; Jiang et al.
2014; Fujio et al. 2015; Li et al. 2015). To overcome the drawbacks of continuous
injections, strategies have been developed where biomaterials have been used as
carriers for PHD inhibitors with the aim to continuously release the PHD inhibitors
which would allow a one-step procedure (Wu et al. 2012; Hertzberg et al. 2013; Agis
et al. 2014; Duscher et al. 2015; Kuchler et al. 2015; Jia et al. 2016). These
approaches will further be discussed below in “In Vivo Hypoxia-Based Strategies
in Tissue Engineering and Regenerative Medicine.”
4 Potential Applications for Hypoxia-Based Strategies
There is a broad spectrum for promising applications of hypoxia-based strategies

from which patients can benefit in the future. Currently, hypoxia-based approaches
for ischemic diseases, inflammatory diseases, and tissue injury are in development
(Fig. 1) (Fraisl et al. 2009). The promising results which span from in vitro to
preclinical studies led to clinical trials which have been initiated. These clinical
studies will reveal if the in vitro results translate into the clinical situation (Fraisl
et al. 2009) (See Sect. 7). Potential applications include ex vivo application including
hypoxia pre-conditioning and in vivo applications have been described (Fig. 1).
4.1 Ischemia
Hypoxic conditions due to restriction of blood supply in ischemic diseases together

with rapid restoration of normoxia can cause tissue damage. High levels of reactive
oxygen species are produced in ischemia. These reactive oxygen species induce cell
damage and induce cell death (Taylor and Pouyssegur 2007; Fraisl et al. 2009).
PHD1-deficiency and HIF-1 stabilization can reduce the amount of ROS generated
under ischemic conditions (Fraisl et al. 2009). Thus, hypoxia pre-conditioning and
PHD inhibitors can protect cells against ischemic reperfusion injury (Bernhardt et al.
2006; Hill et al. 2008; Fraisl et al. 2009). This suggests that hypoxia-based strategies
can be used to increase hypoxia tolerance. HIF and PHD have a central role in
ischemic disease which extends beyond pro-angiogenic signaling and hypoxia
tolerance which have to be considered.
4.2 Inflammatory Disease
Inflammation can induce hypoxia due to increased metabolism and low levels of
oxygen and glucose in the inflamed region (Taylor and Colgan 2007). The cellular
response to hypoxia in particular via HIF-1 and HIF-2 is therefore linked to various
human diseases including inflammatory and autoimmune diseases as bowel disease,
Crohn’s disease, periodontitis, and infectious diseases (Fraisl et al. 2009; Huang
et al. 2011; Ng et al. 2011; Vasconcelos et al. 2016). In addition to hypoxia, also
12 H. Agis
pro-inflammatory factors and bacterial compounds can lead stabilization of HIFα

(Golz et al. 2015). While hypoxia can induce a pro-inflammatory response, HIFα can
induce anti-inflammatory signaling. PHD1 and FIH have been shown to be
pro-inflammatory, suggesting that inhibition of PHD can reduce excessive inflam-
mation. Interestingly, among the PHD inhibitors, there are also candidates with
antibacterial activity such as DFO which has been shown to inhibit Porphyromonas
gingivalis, a gram-negative bacteria involved in oral tissue inflammation (Moon
et al. 2011). The combination of pro-angiogenic and antibiotic activity is a promising
approach to overcome inflammatory diseases in particular when tissue regeneration
is required in tissues which are prone to bacterial infection as in the oral cavity where
periodontitis, peri-implantitis, and pulpitis can occur. However, also
pro-inflammatory effects of hypoxia have been described showing that that hypoxia
does make cells more susceptible to pro-inflammatory cytokines (Cummins et al.
2006).
4.3 Tissue Injury
HIF are central factors in soft tissue and hard tissue healing as it is involved in
cellular responses to hypoxia in the defect, the inflammatory phase, and angiogen-
esis (Fraisl et al. 2009; Zou et al. 2011; Donneys et al. 2013; Duscher et al. 2015).
Hampered HIF signaling is also involved in compromised tissue regeneration as in
diabetes mellitus (Thangarajah et al. 2009; Duscher et al. 2015). Thus, HIF and PHD
are considered promising targets for strategies to stimulate tissue regeneration in
particular under diabetic conditions or elderly patients (Fraisl et al. 2009; Duscher
et al. 2015).
5 Ex Vivo Hypoxia-Based Pre-conditioning Strategies

in Tissue Engineering and Regenerative Medicine
Stem cell isolates of human medical waste material from surgeries, such as dental
pulp stem cells, periodontal stem cells, adipose stem cells, amniotic stem cells, bone
marrow stem cells, and cells isolated from blood, have provided a basis for tissue
engineering (Fan et al. 2010; Kim et al. 2012; Ferraro et al. 2016; Werle et al. 2016;
Yu et al. 2016). The importance for the development of effective approaches depends
not only on the capacity that cells have upon isolation but also on the capacity that
cells maintain and develop during ex vivo expansion and treatment which help them
to fulfill their regenerative function after transplantation into the defect. If stem cells
should be available at their full capacity for therapeutic applications, cost, and time
effective “on demand,” approaches need to be developed that allow isolation,
expansion, storage, and distribution. These approaches should support the capacity
of the stem cells including their potency and ability to adapt fast to the site of
transplantation.
Prevention of hypoxia in large defects is the aim of several approaches for tissue
engineering which include the design of scaffold architectures that allow the diffu-
sion of oxygen and nutrition as well as cocultures with endothelial cells,
pre-vascularization strategies, and even oxygen producing scaffolds (Baldwin
et al. 2014; Sathy et al. 2015; Gholipourmalekabadi et al. 2016). The signaling
initiated by hypoxia, however, can also be exploited to boost the outcome of
regenerative strategies.
Strategies to enhance the capacity of cells for tissue engineering and cell therapy
during the expansion in vitro is one of the focuses of current research (Kono et al.
2013; Proksch et al. 2014; Teramatsu et al. 2014). These strategies include pre-
conditioning with pharmaceuticals, anti-apoptotic molecules, cytokines, growth
factors, and differentiation factors, the incubation of cells under hypoxia, and
more. Pro-inflammatory cytokines have been used for pre-conditioning to stimulate
proliferation in vitro and cell migration in vivo (Fan et al. 2012; Yang et al. 2013).
With this strategy, also immunosuppressive properties of mesenchymal stem cells
were enhanced (Groh et al. 2005). These approaches are clinically relevant to
support the capacity of stem cells and cell-based constructs. Hypoxia
pre-conditioning supports cell viability and migration and modulates the differenti-
ation capacity into osteogenic, chondrogenic, and adipogenic lineage (Kanichai et al.
2008; Liu et al. 2010; Volkmer et al. 2010; Valorani et al. 2012; Beegle et al. 2015).
CXCR4, the receptor for SDF-1, is involved in cell migration (Nagasawa 2014).
Inflammatory or hypoxic pretreatments increase expression of CXCR4 (Shi et al.
2007; Liu et al. 2010; Ziaei et al. 2014). However, simultaneous inflammatory and
hypoxia pre-conditioning did not show a synergistic effect on CXCR4 expression,
suggesting that the approaches work by different mechanisms.
A critical point for cell therapy is the high number of cells needed which requires
cell expansion steps. As the time from cell isolation to cell application needs to be
minimized, the duplication rate is critical for achieving the required cell numbers.
Hypoxia treatment represents a promising tool to shorten the time needed for cell
expansion as proliferation of stem cells can be increased under hypoxic conditions
(Grayson et al. 2007; Nekanti et al. 2010; Basciano et al. 2011; Estrada et al. 2012).
However, hypoxia can also reduce the proliferation capacity of mesenchymal stem
cells, highlighting the fact that culture conditions need to be tightly controlled and
optimized for the desired cell type and purpose (Holzwarth et al. 2010).
Osteogenic differentiation of mesenchymal stem cells and progenitor cells into
bone-forming osteoblasts is required for subsequent bone regeneration.
Pre-conditioning strategies can have a variety of effects on stem cells, including
modulation of osteogenic differentiation. Both inhibition and stimulation of osteo-
genic differentiation have been observed for a variety of stem cells from different
sources upon treatment with hypoxia or PHD inhibitors (Merceron et al. 2010;
Nekanti et al. 2010; Valorani et al. 2012; Wu et al. 2013; Jiang et al. 2014; Zhang
et al. 2014b; Lee et al. 2015; Müller et al. 2015; Yu et al. 2016). It seems that the
conditioning protocol can have an important impact on the outcome.
Short-term hypoxic pre-conditioning improves the migration, proliferation, and
differentiation and promotes in vivo ectopic bone formation, while studies of long-
14 H. Agis
term hypoxia are required to rule out negative effects on cells (Liu et al. 2010; Beegle
et al. 2015; Yu et al. 2016). Overall, a short-term hypoxic stimulus positively modifies
cell behavior (Liu et al. 2010; Beegle et al. 2015; Yu et al. 2016). A novel approach for
cell transplantation is the use of cell spheroids. Several culture systems have been
described from hanging drop cultures to microtissue cultures in agar wells
(Dissanayaka et al. 2014; Kuchler-Bopp et al. 2016). Using per-vascularized spheroids
promotes pulp regeneration (Dissanayaka et al. 2014). Our recent data suggest that
progenitor cells can be pre-conditioned with hypoxia or PHD inhibitors during
spheroid formation (Unpublished observation). Hypoxia pre-conditioning, the suble-
thal exposure of cells to low oxygen and pharmologically simulated hypoxia, can be
used to enhance the pro-angiogenic capacity and to initiate the adaptive mechanism
that supports cell survival and acts anti-apoptotic (Liu et al. 2009; Hadjipanayi and
Schilling 2013). Recently, stem cells grafted as spheroids have improved therapeutic
effects when compared to monolayer cultures with regard to upregulating hypoxia-
adaptive signals, enhancing the release of pro-angiogenic factors, and inhibiting
apoptosis (Xiao et al. 2013; Murphy et al. 2014; Yamamoto et al. 2014). This might
be due to hypoxic conditions in the spheroid core. The use of implantable depots of
hypoxic cells as factory for continuous production and release of angiogenic factors
in vivo could stimulate angiogenesis and regeneration (Park 2011), suggesting that also
transplanted spheroids might be a source for hypoxia-induced paracrine factors.
Hypoxia pre-conditioning of cells has been shown to be a feasible approach to
stimulate a well-balanced pro-angiogenic response; however, various issues with
regard to the application of allogenic cells have to be solved to make this approach
feasible for clinical application and to develop an off-the-shelf product. One
approach is to follow a cell-free strategy by using conditioned medium, also
known as secretome (Di Santo et al. 2009; Cargnoni et al. 2012; Frazier et al.
2013; Mildner et al. 2013; Fujio et al. 2015).
Ex vivo hypoxia pre-conditioning has been applied to stimulate the secretion of
growth factors in various cell types, including amnion fluid-derived stem cells,
adipose-derived stem cells, and dental pulp-derived stem cells (Jun et al. 2014;
Fujio et al. 2015; Lee et al. 2016). Secretome of these hypoxia-treated cells improved
wound healing and tissue regeneration (Jun et al. 2014; Fujio et al. 2015; Lee et al.
2016). In addition, hypoxia pre-conditioning of cells showed improved cell survival
in the tissue grafts involving autocrine and paracrine effects. In particular, cell
spheroids have been applied in cell transplantation approaches (Dissanayaka et al.
2014). Stress response that follows ex vivo exposure to hypoxic conditions will
protect cells against further hypoxia at the site of transplantation (Hadjipanayi and
Schilling 2013; Hsiao et al. 2014). Compared to other pre-conditioning strategies
such as magnetic field, shock waves, and laser, hypoxia pre-conditioning is easier to
implement and does not require uncommon facilities in the laboratory such as
instruments for ultrasound and laser (Hsiao et al. 2014). However, to day there is
no consensus on the optimal hypoxic pre-conditioning protocol as it is highly
dependent on the cell types and species and experiment settings (Hsiao et al. 2014).
Various hypoxia pre-conditioning protocols have been tested, from the classical
cycles of brief hypoxia with intermittent reoxygenation to a single prolonged
exposure to hypoxia (Hsiao et al. 2014). HIF-1α has been found to play a key role in
the effects of hypoxia; therefore, pre-conditioning using pharmacologically simu-
lated hypoxia is a feasible approach. PHD inhibitors that stabilize HIF-1α have been
shown to induce a hypoxia similar paracrine response (Fraisl et al. 2009; Kim and
Yang 2015). Also, HIF-2α can contribute to this increased pro-angiogenic capacity.
An unconventional regenerative strategy utilizes an implantable device for deliver-
ing angiogenic signaling factors on demand based on hypoxic cells (Hadjipanayi
et al. 2011). Overall, the current literature suggests that supernatants of hypoxia- and
PHD inhibitor-treated cells have a great therapeutic potential and that research in this
field will lead to an off-the-shelf cell-free therapeutic option for various pathological
conditions.
Hypoxia pre-conditioning has also been applied to generate blood products such
as hypoxia-conditioned plasma to increase levels of pro-angiogenic factors including
VEGF and angiopoietins via peripheral blood mononuclear cells (Hadjipanayi and
Schilling 2014). Furthermore, a decrease in anti-angiogenic factor levels such as
TSP-1 has been shown (Tenan et al. 2000; Hu et al. 2006; Hadjipanayi et al. 2013;
Hadjipanayi and Schilling 2014). Therefore, hypoxia-conditioned plasma has been
advocated to be a potent alternative to platelet-based approaches for tissue repair and
regeneration (Hadjipanayi and Schilling 2014). Targeting the cellular “oxygen
sensors” ex vivo using prolyl hydroxylase inhibitors to pre-condition cells for
transplantation is an alternative to hypoxia pre-conditioning (Najafi and Sharifi
2013; Mehrabani et al. 2015; Nouri et al. 2016). This approach has the potential to
induce a hypoxia-like response.
6 In Vivo Hypoxia-Based Strategies in Tissue Engineering

and Regenerative Medicine
In vivo hypoxia-based strategies can be divided in two concepts: pharmacological

stabilization of HIF and gene therapy approaches for overexpression of HIF
(Ben-Shoshan et al. 2008; Hadjipanayi and Schilling 2013; Kim and Yang 2015).
Pharmacological stabilization of the labile HIF subunits by application of PHD
inhibitors in vivo is a promising strategy for ischemic diseases, inflammatory
diseases, and regenerative medicine (Hadjipanayi and Schilling 2013; Rabinowitz
2013). In vitro and in vivo studies have shown conserved cellular response to
pharmacological stabilization of HIF-1 using PHD inhibitors (Fraisl et al. 2009;
Rabinowitz 2013). Consequently, strong efforts have been made to develop PHD
inhibitors providing us with a pool of pharmaceutical inhibitors with different modes
of action including mimicking the cofactor 2-oxogluterate, blocking of the active
site, and chelating or replacing iron and their combinations which were discussed
above (Table 1) (Fraisl et al. 2009; Rabinowitz 2013; Kim and Yang 2015).
Although the underlying cellular response with regard to the pro-angiogenic capac-
ity of PHD inhibitors is highly conserved, the different PHD inhibitors vary in their
specificity and thus might also vary in the therapeutic capacity (Fraisl et al. 2009;
Kuchler et al. 2015; Duscher et al. 2017).
16 H. Agis
Stimulation of pro-angiogenic activity by PHD inhibitors has been reported in a

variety of cells including adipose stem cells, progenitor cells from the dental pulp,
fibroblasts of the periodontal ligament and the gingiva, dermal fibroblasts, as well as
osteoclast progenitor cells of the hematopoietic lineage (Leger et al. 2010; Agis et al.
2012; Müller et al. 2012; Ding et al. 2014; Fujio et al. 2015; Müller et al. 2015).
These include enhanced production of VEGF and angiopoietin-2 (Leger et al. 2010;
Agis et al. 2012; Müller et al. 2012, 2015; Trimmel et al. 2015; Fujio et al. 2015).
Also, HIF-1 dependent increase of angiogenin and angiopoietin-4 was observed by
our group in the secretome of dental pulp-derived progenitor cells conditioned with
hypoxia or PHD inhibitors (unpublished observation). Even direct pro-angiogenic
effects of PHD inhibitors on endothelial cells have been described including
enhanced tube formation in vitro (Ikeda et al. 2011). However, some PHD inhibitors
have also shown to reduce cell proliferation due to cell cycle arrest (Hughes and
Cook 1996; Agis et al. 2012). Also, modulation of cellular pro-inflammatory
capacity has been described (Choi et al. 2004, 2007; Rabinowitz 2013; Müller
et al. 2015). The response seems to depend on the cell type. Increase in IL-6
was described upon DFO treatment of epithelial cells, and also increase of IL-8
upon Co2+ treatment in endothelial cells was reported (Kim et al. 2006; Markel et al.
2007). DMOG on the other hand attenuates IL-8 expression in endothelial cells
(Loboda et al. 2009). There was no pronounced increase of IL-6 and IL-8 in cells
from the gingiva and the periodontal ligament (Agis et al. 2012). After treatment
with L-mimosine, an increase in IL-8, but not in IL-6, was detected (Müller et al.
2015). Thus, it is of importance to consider potential modulation of inflammatory
signaling factors upon treatment with PHD inhibitors for treatment of ischemic and
inflammatory diseases with regenerative strategies.
The plasminogen activator system is crucial for regeneration with plasmin being
responsible for resolution of the fibrin-rich blood clot in the early phase of
regeneration and growth factor activation. It is also involved in inflammation
including periodontitis (Wyganowska-Swiatkowska et al. 2014). Plasminogen is
activated by plasminogen activators such as urokinase-type plasminogen activator
and tissue-type plasminogen activator (Wyganowska-Swiatkowska et al. 2014).
The plasminogen activators are tightly controlled by plasminogen activator inhib-
itors (PAIs), including PAI-1 (Wyganowska-Swiatkowska et al. 2014). Hypoxia
and PHD inhibitors such as DMOG and L-mimosine can decrease the capacity of
cells from the periodontal ligament and the gingiva to activate plasminogen,
in chondrocytes and tumor cells via elevating levels of PAI-1 (Zhu et al. 2009;
Wehner et al. 2014). Similar results on PAI-1 were found in adipocytes (Chen et al.
2006). These results highlight the involvement of HIF-1α and the impact of PHD
inhibitors and hypoxia pre-conditioning on cellular plasminogen activation
(Rabinowitz 2013).
For bone and periodontal regeneration, it is important to understand the impact of
hypoxia and PHD inhibitors on differentiation and activity of bone-resorbing oste-
oclasts. Therefore, intensive investigations have been performed on the role of
hypoxia, HIF-1α, and PHD inhibitors on osteoclast formation and resorbing activity.
The key initiator of osteoclast formation is receptor activator of NF-κB ligand
Table 2 An assembly of carrier materials for prolyl hydroxylase inhibitors

Beta-tricalcium phosphate Agis et al. (2014) and Vinzenz et al. (2015)
Deproteinized bovine bone mineral Agis et al. (2014, Kuchler et al. (2015), and
Vinzenz et al. (2015)
Hydroxyapatite Agis et al. (2014) and Vinzenz et al. (2015)
Collagen membranes Hamid et al. (2015)
Demineralized bone matrix Hertzberg et al. (2013)
Gelatin Ulubayram et al. (2005)
Chitosan Rassu et al. (2016)
Alginate Rassu et al. (2016)
Bioactive glasses Wu et al. (2012, 2013) and Min et al. (2015)
Calcium sulfate pellets Hertzberg et al. (2013)
Poly(lactic-co-glycolic acid) Jia et al. (2016)
Poly(3-hydroxybutyrate-co-3- Min et al. (2015)
hydroxyhexanoate) polymers
Complexer devices for transdermal delivery Duscher et al. (2015, 2017)
(RANKL) via activation of RANK. The activity of RANKL is tightly controlled by

its inhibitor osteoprotegerin (OPG). The PHD inhibitor Co2+ can decrease the ratio
of OPG to RANKL in osteoblasts suggesting an increase in osteoclast formation
(Zijlstra et al. 2012). Interestingly, HIF-1α can uncouple osteoclastogenesis and
osteoblastogenesis via increase in OPG (Shao et al. 2015). Hypoxia has been
shown to increase osteoclastogenesis and their bone-resorbing activity (Arnett
et al. 2003). PHD inhibitors such as DMOG, DFO, L-mimosine, and Co2+ have
been shown to reduce the formation of osteoclasts by inhibition of proliferation of
osteoclast progenitors (Leger et al. 2010; Vinzenz et al. 2015). However, the effect
on osteoclast formation seems to be dependent on the time point of exposure and
concentration, as the PHD inhibitors L-mimosine and Co2+ can also stimulate
osteoclast formation and resorption activity (Patntirapong et al. 2009; Knowles
et al. 2010). Thus, hypoxia and PHD inhibitors can have divergent effects on
osteoclast formation and bone resorption depending on timing, concentration, and
type of PHD inhibitors which has to be considered when designing hypoxia-based
strategies for bone regeneration. Application of pharmacological stabilization of
HIF-1 via PHD inhibitors can have “positive” side effects. For certain inhibitors
such as DFO, antimicrobial activity has been shown (Moon et al. 2011). This can be
a benefit in particular in situations which are prone to microbiological colonization
as in regenerative approaches in dentistry but also in traumatology and orthopedics.
Thus, implantation of depots of PHD inhibitors with biomaterials as carriers
which release the PHD inhibitors in an optimized manner (Table 2). Bone substitute
materials, collagen matrices, hydrogels, and more complex devices for bone, peri-
odontal, dental pulp, and soft tissue regeneration can be loaded with PHD inhibitors
to support healing and improve the success rate in challenging clinical situations
while simultaneously stimulating regeneration, inhibiting katabolic processes, and
being antimicrobic.
18 H. Agis
In 2005, already gelatin-based systems were assessed to deliver the PHD inhib-
itors DFO for other therapeutic applications (Ulubayram et al. 2005). Later, DFO
releasing gelatin hydrogels were applied for pro-angiogenic treatment (Saito et al.
2014). Also, hydrogels from chitosan and alginate were developed for controlled
release of DFO, and currently thermos-responsive gels in combination with and
without mesenchymal stem cells are in development (Hastings et al. 2012; Rassu
et al. 2016).
By supplementation of biomaterials currently applied in clinic with well-
characterized PHD inhibitors, an effective novel approach might be easily intro-
duced into the daily clinical practice (Agis et al. 2014; Hamid et al. 2015; Kuchler
et al. 2015). In particular, patients with compromised healing can profit from these
strategies (Thangarajah et al. 2009; Duscher et al. 2015; Kuchler et al. 2015).
Unfortunately, it is not always that easy as combining clinically used biomaterials
with PHD inhibitors. The dental pulp capping material calcium hydroxide does not
release biologically active PHD inhibitors at levels that can induce a pro-angiogenic
response (Müller et al. 2012). Thus, research in endodontology currently focuses on
the development of other carrier materials for PHD inhibitors which can be easily
applied in regenerative endodontology. The abovementioned hydrogels might rep-
resent a feasible approach.
Strategies for bone augmentation and guided tissue regeneration include bone
substitute materials and collagen membranes which release PHD inhibitors (Agis
et al. 2014; Hamid et al. 2015). Bone substitute materials lyophilized with DMOG
induced a pro-angiogenic response but did not stimulate bone regeneration in a
calvarial defect model in diabetic rats (Fig. 5). Deproteinized bovine bone mineral,
hydroxyapatite, and beta-tricalcium phosphate release PHD inhibitors within the first
hours and do not show a controlled release profile (Agis et al. 2014; Vinzenz et al.
2015). Similar collagen scaffolds such as membranes show a burst-like release
profile with a peak in the first hours (Hamid et al. 2015). While a capacity of the
membranes to bind biological activity of growth factors has been described (Miron
et al. 2013; Caballe-Serrano et al. 2017; Stahli et al. 2016), data from our group show
that the PHD inhibitor-loaded membranes have no relevant absorbed pro-angiogenic
activity after 48 h (Al-Habbal et al. 2017). A high release of PHD inhibitors in the
first hours might drive angiogenesis in the early phase where healing is needed;
however, the burst-like release limits the overall amount of PHD inhibitors that can
be loaded onto the scaffolds as high concentrations of PHD inhibitors have been
reported to reduce cell viability and proliferation (Agis et al. 2012; Müller et al.
2012). In addition, high local concentrations on the surface of the biomaterials might
affect their surface properties such as hydrophilicity and the ability to facilitate cell
attachment. Interestingly, the fibrin-rich blood clot in which the biomaterials are
embedded in the defect can modulate the release and activity of the PHD inhibitors
(Agis et al. 2014). When bone substitute materials such as hydroxyapatite, beta-
tricalcium phosphate, and deproteinized bovine bone mineral are embedded in a
fibrin matrix, the release kinetic is modified, leading to a prolonged pro-angiogenic
capacity. Thus, much effort is put into the development of novel biomaterials and
functionalization strategies with the aim to optimize the release kinetics for the
Fig. 5 Bone substitutes loaded with dimethyloxallyl glycine stimulate angiogenesis in calvarial
defects of diabetic rats. Bone substitutes were loaded with PHD inhibitors such as dimethyloxallyl
glycine (DMOG) by lyophilization. In rats with streptozotocin-induced diabetes, calvarial defects
were created and filled with the bone substitutes. Here Levai–Laczko-stained thin-ground sections
from the control group that received unloaded bone substitutes and DMOG group which received
the bone substitute loaded with DMOG are shown. Kindly provided by Uwe Schwarze (Compe-
tence Center for Oral Biology) and Stefan Tangl (Karl Donath Laboratory for Hard Tissue and
Biomaterial Research. Left: The different tissue types were colored according to classification: Dark
blue indicates preexisting host bone; red indicates newly formed bone. Green shows the bone
substitutes and blood vessels are shown in violet. Right: original Levai–Laczko-stained tissue. The
black bar represents 500 μm. For detail, see Kuchler et al. (2015)
respective phases of regeneration (Wu et al. 2012; Hertzberg et al. 2013; Duscher
et al. 2015; Jia et al. 2016).
To improve the release kinetics, biomaterials are in development which allow
controlled release of PHD inhibitors. Overall current preclinical data on biomaterials
which release PHD inhibitors looks promising. In a rodent femoral defect model,
DFO releasing scaffolds increase angiogenesis during bone healing and improve
biomechanical stability (Stewart et al. 2011). Bone healing was also enhanced in a
radial defect model in rabbits (Zhang et al. 2012). Among these carriers are calcium
sulfate pellets containing DFO, outperform collagen sponges soaked in DFO, and
demineralized bone matrix soaked in DFO with regard to pro-angiogenic capacity
and their release profile (Hertzberg et al. 2013). Bioglasses and poly(lactic-co-
glycolic acid)-based constructs were loaded with PHD inhibitors and were devel-
oped for bone and dental pulp regeneration (Wu et al. 2012, 2013b; Jia et al. 2016).
More sophisticated release devices have been developed for the transdermal appli-
cation (Duscher et al. 2015, 2017). To treat diabetic ulcers and overcome the
compromised wound healing in diabetes, a local transdermal drug delivery system
for DFO delivers promising results (Duscher et al. 2015). This system shows a
controlled release of DFO and can be applied as patch for transdermal delivery
(Duscher et al. 2015). In preclinical studies, transdermal DFO treatment improves
diabetic healing and prevents ulcer formation (Duscher et al. 2015). Comparison of
DFO and DMOG in vitro and in compromised healing and aged wound healing
20 H. Agis
supports the capacity of DFO for clinical applications (Duscher et al. 2017). Thus, as
differences in the effects of the various PHD inhibitors have been described for each
application, the optimal release kinetics and the optimal PHD inhibitor candidate
have to be determined to maximize the pro-angiogenic effect to stimulate tissue
regeneration (Kuchler et al. 2015; Duscher et al. 2017). A novel personalized
strategy was designed to produce scaffolds that fit the anatomical needs by 3D
printing of DMOG releasing scaffolds (Min et al. 2015). This strategy would
allow to generate the appropriate scaffold based on computed tomography images
to fit the patient’s needs. Overall the field is working on optimization of carriers for
PHD inhibitors.
The question arises if combination of hypoxia-based strategies might further
boost the regenerative effect. Combination of recombinant proteins with hypoxia-
based strategies can improve the overall impact. Experiments with rat femur seg-
ments have shown that treatment with DFO and bone morphogenetic protein results
in increased vascularization and increased bone stiffness (Stewart et al. 2011).
Although we cannot rule out that there might be positive effects by combining
hypoxia pre-conditioning with PHD inhibitors, data from our group show that
under hypoxic conditions, as present in the early phase of healing, PHD inhibitors
cannot further boost the pro-angiogenic capacity of progenitor cells (unpublished
observation). These data suggest that there are no synergistic effects of PHD
inhibitors and hypoxic pre-conditioning and that hypoxia can affect the cellular
response to PHD inhibitors. This is in line with data from the combination of
inflammatory pre-conditioning and hypoxia pre-conditioning (Yu et al. 2016).
In addition, also combinations of recombinant growth factors with PHD inhibi-
tors have been described. The abovementioned scaffolds can also be applied as
carriers for cell-produced factors. Nanoporous matrices and gels can retain VEGF
from hypoxia-pre-conditioned peripheral blood mononuclear cells more efficiently
than other macroporous hydrogels (Hadjipanayi et al. 2013). It is possible that
combining strategies that use PHD inhibitors with cell secretome might be more
effective than each approach on its own and further boost the pro-angiogenic
response.
Contemplating the role of scaffolds, one might suggest that in addition to their
function as carriers for PHD inhibitors, growth factors, and differentiation factors as
well as cell secretome provide a controllable microenvironment. Thereby, defined
programmable conditions could be established in the ex vivo pre-conditioning phase
and postimplantation in vivo.
In gene therapy, approaches for overexpression of HIF-1α either can be directly
delivered into tissue using vectors or target cells or cells can be treated ex vivo before
cell implantation to deliver the desired effect. Based on preclinical studies, HIF-1α
gene therapy has high potential to promote bone regeneration as HIF-1-
α-overexpressing mesenchymal stem cells dramatically improved the bone healing
in a critical-sized calvarial defect model in rats (Zou et al. 2011). In line with this,
angiogenesis and bone healing were increased by the application of HIF-1α gene
therapy in alveolar bone defects (Zhang et al. 2016). Also, the osseointegration of
implants can be improved as HIF-1α-overexpressing bone marrow stromal cells
increase bone formation in mesi-implant defects and show enhanced

osseointegration in canine mandibles (Zou et al. 2012).
Treatment of myocardial ischemia by increasing hypoxia-inducible factor-1α
expression showed promising results (Jianqiang et al. 2015; Endaya et al. 2016).
Furthermore, synergistic effects of HIF-1α gene therapy and treatment of bone
marrow-derived angiogenic cells with the PHD inhibitor DMOG were found in
limb ischemia (Rey et al. 2009).
An alternative approach is to apply a hypoxia-inducible VEGF expression vector
into mesenchymal stem cells to treat ischemic myocardial injury in a rat model (Kim
et al. 2011). Although fascinating results have been achieved with gene therapy
based on the knowledge on hypoxia-related signaling, the future will show if
pharmacological or gene therapeutic approaches will prove more effective. The
fact that synergistic effects have been reported highlights that the different
approaches involve different mechanisms.
7 Future Directions
Hypoxia pre-conditioning and pharmacological simulation of hypoxia is a promising

tool to stimulate regeneration. A variety of regenerative approaches have emerged
over the past years and this development continues (Figs. 1 and 6). In particular
when healing is compromised, the stabilization of the labile HIF subunits can help to
support angiogenesis and regeneration (Fraisl et al. 2009; Rabinowitz 2013; Duscher
et al. 2015; Jia et al. 2016). This includes soft and hard tissue healing (Duscher et al.
2015; Kuchler et al. 2015; Jia et al. 2016). The here reviewed evidence highlights the
high potential of hypoxia-based strategies for tissue engineering and regenerative
medicine and provides the basis for future clinical trials. Currently in clinicaltrials.
gov, there are 77 studies found for “hypoxia-inducible factor” of which 38 are
completed and 19 are recruiting (clinicaltrials.gov, access date April 22, 2016).
Furthermore, 15 clinical studies registered on “prolyl hydroxylase inhibitor” from
which 11 are completed and 4 recruiting. This underlines the promising capacity of
hypoxia-based approaches and how actively researchers and clinicians are working
on the translation from bench to bedside.
However, target specificity and controlled factor delivery kinetics are required for
optimal pro-angiogenic activity to stimulate tissue regeneration (Fraisl et al. 2009;
Kim and Yang 2015). The PHD inhibitors that are currently used to target PHD and
thereby stabilize HIF are not specific and can induce effects beside their
pro-angiogenic capacity (Fraisl et al. 2009; Kim and Yang 2015). Positive side
effects are antimicrobial activity such as DFO (Moon et al. 2011). However, they
also can reduce proliferation, and differential effects on cell differentiation into
osteoblastic and odontoblastic lineage have been described (Agis et al. 2012; Müller
et al. 2015). It is possible that these side effects are due to unspecific activity of the
PHD inhibitors. Although PHD inhibitors and hypoxia show a similar cellular
response, their effects are not ident (Elvidge et al. 2006). Considering these issues,
there is a need to understand the effect of the different PHD in physiology,
22 H. Agis
Fig. 6 Schematic diagram of the time line showing the development of hypoxia-based strategies.
Following the limited success of therapeutic approaches with the application of single recombinant
proangiogenic factor such as VEGF hypoxia-based strategies have emerged to provide a robust and
physiological angiogenesis (Adopted with modifications from Hadjipanayi et al. 2013)
pathology, and regeneration and develop specific PHD inhibitors or other hypoxia
mimetic agents based on this knowledge.
Significant efforts have been made to develop PHD inhibitors based on a variety
of drug screening assays (Rabinowitz 2013; Kim and Yang 2015). These approaches
led to the identification of candidate compounds which are evaluated for their
therapeutic capacity. However, some of the major concerns have not been entirely
resolved. These include the understanding that HIF-1 is involved in cancers and
tumors. Therefore, strategies that inhibit HIF-1 have been investigated for therapeu-
tic approaches to treat cancer and tumor. How hypoxia-based strategies for regener-
ation interfere with tumor biology is not entirely clear. Interestingly, inhibition of
PHD by PHD inhibitors can also inhibit tumor growth and invasiveness (Seeley et al.
2006; Mazzone et al. 2009; Rabinowitz 2013; Kim and Yang 2015). Another
important issue is that the distinct roles of HIF-isoforms should be considered
when developing novel therapeutic agents. More specific inhibition of PHD could
provoke differential responses of HIF-1 and HIF-2. It is therefore important to
understand the role of the specific PHD and HIF isoforms in physiology, pathology,
and regeneration when developing strategies to modulate their activity. Furthermore,
inhibitory mechanisms of the angiogenic target genes of HIF-1 exist which can
reduce the effectiveness of PHD inhibitors. Although not all issues have been
resolved, hypoxia-based strategies via the local application of isoform-selective
inhibition of PHD seem to be a promising strategy to support tissue regeneration.
Furthermore, the combination of two and more hypoxia-based approaches might
improve the therapeutic outcome and provide more flexibility. Although combined
treatment of progenitor cells of the dental pulp did not lead to enhanced
pro-angiogenic capacity compared to treatment with either hypoxia or PHD inhib-
itors alone, in vitro (unpublished observation) application of hypoxia
pre-conditioned cells in combination with PHD inhibitors or cell secretome together
with PHD inhibitors might be a feasible approach for challenging situations.
A further approach in tissue engineering is the use of scaffolds made from
biomaterials that generate oxygen to overcome the transport limit and thus support
survival and activity of the transplanted cells (Gholipourmalekabadi et al. 2016).
While these novel biomaterials allow the development of new strategies for tissue
regeneration, the relevance of hypoxia signaling for regeneration suggests that the
simultaneous application of PHD inhibitors might further enhance regeneration. This
idea is further supported by the fact that PHD inhibitors do not further boost the
hypoxia-induced pro-angiogenic response (unpublished data). For further details on
hypoxia-based strategies, see Fraisl et al. (2009), Maes et al. (2012), Hadjipanayi and
Schilling (2013), Kim and Yang (2015), Müller et al. (2017).
8 Conclusion
Hypoxia pre-conditioning and pharmacological stabilization of HIF represent a

promising approach for tissue engineering and regenerative medicine which is
based on the cell biological mechanisms underlying wound healing and hard tissue
regeneration. Ex vivo- and in vivo-based strategies have been developed to treat
tissue trauma, ischemic diseases, and inflammatory diseases.
Acknowledgments The author acknowledges that the research of his team on hypoxia, PHD
inhibitors, and pre-conditioning was supported by the Osteology Foundation (Lucerne, Switzer-
land), Grant 10-063; the International Team Implantology (Basel, Switzerland), Grant RCL 653 and
Grant 1085_2015; and the European Society of Endodontics, Research Grant 2015. The author
thanks Barbara Cvikl (Department of Conservative Dentistry and Periodontology, School of
Dentistry, Medical University of Vienna, Austria), Ulrike Kuchler (Department of Oral Surgery,
School of Dentistry, Medical University of Vienna, Austria), Reinhard Gruber (Department of Oral
Biology, School of Dentistry, Medical University of Vienna, Austria), and Heinz Redl (Ludwig
Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria) for inspiration.
The author acknowledges Klara Janjic (Department of Conservative Dentistry and Periodontology,
School of Dentistry, Medical University of Vienna, Austria) and Janina Agis-Blei for proof reading.
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Biomaterials 33(7):2097–2108
Co-Culture Systems for Vasculogenesis
Luba Perry, Shahar Ben-Shaul, Shira Landau, and

Shulamit Levenberg
Abstract
Vascularization is a fundamental aspect of tissue engineering and is one of the
main challenges in the field when trying to construct thick tissues. Co-culture
systems have demonstrated promising potential in construction of vascularized
tissues, and in enhancing graft viability and persistence in vivo. In this chapter,
we discuss pivotal studies integrating co-cultures of endothelial with various
types of supporting cells, aimed to generate vascularized and functional tissue.
The influence of different biomaterial components, construct geometry and exter-
nal mechanical stimulations on the forming vasculature, is reviewed. A compre-
hensive understanding of the processes leading to the formation of mature and
stable vessel networks within engineered tissues will provide guidelines to
enhance current protocols, which will ultimately improve integration prospects
and enable the fabrication of clinically relevant, large engineered tissues.
Luba Perry, Shahar Ben-Shaul and Shira Landau contributed

equally to this work.
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Choice of Cells to Create Vascularized Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1 Endothelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2 Supporting Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
L. Perry • S. Ben-Shaul
Department of Biomedical Engineering, Technion, Israel Institute of Technology, Haifa, Israel
Inter-departmental Program in Biotechnology, Technion-Israel Institute of Technology, Haifa, Israel
e-mail: lubashargo@gmail.com; shahar.ben.shaul@gmail.com
S. Landau • S. Levenberg (*)
Department of Biomedical Engineering, Technion, Israel Institute of Technology, Haifa, Israel
e-mail: shiralevis@gmail.com; Shulamit@bm.technion.ac.il

DOI 10.1007/978-3-319-21056-8_7-1
2 L. Perry et al.
3 Engineering Tissue-Specific Vasculature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

3.1 Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.2 Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.3 Cardiac Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.4 Pancreas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.5 Bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
4 Construct Fabrication Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4.1 Biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4.2 Decellularization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.3 3D–Bioprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.4 Microfabrication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
5 Manipulation of External Forces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
5.1 Cyclic and Static Strain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
5.2 Flow-Induced Shear Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
1 Introduction
Angiogenesis is the physiological process through which preexisting blood vessels

sprout to form new ones, whereas vasculogenesis is the spontaneous assembly of
blood vessels, followed by angiogenesis, which extends the vascular tree (Risau and
Flamme 1995). In both processes, proangiogenic factors activate endothelial cells
(ECs), which then migrate, and participate in sprout elongation and lumen formation
by anastomosing with neighboring ECs. The ECs form the inner lining of the
perfused branch, while supporting cells recruited to stabilize the forming sprout
form the outer layers (Welti et al. 2013). These processes satisfy the dynamic
vascular demands of viable organs and play a crucial role in maintaining tissue
homeostasis through transportation of oxygen and nutrient supplies, together with
circulating cells, and waste products to and from the different tissues.
Establishment of an adequate blood supply to grafts is one of the greatest
challenges of organ and tissue transplantations, and has driven researchers to focus
on generation of functional blood vessel constructs to support large artificial tissues
(Hoch et al. 2014), and to accelerate anastomosis between the graft and host
vasculature (Pill et al. 2015; Rouwkema et al. 2008; Levenberg et al. 2005).
Co-culture systems of ECs and mural supporting cells, such as pericytes, fibroblasts,
mesenchymal stem cells, or smooth muscle cells (Karamysheva 2008; Armulik et al.
2005), are frequently used to promote stable vascular network formation within the
engineered tissue. Endothelial cell responsiveness to a wide range of microenviron-
mental cues supports vessel network formation under organ-specific regulation
(Ribatti et al. 2002). To construct a vascular network within a specific tissue, e.g.,
muscle or bone, a third tissue-specific cell type is included in the cell culture
(Levenberg et al. 2005). Moreover, the in vitro vessel network models can serve
toward investigation of tissue-specific cellular and functional mechanisms and can
be integrated in testing drug delivery strategies (Cantòn et al. 2010; Kirkpatrick et al.
2011). In this chapter, we will present a broad review of engineered construct
Co-Culture Systems for Vasculogenesis 3
vascularization approaches, using various co-culture systems. Numerous endothelial

and supporting cell combinations, biomimetic techniques, and external force manip-
ulation options will be discussed. A number of examples of engineered tissue-
specific vasculature will also be described.
2 Choice of Cells to Create Vascularized Systems
2.1 Endothelial Cells
As endothelial cells line all types of blood vessels, are in direct contact with the
blood flow, and are the principle cells involved in angiogenesis and vasculogenesis,
they constitute the primary cell of choice for construction of a co-culture system for
vascularization studies. Primary isolated ECs are necessary in studies with a clinical
orientation but bring with them limitations and challenges in the form of sample
purity, inter-donor variance, limited passages and isolation and expansion difficul-
ties. Nevertheless, most co-culture systems today use primary ECs instead of
endothelial cell lines. In their comparison of the in vitro attributes of seven endo-
thelial cell lines and three primary EC cultures, Unger et al. (2002) demonstrated that
only a few EC characteristics were observed in each of the tested cell lines, while all
characteristics were maintained in all primary cultures.
Different types of primary ECs are used for the purpose of vascularization.
Human umbilical vein endothelial cells (HUVECs) are most commonly used
(Koffler et al. 2011; Shandalov et al. 2014; Lesman et al. 2011; Chen et al. 2010;
Cheng et al. 2011; Koike et al. 2004; Rao et al. 2012); however, as they are not
available for autologous procedures in adults, they are best suited for basic science
studies only. Human adipose microvascular endothelial cells (HAMECs), mature
primary ECs that can be easily isolated from adults, exhibit impressive angiogenic
and vasculogenic capacities, both in vitro and in vivo (Freiman et al. 2016). The
cephalic and the basilic arm veins present another source of mature adult autologous
ECs (Grossman et al. 2016); however, the vascularization potential of these cells
remains to be evaluated.
Endothelial progenitor cells (EPCs) constitute another promising source of ECs
for clinical application. EPCs isolation from peripheral blood was first described by
Asahara et al. in 1997. Medina et al. conducted a comprehensive study comparing
early EPCs (eEPCs) and outgrowth endothelial cells (OECs), the two main EPCs
subpopulations (Medina et al. 2010). Based on their study, as well as the work of
other groups, eEPCs are categorized as hematopoietic cells of monocyte lineage,
whereas OECs both express endothelial markers and are capable of forming vessel-
like networks in vitro and of interacting with the host vasculature in vivo (Hirschi
et al. 2008; Medina et al. 2010). In fact, Chen et al. demonstrated that cord blood-
isolated OECs had a much greater angiogenic potential, both in vitro and in vivo,
compared to HUVECS (Chen et al. 2010). The team’s three-dimensional
OEC-prevascularized fibrin gel constructs were perfused as early as one day post-
implantation into mice. Sieminski et al. found that OECs formed more extensive
4 L. Perry et al.
networks within collagen gels, as compared to the other tested ECs derived from
three different vessel types (Sieminski et al. 2005). Additionally, Bompais et al.
(2004) found that EPCs are more sensitive to angiogenic growth factors compared to
mature ECs and were significantly superior to HUVECs in repairing an artificial scar.
The same group also demonstrated that EPCs participate in neoangiogenesis and
colonize a Matrigel fibroblast growth factor (FGF)-2 diffusing plug implanted in
immunodeficient mice. Conflicting evidence has been presented by Haug et al.
(2015), who report of significantly more in vivo neovascularization in a co-culture
of HUVECs with adipose-derived stem cells (ASCs) as compared to EPCs-ASCs
co-cultures; HUVECs-ASCs co-implants formed a complex functional neo-
vasculature, while no vessels were detected up to 6 months after co-implantation
of EPCs and ACSs. They found that when subcutaneously injecting a monoculture
of either EPCs or HUVECs into athymic nude mice, no vasculogenesis was observed
after 1 month. This might be due to the fact that when ECs are cultured without any
supporting cells, they form unstable blood vessels and later regress.
Alternatively, vascularized tissue-specific grafts can be generated by culturing
isolated microvessels with different combinations of endothelial cells, mural cells,
and/or stem cells. The simplicity of microvessel isolation from fat tissue, along with
their high angiogenic activity, and rapid vascular network-building potential stand as
significant advantages to this approach. Microvascular fragments can be embedded
within different constructs to form a prevascularized tissue grafts, which, upon
implantation, are invaded by host microvasculature that then anastomose with the
engineered tissue (Laschke et al. 2009, 2015).
2.2 Supporting Cells
Apart from the ECs, all vessel types include supporting mural cells (pericytes,
vascular smooth muscle cells). Capillaries are comprised of an ECs layer forming
the tube and pericytes, which are cells of mesenchymal origin with the ability to
differentiate into fibroblasts and smooth muscle cells (SMCs), as well as to other
mesenchymal cell types (Armulik et al. 2005). Arteries and veins are formed of an
EC layer surrounded by one or more layers of SMCs (Kutcher and Herman 2009;
Karamysheva 2008). In order to create long-lasting, stable blood vessels, ECs must
be co-cultured with one of these mural cell types (Levenberg et al. 2005; Wang et al.
2007; Cheng et al. 2011; Lesman et al. 2011). The co-culturing stimulates the
secretion of proangiogenic cytokines from both ECs and supporting cells, which
results in the formation of a stable and quiescent blood vessel (Welti et al. 2013).
Various co-culture combinations are being explored for tissue vascularization
purposes; this section will concentrate on the most commonly used combinations. In
their comparison of HUVECs versus OECs, co-cultured with fibroblasts seeded at
various densities, Chen et al. (2010) demonstrated that constructs with OECs
displayed a more developed vessel network with higher vessel density compared
to HUVECs constructs. Furthermore, anastomosis was achieved more rapidly upon
implantation of OEC compared to HUVECs constructs. The researchers also noted
that at a high fibroblast:EC ratio (2:1) enhanced vessel network formation in vitro in
both construct types. Moreover, anastomosis with host vasculature occurred
2–3 days earlier in the high density co-cultures. Wang et al. developed a
2-dimensional co-culture system composed of endothelial cells differentiated from
human embryonic stem cells (hESCs), and 10 T1/2 mouse fibroblasts, seeded at a 5:1
ECs:fibroblast ratio. The ECs formed functional vessels within 151 days of implan-
tation into severe combined immunodeficient (SCID) mice (Wang et al. 2007).
Lesman et al. generated constructs from poly(L-lactic acid) (PLLA)/polylactic-
glycolic acid (PLGA) sponges seeded with a 5:1 ratio of HUVECs and human
neonatal dermal fibroblasts (HNDFs), supplemented with fibrin, in order to enhance
vascularization (Lesman et al. 2011). They found that the fibrin concentration
impacted vessel network morphology and maturity. When adding C2 skeletal myo-
blast cells to the HUVECs-HNDFs co-culture, the myoblasts took on a partially
aligned myotube phenotype, which is of a great significance when constructing an
engineered skeletal muscle tissue. A similar combination and ratio of cells was used
by Kofflet et al. to construct tri-culture grafts comprised of HUVECs and HNDFs
and C2C12 mouse myoblasts on small intestinal submucosa (SIS) scaffolds (Koffler
et al. 2011). They showed that in vivo anastomosis with the host vasculature,
perfusion, and vessel maturity improved with increasing in vitro incubation times
of the tri-culture grafts. Building on these findings, Shandalov et al. seeded the same
tri-cell combination on PLLA/PLGA scaffolds to form an engineered muscle flap
(Shandalov et al. 2014), and demonstrated that the grafts contained more host vessels
after implantation compared to grafts populated with myoblasts only. Taken
together, these studies demonstrate that preexisting vascular networks contribute to
postimplantation graft vascularization by host vessels.
Blinder et al. used three-dimensional (3D) co-culture HUVECs-HNDFs con-
structs to characterize vasculogenic dynamics in vitro (Blinder et al. 2015). Using
live confocal microscopy, they observed distinct initial endothelial clustering,
followed by endothelial sprouting and stable endothelial network development.
Evensen et al. used two-dimensional HUVECs and SMCs co-cultures to study the
interactions between endothelial and mural cells (Evensen et al. 2009). As with
ECs-fibroblast co-cultures, they documented spontaneous organization of ECs into
vessel-like networks. In addition, vascular basement membrane-like structures,
indicative of vessel maturation, were noted. Moreover, they demonstrated that
vascular endothelial growth factor (VEGF), secreted by SMCs, is essential for
generation of a stable vessel-like network. Melero-Martin et al. also used SMCs to
support human blood-derived EPCs (Melero-Martin et al. 2007), co-embedded (4:1
ratio between EPCs and SMCs) in Matrigel and then subcutaneously injected into
immunodeficient mice; EPCs organized into mature functional blood vessels within
1 week of implantation.
Mesenchymal stem cells (MSCs) induce endothelial cell quiescence and promote
capillary formation, and have therefore been increasingly applied in tissue engineer-
ing protocols, particularly vascular tissues (Pedersen et al. 2014). In a 14-day analysis
of bone marrow endothelial progenitor cells (BM-EPCs) and MSCs co-cultures (1:1)
seeded on Matrigel-coated plates, Aguirre et al. noted tubelike structure formation,
6 L. Perry et al.
Table 1 Most commonly utilized EC and supporting cells for co culture systems
Cell type Source of isolation References
ESCs Inner cell mass of blastocyst (Levenberg et al. 2005; Wang et al. 2007)
HUVECs Umbilical vein (Blinder et al. 2015; Lesman et al. 2011;
Shandalov et al. 2014; Koike et al. 2004;
Chen et al. 2010; Cheng et al. 2011;
Pedersen et al. 2014)
HAMECs Adipose tissue (Freiman et al. 2016; Czajka et al. 2014)
EPCs Peripheral blood (Medina et al. 2010; Hirschi et al. 2008;
Chen et al. 2010; Sieminski et al. 2005;
Bompais et al. 2004; Haug et al. 2015;
Aguirre et al. 2010; Holnthoner et al.
2015)
Fibroblasts Various tissue sources including (Koffler et al. 2011; Levenberg et al. 2005;
skin and muscle Shandalov et al. 2015; Chen et al. 2010;
Wang et al. 2007; Sekine et al. 2008;
Donovan et al. 2001)
SMCs Blood vessels (Evensen et al. 2009; Melero-Martin et al.
2007; Wang et al. 2012)
MSCs Various tissue sources including (Freiman et al. 2016; Aguirre et al. 2010;
adipose tissue, bone marrow, and Pedersen et al. 2014; Carrion et al. 2010;
umbilical cord Xue et al. 2009; Rao et al. 2012)
even under starvation conditions (Aguirre et al. 2010). A number of angiogenic

markers were shown to be upregulated, while many cytokine genes were down-
regulated. Pedersen et al. (2014) seeded a HUVECs:MSCs co-culture (5:1) on poly
(L-lactide-co-1, 5-dioxepan-2-one) scaffolds and then subcutaneously implanted the
engineered constructs in immunocompromised mice for 1 and 3 weeks. The
researchers found that MSCs induced quiescence in the HUVECs, thereby
supporting tube formation. In a comparative analysis of various co-culture combina-
tions of HUVECs, HAMECs, HNDFs, and MSCs (EC:supporting cell ratios of 5:1)
grown in vitro on PLLA/PLGA scaffolds, Freiman et al. (2016) demonstrated that the
HAMECs:MSCs pair provided for the fastest vessel network formation, best vessel
alignment, and highest vessel complexity and maturity.
When culturing two or more cell types together, a compromise must often be
made when selecting a medium most suitable for growth and differentiation; cali-
bration must typically be made for each individual system (Kirkpatrick et al. 2011).
Levenberg et al. conducted a broad study comparing the appropriateness of myoblast
versus endothelial cell medium in construction of vascularized skeletal muscle
(Levenberg et al. 2005). They showed that use of myoblast medium only promoted
both myoblast differentiation and EC-dependent lumen formation in the constructs,
while EC medium alone inhibited myoblast differentiation and EC lumen formation.
Additional studies conducted by the same group established a 1:1 ratio of myoblast
and endothelial cell media as optimal for incubation of myoblast, fibroblast, and EC
tri-cultures (Shandalov et al. 2014; Lesman et al. 2011; Koffler et al. 2011; Freiman
et al. 2016) (Table 1).
3 Engineering Tissue-Specific Vasculature
3.1 Skin
Bioengineered dermal and skin products were one of the first organo-specific models
researched and provided important tools and resources for studying skin-related
physiological processes, structure-function relations, and skin replacement therapy,
mostly for deep burn wounds and diverse skin-related disorders (Bell et al. 1979,
1981; Asselineau et al. 1986; Huang and Fu 2011).One of the important discoveries
in the field was made by Bishop et al., who developed a two-dimensional
(2D) in vitro micro-capillaries platform which mimics in vivo angiogenesis pro-
cesses. Based on the knowledge that a close association between fibroblasts and
endothelial cells is crucial for angiogenesis, they cultured HUVECs with human
foreskin-isolated dermal fibroblasts and observed spontaneous formation of micro-
vessels. Stimulation and inhibition studies together with EC-specific structural and
expression analysis validated that the observed angiogenic processes closely mim-
icked those occurring in vivo (Staton et al. 2009; Bishop et al. 1999). Using the same
approach, Sorrell et al. cultured micro- and macrovascular ECs together with adult
human dermal fibroblasts as supporting cells, to create three-dimensional thin sheets
bearing tubelike structures. While the sheets were too thin to be implanted, the
method has been used as a basic model to study ECs-mural cells interactions (Sorrell
et al. 2007).
The complex, multilayered nature of skin challenges attempts of engineering full-
thickness skin tissue (Sorrell and Caplan 2004). To address these challenges, Black
et al. engineered a skin graft by co-culturing isolated human keratinocytes, dermal
fibroblasts, and HUVEC on Chitosan/collagen biopolymers. The skin equivalent
constructs were created using a unique sequential seeding protocol; the fibroblast
cells and HUVECs were co-cultured in a 1:1 ratio, and after 10 days constructs were
re-cultured with keratinocytes for another 14 days. Histological analysis revealed
well differentiated dermis and epidermis layers together with a capillary-like net-
work (Black et al. 1998).Those promising results have motivated Tremblay et al. to
culture isolated keratinocytes and fibroblasts from human skin biopsies, with
HUVECs, on collagen sponges for approximately 1 month and then implant the
vascularized skin constructs. Well-perfused vessels were apparent within 4 days of
implantation of the vascularized constructs into a mouse back skin defect (Tremblay
et al. 2005). Thick prevascularized dermo-epidermal skin substitutes (DESSD) were
generated by Klar et al., who co-cultured stromal vascular fraction (SVF), consisting
of both stromal and endothelial cells, isolated from healthy patients, with both
adipose stromal cells and human dermal fibroblasts, on a 3D fibrin/collagen con-
struct for 2 weeks, followed by another week of incubation with keratinocytes. The
dermal compartment of the graft was primarily formed from the stromal cells (with
or without ECs), while the epidermal compartment mainly contained keratinocytes.
Following prevascularization in vitro, the grafts were transplanted into a full-
thickness skin defects surgically formed at the back of immune-incompetent rats.
Monitoring graft anastomosis and integration revealed that the prevascularized
8 L. Perry et al.
constructs were perfused as early as 4 days postimplantation, while the

non-prevascularized grafts showed no perfusion at any of the tested time points.
Moreover, the prevascularized grafts exhibited in vivo vessel maturation, accelerated
wound healing and tissue homeostasis; the human epidermis graft appeared smooth,
white, and continuous with the rat epidermis, the size of the dermis layer exhibited
almost no reduction and the percentage of the epidermal size coverage remained
substantial over time (Klar et al. 2014). Construct prevascularization techniques
have enabled engineered skin tissues to preserve their regenerative properties and
improve postimplantation survival prospects. However, challenges in the creation of
grafts, such as fabrication of fully functional skin that can ultimately replace the live
skin tissue, still remain.
3.2 Muscle
A significant loss of muscle tissue may occur due to tumor ablation, traumatic
injuries, and congenital muscular dystrophies. To date, autologous muscle flaps
provide the most optimal means of addressing significant muscle tissue loss, yet
elicit donor-site morbidity and have achieved only very modest clinical success.
Thus, engineered muscle tissue alternatives are of great importance. In 1988,
Vandenburgh et al. described a contracting engineered muscle tissue (Vandenburgh
et al. 1988). Since then, many developments in the field of muscle tissue engineering
have emerged. Due to the marked thickness, capaciousness, and high oxygen
demand of muscle tissue, implantation of avascular engineered muscle would result
in necrosis and apoptosis of cells located at distances greater than 200 μm from the
nearest capillary (Kannan et al. 2005). Engineered muscle tissue can be vascularized
by co-seeding muscle and endothelial cells, and allowing the endothelial cells to
organize into vessel-like structures that can later anastomose with the host vascula-
ture upon implantation (Fig. 1a, b) (Lesman et al. 2010b; Koffler et al. 2011;
Levenberg et al. 2005). Alternatively, microsurgical techniques can be applied to
create an arteriovenous (AV) loop between the saphenous artery and vein, which is
then incorporated into the engineered muscle tissue (Polykandriotis et al. 2008).
Shandalov et al. also reported implantation of a graft populated with both muscle and
endothelial cells, around the femoral AV of a mouse, which, after 1 week, was
transferred, along with its vascular pedicle, to reconstruct an abdominal wall defect
in the same mouse (Shandalov et al. 2014).
Prior to cell selection, the most suitable matrix for engineered muscle must be
selected. Natural materials, such as collagen I and fibrin, as well as numerous
synthetic materials such as PLLA and PLGA polymers, have been frequently used
for this purpose (Klumpp et al. 2010a, b). Cell selection is a critical element in
optimal muscle tissue design (Fishman et al. 2013). Satellite cells (SCs), which are a
heterogeneous group of adult stem cells, are the most prominent muscle cell type
currently used for skeletal muscle tissue engineering (Sacco et al. 2008; Klumpp
et al. 2010b). Even though it is well established that SCs and ECs interact with one
another and that SCs can be found in close proximity to capillaries in vivo, only a
Fig. 1 Vascularized engineered tissues. Confocal images of engineered muscle graft consist of
HUVEC, HNDF, and C2C12 seeded on a SIS scaffold, 14 days postimplantation into a mouse abdominal
defect. FITC-dextran (Green), Desmin (Red). Bar-100 μm (a) compared to a normal abdominal muscle
(b) (Koffler et al. 2011). Confocal images of a 10-day-old vascularized engineered pancreatic tissue
composed of mouse pancreatic islets, human ECs, and HNDF embedded with Matrigel on 3D PLLA/
PLGA polymer scaffolds. Insulin (red), HUVEC-GFP (green). Scale bar-100 mm (c, d) (Kaufman-
Francis et al. 2012). Confocal image demonstrating the hESC-CMs tissue structural maturation. The
cardiac marker, TnI (red) and DAPI (blue) (e). Confocal images of the vascularized cardiac constructs
transplanted into the rat anterior wall of the left ventricle. hECs marker, CD31 (red), human and rat-vWF
(green), and DAPI (blue) (Lesman et al. 2010b)
10 L. Perry et al.
few studies have investigated co-culture models integrating the two cell types.
Christov et al. (2007) showed that an EC monolayer markedly increased SC growth,
whereas a MSCs or fibroblast monolayer had no effect. The influence of SCs on EC
vessel formation remains to be investigated.
Cardiac and skeletal myoblasts are also commonly used for muscle tissue engi-
neering (Fishman et al. 2013; Klumpp et al. 2010a, b; Gholobova et al. 2015;
Levenberg et al. 2005; Koffler et al. 2011; Lesman et al. 2011; Shandalov et al.
2014; Caspi et al. 2007b). Levenberg et al. were the first to report on in vitro
construction of a vascularized skeletal muscle tissue (Levenberg et al. 2005), by
tri-culture of C2C12 mouse myoblasts, embryonic fibroblasts, and endothelial cells
on PLLA/PLGA scaffolds. Upon implantation, the engineered muscle integrated and
anastomosed to the host vasculature, creating chimeric functional vessels. Lesman
et al. demonstrated the formation of functional blood vessels within differentiated
myotubes generated from HUVECs, HFF, and C2 skeletal myoblasts concomitantly
cultured on 3D polymer scaffolds (Lesman et al. 2011). Upon incubation of a similar
cell combination on SIS 3D polymer scaffolds, Koffler et al. (2011) observed
formation of functional blood vessels within differentiated myotubes, which anasto-
mosed to the host vasculature in vivo. Moreover, they demonstrated that the
engineered vascular muscle was functional and contracted in response to stimulation.
Furthermore, they showed a positive correlation between in vitro incubation times
and vascular organization following implantation. A similar study was conducted by
Gholobova et al., who demonstrated the formation of engineered vessel networks
within aligned human muscle fibers in a fibrin gel (Gholobova et al. 2015).
3.3 Cardiac Tissue
Following myocardial infarction (MI), the substantial loss of cardiomyocytes,

together with the low proliferation capacity of mature cardiomyocytes, results in
heart failure. Vascularized cardiac graft would offer a means of restoring heart
function (Xin et al. 2013; Sun et al. 2016). Caspi et al. generated a 3D vascularized
human cardiac tissue by co-culturing human embryonic stem cell (hESC)-derived
cardiomyocytes (hESC-CMs) with either HUVECs or hESC-derived ECs, with/
without embryonic fibroblasts, on PLLA:PLGA scaffolds together with matrigel.
Scaffolds containing fibroblasts in the cell mixture yielded highly vascularized
engineered cardiac tissue, where vessels were organized in a vast network, which
exhibited distinct lumen formation. The presence of engineered capillaries promoted
cardiomyocyte proliferation without interfering with tissue alignment and orienta-
tion (Caspi et al. 2007a). Two weeks after transplantation of the vascularized scaf-
folds into the anterior wall of the left ventricle of a rat heart, graft integration was
confirmed by observation of chimeric donor-host vasculature within the engrafted
engineered-tissue constructs (Fig. 1e, f). Determination of vessel functionality and
further confirmation of host-graft anastomosis were achieved by intraventricular
injection of fluorescent microspheres or lectin (Lesman et al. 2010b). This unique
3D model of engineered vascularized human cardiac tissue offers a tool for studying
cardiac tissue function, structure, and development, and can also serve as a tissue
replacement agent (Caspi et al. 2007a; Lesman et al. 2010b). Stevens et al. devel-
oped prevascularized human heart grafts by culturing cardiomyocyte-differentiated
hESCs with fibroblasts and either hESC-derived endothelial cells or HUVECs.
Contraction in response to electrical stimuli was greater in the prevascularized
patches, as compared to grafts containing cardiomyocytes only, but these patches
also exhibited more passive myocardial-like mechanical stiffness than the control
patches. Implantation of the prevascularized patches into rat skeleton muscle inci-
sions resulted in larger human cardiomyocyte tissue area than the cardio-only
patches and exhibited human vessel formation when implanted directly into the rat
heart. In general, patches bearing cardiomyocytes only were associated with a poor
survival rate, while the prevascularized patches successfully integrated with the host
coronary system (Stevens et al. 2009). Kreutziger et al. characterized the extracel-
lular matrix (ECM) components produced in vascularized tissue by co-culturing
endothelial cells together with various supporting cell types, to form a diverse
endothelial cell network, and an in vitro platform that can predict in vivo
vasculogenesis potential of each graft type. The group created tri-cultured cardiac
patches using HUVECs, differentiated cardiomyocytes which were derived from
hESc and two different hMSC clones, known to bear diverse gene expression
profiles. While one clone supported EC network organization in vitro, the other
failed to support both vessel formation and EC survival. A week postimplantation
into rat hearts, both patches were still viable, but microvessels were only observed in
the graft that showed network formation in vitro. These results demonstrate that
in vitro vessel assembly can predicate the formation of functional vessel post-
implantation and emphasize the importance of implanting a vascularized graft
(Kreutziger et al. 2011). Sakaguchi and Shimizu et al. engineered a cardiac tissue
with perfusable blood vessels (Sakaguchi et al. 2015; Sekine et al. 2008). Their
vascular platform was formed from resected rat femoral muscle, bearing a connect-
able artery and vein. In parallel, they created media-perfused triple-layer sheets of
neonate-derived cardiac cells, co-cultured with endothelial cells in a bioreactor. The
stacked cardiac sheets, which defined graft thickness, were then placed on top of the
ex vivo-engineered vascular platform, yielding spontaneous formation of blood
vessels throughout the cardiac sheet layer. The neovessels then anastomosed with
the blood vessels residing in the vascular platform, allowing for adequate media
perfusion throughout the cardiac tissue. Graft transplantation revealed that anasto-
mosis between the two engineered platforms increased both cardiac tissue function-
ality and animal survival. In their pioneering work, Zhang et al. used the
biodegradable poly(octamethylene maleate (anhydride) citrate) elastomer to design
the AngioChip, a unique microscaled scaffold consisting of branched hollow micro-
channels. Following chip fabrication, the 3D microchannels were coated with a
co-culture of endothelial cells and parenchymal cells, which enabled vessel assem-
bly while maintaining patent vessels. Cardiomyocytes isolated from neonatal rats
were then added to the precultured Angiochip, yielding cardiac tissue which
12 L. Perry et al.
established immediate blood perfusion upon direct surgical anastomosis with the
femoral vessels of rat hindlimbs (Zhang et al. 2016).
3.4 Pancreas
The success of pancreatic islet transplantation heavily relies on their ability to

anastomose with host vasculature. Reestablishment of new vessels within the grafts
can begin as soon as 1–3 days posttransplantation and concludes on around day
14, through the expansion of both preexisting islet endothelial cells and of recipient
endothelial cells (Daneman 2006; Allegaert et al. 2011). Johansson et al. co-cultured
isolated human islets with human aortic endothelial cells, to obtain endothelial cell-
coated islets that demonstrated both reduced infiltration of immune cells into the
islets and increased survival and functionality following transplantation, compared
to uncoated islets (Johansson et al. 2005). By using a unique 3D PLLA:PLGA (1:1)
scaffold embedded with a multicellular culture of mouse islets, human ECs, and
human foreskin fibroblasts, Kaufman-Francis et al. obtained an extensive, self-
assembled vessel network that effectively supported pancreatic tissue survival
in vitro and improved graft integration and function posttransplantation (Fig. 1c,
b) (Kaufman-Francis et al. 2012). The co-transplanted system reported by Borg
et al., involved concomitant transplantation of isolated mouse islets and isolated
mouse bone marrow-derived MSCs either under the kidney capsule, intraocularly or
intra-hepatically. Upon transplantation under the kidney capsule, glucose blood level
were stabilized and islet survival was higher in the islet-MSCs grafts versus islet-
only grafts. However, in vivo islet revascularization was not improved by MSCs
co-transplantation (Borg et al. 2014). In their paper, Lou et al. reported detection of
human insulin even 4 months after implantation of human islets and BM-derived
cells, co-cultured for 3 weeks in vitro, into the left sub-renal capsule of
Streptozotocin (STZ)-induced mice. Moreover, blood glucose levels increased dra-
matically upon removal of the graft, indicating that the reduction observed upon
transplantation was dependent on the transplant. Immunohistological analysis
showed that islets transplanted with BM-derived cells were located next to
vascularized areas of the kidney, suggesting that the BM-derived cells supported
islet survival and viability and enabled islet migration toward host blood vessels
(Luo et al. 2013).
Buitinga et al. described a reproducible controlled method using nonadherent
agarose microwells, to form a 3D tri-culture of human islets, hMSCs, and HUVECs.
The composite tri-culture maintained insulin-secreting capacities both in vitro and
in vivo. In addition, intra-islet endothelial cell sprouting was more extensive when
hMSCs were included in the construct, with Matrigel# providing better support
than fibrin gel. Subcutaneous implantation of the Matrigel-embedded plugs demon-
strated highest angiogenic activity in islets cultured with both HUVECs and hMSCs,
compared to islets seeded with hMSCs only (Buitinga et al. 2016). Quaranta and
Antonini observed normal blood glucose levels as early as 5 days after transplanta-
tion of isolated rat islets and bone marrow-derived endothelial progenitor cells
(EPCs) into the portal vein of induced-diabetic rats, which was maintained for up to
6 months after transplantation. Graft functionality was demonstrated by insulin
secretion in response to a glucose tolerance test. The degree of islet vascularization
at transplantation correlated with graft functionality, underscoring the importance of
vascularized pancreatic tissue postimplantation to achieve islet functionality and
prevent diabetes over time (Quaranta et al. 2014). The essentiality of
co-transplantation of endothelial cells and islets was also demonstrated upon their
transplantation under the kidney capsules of STZ-induced mice (Oh et al. 2013).
Embryonic stem cells have also been exploited as a source of insulin-secreting beta
cells. Weizman et al. noted significant upregulation of pancreatic progenitor markers
in hESC-derived pancreatic precursor cells cultured for 7 days with human endo-
thelial cells and fibroblasts. Following subcutaneous implantation of the 7-day-old
scaffolds into STZ-induced diabetic mice, promotion of pancreatic progenitor cell
differentiation into β-like-cells was observed, confirming differentiation adequate to
rescue glucose levels in these mice (Weizman et al. 2014; Tuch et al. 2014). Such a
model may set the stage for replacement therapy for diabetic patients.
3.5 Bone
Bone vascularization impacts cell migration, tissue remodeling, and, in pathological

cases, metastatic tumor invasion; its involvement in bone fracture healing is multi-
faceted and dynamic (Barou et al. 2002; Guerrero et al. 2015; Bahney et al. 2015).
During bone damage involving disruption of the vascular supply, hypoxic conditions
prevail, triggering endothelial cell migration to form a vessel network. However,
slow vascular ingrowth may lead to inadequate exchange of nutrients and oxygen to
the affected area (Liu et al. 2015a). Although much progress has been made in thin
tissue engineering, successful thick bone tissue engineering is limited by vascular
insufficiency (Guerrero et al. 2015). In attempt to generate vascularized bone tissue,
Stahl et al. demonstrated contact-dependent, bidirectional gene regulation between
endothelial cell-driven angiogenesis and osteoblast differentiation in a spheroid
co-culture system composed of human endothelial and human primary osteoblast
cells (500 cells/spheroid) (Stahl et al. 2004). Crosstalk between endothelial cells and
bone-derived cells was also described by Hofmann et al., who co-cultured (1:1)
primary human osteoblasts isolated from endosteal cancellous bone fragments, with
HUVECs in cylindrical biodegradable polyurethane scaffolds. Cell attachment was
ensured by the addition of platelet-rich plasma (PRP) and thrombin. The medium
was supplemented with human platelet-released growth factors. Osteoblast-
dependent sprouting and formation of tubelike structures demonstrated the contri-
bution of these cells to EC proliferation, differentiation, and neo-vessel formation
in vitro (Hofmann et al. 2008). While most bone regeneration approaches co-culture
cells at a specific differentiation stage to induce angiogenesis and osteogenesis in
bone constructs (Liu et al. 2015a), Guerrero et al. cultured a heterogeneous popula-
tion of human whole bone marrow-derived cells in a biodegradable 3D pullulan:
dextran macroporous polymeric matrix. Both osteoid and vessel formation were
14 L. Perry et al.
apparent within the constructs after ectopic implantation in mice, emphasizing the
clinical potential of human whole bone marrow in bone regeneration procedures, and
as a tool for microvascular network formation in vitro (Guerrero et al. 2015). Yu et al.
induced differentiation of bone marrow mononuclear cells to endothelial cells and
osteoblasts which were then co-cultured on polycaprolactone (PCL)/hydroxyapatite
scaffolds to yield vascularized osteogenic constructs. When implanted into a rat bone
fracture model, the constructs not only prevented ischemic necrosis but also
increased tissue stiffness, resulting in muscle tissue capable of bearing greater stress
(Yu et al. 2009). Zhou et al. reported that engineered vascularized bone tissue
comprised of MSCs and MSCs-derived endothelial cells, co-cultured within a
porous b-tricalcium phosphate ceramic (b-TCP) and implanted in a rabbit ulna defect
model, promoted MSCs osteogenesis and brought to complete bone repair, where
the bone exhibited natural mechanical properties (Zhou et al. 2010).
Endothelial cell and osteoblast co-cultures have provided an excellent in vitro
model for the discovery of factors and pathways involved in bone formation, repair,
and remodeling. However, the role of inflammation, which initiates angiogenesis
in vivo, was not considered. Using a triple culture setup, consisting of human
outgrowth endothelial cells, primary osteoblasts, and a PMA-activated THP 1 cell
line, Dohle et al. demonstrated increased proinflammatory cytokine expression in
parallel to microvessel formation (Dohle et al. 2014).
4 Construct Fabrication Techniques
4.1 Biomaterials
Both vasculogenesis and angiogenesis are dramatically influenced by the physical

and mechanical properties of their environment (Lesman et al. 2016). Therefore, by
using diverse fabrication techniques various vascularized constructs can be obtained
(Fig. 2). Fibrin and collagen gels are the most commonly used 3D matrices for
vascular formation; fibrin mesh has been shown to support sprouting healing
wounds, and collagen is the most common ECM protein (Brown et al. 2009).
Network formation in EC and MSCs co-cultures within constructs prepared from
varying ratios of collagen and fibrin, positively correlated with the fibrin content. In
contrast, an inverse correlation was observed between matrix stiffness and network
formation (Rao et al. 2012). In contrast, others showed that network density was
enhanced by environmental compliance and was therefore highest in constructs
generated from engineered peptide PuraMatrix hydrogel co-seeded with EPCs and
muscle progenitor cells (MPCs), versus identical cultures seeded PuraMatrix
hydrogels supplemented with collagen and fibrin (Allen et al. 2011).
While these 3D interconnected fibrous structures provide biological cues for the
forming vasculature, they fail to provide control over degradation rate, and porosity
of the cell environment. Synthetic materials can be manipulated to achieve better
control of the mechanical and chemical environment, and tailored for individual
applications (Klumpp et al. 2011). After seeding a co-culture of ECs and fibroblasts
Synthetic scaffolds
Natural gels
Flow-induces shear stress
Decellularized vessels Supporting

Endothelial cells
cell-induced contractile forces
cells
10% strain ,1Hz
Stretching
direction
3D printing
Cyclic stretch
Matrix geometry Mechanical forces

and composition Micro-fabrication
Fig. 2 Construct fabrication techniques and external mechanical stimulation. Different

vascularized constructs can be obtained by applying various fabrication techniques. Use of syn-
thetic polymers provides for high control of mechanical properties such as degradation rate and
stiffness; natural gels provide the natural environment for spontaneous network formation;
decelluarized vessels or organs being the benefit of scaffolds bearing natural shapes; 3D printing
provides high control of construct architecture, crucial for patterning an engineered tissue with
vessel network; microfabrication techniques allow for the design of microstructures for the in vitro
assessment of vessel function, such as in drug delivery. Application of external mechanical
stimulations enables the control of vessel alignment and morphology and induces the secretion of
various growth factors, thus, enabling higher control of vessel formation in vitro
or a tri-culture of ECs, fibroblasts, and myoblasts on synthetic PLLA-PLGA scaf-

folds with fibrin gel, Lesman et al. observed spontaneous network formation, with
network maturation correlating with the fibrinogen concentration. The synthetic
component added mechanical strength to the constructs, which resulted in more
vascular maturation in vitro and more interconnected vessels in vivo (Lesman et al.
2011). Lee et al. observed vessel network formation by seeding a co-culture of
HUVECs and human dermal fibroblasts (HDFs) on electrospun poly(L-lactide-co-e-
caprolactone) constructs, where both collagen deposition and the mechanical prop-
erties increased up to 14 days of culturing. One month after implantation of the
co-culture constructs into mice, massive penetration of the host vasculature, collagen
remodeling, and positive alpha-smooth muscle actin (SMA) staining were observed,
while HDF-seeded constructs showed poor vascularization (Lee et al. 2015). In
16 L. Perry et al.
attempt to better control cellular remodeling in synthetic biomaterials, Hanjaya-Putra

et al. subjected hyaluronic acid (HA) hydrogels to primary and secondary radical
polymerization; primary polymerization facilitated tube formation, while secondary
radical crosslinking inhibited it. These features were exploited to create patterned
vascularized hydrogels (Hanjaya-Putra et al. 2012).
4.2 Decellularization
Controlling the architecture of the forming vasculature can improve integration

prospects of the engineered tissue in vivo; an organized pre-patterned vasculature
will provide for sufficient nutrient supply to the implanted cells. Much work has
focused on the application of decellularized tissues and vessels to pattern engineered
vascular networks, since scaffolds bearing the native 3D anatomical architecture
offer great potential in enhancing the clinical advantage of engineered organ trans-
plants. To this end, Manalil et al. report of viable constructs generated from ECs and
SMCs grown in a decellularized porcine vessel in a bioreactor that exhibited
mechanical strength similar to a native artery. In attempt to avoid atherosclerotic
stenosis in vivo, A20 gene-transfected endothelial progenitor cells, featuring
decreased endothelium-protecting activity and consequentially lower risk of athero-
sclerosis, were used in their model. When implanting the constructs into rats, blood
flow through the vessels was observed for up to 6 months, with no signs of stenosis
or dilatation, while all nontransfected ECs constructs were rejected (Manalil et al.
2015). Ren et al. constructed a mature endothelium by seeding a co-culture of ECs
and perivascular cells derived from induced pluripotent cells on decellularized rat
and human lung scaffolds. During in vitro culture, the endothelium showed high
coverage of the decellularized vessels and high barrier functioning and remained
patent for 3 days posttransplantation in rats. A similar procedure was applied with
decellularized human lungs; however, endothelium coverage was low compared to
healthy lungs (Ren et al. 2015). In attempt to enhance the repopulation of
decellularized porcine arterial tissue, Sheridan et al. first injected HGF-loaded
chitosan b-glycerophosphate into the artery wall, followed by rMSC injection and
ECs seeding within the lumens. Constructs were then subjected to pulsatile flow.
This combination of biochemical and mechanical cues significantly impacted cell
growth compared to the static or unstimulated groups (Sheridan et al. 2014).
4.3 3D–Bioprinting
Additive manufacturing technologies is an emerging technique which brings the

advantage of rapid and accurate production of micro-patterned structures, essential
for the creation of small biomimetic capillaries. Sacrificial inks, such as sugar and
wax, can be used to print the initial micro-patterned architecture of a scaffold.
Carbohydrate glass coated with poly(DL-lactide-co-glycolide) (PDLGA) has been

used as sacrificial material to form different vascular architectures of varying
diameters. Miller, Stevens et al. casted the lattice glass with various biomaterials,
such as alginate, fibrin, and matrigel, and designed dual-compartment constructs,
comprised of 10 T1/2 cells embedded within the bulk material, and ECs, which were
injected upon the carbohydrate glass wash. ECs coated the vascular lumen and
formed sprouts from the main channels into the bulk gel (Miller et al. 2012). In
another study, gelatin was used as a sacrificial material which was then covered with
a mixture of fibrin, a co-culture of HUVECs and human lung fibroblasts (NHLFs),
and a number of layers of collagen placed on top of the entire structure. Incubation of
the construct resulted in liquification of the gelatin, which revealed the micro-
channels and enabled HUVECs seeding through the inlet of the channel. The ECs
ultimately lined the channels and those from the main channel layer created sprouts,
which later connected to the capillary networks forming within the bulk area (Lee
et al. 2014). Although all the materials are biocompatible, the described procedure
eliminates the possibility of printing cells, as it either requires high printing temper-
atures or cytotoxic solvents to remove the sacrificial material. To avoid the chal-
lenges of cytotoxic reaction by-products of sacrificial materials, Bertassoni et al.
bio-printed agarose fibers, which served as a well-defined microchannels template
for generation of photopolymerized hydrogel-based constructs, formed of various
hydrogel types and concentrations (e.g., methacrylated gelatin (GelMA), star poly
(ethylene glycol-co-lactide) acrylate (SPELA), poly(ethylene glycol)dimethacrylate
(PEGDMA), and poly(ethylene glycol) diacrylate (PEGDA) hydrogels). The aga-
rose fibers were then easily removed, since they do not adhere to the hydrogel bulk
(Bertassoni et al. 2014). Rapid and precise manufacture of prevascularized hetero-
geneous 3D tissues was reported by Kolesky et al., who exploited vasculature by
printing 3D structures with varying morphologies. First, Pluronic F127, an easily
printed and dissolved fugitive ink was co-printed with GelMA ink containing
HNDFs, followed by encapsulation of the printed construct into GelMA ink, and
photopolimerization. Pluronic F127 was then liquefied and removed, resulting in
cavities which were then coated with ECs (Kolesky et al. 2014). While these
approaches mainly rely on cell seeding within 3D scaffolds, the use of synthetic
and natural materials may interfere with both the production of ECM by the
constituent cells and with cell-cell interactions and could elicit host immune
responses. To overcome this, Norotte et al. used a layer-by-layer method to bioprint
a scaffold-free hierarchical architecture network, using spheroids and cylinders of
combined tri-cultures of ECs, SMCs, and fibroblasts, printed on an agarose template
(Norotte et al. 2009).
Biological laser printing (BioLP) deposits both biomaterial and individual cells,
with a printing resolution at the cellular level (Hoch et al. 2014). Wu et al. used this
technique to fabricate HUVECs- and HUVSMC-based vessels. In the first stage,
they printed ECs only, and after a 24-h incubation, they printed SMCs on top and
around the ECs. Cells formed cell-cell junctions around the forming lumens (Wu and
Ringeisen 2010).
18 L. Perry et al.
4.4 Microfabrication
Blood vessels multifunction in nutrient, oxygen, and blood cell transport, drug
penetration, and cancer cell metastasis. As these physiological functions are difficult
to evaluate in vivo, an in vitro model is essential to assess and study these processes.
Precise manufacturing in microfabrication allows the construction of micro-assays
which can mimic blood vessel functions on a small scale. Wray et al. designed silk-
based porous microchannels, with a wide range of dimensions, to mimic capillary
branching; hMVECs were seeded on the patterned surface of the constructs and
MSCs in the bulk space. Constructs were reported to support lumen formation and to
sustain tissue structure (Wray et al. 2013). In another approach, Baranski et al.
incorporated a cells-collagen mixture embedded within fibrin, to form aligned
cords of ECs and MSCs. Upon implantation into the parametrial fat pad in the
intraperitoneal space of athymic mice, the cords formed an aligned perfused vascu-
lature (Baranski et al. 2013). In another attempt to fabricate biomaterial-free con-
structs, Rivron et al. used soft lithographic templates to fabricate 3D agarose tissues
which deform autonomously. After seeding a co-culture of hUVEC and hMSCs, the
tissue showed a higher percentage of EC proliferation and an increase in vascular
structures in regions of high deformation. Additionally, a spatial gradient of VEGF
and VEGFR-2 expression was observed, positively correlating with degree of
deformation (Rivron et al. 2012). In an attempt to mimic small capillaries, Ye
et al. constructed a device with poly(glycerol sebacate) (PGS) elastomer microfluidic
channels, that included both vascular and parenchymal compartments, separated by
a permeable membrane. Perfusion of a myotoxic drug through the microchannels
resulted in a 90% decrease in muscle cell counts in the parenchymal space. Implan-
tation of the constructs subcutaneously and intraperitoneally into nude rats led to
biodegradation of the membrane and infiltration of host blood cells into the
implanted microvessels (Ye et al. 2013). In a similar work, endothelialized micro-
channels embedded within type I collagen were created using a lithographic tech-
nique: collagen gel was molded using a silicon stamp with a designed structure
aiming to examine endothelium activity, morphology, and stability. HUVECs
injected through the inlet maintained their phenotype and function as a barrier
between the vessels lumen and the collagen ECM. To study ECs interaction with
perivascular cells, pericytes were added to the collagen bulk which further reduced
vessel permeability and triggered the HUVECs to form long and stable sprouts. In
addition, the constructs served as a platform to investigate the endothelium behavior
during inflammation, in particular, the transition from nonthrombotic state to pro-
thrombotic state (Zheng et al. 2012). To investigate capillary permeability to drugs
and cancer cells, Yoshida et al. extracted silica capillary tubes from biodegradable
hydrogels, which were then covered with a bilayer consisting of ECs and SMCs,
yielding a controllable vascular network. To examine the barrier effect of the
forming network, rhodamine-labeled bovine serum albumin (BSA) was injected
through the vascularized channels. BSA did not leak into the hydrogel bulk in the
bilayer cell structure during incubation time of several hours, while BSA leakage
was detected in constructs with no seeded cells. However, in 1-day-long
experiments, BSA penetration was observed, likely due to albumin endocytosis

(Yoshida et al. 2013). Moya et al. fabricated a high-throughput, micro-
physiologically perfused vessel network made of polydimethylsiloxane (PDMS)
and self-assembled ECs-fibroblast capillaries, embedded within a matrix compart-
ment. The capillaries were connected to fluidic channels with two different pressure
gradients representing arterial and venule conditions, resulting in physiological flow
within them. The system demonstrated applicative potential for drug screening and
in models mimicking vascular permeability (Moya et al. 2013).
To mimic the natural bilayer morphology of blood vessels, Liu et al. engineered
mats consisting of aligned electrospun fibers, which were seeded with SMCs. The
cells aligned with the fibers and formed an organized mat, which was then assembled
on a flat EC mat. ECs spread over the entire mat and produced collagen IV and
laminin to levels significantly higher than those seen in random electrospun fiber
mats. Additionally, encapsulation of plasmid-encoded VEGF and basic fibroblasts
growth factor in the fibers enhanced proliferation, migration, and ECM synthesis of
both ECs and SMCs. Moreover, compliance similar to that of the human saphenous
vein was achieved by wrapping the mats to form a cylindrical vessel and then
culturing them for 3 months (Liu et al. 2015b). In vitro perfusion of micro-
vasculature ensures nutrient and oxygen supply essential for the survival of large
engineered tissue. Sekine et al. used resected rat femoral tissue containing an artery
and a vein, as a vascular bed for ECs and cardiac cells co-cultured in multilayered
sheets. ECs within the sheets anastomosed to the artery and vein sprouts and formed
lumens, ensuring cell survival. Scaled-up engineered tissue, generated by addition of
layers over the primary sheets, displayed high viability when the artery and vein
anastomosed to the carotid artery and the jugular vein of the rat neck (Sekine et al.
2013).
5 Manipulation of External Forces
5.1 Cyclic and Static Strain
Although mechanotransduction plays a central role in EC stimulation in natural

processes, most co-culture constructs are grown under static conditions. In recent
years, there is a growing attempt to include mechanical stimulation, such as shear
stress and strain, in construct engineering protocols, primarily achieved using bio-
reactors (Fig. 2). Ceccarelli et al. used a stretchable multiwell system with a 3D ECs
culture coated onto microcarrier beads embedded in a fibrin gel decorated with a
supporting external monolayer of smooth muscle cells. Capillaries formed in con-
structs exposed to 10% cyclic strain at 0.7 Hz sprouted in a direction parallel to the
applied strain, while capillaries in the unstrained control sample grew radially
outward. Transfer of strained constructs to static conditions led to randomization
of the oriented sprout angles, suggesting that vessel sprouting patterns, even after
formation and stabilization, are plastic (Ceccarelli et al. 2012). Similarly, upon
application of cyclic strain (6% strain, 1 Hz) to isolated microvessels embedded
20 L. Perry et al.
within collagen gels, sprouts aligned parallel to the direction of the applied strain
(Krishnan et al. 2008). However, when using a dextran microcarrier-based model
embedded within fibrin gel, Matsumoto et al. observed sprout growth perpendicular
to the cyclic uniaxial strain direction (Matsumoto et al. 2007), an observation
consistent with that of Rosenfeld et al., who showed that static stretching of 3D
scaffolds seeded with ECs and fibroblasts results in vessels oriented parallel to the
stretching direction, whereas cyclic stretching results in diagonally oriented vessel
structures. Moreover, cyclic stretching imparted a proangiogenic environment,
manifested by reduced VEGF levels, and increased platelet derived growth factor
(PDGF)-ββ levels, when compared to static stretching and control conditions
(Rosenfeld et al. 2016). These results agree with a report of induced autocrine and
paracrine signaling in endothelial and stromal cells exposed to cyclic stretching,
followed by upregulation of Ang-2 and PDGF-ββ protein expression (Yung et al.
2009). In another study, ECs and muscle cells constrained within a hydrogel
construct and exposed to static stretch aligned in the direction of stress. Moreover,
the ECs formed vascular structures without the addition of any growth factors (van
der Schaft et al. 2011). Application of uniaxial stretch on tissue-engineered blood
vessels (TEVs) made out of PGA scaffolds resulted in the development of mature
elastic fibers and in the alignment of collagen fibers within the TEVs. In addition, an
increase in the mechanical strength and compliance was observed (Huang et al.
2016). Chang et al. successfully printed aligned, encapsulated microvessel fragments
within a collagen gel under uniaxial strain. While the spatial pattern disappeared
following implantation, the invading host vasculature took on the same pattern of
alignment. Taken together, vessel architecture is greatly influenced by mechanical
forces (Chang et al. 2012).
The ECM and secreted soluble factors comprise the two major classes of angio-
genic stimulators. Ingber hypothesized that accelerated ECM turnover causes local
thinning in the ECM, which increases its compliance, and subsequently leads to local
cell distortion through tractional forces produced by neighboring cell. This, in turn,
increases tension transfer across cell-surface ECM receptors, which can modify
cellular biochemistry that can locally modify cellular growth and motility and overall
network patterning (Ingber 2002). Kroff and Augustin showed that EC-generated
tensile forces induced ECM alignment, leading to directional sprouting (Korff and
Augustin 1999). Similarly, application of a magnetic field on spheroids embedded
within a fibrin gel led to sprout alignment in a directional fashion (Morin and
Tranquillo 2011). In the microcarrier beads experiment mentioned above (Ceccarelli
et al. 2012), Ceccarelli et al. claimed that capillary alignment was not dictated by
ECM alignment, but rather, by local changes in ECM stiffness caused by the applied
strain, all this, without significant disruption of the fiber architecture.
5.2 Flow-Induced Shear Stress
It has been clearly established that flow-induced shear stress has a marked impact on
the function and morphology of ECs. Ziegler et al. exposed an arterial wall model
containing SMCs, collagen type I, and a monolayer of ECs, to one of two levels of
laminar shear stress; ECs aligned with the flow direction (Ziegler et al. 1995). In a
similar study with an EC-SMCs co-culture within a collagen gel, application of 10%
cyclic strain resulted in an organized aligned vessel wall. In the absence of flow, ECs
aligned in the direction of collagen fibrils and SMCs; however, under shear stress,
ECs aligned in the flow direction. Additionally, ECs exhibited reduced proliferation
under flow conditions, with a greater reduction in proliferation rates when scaffolds
were preconditioned with strain. EC proliferation inhibition was maintained in
cultures grown in conditioned media from the flow experiments (Imberti et al.
2002). Upon exposure of MSCs-embedded collagen cylinders covered with ECs to
flow in a microfluidic chamber, ECs migrated toward the surface and showed
elevated expression of smooth muscle cells markers, such as alpha-SMA and desmin
(Khan et al. 2012). In a computational fluid dynamics (CFD) model, designed to
simulate shear stress within a porous scaffold in a direct perfusion bioreactor,
average shear stress and pressure drop correlated with inflow velocity. In addition,
cell growth was shown to affect shear stress distribution (Lesman et al. 2010a).
Opitz et al. engineered an ovine aorta using vascular smooth muscle cells
(vSMCs) seeded onto porous poly-4-hydroxybutyrate (P-4-HB) scaffolds, subjected
to dynamic stress conditions in a pulsatile flow bioreactor. The cells took on a
multilayered homogenous structure, and displayed higher ECM production, and
significantly higher vSMC protein content, as compared to those grown under static
conditions. Moreover, on day 14 of culture, the mechanical strength of the
engineered aorta was similar to that of native aorta (Opitz et al. 2004). Niklason
et al. developed arteries from PGA tubular scaffolds seeded with SMCs for 8 weeks
followed by ECs seeding; constructs were exposed to pulsatile radial stress. Rapture
strength of the stimulated constructs was shown to be greater than that of native
human saphenous veins. Additionally, upon implantation into a miniature swine,
engineered arteries showed patency up to 24 days post implantation (Niklason et al.
1999).
In pursuit of the cues transmitted by shear stress-exposed ECs to the surrounding
SMCs, Qi et al. performed a comparative proteome analysis of rat aorta cultured
under low versus normal shear stress conditions. They noted upregulation of two
main secretary molecules, PDGF-BB and TGF-β1, and of three linked proteins,
lamin A, lysyl oxidase, and ERK 1/2, in the low shear stress-stimulated group. The
same pattern of protein expression upregulation was observed in a parallel-plate flow
chamber system bearing ECs and vSMCs at both ends of a membrane, after
application of shear stress to the ECs only (Qi et al. 2011). Orsenigo et al. reported
shear stress-induced Src activation in veins, which led to phosphorylation of Y658
and Y685 of vascular endothelial cadherin. HUVECs exposed to 3.5 dynes cm^2
showed high phosphorylation, whereas low phosphorylation was observed follow-
ing exposure to 28 dynes cm^2 and static conditions. These values correspond with
measured shear stress of arteries and veins in vivo. Similar observations were noted
in vivo, where ECs within rat jugular vein were shown to be phosphorylated. ECs
within the carotid artery, however, showed lower levels of phosphorylation
(Orsenigo et al. 2012).
22 L. Perry et al.
6 Conclusions
Despite the complexity of achieving vasculogenesis in co-culture systems, much

progress has been made in recent years. The main challenges remaining include:
(1) selection of an autologous EC source with optimal isolation, expansion, and
vessel-forming properties; (2) selection of autologous mural supporting cells with
optimal isolation, expansion, and endothelial vessel forming support properties;
(3) selection of the ratio of cells in the co-culture providing for optimal endothelial
vessel network formation, stability, and maturation; (4) selection of media that will
best facilitate both the differentiation of tissue-specific cells and endothelial vessel
formation; (5) inclusion of additional cells to create tissue-specific vascularized
grafts; and (6) control of the tissue microenvironment and geometry to set the
stage for large-scale clinically relevant tissue and organ engineering.
Acknowledgments The authors thank Dr. Yehudit Posen for proofreading the manuscript and the
European Research Council under the European Union’s Seventh Framework Program FP7, ERC
Grant Agreement no. [281501 – ENGVASC].
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Approaches for Generation of Lymphatic
Vessels
Sabrina Rohringer, Mira Schaupper, and Wolfgang Holnthoner
Abstract
The lymphatic system plays an important role in fluid homeostasis, immune cell
trafficking, and fat absorption. Due to injury, diseases, or surgery, the lymphatic
system can be disrupted which often leads to lymphedema in the adjacent
extremities. Tissue engineering is an emerging research field dealing with the
substitution of nonfunctional parts of the human body with in vitro engineered
tissues. Regenerative approaches try to stimulate the formation of functional
tissues in situ. During the last few decades, the construction of blood vessels
in vitro to supply engineered tissues with nutrients gained more and more interest.
However, research in the field of lymphatic development stayed behind, but
several approaches for lymphatics engineering were developed so far. Lymphatic
endothelial cells can be seeded to scaffold materials and afterwards implanted into
S. Rohringer (*)
University of Vienna, Vienna, Austria
e-mail: Sabrina.Rohringer@univie.ac.at
M. Schaupper
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
Austrian Cluster for Tissue Engineering, Vienna, Austria
e-mail: mira@schaupper.at
W. Holnthoner (*)
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
Austrian Cluster for Tissue Engineering, Vienna, Austria
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center,
Vienna, Austria
Tissue Regeneration, Ludwig Boltzmann Institute for Experimental and Clinical Traumatology,
Vienna, Austria
e-mail: Wolfgang.Holnthoner@trauma.lbg.ac.at

DOI 10.1007/978-3-319-21056-8_8-1
2 S. Rohringer et al.
sites of disrupted lymphatic vasculature. Several regenerative approaches describe

the stimulation of lymph vessel growth in vivo. Although the methods developed
so far hold promise for the clinical use of engineered lymphatics, the optimal
parameters for lymphatic engineering remain a challenge for future studies.
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Biology and Functions of the Lymphatic System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3 Lymphatic Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4 Lymph-Specific Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5 In Vitro Culturing of LECs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
6 Scaffold-Based Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
7 Regenerative Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1 Introduction
The blood circulatory system transports oxygen, nutrients, and hormones from the
heart to all tissues and organs. The lymphatic system, the second vascular network in
the human body, is responsible for immune cell trafficking, fat absorption, fluid
homeostasis, and “waste” removal from the blood circulatory system. The lymphatic
system was first discovered by Nicolas Massa of Venice (1531–1569) and described
by Bartolomeo Eustachius in 1563, but the function of this newly investigated
system remained unclear at that time. Gasparo Aselli was able to describe the
differentiation between veins and lymphatics in dogs in 1622. Two years later,
Johann Vesling discovered and sketched the lymphatic system in humans. The first
hints on the function of lymphatics were discovered by Olof Rudbeck. He was able
to reveal that the lymphatic system is contained of vessels similar to the blood
vascular network and drains fluid (Chikly 1997). However, the molecular mecha-
nisms of the lymphatic system remained elusive until the last two decades of the
twentieth century due to a lack of lymph-specific markers (Kiefer and Schulte-
Merker 2014; Cueni and Detmar 2006) and reliable models. However, after the
identification of several markers and the increasing knowledge about lymphatic
functions, research in the field of lymphangiogenesis was boosted since lymph-
edema and diseases of the lymphatic system affect a considerable part of the world
population. Lymphatics disruption after injury and the parasitic disease filariasis
which mainly occurs in Southeast Asia are two of the prevalent causes for lymph-
edema. However, in more developed countries, cancer treatment via surgery or
radiation displays the major cause for lymphatic dysfunction. Especially breast
cancer patients often suffer from lymphedema since it is usually necessary to remove
adjacent lymph nodes due to metastasis (Carlson 2014). The state-of-the-art methods
to treat lymphedema are lymph drainage, compression garments, and microsurgery.
Approaches for Generation of Lymphatic Vessels 3
Even though these treatments reduce the edema volume and prevent lymph accu-
mulation, the patients often need to follow long-term therapy to avoid lymphedema
formation thus straining not only the health care system but also the psychological
well-being of the patients and their families (Szuba and Rockson 1998). In recent
years, it became possible to auto-transplant lymphatic grafts into the defective tissue
site where the lymphatic vessels can anastomose and regain physiological function
again. However, the connection of lymphatic vessels is complicated since the walls
of lymphatics are very thin and fragile in comparison to blood vessels (Baumeister
et al. 1985; Baumeister et al. 2015). Due to the disadvantages and problems of
current treatment methods, tissue engineering holds future promise to treat pathol-
ogies of the lymphatic system efficiently.
Tissue engineering is an emerging field of research aiming to substitute human
tissues by in vitro engineered, cell-seeded scaffolds. Tissue engineering enables the
possibility to isolate cells from the host patient, grow them on biocompatible
extracellular matrices under physiological stress conditions, and to reimplant the
developed construct to the patient where it should connect to the surrounding tissue
and fulfill its physiological functions. To ensure sufficient nutrient supply,
engineered tissues have to be supported by blood and lymphatic vasculature. The
aim of vascular tissue engineering is to construct blood and/or lymphatic vessels
which are perfusable and anastomose with the host vasculature. Regarding the
construction and regeneration of blood vessels, numerous studies were performed
so far either using scaffold-based or regenerative approaches. Whereas scaffold-
based approaches use the combination of a biocompatible scaffold material, endo-
thelial cells and often stem cells to induce vessel growth; regenerative approaches
deal with the stimulation of existent vasculature to increase vessel growth and/or
density. In the last few decades, especially the regeneration and engineering of
lymphatics gained importance since injuries, diseases or surgeries, especially
lymph node removal due to cancer metastasis, often lead to insufficient lymph
flow and therefore lymphedema. Although the engineering of blood vessels
in vitro has been performed for numerous years by now, the regeneration and
construction of lymphatic vessels is a relatively new field of research. This review
shall provide the reader with the state-of-the-art of lymphatics tissue engineering by
describing the most promising scaffold-based and regenerative approaches to induce
lymphangiogenesis in vitro and in vivo (Fig. 1).
2 Biology and Functions of the Lymphatic System
The main functions of the lymphatic system are the transport of immune cells,
absorption of dietary fats, and interstitial fluid homeostasis (Tammela and Alitalo
2010). Plasma ingredients are constantly transported from arteries into the interstitial
space where the lymphatic vasculature transports back around 10% to the blood
circulatory system whereas 90% are taken up by the blood system itself again (Földi
and Strössenreuther 2004). The lymphatic system consists of primary (thymus) and
secondary lymphoid organs such as the spleen, lymph nodes, adenoids, Peyer’s
4
Understanding lymphangiogenesis Isolation and culture of LECs

-) embryonic development -) Isolation
VEGFR-3/VEGF-C signaling Dermis, intestine, peripheral blood, lymph nodes, iPSCs
Sox-18 induction -) Culture
Prox-1/LYVE-1 expression Adequate growth factor delivery
-) Adult lymphangiogenesis Immortalization
VEGF-A/C/D stimulation
VEGFR-2/VEGFR-3 signalling
Approaches for engineering lymphatic vessels
Scaffold-based approaches Regenerative approaches

-) Extra-cellular matrices -) Growth factor induced
Fibrin, collagen, PGA VEGF-C overexpression
-) Physiological conditions Growth factor delivery
Interstitial flow TGF-beta blocking
-) Stem cell co-culture
-) Mechanical stimulation
Laser irradiation
Shockwave treatment
Fig. 1 Approaches for engineering lymphatic vessels. Understanding the biological mechanisms of lymphangiogenesis is one of the prerequesites for
developing strategies for lymphatic engineering. Several studies describe the isolation of LECs from human dermis, intestine, peripheral blood, lymph
S. Rohringer et al.
patches, appendix and mucosa, gut- and bronchial-associated lymphoid tissue, as

well as lymphatic vessels maintaining a network between secondary lymphoid
organs (Murphy and Weaver 2016). These organs are responsible for lymphocyte
activation and trafficking (Kesler et al. 2013).
The interstitial fluid enters the lymphatic capillaries and is further transported via
larger collecting vessels to the thoracic duct or the right lymphatic duct. The anatomy
of pre-collector vessels differs from lymphatic capillaries in several points. Whereas
lymphatic capillaries are built by a single layer of lymphatic endothelial cells (LECs)
located on an incomplete basal lamina and have blind ends, larger collecting
lymphatic vessels consist of LECs surrounded by a smooth muscle cell layer. The
endothelial junctions in capillaries are discontinuous to facilitate leukocyte entry and
uptake of lymph components. Moreover, the lymphatic capillaries are connected to
the extracellular matrix by anchoring filaments enabling the opening of the vessel
lumen and increasing the uptake of tissue fluid. Pre-collector vessels have a basal
lamina, “zipper-like” interendothelial junctions and valves and are surrounded by a
smooth muscle cell layer. The contraction of this cell layer together with the
contraction of the surrounding skeletal muscles and arterial pulsations lead to
lymph propulsion (Baluk et al. 2007; Bazigou et al. 2009). Lymph transport is
mediated by hydrostatic and colloid osmotic pressures in capillaries. The colloid
osmotic pressure level has an average pressure level of 28 mmHg which is enough to
keep fluids in the capillaries. On the other side, hydrostatic osmotic pressure, the
surrounding interstitium and the filtration pressure between inside and outside of the
capillary lead to the filtration of fluid from the capillaries. Under physiological
circumstances, a net outward pressure ensures that fluid can flow out of the vessel
into the interstitium (Heldin et al. 2004) The lymph transport is mediated by extrinsic
forces like respiration, muscle contraction or the pulsation of adjacent arteries but
also intrinsic forces (fibroblast contraction or tissue growth (Wiig and Swartz 2012))
and autonomous contraction of lymphangions. Lymphangions are segments of
lymphatic vessels lying between two valves. If the pressure outside of the vessel
exceeds the inlet pressure, lymphangions have to pump to transport fluid. However,
if the inlet pressure exceeds the outlet pressure, lymphangions act as conduits which
passively transport lymph through the vessel (Quick et al. 2007). Valves ensure that
the lymph flow is unidirectional and prevent lymph backflow and therefore the
formation of lymphedema (Jeltsch et al. 2003). It was shown that initial lymphatics
have a primary valve system at the endothelial cell level in addition to secondary
bilayered valves. Open interendothelial junctions in pulsating lymphatic vessels
guide the lymph fluid in a distinct direction and inhibit the backflow of lymph
components into the adjacent areas (Trzewik et al. 2001). The formation of
Fig. 1 (continued) nodes, and recently iPSCs. The culture and isolation of LECS requires sufficient
supply with growth factors and optimal culture conditions. To engineer lymphatic vessels, either
scaffold-based or regenerative approaches can be used. Whereas scaffold-based methods deal with
the optimization of the extracellular matrix to ensure lymphangiogenesis, regenerative approaches
use mechanical or chemical stimulation of pre-existing lymphatics to increase growth
lymphatic valves is mediated by flow, Forkhead box protein C2 (FOXC2), and

prospero homeobox protein 1 (PROX1) expression. These factors influence the
expression of connexin37 and calcineurin/nuclear factor of activated T-cell signaling
activation. Both connexin37 and calcineurin play an important role for valve assem-
bly and confinement of newly formed valves (Sabine et al. 2012). To engineer
lymphatic vessel structures, it is indispensable to pay attention to the physiological
differences between lymphatic capillaries and collector vessels to ensure that the
required properties towards function can be achieved.
3 Lymphatic Development
The formation of lymphatic vasculature during embryonic development in humans

starts around week 6–7 of pregnancy. Endothelial cells undergo arterial-venous
specification after differentiation from so-called angioblasts (endothelial precursor
cells). After this division into the arterial and venous section, the embryonic vascu-
lature highly expresses vascular endothelial growth factor receptor 3 (VEGFR-3). At
the same time, large veins upregulate lymphatic vessel hyaluronan receptor-1
(LYVE-1). Further, the induction of the transcription factor SOX18 leads to the
formation of LYVE-1 positive LEC precursors and the expression of prospero
homeobox protein-1 (Prox-1). Simultaneously, VEGFR-3 is downregulated in
blood endothelial cells (BECs) but remains highly expressed in LEC precursors
which now also start to express neuropilin-2 ensuring that the cells respond to
VEGF-C signals mediated by the lateral mesenchyme. These signals lead to
sprouting of LECs and the formation of lymphatic sacs. LECs now start to express
podoplanin and the lymphatic and blood vasculature separates. VEGF-C/VEGFR-3
signaling leads to further growth of lymphatic vessels. (Tammela and Alitalo 2010).
Post-developmental lymphangiogenesis, also known as adult lymphangiogenesis,
is a process often occurring during inflammation or in lymph nodes after infection,
vaccination, and tissue transplantation (Kilarski et al. 2014). It was shown that
VEGFR-3 in cooperation with VEGFR-2 signaling plays an important role during
adult lymphangiogenesis, especially during metastasis. Interestingly, both VEGFR-2
and VEGFR-3 were responsible for migration and proliferation of LECs, but blocking
of VEGFR-3 did not affect tubulogenesis at all whereas the inhibition of VEGFR-2
just slightly blocked the formation of tubelike structures (Goldman et al. 2007).
Inflammation-induced lymphangiogenesis is mediated by macrophage recruitment to
the inflamed tissue site. There, the immune cells express high levels of cytokines,
peptides and proteins and VEGF-A which lead to the formation of new lymphatic
vessels (Cursiefen et al. 2004). Moreover, tumor-driven lymphangiogenesis can sup-
port metastasis and immune tolerance in melanomas by expression of VEGF-C (Lund
et al. 2012). However, lymphatics can also act as inflammation-reducing mediators.
Huggenberger et al. (2011) showed that the transgenic delivery of VEGF-C and
VEGF-D limits acute skin inflammation in mice by enhancing lymphangiogenesis
and lymphatic drainage. Due to the increased lymph flow and vessel formation also,
edema formation was limited (Huggenberger et al. 2011).
4 Lymph-Specific Markers
Wigle and others could identify the main functions of Prox-1, one of the master
regulator proteins in lymphatic development. Prox-1 is not only required for suc-
cessful budding and sprouting of lymphatic precursor cells but also plays an
important role in determining the lymphatic fate since the expression of other
lymph-specific markers are dependent on Prox-1 expression (Wigle et al. 2002).
Another major lymphatic marker in adults is VEGFR-3. Interestingly, VEGFR-3 is
expressed in both blood and lymphatic endothelium, during embryonic development
and becomes restricted to lymphatics later on (Paavonen et al. 2000). However,
VEGFR-3 expression on blood vessels was observed in breast adenocarcinomas and
numerous other tumor types (Partanen et al. 1999) thus serving as a potential target
for therapies. VEGF-C, a ligand of VEGFR-3, mediates lymphatic vessel growth.
However, overexpression of VEGF-C in tumor cells leads to increased intratumoral
lymphangiogenesis and thus an increased metastasis to adjacent lymph nodes (Skobe
et al. 2001). LYVE-1 is a major receptor for hyaluronan on the lymph vessel wall, a
glycosaminoglycan which facilitates cell migration during various processes such as
wound healing, inflammation, or embryonic development. After fulfilling its func-
tion in one of these processes, hyaluronan is transported via lymphatic vessels to
adjacent lymph nodes for degradation (Banerji et al. 1999). Another lymphatic
marker, podoplanin, is a transmembrane glycoprotein expressed on lymphatic, but
not on blood endothelium. It was further found that podoplanin is expressed on
osteocytes, peritoneal mesothelial cells, glandular myoepithelial cells, ependymal
cells, stromal reticular cells, and follicular dendritic cells of lymphoid organs but also
in specific tumor types. These findings indicate that podoplanin may also have an
effect in tumor progression (Schacht et al. 2005). Deletion of the podoplanin gene in
mice led to death births due to respiratory failure and insufficient lymphatic pattern-
ing which was associated with dilated lymphatic vessels, lymphedema, and dimin-
ished lymphatic transport (Schacht et al. 2003).
5 In Vitro Culturing of LECs
To be able to perform lymphatic vascular tissue engineering, LECs have to be

isolated and characterized. So far, LECs could be isolated from dermis, intestine,
peripheral blood, and lymph nodes (DiMaio et al. 2016; Bruyère and Noël 2010;
Hayes et al. 2003). However, it was recently shown that induced pluripotent stem
cells (iPSCs) can be differentiated into the lymphatic lineage (Lee et al. 2015). The
isolation of LECs from different lymphoid tissues such as lymph nodes, the thymus,
the spleen, lymphatic vessels and palatine tonsils can be performed with Ulex
europaeus Agglutinin 1-coated beads followed by an additional purification step
with an anti-podoplanin antibody (Marsee et al. 2009; Garrafa et al. 2005; Kriehuber
et al. 2001). The isolation from lymph nodes is usually performed by disruption of
the lymph node with a needle and furthermore enzymatic digestion. Moreover, LECs
are then further separated from BECs via LYVE-1+CD31+ podoplanin+ selection
(Link et al. 2007; Fletcher et al. 2011; Broggi et al. 2014). Another source for LEC
isolation is the intestine. Enzymatic digestion of the mucosa from jejunum leads to a
microvascular cell population which can be again further separated into LECs and
BECs (Haraldsen et al. 1995; Jahnsen et al. 1997). However, the isolation of LECs
from skin is often preferred to the isolation from organs inside the human body due
to availability and severity of surgical intervention. LECs can be isolated either from
adults who underwent elective surgery (Kriehuber et al. 2001) or from children’s
foreskins after circumcision (Hirakawa et al. 2003). After removal of the epidermis,
enzymatic digestion is performed to gain human dermal microvascular endothelial
cells (HDMECs) which were then separated into blood and lymphatic endothelial
(podoplanin+CD34+CD45 ) cells via fluorescence-activated cell sorting (Kriehuber
et al. 2001).
After successful isolation of LECs, the cells are usually grown on fibronectin-
coated or collagen-coated tissue culture flasks (Schweighofer et al. 2015; Jiang et al.
2010), although it has been shown that coating materials are not compulsory (Jiang
et al. 2010). Since LECs require specific growth factors and proteins to grow in vitro,
the use of special endothelial cell culture media is required (Breier 2005; Bonvin
et al. 2010; Weitman et al. 2013; Onimaru et al. 2009). These media should contain a
variety of pro-angiogenic growth factors such as VEGFs, fibroblast growth factors
(FGFs)-B and FGF-2, insulin- and epidermal-like growth factor-1 (IGF-1 and
EGF-1), hepatocyte growth factor (HGF), and platelet-derived growth factor BB
(PDGF-BB) (Onimaru et al. 2009; Cao 2005; Cao et al. 2004; Chang et al. 2004;
Kajiya et al. 2005; Saito et al. 2006; Björndahl et al. 2005). Since the proliferation
potential of primary LECs is limited, it is possible to perform retroviral infection
with a virus containing a coding region for human telomerase reverse transcriptase
(TERT) (Nisato et al. 2004; Pegu et al. 2008). These immortalized LECs preserve
their typical morphology and marker expression beyond 40 passages (Pegu et al.
2008). However, the infection with TERT or the reprogramming of iPS cells to LECs
may be useful to ensure a longer time period for culturing LECs but on the other
hand raises questions about safety and long-term effects.
6 Scaffold-Based Approaches
Scaffold-based approaches of lymphatic tissue engineering use cell-seeded bioma-

terials such as collagen or fibrin gels often in combination with flow stimulation to
construct lymphatic vessels in vitro. Several studies have already indicated the
importance of interstitial flow for studying lymphangiogenesis (Ng et al. 2004;
Helm et al. 2007). It was shown that interstitial flow stimulates BECs and LECs to
form capillary-like structures containing lumen in vitro. Bonvin and others devel-
oped a three-dimensional fluidic device with nine chambers allowing the observation
of various conditions in the presence of interstitial flow at the same time. They were
able to show that LECs form structures containing lumen after 5 days of incubation
in the flow chamber under flow conditions in 3 mg/ml fibrin gels. However, without
application of flow, LECs were not able to form structures at all and remained
distributed as single cells throughout the gel. This study does not only highlight
the importance of interstitial flow on the formation of capillary networks in vitro but
does also show that the choice of the right scaffold material is indispensable.
Whereas LECs formed more capillaries in fibrin-only gels, BECs preferred an
equal mixture of fibrin and collagen to form vessels. Although the application of
flow led to structural organization of LECs and BECs in the developed model, the
further addition of VEGF121 increased the structure formation thus indicating that
the addition of growth factors play an important role for lymphangiogenic
approaches in vitro (Bonvin et al. 2010).
Boardman and Swartz also showed that interstitial flow mediates LEC organiza-
tion and capillary formation. They removed a 2-mm-wide circumferential band of
dermal tissue in a mouse tail to induce a disruption of lymphatic vessels which would
lead to lymphedema under physiological conditions. Whereas the major blood
vessels, bone, muscles, and tendons stayed intact, the missing lymph vessels were
bridged via a so-called collagen dermal equivalent (CDE) which provides an effec-
tive part for fluid transport and therefore inhibits lymphedema. Interestingly, the
migration and formation of new lymphatics happened exclusively in direction of the
lymph flow whereas blood vessel formation occurs multi-directional (Boardman and
Swartz 2003).
Another study using growth factors for lymphangiogenic approaches showed that
it was possible to induce lymphatic capillary network growth by seeding LECs on a
feeder layer of fibroblasts. The formation of a lymphatic network was then stimu-
lated by the use of VEGF-C and hepatocyte growth factor (HGF) (Gibot et al. 2016).
Other approaches also used the combination of endothelial cells with fibroblasts to
induce network formation in vitro. Marino et al. showed that a co-culture of human
dermal microvascular endothelial cells (HDMECs), which are composed of BECs
and LECs, with fibroblasts result in both a blood and lymphatic capillary networks in
fibrin hydrogels after 3 weeks (Marino et al. 2014). They were able to construct skin
grafts containing HDMECs, fibroblasts, and keratinocytes which could be success-
fully implanted into rats. There, the engineered blood and lymphatic vasculature
anastomosed with host vessels and were shown to be perfusable with Evans Blue dye
(Marino et al. 2014). Another idea was to seed LECs on polyglycolic acid (PGA)
tubes which were implanted into mice. However, LECs did not form capillary
networks in this model but formed sheets around the PGA tubes (Dai et al. 2010).
Although scaffold-based lymphangiogenic approaches developed so far hold prom-
ising results for the future use of engineered vessels for implantation into patients,
future studies have to prove that the vessels formed in vitro remain stable after
implantation in situ, are perfusable, and do not anastomose with the host blood
vasculature.
7 Regenerative Approaches
Since scaffold-based approaches for the engineering of the lymphatic system need
further development, the focus of several other studies lays on the stimulation of
lymphangiogenesis in situ. Lymphatic regeneration can be achieved by various
different methods like growth factor delivery, growth factor overexpression, stem
cell stimulation, or mechanical approaches.
It was shown that a functional lymphatic network could be generated by the
transient overexpression of VEGF-C. This overexpression leads to proliferation,
differentiation, and maturation of LECs and therefore to a stable vessel network
containing valves and smooth muscle cell coverage (Tammela et al. 2007). However,
Goldman et al. (2005) showed that VEGF-C overexpression leads to transient
hyperplasia, but not to enhanced formation of lymphatic vessels during skin regen-
eration (Goldman et al. 2005). Furthermore, lymphangiogenesis is promoted by the
activation of VEGF-C via the calcium-binding epidermal growth factor domain
1 (CCBE-1) thus indicating that CCBE-1 is a new potential target for
lymphangiogenic studies (Jeltsch et al. 2014, 1). As indicated already in the section
“Lymphatic Development” of this review article, lymphangiogenesis can also be
induced by growth factor delivery of VEGF-C and VEGF-D (Huggenberger et al.
2011). Transforming growth factor beta (TGF-beta) was identified as a negative
regulator of lymphatic regeneration during wound repair (Clavin et al. 2008).
Blocking of TGF-beta thus resulted in an accelerated lymphatic regeneration in
wound healing (Avraham et al. 2010). By the identification of more pro- and anti-
lymphangiogenic factors, growth factor-induced lymphatic regeneration in vivo
could gain more importance.
During the last decade, the potential of using stem cells for tissue engineering
gained importance. Conrad et al. (2009) were able to induce lymphatic regeneration
and restoration of fluid drainage in a murine lymphedema model by stimulation and
injection of mesenchymal stem cells (MSCs) in situ. The supernatants of cultured
LECs were transferred to MSC culture which induced an endothelium-like morphol-
ogy and lymphendothelial marker expression in vitro. The injection of these
pre-conditioned MSCs into sites of lymphedema in a mouse model decreased
edema formation by 20% after 3 weeks compared to a non-treated control. After
7 weeks of injections, the fluid drainage could be fully restored (Conrad et al. 2009).
Apart from growth-factor-induced regenerative approaches and the use of sup-
portive stem cells, also mechanical forces were used to induce lymphangiogenesis to
enhance wound healing and reduce inflammation. Lievens (1991) treated wounds
with a combination of a HeNe and an infrared laser and focused on scar adhesion to
the surrounding tissue, local edema reduction, and the regeneration of the vein and
lymphatic vasculature. The study proved that vessel regeneration and edema reduc-
tion was accelerated in the treated groups and scar adhesion was reduced faster
(Lievens 1991). Another approach to stimulate lymphangiogenesis mechanically is
extracorporeal shockwave treatment (ESWT). In earlier times exclusively used for
kidney stone destruction, ESWT was found to induce regenerative processes in a
variety of tissues recently (Mittermayr et al. 2011, 2012; Furia et al. 2010; Haupt
1997; Davis et al. 2009). One of the first studies describing the positive influence of
ESWT on lymphangiogenesis was performed by Kubo et al. (Kubo et al. 2010).
They disrupted lymphatic vessels in rabbit ears which led to insufficient lymph flow
and therefore lymphedema. The edemic sites were treated with low-level shockwave
treatment which led to increased levels of VEGFR-3 and VEGF-C and decreased
lymphedema thickness (Kubo et al. 2010). Another study showed that not only
VEGF-C but also basic fibroblast growth factor (bFGF) levels were increased after
stimulation with ESWT of rat tails with nonfunctional lymphatic vasculature
(Serizawa et al. 2011). Kim et al. implanted hydrogels into mice suffering from
lymphedema in the mid-thigh which were either stimulated with VEGF-C or ESWT
or both. Interestingly, the combination of both treatment methods was the most
successful to induce lymphangiogenesis (Kim et al. 2013). To understand the
influences of ESWT on cultured LECs in terms of morphology, proliferation, and
marker and gene expressions, Rohringer et al. used ESWT stimulation of LECs with
different energy levels. The performed experiments showed that the proliferation of
LECs was increased by ESWT, although these effects were highly energy dependent.
Furthermore, ESWT led to a morphology change and two subpopulations of LECs
differing in size and podoplanin marker expression which led to the assumption that
ESWT could also lead to podoplanin-mediated lymphangiogenesis in vivo
(Rohringer et al. 2014).
8 Conclusion
Several scaffold-based and regenerative approaches to induce lymphangiogenesis

in vitro and in vivo have been described so far. Using methods already performed for
in vitro blood vessel engineering, it was possible to create lymphatic structures
containing lumen or even lymphatic capillaries in hydrogels which could be
implanted into several animal models. However, the engineering of lymphatic
vessels often requires the additional use of growth factors, feeder cells, specific
scaffold materials, and the application of interstitial flow. Due to the high variety of
lymphangiogenic growth factors and receptors, the numerous available scaffold
materials and the discovery of alternative methods such as ESWT, researchers are
challenged to define the most optimal conditions to engineer and regenerate lym-
phatic vessels.
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In Vitro and In Vivo Approaches for Pre-
vascularization of 3-Dimensional
Engineered Tissues
Geraldine M. Mitchell and Wayne A. Morrison
Abstract
A major hurdle in tissue engineering of organs is the incorporation of a function-
ing blood vessel network integrated throughout the engineered tissue that readily
links to the surrounding host blood vessels to provide the oxygen and nutrients
required by the engineered construct. In the early years of tissue engineering
development, vascularization was not a priority and generally angiogenic
ingrowth from neighboring host capillary networks, a process termed extrinsic
vascularization was used to vascularize implanted tissue engineering constructs.
Extrinsic vascularization takes weeks, and much of the implanted tissue becomes
ischemic and dies before capillary ingrowth is complete. In 2000, intrinsic
vascularization was devised by Tanaka et al. who isolated a macrovascular
pedicle in a plastic chamber which subsequently underwent considerable angio-
genic sprouting. A new arteriovenous capillary network was therefore formed
within the chamber space which was capable of growing with and supporting the
survival of tissue/organ specific cells implanted in the chamber. There was a time
lag to development of this pedicle-based angiogenic network, and in recent years
a new technique termed pre-vascularization has been developed that involves
co-culture of endothelial cells with parenchymal cells or stem cells as they
assemble in vitro. Capillary networks are formed throughout the construct, and
upon implantation inosculate (functionally join) with host capillaries. Inoscula-
tion takes at least 2 days and provides blood flow within this time period within
the construct. The most efficient vascularization technique for thick three-
G.M. Mitchell (*) • W.A. Morrison

O’Brien Institute Department, St Vincent’s Institute of Medical Research, Fitzroy, VIC, Australia
Department of Surgery, St Vincent’s Hospital, University of Melbourne, Fitzroy, VIC, Australia
Health Sciences Faculty, Australian Catholic University, Fitzroy, VIC, Australia
e-mail: geraldine.mitchell@svha.org.au; gmitchell@svi.edu.au

DOI 10.1007/978-3-319-21056-8_13-1
2 G.M. Mitchell and W.A. Morrison
dimensional tissue engineering would be the combination of pre-vascularization

in vitro with vascularization via angiogenic sprouting of a vascular pedicle, this
combination has rarely been successfully utilized.
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1 Extrinsic Vascularization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 Addition of Angiogenic Growth Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.3 Vascular Pedicle Capillary Sprouting and Its Use in Surgical Prefabrication . . . . . . . . . 4
1.4 The Rat Arteriovenous Loop Chamber Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.5 Versatility and Modifications of the AVL Chamber Model . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.6 Alternative Animal Chamber Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.7 The Concept of Pre-vascularization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.8 3D Printing of Capillary Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.9 Combining Vascular Pedicles with Pre-vascularized Scaffolds . . . . . . . . . . . . . . . . . . . . . . . 15
2 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1 Introduction
The hope for the next generation of reconstructive surgeons is Tissue Engineering,
the part-programmed and part self-directed expression of tissues in the body from
minimal core ingredients into a three-dimensional structure. Instead of the current
mutilating transfer of tissue from one part of the body to repair another, tissue
engineering attempts to mimic embryogenesis and grow the missing part de novo.
Autograft cells, usually stem cells, combined with extracellular matrices and growth
factors can be assembled in laboratory conditions and once established, implanted
in vivo where they will expand to replace the required tissue defect. Nature and
clinical precedents support the feasibility of such an idea and include limb regrowth
in the salamander and skin, liver, and bone regeneration in humans. But this
engineering paradigm of initial ex-vivo incubation followed by implantation into
the animal ignores the reality of the hostile leap into the ischemic in vivo environ-
ment. The biologist’s attempts to expand cell numbers in sophisticated bioreactors
only compound the challenge for in vivo survival and highlight the as yet unsolved
problems of upscaling to realistic volumes for human use. The history of grafting
tissues is a long and salutatory one. Myth and legends tell of miraculous grafts of
whole limbs, digits, and noses, but by the late nineteenth century it was understood
that only very small pieces of tissue could reliably survive reattachment or trans-
plantation due to the need for blood vessels to grow into the graft. This process took
some days and in the intervening period, nutrition was derived from tissue fluid. It is
now accepted that cells cannot survive further than 200 μm from a capillary
(Folkmann and Hochberg 1973).
Although largely ignored in early tissue engineering studies, the major limitations
to survival of tissue engineering scaffold/cell/growth factor combinations (termed
In Vitro and In Vivo Approaches for Pre-vascularization of 3-Dimensional. . . 3
“constructs”) when implanted in vivo is the creation of a functioning blood vessel

network throughout the construct which rapidly connects to the circulatory system.
Strategies devised to facilitate graft/cell survival have included in vitro measures
such as cytoprotection (Tilkorn et al. 2012; Mehrabani et al. 2015) and
pre-vascularization (Levenberg et al. 2005), or in vivo manipulations to boost
angiogenesis (Rophael et al. 2007 and others), or lessen ischemic and inflammatory
conditions (Zachmann et al. 2013; Czekanska et al. 2014; Spiller et al. 2014;
Yamahara et al. 2014; Brown et al. 2015).
1.1 Extrinsic Vascularization
Most early studies relied on the local host recipient site’s ability to respond to the
wound space within which the construct was placed. The mammalian body is
capable of providing capillary ingrowth into a tissue engineering construct via
recipient site capillary sprouting that penetrates the construct periphery – this is
termed extrinsic vascularization (Lokmic and Mitchell 2008). Extrinsic vasculariza-
tion takes up to several weeks depending on the size on the construct. Using this
approach many construct cells die if its dimensions are greater than 400μm3 due to
ischemic conditions existing for many days (Auger et al. 2013). Not surprisingly, it
has been established (Tilkorn et al. 2010) that the survival of implanted cells in vivo
is directly related to the vascular volume of the recipient site capillary bed.
Consequently, the majority of successful tissue engineered tissues which have
been applied clinically involve the construction of thin layers of tissue such as skin
(Auger et al. 2004; Shevchenko et al. 2010), parts of the urinary tract (Park et al.
2000; Raya-Rivera et al. 2011), avascular tissues such as cartilage (Paige and Vacanti
1995; Takazawa et al. 2012), or relatively avascular tissues such as heart valves
(Mazzitelli et al. 2015).
1.2 Addition of Angiogenic Growth Factors
Addition of angiogenic growth factors such as vascular endothelial growth factor-A

(VEGF-A), fibroblast growth factor 2 (FGF-2), and platelet derived growth factor
BB (PDGF BB) hastened the process of recipient site angiogenic sprouting.
Although this growth factor stimulated angiogenic response is not permanent
(Rophael et al. 2007), it has the advantage that an early boost to capillary growth
and therefore blood flow will enhance the early survival of construct cells.
Remodeling of any growing capillary network associated with a tissue engineering
construct will always occur (Lokmic et al. 2007; Lokmic and Mitchell 2008), with
the new vascular network stabilizing to a degree that meets the tissue’s metabolic
demands.
More subtle uses of angiogenic growth factors have included their incorporation
into construct scaffolds or onto the surface of degrading beads for gradual release to
boost tissue angiogenesis over the first weeks of implantation (Elcin et al. 1996;
Peters et al. 1998; Tabata et al. 2000; Richardson et al. 2001; Vashi et al. 2006, Nair
et al. 2010; Ayvazyan et al. 2011; Macdonald et al. 2011; He et al. 2012; Matsui and
Tabata 2012; Oliviero et al. 2012; Del Gaudio et al. 2013; Kim et al. 2014; Lai et al.
2014; Go et al. 2015; Lee et al. 2015; Wang et al. 2015; Li et al. 2015; Subbaih et al.
2015; Zhang et al. 2015a, and many other publications on this theme).
In addition various cell therapies/tissue engineering protocols have used a variety
of preconditioning techniques of implanted cells to promote innate pro-survival
capabilities. These techniques have largely been utilized on mesenchymal stem
cells which have been preconditioned with hypoxia or various drugs to promote
implanted cell survival in heart tissues (Hu et al. 2008; Pasha et al. 2008). Pre-
conditioning techniques have also had a pro-angiogenic effect on the recipient site
with significant increases in local angiogenesis. Recently Taylor et al.(2016) dem-
onstrated a significant increase in the production of VEGF-A and a concomitant
downregulation of Angiopoietin 1, by hypoxic preconditioned primary myoblasts
in vitro, resulting in a significant increase in local angiogenesis 2 weeks post-
implantation of preconditioned myoblasts in vivo in a vascularized tissue engineer-
ing chamber.
1.3 Vascular Pedicle Capillary Sprouting and Its Use in Surgical

Prefabrication
For many decades reconstructive surgeons have recognized the significant capillary
sprouting potential of large vascular pedicles. Hori et al. (1979), and Erol and Spira
in 1980 described the use of vascular pedicles (a large artery and its accompanying
vein) which were surgically isolated and moved to adjacent areas of nonvascularized
tissue. In lying the vascular pedicle over or under nonvascularized tissue, the pedicle
demonstrated considerable capillary sprouting potential and was able to
revascularize the previously nonvascularized tissue which could be bone, skin, or
several different tissue layers together (skin, fat, and bone). This process is termed
prefabrication. After the pedicle had successfully vascularized a new tissue area,
this area with its supplying pedicle could be transplanted to an entirely different area
of the body for surgical reconstruction of a tissue deficit after trauma or cancer
resection (Morrison et al. 1990, 1997; Khouri et al. 1992; Takato et al. 1993; Pribaz
et al. 1999a, b; Guo and Pribaz 2009). Prefabrication techniques have been further
developed and used in clinical and experimental reconstructive surgery.
1.4 The Rat Arteriovenous Loop Chamber Model
Khouri et al. in 1994 established an isolated vascular pedicle in a plastic chamber. In

2000, the O’Brien Institute published two papers (Tanaka et al. 2000; Mian et al.
2000) which further developed this concept of vascular pedicle isolation in a plastic
chamber by creating an arteriovenous loop (AV loop) in an empty chamber. The
chamber was circular in shape, constructed of polycarbonate, included a completely
Fig. 1 Diagram illustrating the AV loop and chamber, largely used in the rat and based on the
femoral vessels
flat base and a lid that fitted neatly into one another, and was approximately 1.5 cm in
diameter, with an entrance window cut in its side for the AV loop to enter and leave
the chamber space (Fig. 1). The surgical technique involved severing the femoral
artery and vein in the rat and microsurgically anastomosing the cut vessel ends with a
reversed vein graft harvested from the femoral vein of the opposite leg (Tanaka et al.
2000; Zhan et al. 2016). This arteriovenous loop (AV loop) was continuous with the
circulation and its position in the chamber left a relatively large empty space around
the AV loop. Within 24 h a large fibrin clot formed around the AV loop filling two
thirds of the chamber. Within 72 h macrophages and fibroblasts were seen migrating
into the fibrin and early capillaries sprouted off the femoral vein segment of the AV
loop into the highly angiogenic fibrin scaffold (Lokmic et al. 2007). Capillary
sprouting and growth continued extensively along the length of the loop and
extended into the fibrin converting the fibrin scaffold into a highly vascular collag-
enous connective tissue a “neo-tissue” that reached a peak of angiogenesis (23 per-
cent vascular volume of new construct tissue) at 10 days post AV loop insertion in
the chamber. In the first few weeks, vascular development was always more exten-
sive on the venous side of the loop, but arteriolar sprouting was observed from the
femoral artery section of the loop at 14 days. This process of capillary network
formation from a vascular pedicle in an isolated chamber is termed intrinsic
vascularization.
As well as directly sprouting from the AV loop angiogenesis also occurred in a
“front” of angiogenic tissue around the loop and progressed through the fibrin clot
towards the chamber periphery (Fig. 2). This moving angiogenic front persisted for
several weeks while new tissue was growing into the fibrin. The “front” labeled
positively for hydroxyprobe-1, indicating this tissue was hypoxic (Hofer et al. 2005;
Lokmic et al. 2007). But the hypoxic zone disappeared by 2 weeks post AV loop
Fig. 2 Histological sections through the rat arteriovenous loop at 7 days. (A) Low power figure of
cross section through arteriovenous loop and new tissue at 7 days, stained with Bandeiraea
simplicifolia lectin. Arrows indicate numerous positively stained blood vessels (arrows), around
the AV loop (*). Scale bar̴ 200 μm. (B) Toluidine blue stained paraffin embedded tissue. Cross
section of the chamber contents at 7 days reveals newly formed collagenous connective tissue
containing numerous new capillaries (arrows) at the angiogenic front around the arteriovenous
loop, part of which is seen in cross section (*). Scale bar ̴ 100 μm. (Fig. 2 images provided by
Dr. Zerina Lokmic (previously at the O’Brien Institute, Melbourne and currently at the University of
Melbourne, Department of Paediatrics and Nursing, Melbourne, Australia))
insertion in the chamber, indicating adequate oxygenation of the chamber tissue at

this time and a cessation of angiogenesis in the construct. The extensive capillary
network formed gradually matured and pericytes and smooth muscle cells attached
abluminally to the capillary network to create a mature arterio-capillary venous
network (Lokmic et al. 2007). The new tissue that developed around the AV loop
persisted for at least 16 weeks (Lokmic et al. 2007).
The formation of this extensive vascular bed appears to be driven by a number of
factors including the mammalian body’s drive to fill up the empty chamber space
which is likely to involve altered biomechanical signaling in the chamber which
initiates cell proliferation and migration (O’Connor and Morrison 2012), the
pro-angiogenic temporary fibrin clot (scaffold) (van Hinsbergh et al. 2001) that
largely fills the chamber in the first 24 h, and which when reduced in volume
significantly inhibits vascularized connective tissue growth in the chamber (Lokmic
et al. 2008), and the significant angiogenic sprouting capability of the isolated
arteriovenous loop (Lokmic et al. 2007).
The O’Brien Institute and other groups have been able to grow a variety of tissues
in the AV loop chamber. Stem cells (Choi et al. 2010; Boos et al. 2013; Matsuda et al.
2013; Buehrer et al. 2015), progenitor cells (Simcock et al. 2009; Tilkorn et al.
2010), or differentiated cells or tissues (Messina et al. 2005; Morritt et al. 2007; Tee
et al. 2012) can be seeded into the chamber, and tissues or organoids grown over
several weeks. The range of tissues grown includes: cardiac muscle (Morritt et al.
2007; Choi et al. 2010; Tee et al. 2012), skeletal muscle (Messina et al. 2005; Bach
et al. 2006; Tilkorn et al. 2010; Bitto et al. 2013), adipose tissue (Messina et al. 2005;
Dolderer et al. 2007, 2011), and bone (Arkudas et al. 2007a; Buehrer et al. 2015).
The rat chamber model provides a number of significant advantages in growing

vascularized tissue. The major advantage is its ability to support the concurrent
growth of a vasculature with implanted cells to develop thick three-dimensional
tissue constructs – a significant advancement, when one considers that only very
thin tissues or completely avascular tissues can be engineered currently for human
use. Not only does the AV loop chamber model provide a highly vascularized
environment where the blood vessels can grow with the construct, but the chamber
space protects the growing construct from outside tissue forces, enabling continued
expansile growth of tissue. The provision of a macrovascular pedicle means that the
chamber contents can be transplanted to other sites around the body by surgical
anastomosis to recipient site macro-vessels. This was initially demonstrated in a
rabbit model where the vascularized chamber contents were transplanted to the ear
and skin grafted (Tanaka et al. 2006). Cardiac muscle flaps grown in rat chambers
were successfully transferred to the heart of a recipient syngeneic rat where the new
tissue survived and maintained structure and function (Tee et al. 2012).
1.5 Versatility and Modifications of the AVL Chamber Model
A simpler model than the AV loop is the ligated AV pedicle which has been shown
(Tanaka et al. 2003; Polykandriotis et al. 2007) to grow vascularized tissue almost as
efficiently as the AV loop. Another technique which eliminates the harvest of a vein
from the opposite leg to lengthen the loop involves lengthening of the ipsilateral
femoral vein by including the epigastric vein as an extension. The femoral vein is
ligated distal to the epigastric branch and the branch itself is joined end to end to the
divided artery. This procedure requires only one anastomosis, saves time, reduces
costs, and permits the option of bilateral chambers (devised by Dr. Alberto Bedogni
and used in Tilkorn et al. 2010). Cold-stored allograft veins have been used in the
arteriovenous loop and have been shown to support the AV loop create robust
angiogenic spouting that is not significantly different in the vascular volume gener-
ated in the chamber from control autologous veins (Zdolsek et al. 2011). Although
allograft veins will inevitably thrombose in time, their temporary purpose in the AV
loop would be to supply angiogenic support for example to an overlying skin graft
until the transplanted construct is alternatively vascularized by the surrounding
tissue. Using allograft veins also allows two chambers to be created per rat. Fat
flaps based on the epigastric pedicle can be implanted in the chamber rather than the
AV loop alone. These isolated flaps expand and grow within the empty chamber
space (Dolderer et al. 2007, 2011). This concept has been exploited further by Lu
et al. (2014) in a rabbit fat flap model. The AVL chamber has also been “neurotized”
with the addition of a motor nerve to act as a myogenic stimulator when myoblasts
and mesenchymal stem cells (MSCs) were implanted in the chamber (Bitto et al.
2013). The chamber has been upscaled to the pig and human where an initial small
vascularized fat flap expanded to 80mLs and 210mLs, respectively, and comprised
both fat and fibro vascular tissue (Findlay et al. 2011; Morrison et al. 2016), and in
sheep it has been successfully used to produce bone (Boos et al. 2013).
The chamber design has been modified to include multiple large fenestrations
(termed perforations) which facilitate vascularization from outside the chamber and
significantly increase chamber tissue growth in rabbits and rats (Tanaka et al. 2006;
Dolderer et al. 2011; Zdolsek et al. 2011). The perforated chamber therefore utilizes
both intrinsic vascularization (from the vascular pedicle) and extrinsic (from the
surrounding tissues) to maximize construct tissue growth. The chamber can also be
modified with custom-made cell delivery ports, in which cells can be seeded in a
delayed fashion some days after initial pedicle creation (Tilkorn et al. 2010).
Delayed seeding improves implanted cell survival, as vascular density peaks at
7–10 days and is therefore better able to sustain the viability of implanted cells at
this time. Drugs or growth factors can also be delivered into the chamber via
minipumps. SDF-1 delivered in this way recruited circulating endothelial progenitor
cells to the chamber tissue (Simcock et al. 2009).
A flow-through vascular pedicle chamber model using rat epigastric vessels to
grow fat tissue was described by Walton et al. in 2004. More recently the rat chamber
has been modified to utilize the rat femoral artery and vein as a flow-through pedicle
without any anastomosis or vein graft insertion. Cardiac tissue has been successfully
grown in these flow-through chambers (Lim et al. 2012, 2013; Piao et al. 2014;
Zhang et al. 2015b; Chan et al. 2016; Hung et al. 2016; Zhan et al. 2016).
Tissue growth can be manipulated in the rat chamber by the addition of growth
factors, particularly angiogenic growth factors (Tanaka et al. 2006; Arkudas et al.
2007b, 2009) and differentiation factors that promote the growth of specific tissues:
to date this has largely been bone differentiation (Boos et al. 2013; Buehrer et al.
2015) and muscle differentiation (Bitto et al. 2013). Alternatively specific scaffolds
and matrices have been implanted in the chamber to support tissue growth, including
a collagen sponge (Tanaka et al. 2006); poly(lactic-co-glycolic acid) (PLGA)
(Cassell et al. 2001; Hofer et al. 2003; Cao et al. 2006; Findlay et al. 2011); bioglass
granula matrix filled with fibrin (Arkudas et al. 2013); collagen-chitosan scaffolds
(Zhang et al. 2015a) or to direct differentiation of implanted cells towards a specific
outcome, for example, cancellous bone matrix, or β-tricalcium phosphate-
hydroxyapatite granules used to promote bone differentiation of MSCs (Poly-
kandriotis et al. 2007; Boos et al. 2013; Buehrer et al. 2015); and poly(lactic-co-
glycolic-acid/polyethylene glycol (PLGA/PEG) microspheres delivering VEGF to
promote adipocyte differentiation from MSCs in the chamber (Zhang et al. 2015a).
Although vascularized tissue will generate spontaneously in the temporary fibrin
scaffold produced by the AV loop, various hydrogels including commercially
available fibrin glues (Arkudas et al. 2012) and tissue-specific manufactured gels
such as cardiogel support vascularized tissue growth within the rat AV loop chamber
(Matsuda et al. 2013). Fibrin has also been used as a vehicle for delivery of cells
(Tilkorn et al. 2010; Buehrer et al. 2015) and angiogenic growth factors (VEGF and
bFGF) (Arkudas et al. 2007b, 2009).
Fig. 3 Diagram illustrating the flow-through pedicle chamber model, largely used in the mouse
and based on the epigastric vascular pedicle
1.6 Alternative Animal Chamber Models
The concept of an isolated vascular pedicle in an empty chamber which subsequently

sprouts an extensive capillary network has been employed in a number of surgical
variations in a variety of animal models. The mouse is the animal preferred for
research because of its capacity for genetic manipulation and investigative analysis.
However, its vessels are generally too small to reliably anastomose. A “flow-
through” model where vessels remain in continuity was described by Cronin et al.
(2004) in the mouse (and also in the rat by Walton et al. in 2004). In this model a
1 cm length of epigastric vascular pedicle in the mouse is cleared of connective tissue
and surrounded in a split cylindrical silicon tube chamber 5 mm long and 3.5 mm
diameter, with a volume of 45–50 μL. Once in place the ends of the tube are sealed
with bone wax. The pedicle remains a “flow-through” pedicle and no microvascular
anastomosis is required (Fig. 3).
There are positives and negatives with this model compared to the AV loop rat
chamber model. As no vein graft is involved, the procedure is quicker and does not
require microsurgical skills. Bilateral chambers can be readily created in each
animal, reducing animal numbers and costs when several experimental groups and
controls are involved. Additionally mice are more cost efficient than rats, and many
genetically modified mouse strains are available. A disadvantage of this mouse
model is that the large volume of naturally occurring fibrin that is released from
the rat AV loop is considerably less in the mouse model, and the rate of angiogenesis
somewhat slower. Generally therefore the mouse chamber is filled with an additional
extracellular matrix that is placed in the chamber on the day of chamber creation. A
variety of extracellular matrices (ECM) have been used, most commonly Matrigel™
an ECM developed from a murine sarcoma tissue (Cronin et al. 2004; Kelly et al.
2006; Rophael et al. 2007; Tilkorn et al. 2012; Yap et al. 2013). Other ECMs used
include Cymetra ® and PuraMatrix™ (Ting et al. 2014), Pluronic F-127 hydrogel
(Vashi et al. 2008), collagen (Vashi et al. 2006), and poly(lactic-co-glycolic acid)
(PLGA) (Cronin et al. 2004).
We have chosen the mouse model to investigate the fundamentals of cell differ-
entiation and, tissue development and function. This includes implantation of, or the
stimulation of specific tissue growth, including the following cells and tissues:
pituitary colony forming cells (Lepore et al. 2007), liver progenitor cells (Yap
et al. 2013), pancreatic Islets of Langerhans (Hussey et al. 2009, 2010; Forster
et al. 2011), thymus tissue (Seach et al. 2010), skeletal muscle myoblasts (Tilkorn
et al. 2012), and adipose tissue (Kelly et al. 2006; Hemmrich et al. 2007; Rophael
et al. 2007; Thomas et al. 2007; Findlay et al. 2009, Lilja et al. 2013; Ting et al.
2014). The mouse chamber has been utilized to grow lymphatic malformations
(Lokmic et al. 2014) and zenograft models of high and low mammographic density
human breast tissues (Chew et al. 2012). The mouse vascularized chamber can be
partially dismantled at later time points to implant drugs, cells, or tissue for delayed
seeding such as human Islets of Langerhans to alleviate diabetes in a streptozotocin-
induced diabetes model in C57BL/6 J mice (Forster et al. 2011). It can be utilized as
a screening assay to test scaffolds (Ting et al. 2014), growth factor combinations
(Rophael et al. 2007), and preconditioning cell treatments (Tilkorn et al. 2012;
Taylor et al. 2016).
1.7 The Concept of Pre-vascularization
Pre-vascularization involves seeding endothelial cells with or without vascular

support cells of mesenchymal origin (including mesenchymal stem cells, fibroblasts,
smooth muscle cells, and pericytes) into scaffolds or gels in vitro with subsequent
implantation in vivo. As early as 1998, Black et al. seeded human umbilical vein
endothelial cells (HUVECs) with fibroblasts and keratinocytes onto a collagen
biopolymer in vitro and noted the formation of capillary-like structures within the
scaffold. In 2004, Koike et al. combined HUVECs with mesenchymal precursor
cells in a fibronectin-type-1 collagen gel which was implanted in mice. Mesenchy-
mal precursors were observed to have attached to the outside of the endothelial tubes
and perfusion of host blood within the HUVEC–derived capillaries was evident at
2 weeks. These vessels were maintained for 12 months. The pre-vascularization
approach has the potential to become a successful strategy for facilitating the transfer
and survival of tissue engineered constructs from the laboratory to an in vivo
environment. Pre-vascularization also has applications in establishing laboratory-
based tissue and organoid models for disease modeling, diagnostics, and drug
development.
Pre-vascularization depends not only on the assembly of an interconnected
capillary network throughout the scaffold but also on the functional union of
construct capillaries with host tissue recipient site capillaries to allow blood flow
throughout the construct when implanted in vivo. The functional union of two
capillary beds is termed inosculation and was coined as early as 1975 by Converse
et al. to describe the early process by which a skin graft “takes” when applied to a
wound bed. Nutrient fluids from the wound bed initially diffuse into the graft
(imbibition), followed by the close alignment and joining of open dermal capillaries
of the graft with those in the wound bed (inosculation), facilitating vascular perfu-
sion. This occurs within 48 h (Converse et al. 1975; Tremblay et al. 2005;
O’Ceallaigh et al. 2006). Subsequently blood flow is consolidated by angiogenic
ingrowth of new capillaries. Despite the ischemic insult to the graft during this time
delay, its cells and extracellular matrix usually survive. Similarly for
pre-vascularized constructs that are assembled in the laboratory, the host capillaries
at the recipient site must first inosculate with the construct capillaries and consolidate
their circulatory connections through angiogenesis and vascular remodeling. Con-
currently, the construct is also infiltrated with inflammatory cells and stem cells
(Laschke et al. 2009).
Matrices and scaffolds used in pre-vascularization: Pre-vascularization studies
have generally used a matrix in a “gel” form to support the formation of capillaries.
The matrix gel appears essential for entubulation of endothelial cells into three-
dimensional structures, and for these tube-like capillaries to unite or join with one
another into an interconnected network. The “gels” used include fibrin (Chen et al.
2009, 2010), fibronectin type-1 collagen (Koike et al. 2004), hyaluronic acid
(Kusuma et al. 2013), fibrin hydrogel microfibers (Barreto-Ortiz et al. 2015),
chitosan hydrogel (Lee et al. 2015), a platelet lysate hydrogel (Robinson et al.
2016), and others. Endothelial cells and matrix gels can be mixed and maintained
in vitro and/or surgically transplanted or injected into in vivo sites. HUVECs with
smooth muscle cells (SMCs) have also been delivered on a thermoresponsive
hydrogel patch (constructed from tetronic-tryamine polymer) in vivo (Bak et al.
2016).
Other studies have used highly porous interconnected scaffolds into which both a
matrix gel and endothelial cells are seeded in vitro (Hegen et al. 2011; Lesman et al.
2011). The scaffolds used include poly-L lactic acid (PLLA) (Hegen et al. 2011),
PLLA/PLGA (Levenberg et al. 2005; Lesman et al. 2011), poly(L-lactide-co-
1,5-dioxepan-2-one) (poly(LLA-c0-DXO) (Pederson et al. 2014), silk (Unger et al.
2010), porous collagen (Chan et al. 2016), decalcified discs of cancellous bone
(Goerke et al. 2015), and others. Rarely the porous scaffold itself is used without a
gel matrix (Unger et al. 2010, Goerke et al. 2015). Generally the capillary networks
form in the gel which seeps into the pores of the scaffold. The scaffold provides
construct structure, and protection for the capillary networks from physical damage
during surgical transplantation.
Endothelial cells: Many pre-vascularization studies have used HUVECs [Koike
et al. 2004; Levenberg et al. 2005; Chen et al. 2009; Lesman et al. 2011; Takebe et al.
2013; Bak et al. 2016]. Less frequently endothelial precursor or progenitor cells
(EPCs) (Chen et al. 2010; Goerke et al. 2015), venous and arterial endothelial cells
(Bowers et al. 2015), or primary human microvascular endothelial cells have been
used (Unger et al. 2010; Hegen et al. 2011; Chan et al. 2016; Freiman et al. 2016)
(Fig. 4). All human endothelial cells types have successfully formed capillary
Fig. 4 Images of pre-vascularized scaffolds at 3 days in culture. (A) Human microvascular

endothelial cells (hMEC) were seeded with human fibrin into porous collagen scaffolds. At
3 days numerous capillary cross sections (arrows indicate antihuman CD31 positive human
capillaries) are evident within the pores (*) of the collagen. (B) Another hMEC seeded collagen
scaffold at 3 days. The area surrounded by the black rectangle is seen in higher power in (C) where
anti-CD31 positive capillaries (arrows) are clearly seen in the scaffold pores (*). The collagen
scaffold is indicated by the wavy mauve lines surrounding the pores. (Fig. 4 images provided by
Dr. Anne Kong and Dr. Shiang Lim (O’Brien Institute Department at St Vincent’s Institute,
Melbourne, Australia), Dr. Guei-Sheung Liu (Centre for Eye Research Australia), and Prof Shyh-
Ming Kuo (Department of Biomedical Engineering, I-Shou University, Kaohsiung, Taiwan))
networks in vitro or in vivo, indicating the plasticity of endothelial cells in general.

Several studies have shown that the co-culture of endothelial cells with or without a
vascular support cell of mesenchymal origin, and an organ-specific cell type such as
hepatocytes, osteoblasts, or myoblasts can create tissue-specific organoids in vitro
including skeletal muscle, bone, and liver for in vivo implantation (Levenberg et al.
2005; Unger et al. 2010; Takebe et al. 2013).
Primary endothelial cells harvested from a patient to form a patient-specific
pre-vascularized scaffold in vitro, for example, to transplant into a nonhealing skin
wound is unlikely to be feasible clinically, due to the large number of endothelial
cells required. Alternatives are being investigated. Embryonic stem cell derived
endothelial cells (Ferreira et al. 2007) have been characterized (Glaser et al. 2011;
Sriram et al. 2015), but given safety and ethical issues they are unlikely to be
promoted for clinical translation. Human induced pluripotent stem cell derived
endothelial cells (iPSC EC) offer more promise as they are autologous, and can be
banked in advance. Considering these factors, a timely regenerative approach could
be developed for clinical revascularization. Currently it takes approximately 4 weeks
to generate human iPSCs, and many millions of endothelial cells can be differenti-
ated from these iPSC within 7–10 days (Dr Shiang Lim, personal communication).
This 5 week time frame to develop the millions of patient-specific endothelial cells is
likely to be shortened with the recent utilization of robotic systems which have the
capability of mass expansion of multiple human iPSC cell lines at any one time. The
development of iPSC haplobanks (Solomon et al. 2015) will make iPSC an “off the
shelf” product with stringent quality control where donors are selected based on
major transplant antigens to match a large number of worldwide recipients. This
could reduce the iPSC EC generation time to less than a week. Induced pluripotent
stem cells can develop transcriptional and epigenetic aberrations (Johannesson et al.
2014) particularly when generated with the conventional integrative viral delivery
method potentially resulting in insertional mutogenesis. However, the far safer

nonintergrating episomal vector method is now being used (Hernández et al. 2016).
Induced pluripotent stem cell derived endothelial cells have been developed by a
number of groups (Kusuma et al. 2013; Samuel et al. 2013; Orlova et al. 2014;
Patsch et al. 2015; Zanotelli et al. 2016). These studies have focused on significant
facets of the iPSC to EC differentiation process, cell biology, and vascular assembly.
Limited (N = 1 in some cases) in vivo implantation experiments have been
conducted with iPSC EC, and only (Chan et al. (2015) have used iPSC derived
endothelial cells in a clinically relevant model.
Endothelial cells in co-culture with vascular support cells: In the late 1990s
Benjamin et al. (1998) and Darland and D’Amore (1999) described the mechanism
by which newly formed endothelial cell tubes (capillaries) matured and became
stable permanent structures. VEGF is required to stimulate endothelial cell migra-
tion, proliferation, and survival in the tip and stalk cells of new sprouting capillary
buds. However, these capillaries are not stable and leak blood. Platelet derived
growth factor-BB (PDGF-BB) secreted by the endothelial cells attracts mesenchy-
mal cells in the local interstitial tissue via their receptor, PDGFRβ to migrate towards
the newly formed capillary and wrap around the abluminal surface of the endothelial
cells. This process promotes basement membrane production and consequently the
capillary sprout takes on a mature phenotype, allowing it to be less dependent on
fluctuations in VEGF. As the mesenchymal cell wraps around the capillary, activa-
tion of TGF-β occurs as well as pericyte and smooth muscle differentiation, and at
the same time capillary endothelial cells cease proliferation. The angiopoietin 1 and
2 (Ang 1 and 2) and Tie 2 system also contribute to the process of angiogenesis and
vessel stabilization. Capillaries sprout in response to VEGF secreted by nearby
hypoxic cells. Ang 2 is mostly expressed by sprout tip cells and may have a role
in cell-matrix interactions. The Tie 2 receptor is expressed in endothelial stalk cells,
which become enveloped in basement membrane and covered by pericytes during
vessel stabilization/maturation. During this process Ang 1 produced by the pericytes
interacts with the Tie2 receptor on the endothelial cells. Ang 1 is necessary for vessel
stabilization and decreasing the angiogenic response (Saharinen and Alitalo 2011).
The capillary is now “mature,” no longer leaks blood, and its morphological
structure is stable.
Brudno et al. (2013) have been able to use these growth factors in specific
combinations and temporal pattern to grow and mature capillary networks in vitro
and in vivo. VEGF and angiopoietin 2 (Ang 2) are proangiogenic, and promoted
endothelial cell sprouting and pericyte detachment in a 3D co-culture model and an
in vivo model. The pro-maturation factors PDGF and angiopoietin 1(Ang 1) if
present with VEGF and Ang 2 inhibit angiogenesis, but if added somewhat later,
promote vessel maturation and vascular remodeling without inhibiting angiogenesis.
The authors recommended these growth factor combinations be applied in temporal
sequence to promote tissue engineered vascularization.
In pre-vascularization studies, co-culture of endothelial cells with fibroblasts or
embryonic fibroblasts promotes capillary tube and lumen formation in vitro
(Levenberg et al. 2005; Chen et al. 2009; Unger et al. 2010, Newman et al. 2011).
Less frequently used in co-cultures with endothelial cells are pericytes and smooth
muscle cells (Hegen et al. 2011). Mesenchymal stem cells (MSCs) (Au et al. 2008;
Choi et al. 2010; Pederson et al. 2014; Freiman et al. 2016) and mesenchymal
precursor cells (Koike et al. 2004) can also take on the role of perivascular cells in
growing and maturing tissue engineered capillary networks. MSCs are also a source
of paracrine angiogenic growth factors (Potapova et al. 2007; Hsiao et al. 2012) and
promote human microvascular endothelial cell survival (Butler and Sefton 2012).
Surprisingly, this varied cohort of mesenchymal-derived cells are all capable of
attachment to capillaries in co-culture or in vivo and have been noted to increase
the number of capillaries that form in pre-vascularized constructs (Levenberg et al.
2005; Chen et al. 2010; Hegen et al. 2011; Lesman et al. 2011) and/or the number of
perfused pre-vascularized vessels in vivo.
In the last few years in addition to the development of differentiation protocols to
produce iPSC derived endothelial cells, protocols have been developed to differen-
tiate iPSCs into vascular support cells in particular pericytes and smooth muscle, the
latter into both synthetic and contractile smooth muscle cell phenotypes (Kusuma
et al. 2013; Wanjare et al. 2013, 2014; Orlova et al. 2014; Patsch et al. 2015). iPSC
derived pericytes and smooth muscle cells have been well characterized in these
studies and shown to attach to the capillaries formed from iPSC ECs in vitro and in
limited in vivo studies.
1.8 3D Printing of Capillary Networks
Apart from the pre-vascularization of scaffolds there are other technologies that seek
to create a three-dimensional capillary network throughout a scaffold in vitro. 3D
printing of capillary networks is one such approach.
In 2012, Miller et al. described the printing of 3D rigid filament networks of
carbohydrate glass. The rigid networks (or lattices) were stacked on top of one
another in rectangular molds and the molds filled with a number of different
hydrogels including poly(ethylene glycol) (PEG), fibrin gels, Matrigel and alginate
and agarose. The hydrogels included cells such as HUVECs, human fibroblasts, and
mesenchymal 10T/2 cells. The lattices act as a “sacrificial element” for creating
fluidic channels. The glass filaments were dissolved to form channels, and the
encapsulated cells survived and migrated. Endothelial cells were also infused
through an inlet in the channel network. The endothelial cells reached confluence
as they lined the walls of the channels within a day under fluid flow, and latter
sprouted from the channels to form another order of capillaries. Co-cultures with
10T1/2 cells in the gel demonstrated endothelial cell tubes being externally wrapped
by 10T1/2. Similar 3D printed vascular channeled constructs have also been applied
in vivo with apparent inosculation of the printed vascular networks with host vessels
in an ischemic limb model (Mirabella et al. 2015).
Jia et al. (2016) have developed a one-step 3D printing strategy where the bioink
is composed of gelatin methacryloyl, sodium alginate, and poly(ethylene glycol)-
tetra-acrylate – which enabled the precise deposition of complex multilayered 3D
hollow tubes. Vascular cells – HUVECs and MSC – could be encapsulated directly
within the bioink. Within the printed network ECs spread out and proliferated to line
the printed tubes; while outside the ECs, MSCs differentiated into smooth muscle
cells under the influence of transforming growth factor-β1.
The concept of patterning capillary networks in vitro without using a 3D printing
technique is also being explored. Chaturvedi et al. (2015) micro-patterned parallel
capillaries using a microfabricated poly(dimethylsiloxane) (PDMS) template that
consisted of parallel channels into which collagen and endothelial cells were inserted
and cultured for 4–6 h under a layer of bovine fibrin. The endothelial cells aggre-
gated around the outside of the collagen to form capillary tubes encased in fibrin and
then mixed with other cell types such as hepatocytes, fibroblasts in a 1:1:1 ratio with
the endothelial cells. Six mm endothelial cord constructs were implanted into the
mesenteric fat pad of immunosuppressed mice. All endothelial cell cord types were
able to inosculate with the host vessels and support host blood flow.
Riemenschneider et al. in 2015 described the use of patches incorporating parallel
endothelial lined tubes at a density of 900+/ 200 microvessels/mm2 formed from
endothelial cells and pericytes, which were transplanted into a site of myocardial
infarction in a nude rat model. The networks aligned via cell-induced gel compaction
to achieve this high vessel density. Many of these microvessels inosculated with the
host and were perfused with host blood at 6 days.
1.9 Combining Vascular Pedicles with Pre-vascularized Scaffolds
To date thin pre-vascularized scaffolds or gel matrices have largely been implanted
under the skin, and survival and inosculation to the host vasculature has been
demonstrated. Rat skin dermal tissue has a percent vascular volume of 1–2 percent
(Dr Anne Kong, personal communication) and provides an adequate host capillary
bed capable of inosculation with the pre-vascularized scaffold capillaries within
2–3 days. Due to the 200 μm limitation for oxygen diffusion, and this critical time
delay for inosculation, the thickness of the pre-vascularized scaffold graft is quite
limited.
Potentially quicker vascularization could be achieved by combining the
vascularized chamber models with pre-vascularized scaffolds. The AV loop rat
chamber model (without a scaffold or growth factors added) is a highly angiogenic
expanding vascular bed with approximately 3% vascular volume at 3 days, 15%
vascular volume at 7 days, and a maximal vascular percent volume of 23% at
10 days (Lokmic et al. 2007), more than seven times that of rat skin. Using delayed
implantation techniques – which would involve re-opening of the chamber – a
pre-vascularized scaffold implanted in the chamber at 7–10 days will be immediately
exposed to an environment where active angiogenesis is established. Inosculation of
the actively sprouting chamber vessels to an implanted pre-vascularized scaffold
should be more rapid than to those in skin and reduce cell death in the construct.
However, using the chamber model is cumbersome and involves significant
surgery to establish the capillary bed. We know from our in vivo studies the capacity
for capillary sprouting from large vessels particularly large veins, and recently a
number of studies have utilized vascular pedicles to seed stem cells, or grow
capillary networks ex vivo. One study, Chang et al. (2009), explanted a fat flap
with its microcirculatory bed (termed an EMB) and its afferent artery and efferent
vein based on the inferior epigastric vessels. The EMB was perfused ex vivo in a
custom designed bioreactor at 60–120 mmHg at 0.2 mL/min with a combination
medium in DMEM, and 5% rat serum for up to 24 h. Included in the perfusion
mixture were a number of stem cell populations including multipotent adult progen-
itor cells from the bone marrow, as well as bone marrow and adipose derived
mesenchymal stem cells. The stem cells were shown to egress from the capillary
network into the extracellular tissue where they formed proliferative clusters. The
entire EMB was then transplanted back into the original donor rat. Maximum
survival of EMBs occurred in those perfused ex vivo for 12 h or less.
Sekine et al. in 2013 harvested a muscle flap from the leg of rats together with its
supplying femoral artery and vein and perfused the flap in a bioreactor system with
culture media and 6% fetal bovine serum at a rate of 50 μL/min. The authors
manufactured cardiac cell sheets incorporating neonatal cardiomyocytes with rat
cardiac endothelial cells and layered the sheets over the skeletal muscle flap with and
without FGF-2. The entire construct was perfused in a bioreactor for 3 days and later
transplanted into F344 athymic rats as an allograft via microvascular anastomosis of
the femoral vessels to the carotid artery and jugular vein in the neck. The authors
were able to demonstrate that the endothelial cells in the cardiac sheets connected to
capillaries in the muscle vascular bed creating an integrated perfused circuit in vitro
which required FGF-2 to promote reasonable degrees of inosculation, additionally,
the cardiac cells were beating. The entire construct (muscle flap and cardiac sheets)
could then be successfully transplanted in vivo with survival of all elements of the
construct.
In 2012, Chiu et al. used isolated segments of 5 week old mouse thoracic artery
and inferior vena cava at either end of micro-patterned substrates containing thymo-
sin β4-hydrogel, and generated capillaries (with identifiable lumens) which were
derived and grew from the sectioned ends of the macro-vessels towards the opposite
macro-vessel. The capillary outgrowths from each end connected to each other at
21 days. The study also demonstrated similar capillary network formation from
human umbilical arteries and veins in this in vitro system. The period to capillary
outgrowth connection was reduced to 14 days with the addition of VEGF and
hepatocyte growth factor. Cardiomyocytes which retained functional features includ-
ing beating were also successfully grown in vitro over this capillary bed originating
from the macro-vessels in culture. In vivo transplantation was not attempted, but
Chiu et al.’s study demonstrates the ability of capillary networks to sprout from
macro-vessels in culture, while the study of Sekine et al. demonstrates that a macro-
vessel-capillary network can inosculate with pre-vascularized construct capillaries.
Shandolov et al. (2014) seeded a porous biodegradable PLLA/PLGA scaffold
with HUVECs and fibroblasts with and without myoblasts and cultured this
pre-vascularized scaffold in vitro. Ten days post-seeding the seeded scaffold was
implanted around the femoral artery and vein in a mouse. One to two weeks later, the
femoral vessel/pre-vascularized scaffold construct was moved whilst connected to

its pedicle to reconstruct an abdominal wall defect in the same mouse. The authors
demonstrated capillary sprouting from the vascular pedicle into the scaffold in vivo
before and after transplantation to the abdominal defect. Host capillary growth into
the scaffold was maximal when all three cell types were implanted in the scaffold.
However, only minimal survival of the human capillaries in the scaffold occurred
in vivo, although inosculation of human and mouse capillaries in the scaffold was
demonstrated. The vast majority of capillaries in the transplanted scaffold in vivo
were of mouse origin.
These studies demonstrate a number of potential applications of pre-vascularized
scaffolds with macrovascular pedicles. Combining the capillary sprouting potential
of macrovascular vessels in vitro or in vivo with pre-vascularized scaffolds holds
enormous potential for rapid vascularization of skin wounds and deeper or avascular
defects that might occur over bone or tendon or in certain ischemic pathologies such
as diabetes and peripheral vascular disease. However, much work still needs to be
completed to ensure the creation of the many millions of patient-specific endothelial
cells required to create a human pre-vascularized scaffold and that efficient human
capillary formation in scaffolds occurs to a level comparable to human tissue vessel
density. The linkage of pre-vascularized scaffold capillaries to sprouts from a
macrovascular pedicle is also yet to be demonstrated on a significant scale.
2 Conclusion
The use of macrovascular pedicles isolated in a tissue engineering chamber to

generate “intrinsically derived” capillary networks in vivo which grow with
implanted cells/tissues is well established in a large number of experimental studies
in rat and mouse models and encouragingly in large animal models and a human
clinical trial. This form of vascularization is more efficient than the traditionally used
“extrinsic vascularization” approach where tissue engineering constructs were
implanted directly in sites such as under the skin or under the kidney capsule.
These host sites were then relied on to sprout capillaries into the construct – a
relatively slow and inefficient process.
However, the intrinsic vascularization approach is not able to supply a capillary
network around the pedicle for 3–5 days post-vascular pedicle isolation in the
chamber, and for tissue/organ specific cells implanted with the pedicle ischemic
necrosis will still kill many construct cells before capillaries grow.
Recent alternative vascularization techniques such as scaffold
pre-vascularization, combined with a vascular pedicle – particularly if the pedicle
can be induced to sprout prior to application of the pre-vascularized scaffold would,
after inosculation of the two capillary networks, provide an almost immediate blood
supply to the scaffold.
Acknowledgments The authors acknowledge funding from the National Health & Medical
Research Council of Australia, funding from the Australian Catholic University/O’Brien Institute
Tissue Engineering Centre, the Stafford Fox Foundation Australia; the Jack Brockhoff Foundation,
Australia; the Research Endowment Fund, St.Vincent’s Hospital, Melbourne, Australia; and the
Victorian State Government’s Department of Innovation, Industry and Regional Development’s
Operational Infrastructure Support Program.
We also acknowledge the assistance of Dr. Anne Kong, Dr. Shiang Lim, and Dr. Kiryu Yap
(O’Brien Institute Department of St Vincent’s Institute, Melbourne, Australia); Dr. Guei-Sheung
Liu (Centre for Eye Research Australia); Dr. Zerina Lokmic (University of Melbourne, Department
of Paediatrics and Nursing, Melbourne, Australia); and Prof Shyh-Ming Kuo (Department of
Biomedical Engineering, I-Shou University, Kaohsiung, Taiwan).
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Perfusion Bioreactors
for Prevascularization Strategies in Cardiac
Tissue Engineering
Ingra Mannhardt, Anna Marsano, and Andreas Teuschl
Abstract
Cardiac tissue engineering is currently being pursued with three different appli-
cations in mind: drug safety screening, disease modeling, and cardiac repair.
Mini- and microengineered heart tissues are well suitable for drug safety screen-
ing and disease modeling. But generation of large cardiac patches of clinically
relevant thickness, to functionally support the injured heart after myocardial
infarction, still needs improvement. The high oxygen and nutrient demand
request prevascularization of the engineered tissues in vitro prior to implantation.
Vascularization and cardiac tissue development are influenced by several factors
such as perfusion velocity, shear stress, coculture, extracellular matrix, mechan-
ical strain, electrical stimulation, and many more. As engineering approaches get
ever more sophisticated and bioreactors increasingly complex, cardiac tissue
engineering evolves and quality control becomes more prominent. This chapter
will focus on different perfusion bioreactors that aim at cultivating highly
vascularized and functional engineered heart tissues by, e.g., direct perfusion
through the tissue or cultivation on top of an engineered vascular bed.
I. Mannhardt (*)
Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-
Eppendorf, Hamburg, Germany
e-mail: i.mannhardt@uke.de
A. Marsano
Cardiac Surgery and Engineering Group, Departments of Biomedicine and Surgery, University of
Basel and University Hospital of Basel, Basel, Switzerland
e-mail: Anna.Marsano@usb.ch
A. Teuschl
Department of Biochemical Engineering, University of Applied Sciences Technikum Wien, Wien,
Austria
e-mail: andreas.teuschl@technikum-wien.at

DOI 10.1007/978-3-319-21056-8_14-1
2 I. Mannhardt et al.
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Factors Influencing Vascularization and Tissue Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2 Oxygen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.3 Perfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.4 Artificial Lumen Versus Donor Vessel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.5 Shear Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3 Additional Bioreactor Features for Promoting Tissue Development . . . . . . . . . . . . . . . . . . . . . . . . 10
3.1 Mechanical Strain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.2 Quality Control in Advanced Bioreactor Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1 Introduction
Ever since the first reports on engineered heart tissue almost 20 years ago
(Eschenhagen et al. 1997), cardiac tissue engineering has come a long way. Gener-
ation of mini- and microtissues is established and investigated for strategies of
preclinical drug safety screening (Mannhardt et al. 2016) or disease modeling studies
(reviewed in Eschenhagen et al. 2012; Eder et al. 2015). The creation of thick tissues,
densely packed with vital cells, as they are required for regenerative medicine, is a
major challenge though. Intense vascularization is a prerequisite for these tissues
engineered from cells that are metabolically active and have a high energy and
especially oxygen demand. Provision of nutrients and gas supply as well as elimi-
nation of waste products is essential to ensure maximal viability and optimal
function of these tissues. Bone, muscle, and cardiac tissue engineering are the
major fields in regenerative medicine that have to deal with this obstacle. This
chapter will focus on cardiac tissue engineering.
In the human heart, the average capillary distance is 12 μm (Rakusan et al. 1992)
implicating that each cardiomyocyte is supplied by its own small capillary. In vitro
the oxygen diffusion into the tissue is limited to ~150 μm, resulting in necrotic core
regions in thicker tissues (Folkman and Hochberg 1973). In highly dense engineered
cardiac tissue generated with the cell sheet technique, this limit is reached even at
~80 μm tissue depth (Shimizu et al. 2006). Upon implantation, most implanted cells
die within the first few days post-transplantation, likely due to the harsh ischemic
and inflammatory condition at the recipient site. Several strategies have been
attempted to enhance the transplanted cell survival, e.g., by heat shock (Zhang
et al. 2001) or overexpressing pro-angiogenic proteins (Marsano et al. 2013).
Although the combination of such interventions significantly enhances graft cell
survival, death still remains a significant issue (Robey et al. 2008). A prompt and
efficient vascularization upon implantation of several mm-thick constructs is there-
fore crucial to ensure cell survival and contractility of the graft. Hundreds of micro-
to millimeter-thick engineered heart tissues might be readily infiltrated by donor
vessels, taking care of the cells in the graft (Zimmermann et al. 2002, 2006; Shimizu
Perfusion Bioreactors for Prevascularization Strategies in Cardiac Tissue. . . 3
et al. 2006; Stevens et al. 2009; Lesman et al. 2010). Unfortunately this spontaneous
host blood vessel invasion is not fast enough (Clark and Clark 1939) to ensure
survival of cells in grafts with clinically relevant thickness (Shimizu et al. 2006);
therefore, prevascularization in vitro is esteemed necessary.
Vascularization of engineered tissues is pursued by various techniques (please see
also other chapters of this book): Stimulation with growth factors, co-cultivation
with endothelial cells and/or pericytes, functionalized biomaterials (e.g.,
crosslinking with VEGF, inclusion of specific cell attachment sequences, e.g.,
REDV), adaptation of the scaffold morphology (e.g., porous sponges, decellularized
tissue) for maximal permeability, or even prevascularization in vitro or in vivo by
native host vessels are the most frequently pursued strategies (reviewed in Sun et al.
2016). The biomimetic approaches aim at mimicking in vitro the dense capillary
network of the native myocardium and perfusion-based culture could provide an
in vivo-like oxygen supply to cells, overcoming limitations of diffusional transport
intrinsically present in conventional culture system. In addition, hydrodynamic
shears might also positively affect the endothelial differentiation and preorganization
of progenitor cells in engineered tissues, since it mimics the in vivo pulsating blood
flow (Volz et al. 2016). Therefore, perfusion is an important factor promoting
vasculogenesis, increasing cell viability, organization, and differentiation and
hence is often included in various prevascularization strategies.
As bioreactors offer the possibility of cultivating the engineered tissues in closed,
highly controlled environments, their integration in tissue engineering is more and
more state of the art. With regard to cardiac tissue engineering, continuous perfusion
of the developing vascular networks already in vitro prior to implantation is
envisioned. Table 1 gives an overview about studies combining abovementioned
vascularization strategies with perfusion bioreactor systems.
2 Factors Influencing Vascularization and Tissue

Development
Designing the setup of the bioreactor one has to take several aspects influencing
tissue structure into account.
2.1 Cells
The native human heart is composed of 20–30% cardiomyocytes (Jugdutt 2003) as

well as nonmyocytes, such as fibroblasts, endothelial cells, pericytes, and smooth
muscle cells. The nonmyocytes have important influence on tissue development and
cardiac tissue engineering approaches have hence picked up this physiological
mixture and combine several cell types to build engineered heart tissue that resembles
the natural organ as closely as possible (review in Hirt et al. 2013). The native vessel
as well does not only consist of endothelial cells lining the inner luminal wall, but
these cells are additionally surrounded by so-called perivascular cells, such as smooth
4
Table 1 Overview about vascularization strategies implemented in perfusion bioreactor systems. PGA polyglycolic acid, PGS poly(glycerol)sebacate,
NRHC neonatal rat heart cells, C2C12 murine skeletal myoblasts, dia diameter, AV atriovenous, CM cardiomyocytes, EC endothelial cells, HUVEC human
umbilical vein endothelial cells, hMSC human mesenchymal stem cells, hCMPC human cardiomyocyte progenitor cells, hESC-CM human embryonic stem cell-
derived cardiomyoytes, POMaC poly(octamethylene maleate (anhydride) citrate), " parameter augmented, # parameter reduced
Perfusion
Study Matrix Cells Vessel Volume/velocity Duration Tissue size Outcome
Carrier PGA mesh 24 x 106NRHC / Internally, 0.3/1/3 ml/min 10 days 9.5 mm dia x 0.2 mm In vitro: Cell distribution
et al. 2002 scaffold through mesh (140–700 μm/s) ", histomorphology in
center of tissue "
Kofidis Fibrin 0.5 x 107NRHC / Rat aorta 100 ml/h 14 days 8.5 mm dia x 1.2 mm In vitro: Viability " (esp. in
et al. 2003 hydrogel ml vicinity to core vessel),
metabolic activity "
Radisic Collagen 1.35 x 108NRHC / Interstitial flow 500 μm/s 7 days 11 mm dia x 1.5 mm In vitro: Cell viability ",
et al. 2004 sponge ml (0.5 ml/min) aerobic metabolism ", cell
damage #, CM below
100 μm border zone,
synchronized paced tissue
contractility, no
spontaneous contractility
Radisic PorousPGS 2.3 x 106NRHC Microchannels in 0.1 ml/min 3 days 5 mm dia x 2 mm In vitro: Oxygen carrier
et al. scaffold (500 μm/s) enhance oxygen supply,
2006b DNA ", contractility "
Moritt Matrigel 6.5 x 106NRHC / Rat AV-loop Anastomosis 1/4/10 weeks 13 mm dia x 5 mm In vivo: Spontaneous
et al. 150 μl in vivo construct contractility,
Morritt physiological force
et al. 2007 responses
I. Mannhardt et al.
Dvir et al. Porousalginate 2.3 x 106NRHC / Pulsatile through 100–300 ml/min 1/13 days 5 mm dia x 2 mm In vitro: Pulsatile
2007 scaffold pores perfusion led to ERK1/2
activation, shear stress
(>2.4 dyn/cm) resulted in
p38 activation and
initiation of apoptosis
Brown Collagen 8–8.8 x 106NRHC (Pulsatile) 1.5 or 0.3 ml/min 5 days 11 mm dia x 3 mm In vitro: Higher
et al. 2008 sponge / scaffold through pores of contraction amplitude at
scaffold higher flow rate, low flow
and pulsatile perfusion
favor hypertrophy of CM
Cheng Collagen 6 x 106NRHC / Internally, 0.2 mm/s 8 days 7 mm dia x 1.5 mm In vitro: Apoptotic cells #,
et al. 2009 sponge 40 μl through sponge viability ", aerobic cell
metabolism ", elongation/
cross striation of cells,
spontaneous contractility "
Maidhof Porous PGS 1.5 x 108C2C12 or Endothelialized 0.1 mm/s 5 days 8 mm dia x 1 mm In vitro: Generation of
et al. 2010 NRHC / ml and EC channels contractile cardiac tissues
with endothelialized
channels
Maidhof PorousPGS 1.5 x 108NRHC / Channels in 18 μl/min 5 days 11 mm dia x 1.5 mm In vitro: Electrical
et al. 2012 ml scaffold stimulation increases
contraction amplitude
Tee et al. Matrigel 6 x 106NRHC / Rat AV-loop Anastomosis 14 days 13 mm dia x 5 mm In vivo: 8 weeks post
2012 250 μl in vivo implant: vascular patency,
Perfusion Bioreactors for Prevascularization Strategies in Cardiac Tissue. . .
spontaneous tissue
contractility
(continued)
5
6
Table 1 (continued)
Perfusion
Study Matrix Cells Vessel Volume/velocity Duration Tissue size Outcome
Sekine – 2x triple NRHC + In vivo 50 μl/min 6 days 35 mm dia x 0.1 mm In vitro: Synchronous
et al. 2013 EC cell sheet pre-engineered pulsation in vitro. In vivo:
vascular bed (rat 14 days post implantation
artery + vein) via anastomosis still
beating
Sakaguchi Collagen gel 4x triple NRHC Microchannels in 0.5 ml/min 3 days 20 20 0.1 mm3 In vitro: Vascularized
et al. 2013 cell sheet on top of collagen gel 120 μm thick cardiac tissue
micro-channel gel
Vollert Fibrin 4.1 x 106NRHC / Microchannel in 20 μl/h 21 days 25 15 3 mm3 In vitro: Improved tissue
et al. 2014 hydrogel ml fibringel viability, no cell-free core
Zhang Fibrin 30–40 x 106 cells / Microchannels in 0.7 μl/min 7 days 3 5 x 2 mm3 In vitro: No necrotic core.
et al. 2016 hydrogel on scaffold (HUVEC, POMaC scaffold In vivo: Surgical
POMaC lattice hMSC, hESC-CM) anastomosis possible
scaffold
I. Mannhardt et al.
muscle cells or pericytes (Gerhardt and Betsholtz 2003; see also chapter “▶ Angio-
genesis: Basics of Vascular Biology”). The past has shown that simple coculture with
endothelial cells (EC) is clearly not enough. ECs have the capacity to form tubes
(Folkman and Haudenschild 1980), plaster the walls of lumina and migrate through
the tissue towards lumina (Sakaguchi et al. 2013), but spontaneous growth of EC
network takes several days. Differentiation, growth, migration, and survival of EC are
influenced by several factors such as surrounding ECM, cell-cell contacts, growth
factors, and mechanical cues (review in Baiguera and Ribatti 2013). Research groups
worldwide have tried to influence these parameters to enable for the best possible
vascularization of the engineered tissue. Coculture with perivascular cells and fibro-
blasts are known to promote angiogenesis in vitro and in vivo (Riemenschneider et al.
2016). Furthermore, different sources of ECs are investigated to find the optimal
source for clinical application: (i) autologous, (ii) mature enough for sprouting and
vessel formation, and (iii) paracrine effect by synthesis of humoral substances.
2.2 Oxygen
The limited oxygen supply to the deeper regions of the tissue is likely the major
obstacle diminishing the viability in the core regions of larger tissues. To minimize
this issue, some groups prefer cultivating the engineered heart tissue in high oxygen
(40%) incubators (Vollert et al. 2014), whereas others have used artificial oxygen
carriers such as perfluorocarbons (Radisic et al. 2005) to increase oxygen supply and
create constructs of high cell density and clinically relevant thickness. Immediately
after tissue generation, lack of oxygen might not be a critical issue, as the metabolic
activity of the cells is likely still low. At this early stage, lower oxygen could even
favor endothelial network formation (Adair et al. 1990; Shweiki et al. 1992; see also
chapter “▶ Targeting the Cellular “Oxygen Sensors”: Hypoxia Pre-conditioning and
Stabilization of Hypoxia Inducible Factors”). But along with tissue development,
oxygen demand increases as the engineered heart tissue is contracting and
performing work. With modern bioreactor setups, oxygen concentration in the
medium could be measured online and adjusted depending on the lactate production
(anaerobic glycolysis) and changes in pH (Radisic et al. 2006a; Sekine et al. 2013).
2.3 Perfusion
In the past, nutrient supply and waste removal depended on mere diffusion and
frequent medium change. Nowadays, this is regulated by perfusion around or ideally
through the tissue itself.
For tissue perfusion, two main techniques have advanced: direct perfusion
through the tissue or cultivation of the tissue on top of a vascular bed. Lumina for
direct tissue perfusion through the tissue can be created in three different ways:
(i) specially designed scaffolds, (ii) removable template or soluble templates, or (iii)
embedding of native graft vessels.
The group of Vunjak-Novakovic presented one of the first perfusion studies with
porous scaffolds (Carrier et al. 2002). They seeded cardiomyocytes onto pre-
fabricated fibrous polyglycolic acid scaffolds and cultured these constructs with
direct perfusion through the scaffold. Tissue development in the bioreactor was
monitored by measurement of pH, CO2, O2, glucose consumption, and lactate
production. Perfusion culture resulted in better cell distribution and increased
histomorphology throughout the entire construct. The porous scaffold technique
was further modified by Radisic et al. (2004), who demonstrated in a similar setup
that perfusion of cardiomyocytes seeded onto a collagen sponge could in addition
affect the contractility of these cardiomyocytes, decrease cell damage, and increase
aerobic metabolism. Two years later the same group demonstrated that oxygen
carriers such as perfluorocarbon can increase the oxygen supply to the cells (Radisic
et al. 2006b). The cardiomyocytes seeded onto porous poly(glycerol)sebacate were
perfused through microchannels in the scaffold which resulted in higher amounts of
DNA and cardiac markers (troponin I, connexin-43) and better contractility. But
perfusion could also damage the cardiomyocytes: Low pulsatile perfusion has
proven to be beneficiary and led to the activation of the ERK1/2 signaling cascade
and finally elongated and aligned cardiomyocytes with good sarcomeric structure,
but high shear stress (>2.4 dyn/cm) on the other hand resulted in p38 activation and
initiation of apoptosis (Dvir et al. 2007). Brown et al. (2008) compared pulsatile
versus nonpulsatile perfusion through collagen sponge scaffolds and noted beneficial
effects of pulsatile perfusion on contractile properties with augmented cell morphol-
ogy (cardiomyocyte elongation and hypertrophy) at low flow (0.3 ml/min) condi-
tions. Slow bi-directional perfusion through porous collagen sponges enhanced
survival, differentiation, and contractility of engineered cardiac tissues (Cheng
et al. 2009). In a further refinement of their previous setup, electrical stimulation
was integrated into the bioreactor and led to increased contraction amplitudes of the
engineered heart tissues (Maidhof et al. 2012). Recently, a complex designed
scaffold of poly(octamethylene maleate (anhydride) citrate) was introduced as the
“AngioChip, a stable biodegradable scaffold with a built-in branching microchannel
network” (Zhang et al. 2016). The lumina of this sophisticated construct were seeded
with endothelial cells and cardiomyocytes emerged in a fibrin hydrogel on top of this
scaffold. Perfusion through a single inlet and outlet enhanced cell survival and even
allowed for anastomoses, as the AngioChip burst pressure was ~7-fold higher than
the normal systolic blood pressure.
The embedding of soluble or otherwise removable templates into the matrix
during tissue casting is another approach to build artificial lumina. By embedding
thin alginate structures into the cardiomyocytes containing fibrin hydrogel, Vollert
et al. (2014) created small microchannels within the engineered heart tissue. Perfu-
sion of the tissue through these channels improved cell distribution and viability in
the core regions of the tissue and resulted in large, macroscopically contracting
engineered heart tissue. High perfusion pressure led to enlarged lumina as the soft
hydrogel matrix could not stand this pressure. Though oxygen concentration in the
tissue was elevated, mechanical strain via perfusion likely influenced tissue mor-
phology as well. Tocchio et al. (2015) stacked multiple polyvinyl alcohol templates
to create a hollow tube network inside the tissue, but both strategies created only
planar lumina. They did not enable for medium perfusion in the third dimension of
the tissue, but would depend on vessel sprouting. After creating several lumina with
removable steel wires in a collagen gel, Sakaguchi et al. (2013) stacked endothelial
cell and cardiomyocyte-containing cell sheets on top of this perfused microchannel
bed. Endothelial cells readily migrated towards the lumina connecting the cell sheets
to the perfused artificial microchannels. As this sprouting took about 5 days, stacking
of triple-layer cell sheets onto each other had to be performed with delay. But finally
this technique created a highly dense and vital engineered cardiac tissue.
Embedding a native vascular graft into the tissue was one of the first attempts on
prevascularization strategies in perfusion bioreactors. Perfusion of a fibrin-based
engineered cardiac tissue through an explanted rat aorta showed increased cell
viability in the vicinity to the core vessel (Kofidis et al. 2003). The idea of vessel
sprouting from the original host graft was further supported, when atriovenous
(AV) loops were incorporated in Matrigel®-cardiomyocyte mixtures and cultivate
after subcutaneous implantation in vivo (Morritt et al. 2007). Constructs contracted
spontaneously and showed typical muscle-length relationship, positive chronotropic
response to norepinephrine, and positive inotropic response to beta-adrenergic stim-
ulation with isoprenaline. Engineered cardiac muscle flaps generated around such
AV-loops demonstrated good patency and contractility even 8 weeks postimplantation
(Tee et al. 2012). In 2013, Sekine et al. demonstrated a promising combination of
these prevascularization strategies: After resecting a tissue graft with connectable
artery and vein, they seeded triple cell sheets on top of this vascular bed. Perfusion of
the vascular bed in vitro resulted in microvessel sprouting from the artery and vein
and infiltration of the beating cardiomyocyte cell sheet. These created cardiac tissues
survived 2 weeks after transplantation and anastomosis in the neck of nude rats.
Pushing it one step further, are the recent reports on perfusing thick vascularized
cardiac constructs based on decellularized extracellular matrix (Sarig et al. 2015).
2.4 Artificial Lumen Versus Donor Vessel
The most prominent difference between these prevascularization strategies is the use
of native grafts or the generation of artificial lumina. Using autologous grafts as
donor vessels is a technique very well established in clinical cardiology (bypass
surgery). Given there is a suitable donor vessel available, these grafts have several
advantages on hand: (i) anastomoses and suturing is rarely a problem, (ii) burst
pressure is rather high (7-times systolic blood pressure) and perfusion in vitro as well
as after transplantation in vivo is likely successful, (iii) clot-formation is rather rare,
as the endothelial cells of the intima help prevent this (Zhang et al. 2016), and
(iv) intensive sprouting of the vessel in vitro and in vivo. The studies with explanted
native vessel grafts (Morritt et al. 2007; Tee et al. 2012; Sekine et al. 2013)
demonstrated the advantage of the native vessel as they achieved successful anasto-
moses to the host vasculature (see also chapter 17).
On the other hand a biological graft might cause complications due to immuno-
genicity and less standardization. The problem with artificial lumina though is their
creation. Building a simple tube inside a cell-laden hydrogel or porous scaffold is not
too difficult, but forming a branched network of microtubular structures is anything

but trivial and usually fails at the third dimension (see also chapter “▶ In vitro and in
vivo Approaches for Pre-vascularization of 3 Dimensional Engineered Tissues”).
Only recently did the group of Radisic present a complex engineered skeleton with a
branched network of microchannels that allowed for perfusion of vascularized
cardiac tissue and direct anastomosis (Zhang et al. 2016).
2.5 Shear Stress
Perfusion does not only improve medium transports but can also influence cell
behavior and phenotype. The frictional force that perfusion creates on the surface
of the cells is called shear stress. As endothelial cells line the inner wall of the
vessels, they are continuously exposed to the shear stress exerted on them by the
blood rushing by. This shear stress has been shown to be of major importance for
endothelial cell survival and can influence their metabolism, differentiation and
function (Rotenberg et al. 2012; Baiguera and Ribatti 2013; Kang et al. 2013).
Perfusion can stimulate migration of endothelial cells and therefore promote angio-
genesis (Sakaguchi et al. 2013). Cardiomyocytes on the other hand are originally not
exposed to shear stress. Direct exposure of cardiomyocytes to high perfusion
pressure and/or perfusion velocity and therefore high shear stress (>2.4 dyn/cm)
resulted in the activation of p38 and initiation of apoptosis (Dvir et al. 2007). With
this in mind, most studies tried to seed endothelial cells into the artificially created
vessels (see also chapter “▶ Technically Preformed Channels for Vascularisation”).
For example, Maidhof et al. (2010) established a method of sequential seeding to
generate contractile cardiac tissues with endothelialized channels. The idea is to
protect the parenchymal cells from shear stress and in addition allow for vessel
sprouting from the main artificial lumen into the surrounding tissue, stabilize the
artificial vessels, and prevent clot formation.
When designing bioreactor and perfusion setup, there are a number of variables
that differ between the studies (see Table 1). Pulsatile perfusion is mimicking the
heart rate of the human body and was used in one of the first studies by Kofidis et al.
(2003) to perfuse neonatal rat cardiomyocytes in a fibrin hydrogel through an
embedded aorta explant. In some studies this more physiological, pulsatile perfusion
has proven to be superior to steady perfusion (Dvir et al. 2007; Brown et al. 2008).
3 Additional Bioreactor Features for Promoting Tissue

Development
3.1 Mechanical Strain
In addition to oxygen/nutrient supply, waste removal, and endothelial cell migration,

perfusion influences other aspects of tissue development, such as the
histomorphology that can be augmented by development of force lines through the
tissue (Vollert et al. 2014). Mechanical strain is essential for the proper development
(alignment and maturation) of cardiomyocytes in vitro. Modification of the matrix
biomaterial has tremendous effect on cardiomyocyte morphology and function
(Huyer et al. 2015). Stretching of the cardiomyocytes can be applied by either static
tension or phasic stretch. Static tension is the easiest form of stretch, where the
tissues are, e.g., fixated between glass rods (Eschenhagen et al. 1997). Motorized
stretch devices (Fink et al. 2000) as well as elastic anchoring points (Hansen et al.
2010; Vollert et al. 2014) have been applied for phasic stretching of the tissues.
Phasic stretch has proven to increase contractile function of the tissue (Fink et al.
2000) and has been applied for many different models of cardiac (Hansen et al. 2010;
Kensah et al. 2011; Tulloch et al. 2011) or skeletal muscle (Heher et al. 2015) tissue
engineering and eventually incorporated into bioreactor setups. Most likely, rhyth-
mic stretching would facilitate nutrient and oxygen supply and exchange of
byproducts through the engineered constructs mimicking the direct perfusion
through the scaffold voids. Therefore, as the perfusion, this mechanical stretch
also augments endothelial cell migration (Joung et al. 2006) and spatial distribution
of living cell throughout the scaffold (Akhyari et al. 2002) and promotes blood
vessel formation.
3.2 Quality Control in Advanced Bioreactor Systems
Elaborate bioreactors include additional features to optimize development and

function of the tissue and control the quality of the tissue in vitro prior to implan-
tation. Electrical stimulation improves structure, function, and maturation of
cardiomyocytes in vitro (Hirt et al. 2014; Godier-Furnemont et al. 2015) and is
integrated in advanced bioreactor systems (Maidhof et al. 2012). Quality control in
most bioreactors is based on control of culture conditions (CO2, O2, temperature,
pH, perfusion pressure/velocity) and markers of metabolic activity and cell death
(glucose, pH, lactate, LDH; e.g., Radisic et al. 2006b, 2008; Sekine et al. 2013).
Some bioreactors consist of separate parts that allow for the sterile, noninvasive
removal of the sample for histological evaluation of the tissue under the microscope
in the middle of the culture cycle (Kensah et al. 2011). But the ideal quality control
for cardiac tissue engineering is of course the key function of the tissue – the
contractility. Integrated force measurement of the tissues would allow for the best
readout, correlation to culture conditions, adaptation of the latter and eventually
improved engineered heart tissue best suitable for subsequent implantation in vivo.
4 Conclusion
Perfusion bioreactors are up and coming for prevascularization of cardiac tissues

in vitro. The current limitation of the different prevascularization strategies is
unfortunately their complexity. Either integration of artificial lumina in the third
dimension, or slow vessel sprouting – unfortunately we are not quite there yet. The
controlled culture of cells with culture conditions readily adjustable to the specific
cell requirements holds great promise though and could finally lead to the large,
functional engineered heart tissue regenerative medicine is waiting for.
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From Autologous Flaps to Engineered
Vascularized Grafts for Bone Regeneration
Alexander Haumer, Tarek Ismail, Alexander Lunger, Rik Osinga,

Arnaud Scherberich, Dirk Johannes Schaefer, and Ivan Martin
Abstract
Replacement of damaged or lost tissue typically relies on the availability of
living, functional substitutes and the rapid development of a stable and efficient
vascularization upon transplantation, in order to guarantee their survival. These
requirements challenge current surgical reconstruction techniques in the clinical
practice.
In the past decades, the field of tissue engineering has introduced the possi-
bility to combine materials and living cells to generate functional substitutes,
which can be tailored to specific requirements of the implantation site. At the
same time, plastic and reconstructive surgery has developed a large armamentar-
ium of grafting possibilities and flaps supporting vascularization of native tissues,
especially through progress made in microsurgical techniques.
In this chapter, we describe advances in the two fields and discuss how the
principles and techniques independently developed could be combined towards
A. Haumer (*) • I. Martin

Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland
Department of Biomedical Engineering, University of Basel, Allschwil, Switzerland
e-mail: alexander.haumer@usb.ch; ivan.martin@usb.ch
T. Ismail • A. Lunger • R. Osinga • D.J. Schaefer
Department of Plastic, Reconstructive, Aesthetic and Hand Surgery, University Hospital Basel,
Basel, Switzerland
e-mail: tarek.ismail@usb.ch; alexander.lunger@usb.ch; rik.osinga@usb.ch; dirk.schaefer@usb.ch
A. Scherberich
Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland
Department of Biomedical Engineering, University of Basel, Allschwil, Switzerland
Department of Plastic, Reconstructive, Aesthetic and Hand Surgery, University Hospital Basel,
Basel, Switzerland
e-mail: arnaud.scherberich@usb.ch

DOI 10.1007/978-3-319-21056-8_16-1
2 A. Haumer et al.
the prefabrication of vascularized tissues. The resulting paradigm of “regenera-

tive surgery,” here exemplified in the specific context of bone regeneration, could
represent the future standard for the reconstruction of complex body parts.
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Approaches in Plastic and Reconstructive Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1 Description of Flaps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2 Categories of Flaps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.3 The Donor Site Problem and the Drawbacks of Grafting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3 Bone Grafting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.1 Fibula Bone Graft . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.2 Iliac Crest Bone Graft . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3.3 Scapula Bone Flap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.4 Costal Bone Graft . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.5 Distal Radius Pedicled Bone Flap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.6 Medial Femoral Condyle Flap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4 Engineering Vascularized Grafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
5 Ectopic Prefabrication of Engineered Bone Grafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
5.1 Angiogenic Ingrowth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
5.2 Flap Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
5.3 AV Loop Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
5.4 AV Bundle Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
6 Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
6.1 Graft Prefabrication Using Adipose-Derived Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
6.2 Graft Prefabrication Using Microvascular Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
6.3 Graft Prefabrication Using 3D Printing and Microfluidic Systems . . . . . . . . . . . . . . . . . . . 26
7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
1 Introduction
Infection, trauma, tumor, and burn sequelae can lead to varying degrees of tissue
damage, ranging from skin only injuries to complex defects including muscle and/or
bone. Not even 100 years ago, the consequence of such damage was often amputa-
tion of a limb or organ, or even death of the patient. This challenge could be
addressed by autologous tissue grafting, namely transferring tissue from a part of
the own body in which it is not essential to the injury site in order to restore crucial
body function or coverage. The solution, however, has not been straightforward in its
implementation, due to the difficulty to provide fast and efficient vascularization to
the transferred tissue. This changed with one of the most important medical advances
of the twentieth century – the discovery of defined body territories with constant,
independent, and reliable blood supply, which in turn made graft and flap surgery
possible. With improvements in microsurgery principles over the last 50 years, the
routine use of grafts and flaps has dramatically broadened the surgical options in
reconstructive surgery.
From Autologous Flaps to Engineered Vascularized Grafts for Bone. . . 3
In this chapter, we will first present current approaches in plastic and reconstruc-
tive surgery to replace damaged tissue with vascularized grafts. We will then discuss
how the concepts and surgical methods to preserve or induce tissue vascularization
could be adapted to tissue engineered implants. Beyond the description of general
paradigms pursued for a variety of indications, the chapter will specifically focus on
the context of bone tissue regeneration.
2 Approaches in Plastic and Reconstructive Surgery
As one of the broadest surgical specialties, plastic surgeons are confronted with
defects in virtually all regions of the body including the integument, craniomax-
illofacial region, extremities, breast, and trunk. In the process of reconstructing
damaged tissue, plastic surgeons must often restore adequate blood flow first and
are frequently involved in revascularization procedures. If vessels are damaged
extensively, autologous substitutes such as the saphenous vein continue to be the
gold standard for replacement of both artery and vein (Bayramiçli et al. 2002;
Gurunluoglu and Rosen 2017; Nemoto et al. 2015). While synthetic grafts are
readily available for large vessels such as the aorta, iliac arteries, and femoral
arteries, grafts for vessels with a diameter of less than 6 mm often fail due to
thrombosis, arteriosclerosis, or intimal hyperplasia (Conte 1998). When treating
large defects which are unlikely to heal within a reasonable amount of time, plastic
surgery has developed a large armamentarium of tools for reconstruction consisting
of (i) autologous grafts, including so-called local flaps, regional flaps, and
microvascular-free tissue transfer, (ii) allogeneic substitutes, and (iii) synthetic
materials. The use and indications are set by the individual clinical scenario, but
every successful treatment is strongly dependent on vascular supply. In the remain-
der of the section, the issue of vascularization will be dealt with in the case of
autologous grafts.
2.1 Description of Flaps
In order for transferred tissue to survive, it must be reached by blood vessels. If the
graft is small enough and the quality of the recipient site is adequate, transferred
tissue may survive with vessel ingrowth from the recipient site only. Two examples
that illustrate this possibility are sheet (split thickness) skin grafts for burns, which
only include the superficial layer of skin, and cancellous bone grafts for long bone
and skull reconstruction.
Nonvascularized tissue survives in the recipient site as a result of neo-
vascularization and/or inosculation (see below), after initial (first 48 h) oxygen
diffusion, and fluid imbibition enables graft survival (Converse et al. 1975). Whereas
in neovascularization new vessels sprout, invade, and eventually supply the new
tissue from the wound, inosculation is the random direct connection of preexisting
vessels in the graft with vessels of the recipient site. This process allows reperfusion
4 A. Haumer et al.
Fig. 1 Flap vascularization. Four different vascularization patterns can be distinguished: Random
pattern supply through unnamed nutrient vessel of the subdermal plexus (a). Axial pattern supply
through one distinct artery (b). Axial pattern supply as described in (b) but detached from its
original nourishing vessel and revascularized through microsurgical anastomosis to a different
feeding vessel (c). Supply through one distinct, isolated perforator vessel or vessels (d)
within a short period of time (Laschke and Menger 2012; Laschke et al. 2009).
Larger tissue transfers, however, cannot rely on vascularization from the recipient
site only, because the vessel ingrowth would be too slow to provide cells in the center
of the tissue with the nutrients necessary for survival, increasing the risk of graft loss
and necrosis. Instead, the tissue is transferred along with the corresponding vascular
structures to the injury site. This type of operative technique is called “flap surgery.”
Flap is a term commonly used to describe transferred tissue with its original
blood supply. Because they are transferred with their vascular structures intact, flaps
do not depend on the recipient bed to vascularize, and the volume of transferred
tissue can be significantly greater than that of a nonvascularized graft. Flaps can also
contain multiple types of tissue, including skin, muscle, nerve, fascia, and bone.
Thus, vascularized tissue can be used to fill larger defects or recreate structures such
as the breast or mandible and provide coverage over areas with special demands,
e.g., joints.
With preserved original vascularization, the flap is immediately perfused after the
inset into the defect. As a result, graft-survival and engraftment as well as tolerance
to infection and mechanical stress are improved compared to nonvascularized grafts
(Rouwkema et al. 2008).
There are several specific patterns of blood supply to the flap, which help to
determine its possible use (Fig. 1).
Fig. 2 Angiosome map of the

human body. Originally
40 angiosomes of the human
body were defined as
composite units of skin and its
underlying deep tissue
supplied by a single source
artery and its branches. This
knowledge of angiosome
boundaries and locations of
source vessels guided flap
designs improving flap
viability
A random patternvascular supply (Fig. 1a) to the flap is provided by small

unnamed vessels of the subdermal plexus. It is the most common supply to local
skin flaps.
An axial vascular supply (Fig. 1b) indicates a distinct artery or group of arteries.
Vascular territories that are supplied by individual known blood vessels are defined
as angiosomes (Fig. 2). The concept of angiosomes revolutionized plastic surgery by
adding anatomic understanding to prior clinical experience and led to the introduc-
tion of microsurgery (Houseman et al. 2000; Taylor et al. 2011; Taylor and Palmer
1987). A constantly present, distinct, and reliable vessel, feeding a defined tissue
area, may become a flap’s pedicle (Fig. 1c). A pedicle usually branches to nourish
bone, muscle, and skin territories. Vessels that pierce anatomical structures on the
way to their destination are defined as perforators (Fig. 1d). A pedicle includes all
consecutive vascular structures such as distant arterioles capillary bed, venules, and
draining veins.
6 A. Haumer et al.
Perforator flaps rely on direct skin arteries or septal perforators as vascular

pedicle that leads to the overlying fascia and/or skin only (Sinna et al. 2010). The
vessels are dissected from the muscle, minimizing muscle sacrifice. Consequently,
perforator flaps cause less donor site morbidity. However, they are more technically
demanding (Blondeel et al. 2003; Gutowski 2009).
2.2 Categories of Flaps
There are two broad characterizations of flaps, delimited by whether or not they keep
their original vascular supply. Pedicled flaps that are used without division of the
feeding vascularization can be local (tissue directly abutting the defect that requires
coverage) or regional (tissue in the vicinity of the defect without actually abutting
the defect). An advantage of this type of flap is that the procedure does not require
the use of microsurgery. Two major drawbacks are that these flaps have a restricted
arc of motion, and their use depends on the availability of local intact vessels. This is
often not the case in clinical situations with large complex defects, such as those
resulting from soft tissue trauma or irradiation.
Flaps whose feeding vascularization is divided are called free flaps. These types
of flaps are used for more remote defects in areas with limited or no skin pliability, in
which it is not possible to use a local or regional flap. The flap must have a distinct
axial artery or vessel, called a pedicle, which can be divided and connected to a
recipient site donor vessel. This requires highly sophisticated microsurgical tech-
niques for vascular anastomoses (Table 1).
Depending on the tissue needed, there is a wide range of reconstructive options.
Among the most frequently used are flaps of skin, skin and fascia (fascio-cutaneous),
muscle, fascia and muscle (musculo-cutaneous), bone, bone and skin (osteo-fascio-
cutaneous). Even innervation can be transferred with the flap providing sensate
tissue substitutes or motor function. Flaps composed of more than one tissue type
are defined as composite – flaps.
Customized flaps seem to provide the link between classic autologous tissue
transfer and tissue engineering. While this would be the ideal treatment for tissue
damage, it is still far away from routine use in clinical practice. Prefabricated flaps
and prelaminated flaps are two modern innovative concepts in plastic surgery. They
are especially used for restoration for complex defects, where conventional recon-
structive methods fail. These two concepts are closely related to each other and often
undifferentiated used in the literature. However, they are two clearly distinct con-
cepts, as explained below.
An axial vascular pedicle is introduced into the desired tissue block. This can be
achieved by insertion of an arterio-venous bundle (AV bundle) or by an arterio-
venous loop (AV shunt). After a period of neovascularization (usually 8 weeks), this
prefabricated flap may be transferred, based on its newly acquired vascular pedicle.
The transfer may be local transposition or by microsurgical transfer (Guo and Pribaz
2009; Pribaz and Fine 2001).
Table 1 Flap vascularization patterns. The reconstructive ladder refers to the widely accepted principle that less invasive treatment options for defect closure
should be explored first in order to provide less harm to the patient
Reconstructive
Flap Vascularization Indication Example Invasivity ladder
Pedicled Random Small unnamed vessels of Defect with abundant surrounding Transposition (e.g.,
pattern the subdermal plexus. tissue to assure primary closure of Limberg) flap after
donor site, appr. maximum of flap base skin tumor excision
to flap length ratio 1:3
Axial Distinct artery feeding Defect with no tissue directly abutting “Indian” flap based
pattern known vascular territories the defect on supratrochlear
located along the flap’s Flap length can be extended artery for large
longitudinal axis nasal defects
Perforator Transmuscular/ Large defects with relevant functional SGAP (superior
(local) transfascial vascular impairment expected at muscle harvest gluteal artery
pedicle site perforator) flap for
sacral pressure ulcer
Free Free (can Distinct reliable Defects with no adjacent tissue DIEP (deep inferior
From Autologous Flaps to Engineered Vascularized Grafts for Bone. . .
be independent vascular available or damaged recipient site epigastric

perforator supply (irradiation), e.g., extremities perforator) flap for
based) breast
reconstruction
7
8 A. Haumer et al.
Clinical Scenario #1 To prefabricate a medial thigh skin flap, the pedicle of the
descending branch of the lateral circumflex femoral vessels are locally transposed for
head and neck reconstruction. Prelaminated flaps offer solutions for reconstruction
of a three-dimensional multiple layer structure, e.g., the nose. By conditioning newly
introduced tissue to a preexisting vascular supply, tissue transfers can be engineered
such that plastic surgeons can transfer tissue with layers normally not available at the
original site (Guo and Pribaz 2009). Examples involve reconstructing a nose defect
or a mandibular defect.
Clinical Scenario #2 A radial forearm fasciocutaneous flap is used to reconstruct a

full layer nose defect by partially raising the flap in a first step and placing ear
cartilage and a skin graft on the deep surface. Three to 6 weeks after vascularization,
in a second step, the construct is transferred to the defect as a free microvascular graft
(Chiang 2006).
Clinical Scenario #3 A maxillofacial reconstruction is performed with prelaminated

fibular osseous free flaps (Fig. 3) (Rohner et al. 2003). Prefabrication constituted
insertion of dental implants and split-thickness skin grafting. The mean delay
between prefabrication and flap transfer was 6 weeks. While the flap was harvested,
a bar construction with overdentures was mounted onto the implants. The over-
dentures were used as an occlusal key for exact three-dimensional positioning of the
graft within the defect. The bar construction also helped to stabilize the horseshoe
shape of the graft.
2.3 The Donor Site Problem and the Drawbacks of Grafting
There are several drawbacks to grafting in its current clinical practice.

Firstly, plastic surgery is still unable to operate without leaving scars. As every
injury will create a scar, every tissue transferred will leave a new defect in need of
coverage. Grafting can lead to varying degrees of donor site morbidity, including
impairment of locomotor function or aesthetic disfigurement, because it requires
sacrificing healthy tissue (Ling and Peng 2012). For this reason, one should always
try to proceed from less invasive reconstructive options to more extensive ones. This
concept is usually referred to as the reconstructive ladder depending on the needs of
the recipient site. In rare cases, there is actually an unintended benefit from the
grafting procedure – as in the case of autologous breast reconstruction with abdom-
inal flap (DIEP deep inferior epigastric perforator flap). Here, closure of the donor
site results in an abdominoplasty, which is a positive side effect for the patient.
Secondly, dissection of free grafts is a difficult, technically highly sophisticated
microsurgical procedure. It requires years of special training and practice and is not
available at all clinics. In addition, anastomoses intrinsically bear the risk of anas-
tomotic failure, leading to partial or even complete graft loss due to venous conges-
tion or arterial occlusion.
Fig. 3 Flap prefabrication. The concept of flap prefabrication is a two-stage technique in which a
flap is surgically altered by partial elevation, structural manipulation, and incorporation other tissue
layers at the first stage to create a specialized composite flap. The example above shows dissection
of a patient’s fibula and fixation of the bar construction after osteotomy to form a neo – maxilla (left)
as a first step. After a delay of typically 6–8 weeks the attachment of the overdenture (right) as a
second step was performed prior to final grafting (From Rohner D, PRS 2003)
Apart from these technical issues, several patient factors contribute to success-
ful engraftment. These include the patient’s general health status, compliance with
implementing postoperative instructions, and medical conditions affecting vessel
quality (e.g., excessive smoking, irradiation, diabetes, thrombogenic disorders, or
peripheral artery disease). These factors must be considered before planning
autologous tissue transfer. Importantly, surgical tumor or infection treatment
should be completed prior to reconstruction. Additionally, in many cases, the
vascularization of the recipient site area is damaged by the medical conditions of
the patient. In order for the surgery to be successful, the planned recipient vessels
for anastomosis should not compromise the vascularization of the originally
supplied organ.
For free vascularized bone grafts, even with careful patient selection, compli-
cation rates cannot be neglected with up to 40%, e.g., hematoma/bleeding seroma,
infection, wound dehiscence (Hanasono and Skoracki 2010; Lodders et al. 2016;
Lpez-Arcas et al. 2010; Van Gemert et al. 2012). Total flap loss varies in relation
to the reconstructed area of the body, with failure rates as high as 15.5%, mostly
due to venous congestions and/or thrombosis, particularly in the head and neck
area (Brands et al. 2010; Fukuiwa et al. 2008; Rosado et al. 2014; Yazar et al.
2004). In some cases, it is not even possible to close a defect with free tissue
transfer.
This plethora of possible risks and complications arising from traditional methods
of grafting explains the need for tissue-engineered alternatives.
10 A. Haumer et al.
3 Bone Grafting
Bone possesses the intrinsic capacity for regeneration as part of the repair process in
response to injury, as well as during skeletal development or continuous remodeling
throughout adult life (Einhorn 1998, 2005). Bone regeneration is comprised of a
well-orchestrated series of biological events of bone induction and conduction,
involving a number cell types and intracellular and extracellular molecular signaling
pathways, with a defined temporal and spatial sequence. The most common form of
bone regeneration is fracture healing, during which the pathway of endochondral
ossification is recapitulated (Lenas et al. 2009). Unlike many other organs, injuries
and fractures to the bone heal without any scar formation. However, in large bone
defects or avascular necrosis, this process often fails. This can be due to insufficient
blood supply, extensive soft tissue injury around the bone, infection, neoplasm,
radiotherapy, systemic disease, or congenital disorder, resulting in delayed union
or nonunion.
In treating these defects, the reconstructive surgeon has to select a bone graft
depending on several factors, such as viability of the surrounding tissue, size, shape
and volume of the defect, desired biomechanical and biological characteristics of the
graft, possible complications at the donor site, costs, and ethical considerations. In
general, graft tissues used can either be autografts, allografts, or xenografts. Some-
times solely or in addition to these bone grafts, substitutes are used. These applied
materials are either synthetic, biologically based, tissue-engineered, or a combina-
tion of these (Oryan et al. 2014).
Following is a description of flaps typically used for bone reconstruction in
plastic surgery are listed below and summarized in table.
3.1 Fibula Bone Graft
Free fibula transfer was first reported in the literature in 1975 (Taylor and Watson
1978; Taylor 1978). It has since become the working horse in autologous bone
reconstruction because of the relative dispensability of the bone, bone size, pliability,
accessibility, predictable vascular pedicle, and mechanical strength. A segment of
bone between 26 and 30 cm can be harvested, providing the largest possible graft
from the patient’s native bone (Fig. 4).
As an example for this graft’s versatility, a transverse osteotomy of the fibular
bone graft can be performed, forming the so-called “double barrel” free vascularized
fibular graft, when it is folded. The two bone struts are still connected via the
periosteum, which ensures blood supply by the fibular artery (Horiuchi et al. 1995;
Jones et al. 1988). This technique overcomes the limitations of the linear shape and
width of the native bone. A classic indication for a “double barrel” fibula is
reconstruction of anterior defects of the mandible, where the cross-section of the
defect is twice that of the fibula. A double barrel fibula provides optimal architecture
for dental implants and ensures excellent functional and aesthetic outcomes (Chang
et al. 2014; Jacobson et al. 2015).
Fig. 4 Bone flaps. The fibula

flap. Schematic anatomical
relationships: The peroneal
artery originates from the
tibio-peroneal trunk
nourishing the fibula graft. To
protect the vascular inflow to
the bone flap, a muscle cuff of
1–2 mm is usually harvested
with the bone. The flap can be
harvested including
perforating vessels to the
overlying skin island (osteo-
myo-cutaneous composite
flap) if needed
Also in the area of trunk, extremity and spinal reconstruction, “double barrel”
fibula grafts become more important (Winters et al. 2010; Clemens et al. 2012).
The presence of several peroneal artery fasciocutaneous perforators also makes
the inclusion of a skin island possible. Therefore, it can be used as a composite graft.
A frequent indication is the treatment of larger segmental bone defects, such as
maxillar and mandibular, as well as lower extremity reconstruction. Unfortunately,
free fibula transfer is not available to some patients, such as a patient for whom a free
fibular harvest would cause insufficient lower extremity blood flow.
In these cases, alternative autologous tissue graft options are outlined below.
3.2 Iliac Crest Bone Graft
Bone from the iliac crest (Fig. 5) can be used as nonvascularized bone graft in case of
smaller defects in nonunion of long bone and carpal bone fractures or segmental
12 A. Haumer et al.
Fig. 5 Bone flaps. The iliac crest bone flap. It can either be grafted nonvascularized or
vascularized. If harvested vascularized, its nourishing vessel the deep circumflex iliac artery
(DCIA) coming from the external iliac artery allows the flap to be up to 15 cm long and 6 cm wide
bone defects (Calori et al. 2014). It can also be harvested as a vascularized bone
graft, named “deep circumflex iliac artery (DCIA) bone flap”. This flap provides a
large concave segment of bone suitable for reconstruction of lower and upper
extremity as well as in the maxillofacial area as the contour of the iliac crest is
similar to the alveolar crest and allows good esthetic and functional results (Brown
et al. 2013; Kumar et al. 2016; Markiewicz et al. 2015; Peek and Giessler 2006).
The DCIA flap is used for intermediate sized bone defects. It can be used with or
without skin paddle and muscle.
There is significant donor side morbidity, including neurosensory deficit in the
territory of the lateral cutaneous nerve of the thigh, chronic pain, gait abnormality,
impaired locomotor function of the hip, and abdominal wall weakness/herniation.
The DCIA flap is a reliable flap for bone reconstruction in patients with vascular
disease or a history of leg injury. It also provides cancellous bone, inconspicuous
scars in the groin and requires less osteotomies compared to the free fibular transfer
(Ling et al. 2013; Tang et al. 1998).
From Autologous Flaps to Engineered Vascularized Grafts for Bone. . .
Fig. 6 Bone flaps. The scapular bone flap. The flap is supplied by a transverse branch of the circumflex scapular artery coming through the triangular space and
originating from the subscapular artery. It can be raised as anosteo-myo-cutaneous flap or as a chimeric flap including the descending parascapular branch of the
13
circumflex scapular artery which can also be raised osteo-myo-cutaneous or with only some of the components mentioned
14 A. Haumer et al.
3.3 Scapula Bone Flap
Scapular osteo-myo-cutaneus flap (Figs. 6 and 7) is mostly used for head and neck
reconstruction, e.g., palatomaxillary reconstruction (Clark et al. 2008; Patel et al.
2008). Chimeric combinations of the scapular skin flap with muscle and/or bone
components provide multiple reconstructive options for complex composite tissue
defects. It features a long vascular pedicle, which is usually spared by atherosclerosis
and large amount of soft tissue available. This is of particular benefit in elderly
patients with peripheral vascular disease and assumption of postoperative impair-
ment of mobilization. Main disadvantage is the accompanying shoulder /arm
dysfunction (Ferrari et al. 2015).
3.4 Costal Bone Graft
The rib is composed of membranous bone that possesses a dual blood supply, from
the posterior intercostal artery and a periosteal blood supply from the serratus
anterior muscle (from the thoracodorsal artery). Rib grafts can be used to reconstruct
both lower and upper extremity (e.g., Humerus and clavicula) and maxillofacial
defects. Their natural bending makes the rib a suitable jawbone substitute. To
reconstruct clavicular or humeral defects, the rib can be transferred either as a
pedicled graft or as a free flap (Dumont et al. 2007; Onishi and Maruyama 1996;
Sawaizumi et al. 1996). An advantage of the costal flap is the possible integration of
a soft-tissue-envelope including serratus- and /or latissimus muscle, depending on
the same vascular pedicle. For smaller defects, e.g., of the mandible, non-
vascularized rib bone can be harvested (Bachelet et al. 2015). Advantages of
nonvascularized rib grafts are ease of harvest without requirement of microsurgical
techniques and low invasivity. However, like all nonvascularized grafts, it can only
be used if the graft is covered by well-vascularized tissue and the wound is free of
infection (Houdek et al. 2015). In case of multilayer defects, e.g., accompanying soft
tissue trauma, or planned postoperative irradiation, nonvascularized costal grafts are
less successful.
3.5 Distal Radius Pedicled Bone Flap
Dorsal distal radius bone graft is mostly used as a vascularized pedicled bone graft
for carpal bone reconstruction in hand surgery. Nonunion after scaphoid fracture
(most common fracture of the carpal bones) and avascular bone necrosis (degener-
ative disease caused by impaired blood supply) are main indications (Al-Jabri et al.
2014; Larson et al. 2006; Saint Cast et al. 2012). Limiting factors are the small
amount of bone tissue available due to the risk of fracture of the remaining radius and
the restricted arch of rotation of the pedicle.
Fig. 7 Bone flaps. The medial femoral condyle flap. It is pedicled on the descending genicular
artery, a branch of the superficial femoral artery. The medial superior geniculate artery can be used
as an alternative, but has a much shorter vascular pedicle. The medial collateral ligament (MCL) and
joint capsule is spared and cannot be incorporated into the flap
3.6 Medial Femoral Condyle Flap
The medial femoral condyle flap (Fig. 8) provides cortico-cancellous bone, suited to
reconstruct mainly defects of the extremities (Bakri et al. 2008; Haddock et al. 2013).
In scaphoid nonunion or avascular necrosis of the carpal bones, it is well established.
Due to the consistent and robust vascular supply in the medial femoral condyle, the
flap can be raised as a composite flap including overlying skin and muscle. In
contrast to the distal radius, the medial femoral condyle flap can also be used in
case of advanced scaphoid degeneration with destroyed articular surface (Jones et al.
2008) since it can be harvested as an osteocartilaginous flap, by including the medial
femoral trochlea, to restore the articular surface of the destroyed carpal bone.
Furthermore, restoration of clavicular and humeral defects is possible indications
(Fuchs et al. 2005). Donor side morbidity is rare, but knee pain in the first few weeks
after surgery is common (Bakri et al. 2008; Friedrich et al. 2012; Jones et al. 2008;
Rao and Davison 2012) (Table 2).
The description of the current options for autologous bone flaps indicates the
variety of possibilities developed in the last decades, but also highlights that no
solution is yet ideal. This represents the rationale for the introduction in plastic and
reconstructive surgery of engineered graft materials, which would be available in the
16 A. Haumer et al.
Fig. 8 The in vivo bioreactor: tissue engineering has proposed the in vivo bioreactor to overcome
the hurdles which in vitro culture poses. Generation of bone grafts in the in vivo bioreactor system
involves i) generation of an osteoconductive scaffold, addition of osteogenic cells, and
osteoinductive proteins. (ii) Subsequent ectopic implantation for cultivation. (iii & iv) Increasing
vascularization of the graft and concomitant scaffold degradation leads to stem cell homing and
bone tissue formation. (v) The graft is functional in situ or can be transferred to the defect site.
Adapted from (McCullen et al. 2011)
necessary quantity and quality without extended donor site morbidity. This topic will
be addressed in the following section.
4 Engineering Vascularized Grafts
Many sophisticated reconstruction techniques exist to repair tissue defects, but each
of them has inherent problems. Grafting in plastic and reconstructive surgery almost
entirely relies on native tissues, which have limited availability and are not always
easy to adapt to the defect site. In need to overcome these shortcomings, the field of
tissue engineering has endeavored to develop alternatives to the traditional methods,
with the goal to avoid the above-mentioned bottlenecks.
To allow a long-term survival and function of tissue engineered constructs,
sufficient vascularization has to be guaranteed to supply the graft with the necessary
oxygen and glucose when transplanted in vivo (Deschepper et al. 2013). Consider-
ing the small diffusion distance of oxygen and the maximum physiological growth of
new blood vessels, which has been shown to be limited to approx. 5 μm/h, large
constructs, where neither oxygen diffusion nor new blood vessel formation is quick
enough, will result in necrosis of cells in the center (Orr et al. 2003; Santos and Reis
2010).
Establishment of avascular network through the entire construct can be achieved
by two means. One principle, called inosculation, relies on a preformed, stable
vascular plexus within the construct which is then transplanted. After transplanta-
tion, this preformed microvascular tree connects to the recipient’s vasculature and is
then rapidly perfused (Laschke et al. 2009). The process of inosculation does not
depend on the size of the construct, which opens an interesting opportunity for
engraftment and survival of large three-dimensional constructs, an aim which tissue
engineering is strongly pursuing.
The other vascularization strategy exploits rapid ingrowth of blood vessels from
the recipient by stimulating angiogenesis into the implanted construct (Laschke et al.
2006). There have been several approaches trying to stimulate vascularization, from
changes in chemical composition of the biomaterial to bioactivation of the scaffold
by incorporation of proteins (Laschke et al. 2008, 2009; Nillesen et al. 2007; Rücker
et al. 2006, 2008; Richardson et al. 2001; Rocha et al. 2008; Yarlagadda et al. 2005).
Since other chapters of the book have more extensively described these strategies or
those implying co-culture of different cell types, here we will describe “surgical
manipulation” strategies for vascularization of engineered grafts, again with a
specific focus on bone regeneration.
Transplanting an engineered construct which contains a preformed microvascular
plexus is an emerging concept in tissue engineering (Laschke et al. 2009; Lokmic
and Mitchell 2008). Vascularized bone grafts seem to provide a higher engraftment
rate in large defects (e.g., mandibular defects >9 cm) and in cases of severely
damaged tissue with impaired vascularization, e.g., irradiated, infected tissue (Foster
et al. 1999; Pogrel et al. 1997). Additionally, vascularized grafts offer composite
tissue solutions (bone including muscle, skin, nerves) for complex defects where
these components also need to be reconstructed.
Different strategies have been developed to engineer prevascularized constructs,
involving in vitro or in vivo prevascularization. While in vitro culture and co-culture
of different cell types such as endothelial cells, stem cells, HUVECs, pericytes, etc.,
can result in a prevascularized construct, other approaches have involved the organ-
ism itself as natural “bioreactor,” preimplanting the construct in good-perfused areas
to achieve high vascularization (Arkudas et al. 2007; Black et al. 1998; Koike et al.
2004; Levenberg et al. 2005; Shepherd et al. 2006; Wang et al. 2007).
Warnke et al. published the first successful clinical application of reconstructing a
large mandibular defect in 2004 through ectopically prevascularizing a titanium
mesh cage filled with bovine bone material blocks coated with recombinant
human bone morphogenetic protein-7 and enriched with the patient’s bone marrow
cells (Warnke et al. 2004). Seven weeks after prevascularization in the latissimus
dorsimuscle, the construct was transplanted as a free composite bone-muscle graft
into the mandibular defect with satisfactory outcome until the patient’s death
15 months later. Despite the great surgical success, this approach has not been
broadly distributed into clinical practice so far (Springer et al. 2006). First, the
external titanium mesh scaffold used was suboptimal. Despite not being degradable,
it did not retain the desired customized shape for individual fit of the bone regenerate
and failed upon increased load. Second, the mineralization within the graft did not
occur homogenously although using the maximum dose of growth factor (rhBMP-7).
Third, the mental state of the patient treated was not properly considered, so that the
Table 2 Most commonly used bone grafting options
18
Bone
flap Soft Tissue used for composite graft Vascularization Indication Morbidity
Fibula Musculo /cutaneus (flexor hallucis Peroneal artery Segmental bone defects of long Stress fracture, peroneal
longus muscle) Skin bones and head and neck (especially palsy, ankle instability
mandibula) > 6 cm and residual ankle pain,
flexion/extension
difficulty
Iliac Musculo /cutaneus (internal Deep circumflex iliac artery Reconstruction of moderate size Chronic donor site pain,
crest oblique muscle) bone defect (5-12 cm) fracture of the ilium,
numbness
Scapula Chimeric nature of the subscapular Angular branch of the toracodorsal Complex 3-dimensional defects in Shoulder dysfunction,
vascular system, allowing for a artery (TDa) head and neck area, (especially Seroma
harvest of multiple, independent skin maxilla and scalp), with need for
paddles, serratus muscle with or long pedicle
without rib, latissimus dorsi muscle,
and up to 14 cm of lateral scapular
bone
Costa Musculo /cutaneous (serratus Posterior intercostal artery, branch Nasal bridge reconstruction, Infection, chronic pain,
anterior muscle, latissimus dorsi of the toracodorsal artery Nonunion after fracture of carpal scarring
muscle) bones of the hand, mandibular
reconstruction
Distal None Intercompartmentalsupraretinacular Nonunion after fracture, avascular Chronic pain, scarring
radius artery bone necorosis of Carpal bones
Medial Skin, cartilage Descending genicular artery Nonunion after fracture of carpal Femur instability,
femoral bones of the hand, clavicula and chronic pain
condyle ankle/foot region, avascular bone
necorosis, where corticoperiosteal
bone is needed
A. Haumer et al.
postoperative instructions were not properly implemented. It remains that the study was
pioneer towards the combination of reconstructive surgical procedures with pre-
fabricated engineered grafts and it sparked enthusiasm to the further development of
multiple techniques, based on the principle of using the own body as the bioreactor for
engineered tissue vascularization prior to transplantation.
5 Ectopic Prefabrication of Engineered Bone Grafts
In vitro tissue engineering allowed to gain the fundamental knowledge and crucial
understandings in the field but has often not been able to recreate the important
systemic and hierarchical organization of in vivo tissues. In this regard, the micro-
environmental niche, highly specific for each tissue, or even subregions within one
tissue, plays a critical role. Tissue engineering aims to recreate this sentient, tissue-
intrinsic balance of microenvironmental factors, such as oxygen concentration,
cytokine gradients, pH, ionic and electrical potential, available nutrients, and
mechanical stimulation, in ex vivo systems such as artificial bioreactors (McCullen
et al. 2011). In vitro resemblance of the complex interplay of these factors represents
a big challenge, which often has led to unsatisfying results (Badylak and Nerem
2010). To tackle these drawbacks, tissue engineering has proposed to combine the
basic elements of bone, namely an osteoconductive scaffold, osteoinductive pro-
teins, and osteogenic cells, within a living organism to recapitulate skeletogenesis
within an isolated in vivo bioreactor (Fig. 5) (Holt et al. 2005). The rationale behind
this approach was to exploit the body’s own regenerative capacity for the regrowth of
tissues and generating the physiological niche that naturally occurs in the bony
environment (McCullen et al. 2011) (Fig. 9).
Whereas the underlying rationale of the in vivo bioreactor is clearly defined, its
experimental translation can assume various forms. In 2005 Stevens et al. generated
bone by deliberately creating and manipulating an artificial space between the tibia
and its periosteum, defining this space as an in vivo bioreactor. The group showed
that the bone, which was biomechanically identical to native bone and generated
through the intramembranous route could be engineered in large volumes and in a
predictable manner. Moreover, they demonstrated that by inhibiting angiogenesis
within this in vivo bioreactor, cartilage could be generated (Stevens et al. 2005). This
work paved the way for the idea of applying the traditional triad of tissue engineering
biomaterials, growth factors, and cells to the in vivo bioreactor (Holt et al. 2005;
McCullen et al. 2011; Warnke et al. 2004).
5.1 Angiogenic Ingrowth
Implanting a scaffold into a well-vascularized and easily accessible part of the body
induces a strong angiogenic tissue response which is “characterized by the random
ingrowth of newly developing microvessels from the surrounding host microvascu-
lature” (Fig. 10a) (prevascularization in tissue engineering: Current concepts and
20 A. Haumer et al.
Fig. 9 Strategies for prevascularization of grafts. A prevascularized graft is generated by angio-

genic ingrowth (a) when a scaffold (gray) is implanted in a well vascularized, easy accessible tissue
in the body (orange) and random vessel ingrowth into the scaffold happens. This graft can then be
transferred freely. (b) The flap technique, also referred to as prelamination, consists in scaffold
implantation into a muscle flap. After vascular ingrowth, the entire flap is harvested, transferred to
the defect site, and anastomosed to the recipient vessels. Similarly, the AV loop technique (c) applies
vascular ingrowth into a protected growth chamber which contains an AV loop. After new vessels
sprout into the chamber, the system can be transferred and anastomosed. Adapted from (Laschke
and Menger 2015)
future directions). This application of the in vivo bioreactor system leads to gener-
ation of fully functional microvasculature and thus represents a very effective in situ
prevascularization method. (Improvement of vascularization of PLGA scaffolds by
inosculation of in situ-preformed functional blood vessels with the host microvas-
culature.) The fully vascularized graft can then be excised and implanted into the
defect site, where it connects to the recipient vasculature by inosculation (Laschke
et al. 2009).
5.2 Flap Technique
The flap technique is an advancement of in situ prevascularization by angiogenic

ingrowth (Fig. 10b). It is also referred to as the process of prelamination. A scaffold
or tissue construct is firstly implanted into a muscle flap to allow the stepwise
ingrowth of newly developing microvessels from the surrounding tissue (Kaempfen
et al. 2015). Subsequently, the entire flap with the incorporated prevascularized
implant can be freely transferred to the tissue defect, where the vascular pedicle of
the flap is surgically anastomosed to appropriate host vessels.
5.3 AV Loop Technique
Since almost four decades arteriovenous fistulas have been used to achieve neo-
vascularization in tissues. Erol and Sira demonstrated already in 1980 that generating
a loop-shaped arteriovenous fistula results in spontaneous sprouting of vessels out of
this loop (Fig. 10c). They created a vascular bed in skin by inserting this arteriove-
nous loop between two dermal layers, using interpositional vein grafts. It was
demonstrated that arranging the fistula in a loop-like shape allows generating skin
flaps of virtually unlimited size, which could be used even as possibility for digital
replantation with severely damaged vessels and provide enough additional vascu-
larity to facilitate the take of bone grafts and tissue composites (Erol and Sira 1980).
Twenty years after Erol and Sira had shown that the AV-loop was a possibility to
restore vascularization, Mian et al. were able to show that not even a particular
extracellular matrix is required for revascularization. A simple transparent plastic
chamber containing the AV loop, without added extracellular matrix, implanted
subcutaneously, is sufficient for the generation of a vascularized tissue matrix.
After an initial phase where most of the mass consists of coagulated inflammatory
exudate and granulation tissue, an intermediate phase, after 4 weeks, with mostly
young scar tissue, by 12 weeks the tissue becomes mature collagenous connective
tissue. In contrast to the flap technique, this in situ strategy allows the generation of a
prevascularized tissue construct, which is not embedded in surrounding muscle
tissue. Hence, the transfer of the construct is not associated with major tissue loss
and deformity at the donor site (Mian et al. 2000). Dependent on the local conditions,
it can even be directly applied at the defect site, avoiding donor site morbidity
completely (Eweida et al. 2014).
Extracellular Matrix Embedding In order to improve the AV loop as a grafting

possibility, studies have investigated on factors that influence graft quality and
quantity within the chamber (Weigand et al. 2016). Concerning the extracellular
matrix, it was shown that matrigel performed superiorly to fibrin gel, because latter
did not integrate with the generating vascular tissue. So one limiting factor to growth
was defined as the capacity of the newly formed tissue to integrate with the matrix,
whereas the second limiting factor to tissue growth naturally is the chamber size.
Encapsulation and regression follow when the sides of the chamber are reached or
tissue fails to integrate (Cassell et al. 2001).
Growth Factor Delivery Arkudas et al. further improved the AV loop system by
implementing a player who finds large use in tissue engineering: growth factors. The
extracellular matrix can be augmented with multiple and different growth factors
also at the same time. For example, when the matrix is enriched with VEGF and
bFGF, significantly higher absolute and relative vascular density and a faster
22 A. Haumer et al.
Fig. 10 In the AV bundle, an

artery (A) and a vein (V ) are
ligated to form a vessel
bundle, without microsurgical
anastomosis of the lumen. The
bundle is then inserted into the
desired tissue structure, e.g.,
in bone segments, where
capillaries and fibroblasts
proliferate and provided
osteoclasts and osteoblasts
take part in bone remodeling.
Adapted (Tanaka et al. 2003)
resorption of the fibrin matrix can be observed. Stimulation of vascular sprouting by

VEGF and bFGF in the AV loop system allows efficient generation of axially
vascularized, tissue-engineered composites (Arkudas et al. 2007).
Cell Incorporation As next logical step in the tissue engineering concept, the
matrices within the AV loop chamber have been seeded with different cell types to
generate different tissue types. Besides skeletal muscle, cardiac tissue, and cartilage,
the AV loop model enhances bone formation, as demonstrated by Arkudas et al.
(2007; Bach et al. 2006; Burghartz et al. 2015; Morritt et al. 2007; Tee et al. 2012).
The AV-loop not only increases osteoblast survival within a porous matrix and
triggers the expression of bone-specific genes, but eventually is able to form bone
compared to AV-loop-free control, which does not form bone (Arkudas et al. 2007).
5.4 AV Bundle Technique
As the name suggests, the arteriovenous (AV) bundle is created by ligation of an

artery and a vein to form a vessel bundle, without microsurgical anastomosis of the
two vessel lumen, as it is performed in AV loops (Fig. 11). Clinically, the AV bundle
plays a major role, since muscle or fascia flaps are harvested with AV bundles as
vascular carriers which supply nutrients to the flap (Pribaz et al. 1999; Shintomi and
Ohura 1982). As clinically relevant model, it had to be compared to the AV loop,
which had been demonstrated to be an efficient mode of prevascularization, as stated
in the previous paragraph. So Tanaka et al. designed a first experiment in 2003 to
compare the potential of tissue generation and angiogenesis between the two models
(Tanaka et al. 2003). Even if both models showed marked angiogenesis, arising from
the femoral vein, which implicated luminal sprouting, 4 weeks after implantation the
tissue in the AV bundle system had already been reorganized, whereas it was still
undergoing reorganization in the AV loop model. Eventually, the authors conclude
that the AV loop is more demanding to construct surgically compared to the bundle
and entails problems such as thrombus formation (Tanaka et al. 2003). Already
25 years earlier, Hori et al. had recognized the importance of the AV-bundle for
clinically challenging scenarios, such as avascular bone necrosis. In this regard, the
group conducted a study to “demonstrate experimentally and clinically the feasibility
of using a vascular bundle to create bone regeneration in isolated bone segments,
necrotized bone, and homografts of bone and to compare the results with pure
arterial pedicles and venous pedicles anastomosed to an artery.” The authors con-
cluded that the surrounding tissue transplanted together with the AV-bundle, where
capillaries and fibroblasts proliferate and provide osteoclasts and osteoblasts which
resorb necrotic bone and at the same time stimulate bone formation and bone
remodeling. This process results in the recipient bone (isolated segment, necrotized
segment, or homograft) taking active part in this remodeling process. The proof that
vascular bundle transplantation leads to active revascularization and bone
remodeling opened the chance to use this method for revascularization of large
bone defects or the treatment of avascular necrosis of the bone, two highly chal-
lenging clinical scenarios (Hori et al. 1979).
6 Future Directions
Almost 60 years after the findings of Hori and his group, treatment for clinically
challenging scenarios such as avascular necrosis of the bone is still to be improved.
Even if an efficient and stable vascularization can be mostly ensured through highly
sophisticated microsurgery, bone formation needs to be improved.
6.1 Graft Prefabrication Using Adipose-Derived Cells
Cells from the stromal vascular fraction (SVF) of adipose tissue are an attractive
source for tissue engineering purposes. As a combination of endothelial and skeletal
progenitors, as well as cells of the immune compartment (Weisberg et al. 2003; Xu
et al. 2003), they have been previously used for the manufacturing of osteogenic and
vasculogenic grafts (Scherberich et al. 2007; Yoshimura et al. 2009). SVF cells also
offer the important benefit that they are easily obtained from fat tissue and their
harvesting through liposuction is associated with low donor-site morbidity.
During autologous fat harvesting, processing, and grafting, different parameters
such as tumescence solution, diameter of the cannula and its lateral perforations, and
centrifuge settings may play an important role. Therefore, these parameters are
currently assessed in order to improve cell viability and amount of grafted adipose
tissue.
24 A. Haumer et al.
Fig. 11 Graft prefabrication using fat-derived cells. i): isolation SVF cells, (ii) fabrication of the
construct, (iii) In vitro culture, and (iv) in vivo implantation in the rat model. i) SVF cells, isolated
from human adipose tissue are (ii) seeded on a hydroxyapatite block (a) and cultured in a perfusion-
based bioreactor system. This block is then inserted into a hollow cylinder of bovine cancellous
bone (b), which simulates the avascular and acellular bone in osteonecrosis. (iv) Upon distal
ligation, the bundle of the left superficial inferior epigastric artery and vein of the nude rat is then
inserted through a drill hole in the center of the graft and implanted subcutaneously
Different liposuction techniques are available and routinely used: classic hand-
assisted liposuction, ultrasound-assisted, and laser-assisted liposuction. The last two
techniques where developed to facilitate liposuction for the surgeon and reduce
morbidity, e.g., bleeding. However, regarding autologous fat transfer, laser-assisted
liposuction impairs biology of the ASCs and decreases their differentiation potential
as well as viability (Chung et al. 2013). In contrast, ultrasound-assisted (third
generation) liposuction seems to have no negative effect on proliferative capacity
or osteogenic differentiation (Duscher et al. 2016; Panetta et al. 2009).
The endothelial cells, monocytes, and mesenchymal stromal cells contained in the
SVF potentially enhance tissue vascularization, osteoclast-mediated remodeling, and
bone formation (Riordan et al. 2009). More recently, SVF cells loaded onto
devitalized, hypertrophic cartilage were shown to enhance bone formation at ectopic
(subcutaneous) and orthotopic (calvarial defect) implantation sites (Todorov et al.
2016). These data opened the attractive possibility of enhancing the potency of bone
grafts by using intraoperatively gained, autologous fat-derived SVF cells (Saxer
et al. 2016).
Previous studies also showed that freshly isolated SVF cells seeded and cultured
within a large porous hydroxyapatite scaffolds in a perfusion bioreactor system can
generate in vivo osteogenic grafts with intrinsic vascularization and rapid engraft-
ment capacity (Güven et al. 2011).
For the treatment of challenging clinical scenarios, such as avascular necrosis of
the bone, the vasculogenic and osteogenic properties of SVF cells could be
combined with other prevascularization techniques to ensure rapid and thorough

vascularization of the implanted graft and enhanced bone formation.
The high complexity of the clinical scenario and the scientific questions related
requires interdisciplinary teams and tight collaborations between basic research and
surgical units.
An AV bundle, created in the rat by ligation of the superficial inferior epigastric
artery and vein (Fig. 8), has emerged as the least technically demanding and prone to
complications (as stated above) (Tanaka et al. 2003). This bundle can then be
combined with hydroxyapatite blocks seeded with SVF cells (Akita et al. 2004;
Kloeters et al. 2011; Willems et al. 2011) (Fig. 12).
To investigate the effectiveness, the dynamics, and possible advantages of this
innovative combined approach, the authors are currently carrying out animal exper-
iments. To better understand and possibly control this process, it has been investi-
gated which effects seeding of SVF cells has on prevascularization strategies such
the AV bundle or AV loop (Haumer et al. 2016). It is now critical to investigate
whether the SVF cells can directly contribute to tissue formation and vascularization
or if they would enhance endogenous cell recruitment by delivering paracrine
factors.
6.2 Graft Prefabrication Using Microvascular Fragments
Exploiting the own body in the process of tissue vascularization has become an
increasingly valuable approach in tissue engineering. Not only using it as a natural
bioreactor (see above), but also directly harvesting native, fully functional tissue
modules represents an intriguing option for tissue engineers (Laschke and Menger
2015). In this regard, the isolation of so-called microvascular fragments from fat
tissue has been studied lately. While the technique for collecting of the fat tissue
remains the convenient minimally invasive liposuction (Banyard et al. 2016;
Choudry et al. 2008) in contrast to the above-mentioned cell isolation techniques
from fat tissue, such as SVF cells (Klar et al. 2014; Mehrkens et al. 2012; Wittmann
et al. 2015), harvesting and transplantation of microvascular tissue fragments offer
the important advantage of an intact tissue microenvironment. Having physiological
vessel morphology allows “rapid developing stable, blood-perfused microvascular
networks avoiding complex and time-consuming in vitro or in situ incubation
periods” (Laschke and Menger 2015). Moreover, the much shorter enzymatic
digestion of the tissue (5–10 min) increases cell viability and yield (Laschke and
Menger 2012).
The few experimental studies that have been conducted in this regard are
widespread over the entire field of regenerative medicine. Microvascular tissue
transfer has been applied from (Laschke and Menger 2012, 2015; Pilia et al.
2014) to cardiology (Shepherd et al. 2007) and diabetes research (Hiscox et al.
2008).
26 A. Haumer et al.
6.3 Graft Prefabrication Using 3D Printing and Microfluidic

Systems
The promising developments in the microscale field, involving microfluidic systems,

allow investigating highly organized microvascular network formation within tis-
sues under defined physical, mechanical, and biological conditions (Hasan et al.
2014; Laschke and Menger 2015). By combining hydrogels, which can resemble
defined microenvironments, specific cell types, such as endothelial cells, the con-
trollable microfluidic system represents animportant tool in basic research (Leung
et al. 2012). For the successful clinical translation of these systems, “it will be
necessary to further adapt current concepts to the physiological requirements of
implantable tissue substitutes. This, for instance, involves the generation of func-
tional microvessels, which do not only consist of endothelial cell-lined channels but
resemble the multicellular architecture of natural blood vessels, and their incorpo-
ration into adequate biocompatible extracellular matrices” (Laschke and Menger
2015).
The upcoming of 3D printing has allowed creating custom-made prosthetics
which eased the way to a more personalized medicine. Tissue engineering and
regenerative medicine have developed biomaterials with implemented cells to gen-
erate 3 dimensional macroscopic tissues and thus fabricate a living scaffold
(Bertassoni et al. 2014a; Bertassoni et al. 2014b; Tasoglu and Demirci 2013).
So far 3D printing has been used for vascularization strategies by a simple
sacrificial template approach. Basically, a primary template is generated by 3D
printing, which then serves as a mold for the secondary biomaterial. The primary
scaffold is then dissolved by chemical, physical, or thermal means, which leaves a
3D scaffold with interconnected channels (Engineering Pre-vascularized Scaffolds
for Bone Regeneration). In case the secondary material is loaded with cells, there
have been developed ways to avoid cell injury during the dissolution process, such
as PLGA coating (rapid casting of patterned vascular networks for perfusable
engineered three dimensional tissues; hydrogel bioprinted microchannel networks
for vascularization of tissue engineering constructs). Bertassoni et al. showed that
the interconnected microchannels of 3D scaffolds allow for a better differentiation of
osteoblastic cells (by increased alkaline phosphatase) as well as of endothelial cells
which use the lumen as supporting matrix. (hydrogel bioprinted microchannel
networks for vascularization of tissue engineering constructs). This technique
bears great potential in tissue engineering since it has shown to lead to a well-
defined vascular network without the need to print cells in the hydrogel (creating
perfused functional vascular channels using 3D bio-printing technology).
7 Conclusions
Tissue loss represents a big challenge in modern medicine. The bigger the defect or
the higher the complexity, the more sophisticated techniques have to be developed.
We have seen that plastic and reconstructive surgery can propose a wide spectrum of
different flaps and grafts which can be used for virtually any shape, size, and
complexity of the defect. However, there are several bottlenecks associated with
current surgical methods, such as donor site morbidity, significant complication
rates, and the highly technically demanding surgeries (especially microsurgery).
Tissue engineering has tried to tackle these hurdles by trying to understand the
fundamental processes and mechanisms which underlie vascularization and engraft-
ment, by enhancing these through development of new methods and drugs and by
elaborating adequate animal models. Nevertheless, tissue engineering has its limita-
tions when it comes to clinical translation and impact. It appears logical to conclude
that a joint venture between both surgery and tissue engineering is necessary for
further progress in the field. The new field which results out of this process, the
so-called Regenerative surgery, could provide the tools to successfully overcome the
above-mentioned hurdles. In this chapter, we have focused on techniques for bone
tissue replacement and the area of plastic and reconstructive surgery, but the
principles of regenerative surgery can be applied to multiple other tissues and
surgical fields.
Acknowledgments This work was partially supported by The Swiss National Foundation (Grant
number 310030_156291), The European Union, FP7 Marie Curie Actions (Project iTERM) and
The Osteology Foundation (Project No. 13-059).
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Microsurgical Approaches for In Vivo
Prevascularization
Christoph Koepple, Ulrich Kneser, and Volker J. Schmidt
Abstract
The rapid and sufficient vascularization of large tissues is the main obstacle to the
broad implementation of tissue engineering (TE) into clinical practices. Typically,
the vascularization of engineered tissues is achieved after implantation, by stim-
ulating the ingrowth of surrounding blood vessels via the delivery of angiogenic
factors, the addition of angiogenic cells, and the optimization of scaffold proper-
ties. Although these approaches showed promising results, the ingrowth of the
host’s vasculature into the implant remains slow. In a parallel effort, various
prevascularization approaches were developed, which aim at inducing the forma-
tion of a vasculature within engineered tissues, before implantation. Such a
prevasculature can connect to the host’s vasculature and rapidly perfuse the
implant. However, building a patterned, hierarchical, functional vascular tree
that can be hooked to the host, possibly via microsurgery, is a long-lasting
challenge. Current approaches of prevascularization include the in vitro induction
of endothelial cells organization into a microvascular network and the in vivo
incubation of an engineered tissue within a surgically prepared angiogenic site
(e.g., arteriovenous loop). This last approach, rooted in surgical practices, allows
for the ingrowth of a hierarchical, functional vasculature within the construct,
which can connect to the host upon transfer to the secondary site of defect. Here,
we outline this family of promising surgical strategies aiming at the in vivo
formation of vascular networks within engineered tissues.
C. Koepple (*) • U. Kneser • V.J. Schmidt

Department for Hand-, Plastic- and Reconstructive Surgery, BG Unfallklinik Ludwigshafen,
University of Heidelberg, Heidelberg, Germany
e-mail: christoph.koepple@bgu-ludwigshafen.de; ulrich.kneser@bgu-ludwigshafen.de;
schmidtvolker@gmx.net

DOI 10.1007/978-3-319-21056-8_17-1
2 C. Koepple et al.
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Prevascularization Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1 Surgical Approaches for In Vivo Prevascularization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3 Prefabrication and Prelamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
4 The Arteriovenous Loop Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4.1 AV Loop Chamber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4.2 Chamber Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4.3 Matrix Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.4 Quantification of AV Loop Associated Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.5 AV Loop as a Model of De Novo Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
4.6 Clinical Implications and Translational Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5 Further Microvascular Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
6 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1 Introduction
A sufficient oxygen and nutrition supply immediately after implantation of large and
engineered tissues remains a main obstacle for their long-term survival. Hence,
avoiding ischemia time of implantation is a vital aspect for the broad implementation
of tissue engineering (TE) into the clinical practice. Classical strategies to optimize
vascularization focus on modification of the engineered tissue itself. Examples are
delivery of angioinductive factors, application of angiogenic cells or modification of
chemistry, and surface and architecture of the scaffolds (Bleiziffer et al. 2011;
Arkudas et al. 2007a; Brudno et al. 2013; Shandalov et al. 2014; Mehdizadeh
et al. 2013). Although many of these concepts showed promising results, the
invasion of the host’s microvessel is still a time-consuming process. Dynamic
quantification of growing neovessels suggests an elongation rate of 5 μm/h (Utzinger
et al. 2015). To overcome these limitations, tremendous efforts were undertaken to
prevascularize implants by forming a vasculature within the implant that can rapidly
connect to the host vasculature. Prevascular networks decrease ischemia time within
TE constructs (Levenberg et al. 2005) and contribute to the formation of the tissue
(Rivron et al. 2012). It remains however challenging to use this approach to form a
patterned, hierarchical, functional vascular tree. A second approach, rooted in
surgical practices, aims at incubating the implant in vivo, within a surgically
prepared angiogenic site, to promote the ingrowth of a vasculature. This leads to
the formation of complex, functional vascular trees within implants that can then be
transferred to the final site of defect and directly connected to recipient vessels by
means of microsurgical techniques. In the following, we outline several promising
strategies for the in vivo formation of hierarchical vasculature with clinical rele-
vance. These strategies add to the current understanding of angiogenesis and safe
clinical implementation of artificial tissue constructs for coverage of complex tissue
defects.
Microsurgical Approaches for In Vivo Prevascularization 3
2 Prevascularization Strategies
2.1 Surgical Approaches for In Vivo Prevascularization
In vivo prevascularization occurs whenever a construct is implanted into a well-

vascularized tissue with the intention of inducing the ingrowth of surrounding blood
vessels, for future local modifications (e.g., addition of bioactive factors or cells) or
transplantation into a defect site. Surrounding extrinsic vessels will eventually grow
into the newly introduced structure depending on the scaffold’s properties and the
local conditions. After a distinct period of angiogenic ingrowth, the prevascularized
scaffold can be loaded with bioactive cells (Takeda et al. 1995; Arkudas et al. 2007b)
and eventually transferred into an ectopic tissue defect.
Cassell et al. described two different modes of vascularization of scaffolds after
implantation: The vascularization of constructs from the outside was defined as
“extrinsic vascularization.” In contrast, vascularization from vessels placed inside
the implanted scaffolds, and originating from an implanted vascular axis such as an
AV bundle or an AV loop, was defined as “intrinsic” (Cassell et al. 2002).
This surgical approach is successfully applied for specific clinical problems (see
below). At the recipient site, the host vasculature eventually develops interconnec-
tions with the preformed microvessel network of the transferred construct. This
process – also termed inosculation (Laschke et al. 2008) – is, however, still time-
consuming. Several challenges thus remain including the rapid connection of the two
vasculatures, the control of the spatial distribution of the newly formed microvas-
culature, which is typically random and, most importantly, the restricted ingrowth of
the vasculature, which is, in the extrinsic mode especially, restricted to the external
parts of the construct.
The intrinsic axial vascularization of a construct along a main vessel axis proved
very efficient and allows instant reperfusion of the in vivo prevascularized construct
after transplantation to the defect site (Tanaka et al. 2000). In general there are two
different options for a central vascular axis: arteriovenous loops and arteriovenous
bundles (Polykandriotis et al. 2006, 2007a; Dolderer et al. 2007; Kawamura et al.
2006; Tanaka et al. 2006). The use of arteriovenous loops for vascularization has
been first described by Erol and Spira (1979). They successfully constructed a
prefabricated full-thickness skin graft dependent on a centrally located vascular
axis. Since then, many modifications of this approach were developed in which
axial blood vessels are embedded into the center of the scaffold and serve as a
starting point for its intrinsic vascularization (Tanaka et al. 2003). After pre-
vascularization, the construct can then be freely transferred by standard microsurgi-
cal techniques. This results in an instant reperfusion of the construct. Since these
early reports, this approach has been used to generate connective, functional, and
organ-specific tissues and has in a few selective cases found its way into clinical
practice, i.e., when treating patients suffering from large bony defects after osteo-
myelitis (Horch et al. 2014).
4 C. Koepple et al.
3 Prefabrication and Prelamination
Prefabrication and prelamination are two distinct but related surgical strategies to
induce tissue vascularization that are also utilized in different tissue engineering
approaches. Both concepts involve a primary operation in order to generate a distinct
axially vascularized flap composed of autologous tissue (Pribaz et al. 1994, 1999).
These multistep approaches have already found their way into clinical practice
(Germann et al. 1998). In its most simple form, any insertion of a biocompatible
material at the primary surgical site be it for generation of vascularized constructs or
to achieve expansion or tissue enlargement can be regarded as a type of pretransfer
flap modification. Tissue expansion is a commonly used method in plastic and
reconstructive surgery to achieve additional tissue growth (Argenta et al. 1983).
The most frequently used clinical example is the expansion of skin through mechan-
ical overstretch. The newly grown skin is virtually indistinguishable from the
surrounding tissue regarding its texture, thickness, and color while simultaneously
minimizing scars and risk of rejection. More complex examples of flap modifications
involve 3D shaping and placement of osseointegrated implants into osteocutaneous
flaps for fast and functional maxillofacial reconstruction (Holle et al. 1996).
The term prelamination describes a multiple-step procedure where the implanta-
tion of different tissue layers or components into an axially vascularized territory
represents the first step. The composite flap is then, in the final step, transferred to the
defect site based on its axial vascular pedicle.
After implantation of an engineered construct into a region with adequate perfu-
sion (i.e., muscle or the muscle encasing fibrous fascia) de novo vascularization of
the construct emerges from the surroundings. This ultimately enables the
reimplantation into the host defect by either a pedicled or free microsurgical transfer.
Instant reperfusion of the construct is achieved and time-to-reperfusion is kept to a
minimum. Originally described by Erol (1976) in a dog model, prelamination
techniques rapidly found their way into clinical practice when surgical reconstruc-
tion requires multiple functional tissue types as it is often the case in oto-rhino-
laryngeal and penile defects (Guo and Pribaz 2009; Warnke et al. 2004). Utilizing the
angiogenic potential of the implantation site, the construct becomes extrinsically
vascularized by vessel sprouts from the surroundings. The radial forearm flap with
its ease of access or the medial thigh region is preferentially used in prelamination
techniques. Their long and reliable pedicles have proven their use in many multi-
layered constructs for ear, tracheal, esophageal, and penile reconstruction.
The term prefabrication was primarily coined by Yao (1982) as well as Pribaz and
Fine (1994) for the implementation of vascular pedicles in two-staged free tissue
transfers. These early reports paved the way for subsequent comparative studies on
intrinsic properties of various types of vascular carriers to induce neovessel
sprouting. Prefabrication provides an axial blood supply to a given donor site by
implanting a vascular carrier (either an arteriovenous bundle or an arteriovenous
loop) into the donor tissue, thus rendering that tissue unit transferable once neo-
vascularization has occurred (Germann et al. 1998; Guo and Pribaz 2009). Alterna-
tively, new axially perfused tissue constructs can be generated in vivo by implanting
autonomously vascularized fascia (the muscle encasing fibrous tissue) into previ-
ously randomly perfused tissue units. Khouri et al. (1995) even combined pre-
expansion with a fascia-dependent vascularization method in a so-called
prefabricated-induced-expansion (PIE-) flap. In this technique, a vascularized
temporoparietal fascia serves as a vascular carrier. By implementing a tissue
expander beneath the fascia, the size of the newly formed vascularized construct
can further be increased and ultimately used for, e.g., facial reconstruction as a
composite capsulofasciocutaneous flap.
In the following section, the application of these above-mentioned techniques
will be discussed with a focus on tissue engineering and regenerative medicine.
4 The Arteriovenous Loop Model
In 1979 Erol and Spira published their highly regarded work based on a micro-
surgically constructed arteriovenous fistula (Erol and Spira 1979). After creating an
arteriovenous fistula by the use of an interpositional vein or arterial graft, they
observed the formation of a new capillary bed. It has been shown that an arteriove-
nous (AV) loop with an interposed vein graft was least likely to occlude and had the
highest potential to induce luminal vessel sprouting (Tanaka et al. 2003).
Rats are the most frequently used animal model to elucidate the full potential of
the AV loop and its use in flap prefabrication. In this model, the saphenous
neurovascular bundle of the anesthetized rat is exposed by a midventral incision.
The contralateral saphenous vein is then dissected, and an approximately 20 mm
long venous graft is harvested and interposed between the saphenous artery and vein
creating an arteriovenous shunt (Fig. 1a). The AV loop model has already been
transferred into several larger animal models (Tanaka et al. 2006; Beier et al. 2010;
Eweida et al. 2012).
4.1 AV Loop Chamber
In order to demonstrate the feasibility of skin grafted island flaps, Tanaka and
Morrison et al. (2000) were the first who introduced an additional cylindrical
chamber consisting of polyestercarbonate which isolated an AV loop-based matrix
flap from the surrounding tissues. This group also characterized tissue formation in
the isolation chamber following implantation of different types of scaffolds in detail
(Tanaka et al. 2000, 2003; Mian et al. 2000). By incorporation of a vascular chamber,
artificial tissue constructs can easily be raised as separate axially vascularized flaps
without the need to further dissect the adjacent tissue. Additionally, a steeper oxygen
gradient can be maintained. Hypoxia has recently been identified to be an essential
factor for flow-driven angiogenesis and capillary network formation (Watson et al.
2013).
The material used for the isolation chamber should be inert, nonresorbable, and
biocompatible in order to reduce foreign body reactions and to maintain an angio-
6 C. Koepple et al.
Fig. 1 Experimental setup of the AV (a) and AA loop (b). The vein graft is placed in an isolating
Teflon chamber (c) and fixed to the underlying fascia (d). Ultrasonic transit-time flow probes were
used to quantify blood flow characteristics (e) (Reproduced in modified form, with permission, from
(Schmidt et al. 2015))
inductive environment for a distinct period of time. Most frequently, Teflon (Kneser
et al. 2006) and polycarbonate (Lokmic et al. 2007) chambers are used. Recently,
titanium cages have also been investigated in the AV loop model with the intention to
combine extrinsic and intrinsic vascularization (Arkudas et al. 2012a). The particular
dimensions of the chamber vary with the experimental setups, specific scientific
endpoints, and the used animal models.
Based on the author’s experiences, perpendicular pins within the chamber are
useful in order to prevent secondary vessel dislocation, twisting, kinking, and
thrombosis of the vascular pedicle (Fig. 1c; Arkudas et al. 2009). However, other
authors preferred anchoring the AV loop to the chamber by means of ligated
superficial epigastric vessels (Lokmic et al. 2007).
4.2 Chamber Modifications
As the chamber is a key element for tissue generation in the AV loop model, varying
its shape and form can have profound effects on tissue formation.
The amount of extrinsic vascularization can be controlled by using a pierced
chamber design. These modifications enable interconnections of the adjacent
environment with the newly formed tissue enhancing nutrient supply as well as
tissue vascularization and growth (Dolderer et al. 2007). Enlarging the chamber
volume while simultaneously increasing pore size can improve tissue growth
(Tanaka et al. 2006). However, allowing extrinsic vessel ingrowth simultaneously
hampers oxygen gradients within the chamber construct and might impede flap
harvest. Arkudas et al. (2012a) quantified the increase in neovessel formation in
axially vascularized tissue constructs with additional extrinsic vascularization. After
2 weeks 83% of the vessels showed a connection to the centrally located AV loop.
By allowing extrinsic vascularization to interconnect with the axial AV loop, con-
struct weight and volume can be significantly enhanced (Zdolsek et al. 2011).
Although beneficial and in wide-spread practice, it is important to state that the
AV loop chamber is not a prerequisite for AV loop-dependent tissue growth. Several
authors completely abandoned the use of a chamber by folding bilayered artificial
skin substitutes around the vascular axis. Combining bioresorbable materials with an
isolating silicone membrane, a proangiogenic setting can, however, still be
maintained within these constructs (Tanaka et al. 2000; Manasseri et al. 2007).
4.3 Matrix Modifications
Although tissue formation is not crucially dependent on an extracellular matrix

within the AV loop chamber (Mian et al. 2000), it is frequently added serving as a
scaffold for cell invasion and tissue growth. In its absence, a temporary fibrin
exudate originating from the AV loop needs to be gradually replaced by migrating
inflammatory, endothelial, mesenchymal cells, and mature connective tissue
(Lokmic et al. 2007). Matrices in the chamber serve as a three dimensional structure
providing a protected environment for a homogenous development of a microcircu-
latory system. Any scaffold has to meet specific requirements apart from being
nontoxic and biocompatible, which will vary dependent on the desired functional
outcome: ideally, avital scaffolds resemble the extracellular matrix of the desired
functional, organ-specific tissue and will affect microenvironments within the con-
struct to mimic in vivo milieus. Dependent on the tissue type and its metabolism rate,
regulating the biodegradability of the scaffold may be an essential step in providing
sufficient time to incorporate new cells without the necessity of a subsequent surgical
removal. As the extracellular matrix is the structural basis for tissue formation,
several attempts to manipulate the microenvironment have shown great promise in
influencing resulting tissue types. Several cell types and delivery vehicles incorpo-
rated into the scaffold have been used to deliver proangiogenic macromolecules or
maintain a suitable microenvironment (Bouhadir and Mooney 2001; Kohane and
Langer 2008).
As a biopolymer matrix fibrin has been extensively studied in the context of tissue
engineering. When combining fibrinogen and thrombin, a fibrin hydrogel is formed.
As fibrin is inexpensive and off the shelf ready to use, it is regularly used to
investigate angiogenesis and cell migration. Furthermore, it can be used as a release
system for bioactive macromolecules (Arkudas et al. 2007a; Fiegel et al. 2010; Jeon
8 C. Koepple et al.
et al. 2005). By varying the concentrations of the components, scaffold stiffness and
microporosity can be tightly regulated and may ultimately effect tissue vasculariza-
tion. Reducing scaffold density by lowering the concentrations of fibrinogen corre-
lates with a superior vascularization (Arkudas et al. 2012b). However, reducing
fibrin concentration also effects the degradation of the tissue by proteolytic enzymes
and its tendency to contract (Cassell et al. 2001). Although not ideally suited as a
tissue engineering matrix alone, combining it with solid co-matrices can generate
more complex and functional tissue constructs (Rath et al. 2012). Cassell et al.
(2001) demonstrated the superior properties of poly-D,L-lactic-co-glycolic acid
(PLGA) and matrigel for fast development of a functional microcirculatory network
in the AV loop model. However, from a translational standpoint, matrigel possesses
significant shortcomings. Matrigel is a reconstituted basement membrane prepara-
tion consisting of structural macroproteins like laminin, entactin, collagen, and
heparan sulfate proteoglycans. As it is derived from Engelbreth-Holm-Swarm
(EHS) mouse sarcoma cells, it still lacks FDA approval. PLGA on the other hand
is highly customizable by production technologies, variations in molecular weight,
copolymer ratio, and porosities. A foreign body reaction as reported may, however,
ultimately hamper its clinical benefit (Cao et al. 2006).
In order to stimulate sprouting of blood vessels, Arkudas et al. (2007a) applied
fibrin-immobilized recombinant VEGF and bFGF into the loop chamber. While both
factors significantly improved absolute and relative vascular densities, they also
negatively affected tissue volume by an accelerated matrix degradation and prote-
olysis. These observations may ultimately limit the use of recombinant pro-
angiogenic growth factors in fibrin gels. However, combinations of mechanically
stable scaffolds and fibrin gel might overcome this limitation (Rath et al. 2011).
Several novel strategies are currently pursued to maintain a defined protein concen-
tration over a long period of time. By various matrix modifications and implemen-
tation of individual binding sites, stable protein release and long-lasting and constant
protein concentrations can be realized. Other concepts pursue various gene delivery
strategies (Nowakowski et al. 2015; Bleiziffer et al. 2007). However, all additive and
angio-inductive factors may eventually bear oncogenic potential in vivo and may
aggravate translational approaches and clinical implementation.
4.4 Quantification of AV Loop Associated Angiogenesis
Initially, quantification of AV loop dependent angiogenesis was performed manually

by analyzing histological cross sections. Quantification of neovessel formation is
obtained by morphometric analysis of hematoxylin and eosin or Masson’s Trichrome
stained histological cross-sections. Small caliber vessels, however, are more easily
and conveniently detectable with prior intravascular perfusion of India ink or
Microfil. Standard immunohistochemical staining protocols are then applied for
further molecular analysis of different types of neovessels and protein expression.
Manual assessment of neovessel formation is time-consuming and user-dependent
and the amount of analyzed data is limited. In order to overcome these limitations,
our group developed an automatic and observer-independent quantification algo-

rithm to analyze axial vascularization (Weis et al. 2015). However, vascularization of
the AV loop matrix is a highly dynamical process. Histological cross sections can
only provide a glimpse of its structure and three dimensional process. By analyzing
corrosion casts of vascularized constructs with a scanning electron microscope, a
more accurate representation of the spatial distribution of the formed neovessels and
their three dimensional morphology could be obtained. We provided evidence that
several subtypes of angiogenesis and vessel formation like sprouting, branching, and
intussusceptive angiogenesis occur simultaneously in the AV loop construct (Poly-
kandriotis et al. 2007b). Quantitative 3D analysis of axial vascularization patterns is
also feasible using micro-CT angiography techniques following intraarterial microfil
injection (Arkudas et al. 2010). However, all of these methods rely on postmortem
quantification. Quantification of angiogenesis is a new field of scientific interest and
especially in Tissue Engineering of vital importance. Several quantification methods
have their own ranges of applications and drawbacks. Thus, micro-PET and high
resolution MRI as well as combined multimodal imaging strategies may provide
quantification of a functional microvascular and angiogenic environment ante
mortem (Beier et al. 2010; Nam et al. 2014).
4.5 AV Loop as a Model of De Novo Angiogenesis
The underlying basis and mediating pathways may differ in different angiogenesis
subtypes. The current understanding of angiogenesis is thereby continuously chal-
lenged (Semenza 2007). In the adult organism, physiological angiogenesis is only
observed under tightly regulated and specific conditions such as wound healing, in
the course of the female menstrual cycle and in tumor growth (Carmeliet 2003).
However, as stated above, hypoxia-driven angiogenesis is closely dependent on
persistent blood flow (Watson et al. 2013). Although the AV loop model was
extensively studied in a descriptive and morphometrical manner, data on underlying
proangiogenic trigger factors and mediating signaling pathways are still lacking.
There is, however, an ongoing dispute whether or not the venous graft and especially
the graft endothelium is decisive for vessel sprouting. Zdolsek et al. (2011) stated
that even decellulized, cold-stored allograft veins are able to sufficiently trigger
neovessel formation. Our group clearly demonstrated in microdissected vascular
corrosion casts that neovessel formation predominantly originates from the graft
which is thought to serve as the main effector for flow-dependent angiogenesis
(Polykandriotis et al. 2008).
The mechanical forces of shear stress have previously been considered to be the
major stimulator of angiogenesis in the AV Loop (Asano et al. 2005). By
establishing a flow-modified arterioarterial model (AA loop), which serves as a
corresponding negative model, we recently provided evidence that increased blood
flow and most likely vascular wall shear stress is required to initiate neovessel
formation in the vein graft (Figs. 1 and 2; Schmidt et al. 2015). Intraperitoneal
application of pimonidazole – a hypoxia marker – revealed intense tissue hypoxia
10 C. Koepple et al.
Fig. 2 Micro CT angiography (a–d) and vessel formation (e–h) in the AV and AA loop. In marked
contrast to the AV model, neovessel sprouting is not originating from the venous graft in the AA
model. Scale bars: 1000 μm (Reproduced with permission, from (Schmidt et al. 2015))
within the chamber (Hofer et al. 2005). In line with these results, we recently could
demonstrate that HIF-1a upregulation by systemical application of DMOG
(dimethyloxallyl glycine), a PHD (prolyl hydroxylase) inhibitor, promotes vessel
density (Yuan et al. 2014). Watson et al. (2013) showed that shear stress is required
for angiogenesis in response to hypoxic signaling. Thus, it is likely that both factors
are simultaneously critical for neovessel formation within the AV loop construct.
Interestingly, the hemodynamical alterations within the AV loop specifically increase
the expression of endothelial gap junction protein Connexin43 (Cx43). Contrary to
other endothelium expressed connexins, Cx43 seems to play a crucial role in flow
dependent and adult, nontumor-associated angiogenesis (Wang et al. 2008; Laws
et al. 2008). Cx43 may at least be partially responsible for triggering a flow-
dependent, hypoxia-driven de novo angiogenesis (Cowan et al. 1998).
4.6 Clinical Implications and Translational Approaches
Different concepts of tissue engineering have evolved in recent years, and with
multidisciplinary research using innovative technologies and methods, clinical
application is imminent.
Tissue engineering strategies based on induction of axial vascularization provide
direct restoration of perfusion within the artificial constructs immediately after
anastomosis to the host microvasculature. After demonstrating the feasibility of
AV loop-based engineering approaches in a variety of small and large animal
models, the ongoing research is currently focusing on the successful formation of
functional and organ-specific tissue types. Mian et al. (2001) described the stimu-
lating effect of an AV loop on the growth and proliferation of isografted fibroblasts.
Matsuda et al. (2013) could outline the proangiogenic benefits and stimulating effect
of human adipose-derived stem cells implanted into the AV loop chamber. The
formation of adipose tissue might ultimately benefit novel breast reconstruction
strategies. It has been shown that large-volume adipose tissue can be generated by
implementing a preexisting axially vascularized fat flap into an isolation chamber

(Dolderer et al. 2007). Furthermore, combined application of proangiogenic growth
factors like VEGF, FGF-2, and PDGF-BB could synergistically promote adipose
tissue production (Rophael et al. 2007). Besides these attempts to generate a
sufficiently large amount of connective tissue, several efforts are currently under-
taken to build functional tissue types. Promising results of fetal hepatocyte trans-
plantation and vascularization (Fiegel et al. 2010) as well as hormone-producing
pancreatic islet cells (Brown et al. 2006) have been published. Bitto et al. (2013)
were able to induce myogenic differentiation of mesenchymal stem cells via
neurotization of a primarily axially vascularized tissue construct. Primary cultured
myoblasts mature into functional striated muscle fibers when inserted into a chamber
with an AV loop (Messina et al. 2005). In a groundbreaking study of Morritt et al.
(2007), implantation of neonatal rat cardiomyocytes into an AV loop chamber
generated a contractile and functional cardiac muscle flap. Furthermore, the feasi-
bility of a functional transfer of an engineered cardiac muscle flap (ECMF) onto the
epicardium of syngeneic rats has been successfully performed. Despite being fully
functional and viable, the newly transplanted myocardial tissue flap was an auton-
omous unit without electrical integration into the recipient myocardium (Tee et al.
2012). Nevertheless, the concept of axial vascularization has great value in func-
tional tissue engineering, and we are only beginning to understand its full potential.
Coverage of large tissue defects after trauma or tumor excision frequently
involves autologous bone transfer or free vascularized bone flaps. Bioartificial
generation of bone could at least partially solve some of the shortcomings and
complication like limited tissue availability, wound infections, and donor side
morbidity. Furthermore, by combining various tissue engineering strategies within
a single AV loop chamber, custom-tailored artificial flaps consisting of multiple
tissue types can be engineered. A processed bovine cancellous bone matrix (PBCB)
has already been successfully vascularized by means of an AV loop (Kneser et al.
2006). In a succeeding study, our group has successfully demonstrated that
vascularized PBCBs promote survival of transplanted osteoblasts and expression
of bone-specific marker genes (Arkudas et al. 2007b). In order to vascularize and
generate large stable bone substitutes, biphasic calcium phosphate ceramic (Beier
et al. 2010) and PBCBs (Beier et al. 2011) have been used in a sheep model. The
generation of artificial bone substitutes can be further optimized by adding multiple
proangiogenic and tissue-specific, extrinsic factors like bone morphogenetic protein-
2 (BMP-2) and mesenchymal stem cells (MSC) (Boos et al. 2013; Buehrer et al.
2015).
Using a ligated vascular bundle consisting of the saphenous artery and its
accompanying vein, an intrinsically vascularized multilayered skin flap has already
been validated for successful coverage of soft tissue defects in an animal model
(Tanaka et al. 2006). Vascular bundles were also frequently used in revascularization
strategies as for example in the treatment of osteonecrosis (Tamai et al. 1993). Flow-
dependent and AV associated angiogenesis has also been reported in distal digit
replantation using an arterial-to-distal arteriovenous fistula (Inoue and Tamura
1991).
As stated earlier, when comparing the angiogenic potential of different types of

vascular carriers for tissue generation, Tanaka et al. (2003) found that the AV loop
bears the greatest proangiogenic features. This finding has been confirmed by our
group in a processed bovine cancellous bone matrix (Kneser et al. 2005). However,
until now clinically relevant revascularization strategies are most commonly based
on AV bundles. From a clinical point of view, the use of blood vessels may be limited
by location and length. The creation of AV loops may solve this issue as they can be
created at virtually any surgical site without regarding the vascular pedicle length.
Furthermore, the use of AV loops ensures minimal morbidity at the donor site. In the
future, autologous vein grafts might even be replaced by tissue engineered vascular
constructs (Naito et al. 2011; L’Heureux et al. 2006). Regardless of their potential
use for intrinsic tissue vascularization, AV loops are already frequently used in
reconstructive strategies in order to create extra-anatomical, high-flow,
low-resistance shunts within a distant defect area when local vessels are either
damaged by trauma, secondary comorbidities, or prior radiation (Cavadas 2008;
Oswald et al. 2007; Brüner et al. 2004; Kneser et al. 2013). Besides solely serving as
vascular supplies for free flap transfer, these AV loops might offer additional value
when utilizing their intrinsic potential for tissue generation and may contribute to
novel and complex multistep reconstructive strategies.
5 Further Microvascular Approaches
In an attempt to combine the benefits of viable bone autografts with improved

geometric matching and most importantly absence of donor morbidity, Pelzer and
Bishop (Pelzer et al. 2007) proposed a novel model of vascularized bone allograft
transplantation. In addition to microvascular reconstruction of the supplying nutrient
vessel, a recipient-derived AV bundle was intramedullary implanted to promote host-
specific osseous neoangiogenesis. During surgical vascularization, short-term immu-
nosuppression is applied. After a new functional host-derived blood supply has been
developed, immunosuppression may then be stopped, deliberately accepting nutrient
vessel occlusion. After 2 weeks, blood flow and capillary density was significantly
increased in AV pedicled bone allotransplants originating from the autologous vessel
bundle (Ohno et al. 2007). In a subsequent study in a larger animal model using
rabbits, Giessler and Bishop (Giessler et al. 2009) focused on healing and transplant
characteristics as well as biomechanical features. Using radiographic-based ana-
lyses, healing characteristics in immunosuppressed host-derived neovascularized
femura were comparable to pedicled autotransplants. These results might pave the
way for functional reconstruction of complex musculoskeletal defects (joints and
adjacent bone) without the drawbacks of long-term immunosuppression.
6 Future Perspectives
In summary, concepts of axial vascularization bear great clinical and interdisciplin-

ary research potential in order to generate large and multilayered artificial tissue
constructs. Axially induced vascularization is flow-dependent and most importantly
occurs in the absence of any added proangiogenic growth factors. Although growth
factors are able to modify and optimize tissue growth, they might, dependent on the
factor and origin, additionally bear oncogenic potential and might hamper fast
implementation into clinical practice. By means of its intrinsic abilities to induce
de novo angiogenesis, the AV loop model is an attractive way to circumvent these
limitations.
We and others are currently focusing on the formation of soft tissue and its free
vascular transfer to multilayered and critical wound defects as an alternative for free
conventional reconstructive flaps. While promising results of functional tissue types
like hepatocytes, cardiac, and skeletal muscle have been reported, there are still
obstacles we need to overcome for structural and functional integrity after
transplantation.
Free and axially vascularized soft tissue flaps have become a routine for complex
defect coverage and functional reconstruction in trauma patients. Although possible,
free soft tissue flaps in small animal models are extremely challenging because of the
experimental requirements of a prolonged two-staged microsurgical procedure on
the one hand and a small vessel diameter on the other. Additionally, wound healing
and blood clotting may fundamentally differ compared to humans. For this reason,
translation of these small animal experiments into larger animal models is necessary.
After confirmation in a large animal model, patients with extensive burn injuries
may be the best candidates for an AV-loop-based soft tissue free flap, when reducing
donor site morbidity and an increased therapeutic flexibility is mandatory. In the
future, these treatment concepts may represent an alternative treatment to conven-
tionally used autologous free flaps.
7 Conclusion
The underlying mechanisms of various types of angiogenesis are only beginning to

emerge. AV loop vascularization is fundamentally based on an increased flow rate
and a sufficient hypoxia gradient. As such it might constitute a useful tool to
decipher molecular and mechanosensitive endothelial proangiogenic signaling
pathways.
In our opinion, microsurgically generated AV loops represent a very promising
strategy to induce functional vascularization in bioartificial constructs. Dissecting its
intrinsic molecular properties will provide a better understanding of adult de novo
angiogenesis, which may not only be of critical importance in providing sufficient
oxygen supply in large bioartificial tissue constructs but may also lead to new and
alternative treatment options for a variety of vascular pathologies like critical limb
ischemia and peripheral arterial occlusive disease.
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Angiogenesis: Basics of Vascular Biology

Victor W. M. van Hinsbergh

V.W.M. van Hinsbergh (*)

# Springer International Publishing AG 2016 1

2 Neovascularization in Early Development

During early embryonic development, progenitors of vascular endothelial cells,

3 Different Forms of Angiogenesis

The expansion of the primitive vasculature occurs by sprouting and intussuscep-

Intussusceptive angiogenesis Sprouting Angiogenesis

Fig. 1 Intussusceptive (splitting) angiogenesis (a) and sprouting angiogenesis (b)

proliferative potential. These cells may harbor an important regenerative capacity

While sprouting angiogenesis refers to the expansion of microvessels that radiates

5 Dominant Growth Factors in Angiogenesis

The mechanisms of sprouting angiogenesis have become better understood and

6 Balancing Angiogenesis: Endogenous Inhibitors

Once a vessel is established it becomes less responsive to angiogenic factors.

7 Sprouting Angiogenesis: Initiation and Invasion

The endothelium of a quiescent vessel is enwrapped on its basolateral side by a

motifs), and plasminogen activator inhibitor-1 (PAI-1) (Handsley and Edwards

induce angiogenesis without VEGF/VEGFR-2 signaling as had been suggested

8 Extension and Stabilization of Sprouts: Dll4/Notch

Interestingly, activation of VEGFR-2 and VEGFR-3 induces the expression of

VEGFR-2 and VEGFR-3 on Filopodia

Internalization of VEGF:VEGFR-2 and -R3

9 Guidance of Tip Cells

It has been anticipated that the VEGF-induced response of sprouting endothelial

10 Metabolic Control of Sprouting

Important regulators of perfusion control in a microvascular bed are the shear

FOXO-1 accumulation, which makes FOXO-1 available for inhibition of nuclear

As soon as the sprout consists of several endothelial cells, a vascular lumen is

12 Anastomosis and Vessel Stabilization

Time-lapse movies of zebraﬁsh with green-ﬂuorescent labeled endothelium illustrate

connected vessel. Indeed, in vivo coverage by pericytes, which represent or derive

Once circulation is established, lymphatic vessels start developing, initially by

Late outgrowth EPC = ECFC

Fig. 3 Postnatal vasculogenesis is characterized by the incorporation of circulating endothelial

in the chapter on lymphangiogenesis in this book (Holnthoner), to which the reader

16 Generation of a Functional Microvascular Bed

The efﬁcient transport of ﬂuid requires a hierarchy of vessels throughout the

Optimal fluid low

Much stimulated by the initial studies on the vascularization of tumors, research on

redistribution (remodeling), and pruning of endothelial tubules into a functional

Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, Jeltsch M,

S. Chaudary, S. Rieger, H. Redl, and P. Dungel

S. Chaudary • S. Rieger • H. Redl • P. Dungel (*)

# Springer International Publishing AG 2017 1

As the research to enhance and improve tissue-engineered products is ongoing,

2 History of Low-Level Light Therapy (LLLT)

developing a “chemical rays” lamp to treat tuberculosis. He became famous for

3 Coherent Light (Laser) Versus Incoherent Light (LED)

oscillate in different directions. Also, a frequent and random change in phases

4 Biphasic Dose Response

To comprehend and reconstruct the “Arndt-Schulz law” in the ﬁeld of photobi-

5 Interaction of Light with Tissue

6 General Mechanism of LLLT

wavelength observed ranged in different parts of the red to near-infrared spectrum

observed formation of capillaries and neovascularization effect was diminished after

cytochrome c oxidase after exposure to 595–597 nm light was conﬁrmed, increasing

7.1 Secondary Angiogenic Effects of Supporting Cells After

As mentioned earlier, wound healing is a dynamic process, where multiple cells

VEGF, which guides angiogenesis in the injured tissue through upregulation by NO

(ASC) transplantation efﬁcacy by diminishing apoptosis of transplanted ASCs and

As organ regeneration techniques in the ﬁeld of tissue engineering are advancing,