Bringing new life to damaged bone: the importance of angiogenesis

in bone repair and regeneration

Steve Stegena,b, Nick van Gastela,b and Geert Carmelieta,b

aLaboratory of Clinical and Experimental Endocrinology, Department of Clinical and Experimental

Medicine, KU Leuven, 3000 Leuven, Belgium

bPrometheus, Division of Skeletal Tissue Engineering, KU Leuven, 3000 Leuven, Belgium

Corresponding Author:

Geert Carmeliet, M.D., Ph.D.

Laboratory of Clinical and Experimental Endocrinology, Department of Clinical and

Experimental Medicine, KU Leuven

O&N1 Herestraat 49 bus 902, 3000 Leuven, Belgium

Tel.: 0032 16 330 731

Fax: 0032 16 330 718

e-mail: geert.carmeliet@med.kuleuven.be

Disclaimer: The authors declare no conflict of interest

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Abstract

Bone has the unique capacity to heal without the formation of a fibrous scar, likely because

several of the cellular and molecular processes governing bone healing recapitulate the

events during skeletal development. A critical component in bone healing is the timely

appearance of blood vessels in the fracture callus. Angiogenesis, the formation of new blood

vessels from pre-existing ones, is stimulated after fracture by the local production of

numerous angiogenic growth factors. The fracture vasculature not only supplies oxygen and

nutrients, but also stem cells able to differentiate into osteoblasts and in a later phase also

the ions necessary for mineralization.

This review provides a concise report of the regulation of angiogenesis by bone cells, its

importance during bone healing and its possible therapeutic applications in bone tissue

engineering.

Keywords: bone healing, fracture, angiogenesis, VEGF, hypoxia, tissue engineering

Highlights:

- An adequate angiogenic response, together with abundant mesenchymal stem cells

and mechanical stabilization, is crucial for successful bone healing.

- The vasculature supplies oxygen and nutrients during bone repair, and may serve as

a niche for osteoprogenitor cells.

- The timely establishment of a vascular system in tissue-engineered constructs

ensures cell survival and thus improves ultimately bone formation.

- Preclinical data indicates the benefit of angiogenic factors for bone repair, but

translation into the clinic remains challenging.

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Abbreviations

αSMA alpha Smooth Muscle Actin

BMP Bone Morphogenetic Protein

BOEC Blood Outgrowth Endothelial Cells

Dll4 Delta-like protein 4

EC Endothelial Cells

EPC Endothelial Progenitor Cells

FGF Fibroblast Growth Factor

FGFR Fibroblast Growth Factor Receptor

HIF Hypoxia Inducible Factor

HRE Hypoxia Responsive Element

miRNA micro RNA

MMP Matrix Metalloproteinase

MSC Mesenchymal Stem Cells

PHD Prolyl Hydroxylase Domain protein

PlGF Placental Growth Factor

VEGF Vascular Endothelial Growth Factor

VEGFR Vascular Endothelial Growth Factor Receptor

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Introduction

The establishment of a functional vascular system is crucial during organ development as

well as during tissue repair. In bone, blood vessels not only serve as a source of oxygen and

nutrients, but also supply calcium and phosphate, the building blocks for mineralization. In

addition, blood vessels in the bone marrow appear to have a major role as a niche for both

the bone-forming skeletal stem cells and blood-forming hematopoietic stem cells [1]. The

connection between angiogenesis and osteogenesis is also evident during the healing of

bone fractures, as a timely and coordinated angiogenic response is of vital importance for

successful bone repair [2]. Increasing insight into the molecular and cellular processes

orchestrating the angiogenic cascade may help to develop novel treatments for fracture

healing, especially for clinical situations with a limited angiogenic host response such as

large bone defects or fractures with severe soft tissue trauma. In this review, we will highlight

the current knowledge of developmental skeletal angiogenesis and its regulation by

angiogenic growth factors, as well as the importance of the vascular system during bone

regeneration.

Angiogenesis: a critical process during facture repair

The molecular basis of physiological angiogenesis

When an organism or tissue grows beyond a size where passive diffusion for the exchange

of oxygen, nutrients and metabolic waste products becomes insufficient, the need for the

development of a vascular system emerges. Angiogenesis is the process by which the

organism establishes new blood vessels from pre-existing ones. Recent studies have

highlighted the fundamental aspects of vessel formation, including vasculogenic assembly,

vessel sprouting, lumen formation and vascular remodeling [3, 4].

Sprouting of endothelial cells (EC) is the first in a sequence of events during angiogenesis

[5]. In a resting vessel, both endothelial and mural cells form a basement membrane around

the vessel tube, preventing resident EC to migrate. When new blood vessels are needed in a

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growing tissue, local production of angiogenic growth factors is enhanced, which trigger the

EC to degrade the basement membrane extracellular matrix, a process mediated by matrix

metalloproteinases (MMPs). The action of these MMPs further fortifies the angiogenic

response by releasing proangiogenic factors that were stored within the matrix. Attracted by

the angiogenic signals, some EC become motile and express filopodia. These so-called tip

cells form the leading front of the newly developing vessel; the trailing EC are called stalk

cells. The key angiogenic factor Vascular Endothelial Growth Factor (VEGF) stimulates tip

cell induction and filopodia formation via VEGF receptor (VEGFR) signaling [4]. The filopodia

on the tip cells ‘sense’ the environment for attractive cues, guiding the sprouts into the tissue

stroma. Stalk cells are equipped to form tubes and branches, mediated by their high

proliferative capacity and the ability to stabilize the newly formed lumen. The priming of EC

into tip or stalk cells is also dependent on the Notch pathway, in close intimacy with VEGFR

signaling. In tip cells, VEGF signaling enhances Delta-like protein 4 (Dll4) expression, a

Notch ligand. Dll4 activates Notch signaling in the neighboring EC, thereby preventing their

switch to a tip cell phenotype and favoring a stalk cell phenotype. Novel vessel circuits are

created via the interaction of two neighboring tip cells (i.e. anastomosis). These connections

are then stabilized by several processes including the deposition of extracellular matrix, the

recruitment of pericytes, reduced endothelial cell proliferation and increased formation of cell

junctions [6]. In addition to the maturation of the endothelium, vascular branches are

remodeled to match its rigidity to the local tissue needs.

Angiogenesis and bone repair

The timely formation of new blood vessels is a critical process during embryonic and fetal

development [7, 8]. In the adult, angiogenesis occurs during physiological processes like

wound healing, the menstrual cycle, and pregnancy but also in specific diseases such as

intraocular neovascular disorders and tumorigenesis. As opposed to soft-tissue healing,

bone can regenerate itself without the formation of fibrous scar tissue and hereby maintains

its physiological and mechanical characteristics. Normal fracture healing in adults occurs

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through intramembranous or endochondral bone formation, closely mimicking skeletal

development in the embryo [9]. Intramembranous ossification is characterized by direct

formation of bone by committed osteoprogenitor cells and mesenchymal stem cells (MSC)

from the periosteum. During endochondral ossification, MSC differentiate into chondrocytes,

which in turn produce a cartilaginous matrix. Consecutively, this matrix undergoes

calcification and is eventually replaced by bone [10]. The scar-less regeneration of fractured

bones through the endochondral pathway might be attributed to the avascular nature of the

cartilage template. In fact, chondrocytes are metabolically well adapted to survive in poorly

oxygenated regions and still produce the extracellular matrix needed for mineral deposition,

and hereby likely contribute to optimal fracture healing.

Despite the remarkable regenerative capacity of bone tissue, fracture healing fails in about

10% of the cases leading to delayed union or non-union. Adequate vascularization has been

shown to be critical for successful bone healing, next to the presence of osteoprogenitor cells

and mechanical stabilization. Indeed, inappropriate blood vessel supply is a major cause of

delayed union or non-union during fracture healing [11]. More precisely, when fracture is

associated with large vascular injuries, the rate of impaired healing is as high as 46%,

exceeding by far the global 10% non-union rate [2]. In addition, inhibition of angiogenesis

during fracture repair in animal models resulted in the formation of fibrous scar tissue,

resembling human atrophic non-union [12].

Therefore, treatment modalities that promote tissue vascularization possibly provide a central

strategy to accelerate the healing response and tissue regeneration.

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Factors mediating angiogenesis during fracture healing

During the last years, extensive research has lead to the discovery of a plethora of

angiogenesis-stimulating growth factors that are important during bone development and

normal fracture healing. Some of these factors stimulate angiogenesis directly by inducing

neoangiogenesis and include factors such as VEGF, Placental Growth Factor (PlGF),

Fibroblast Growth Factor (FGF) or members of the Transforming Growth Factor beta family.

Others have angiogenic properties and mainly regulate the production of angiogenic

molecules; examples are Bone Morphogenetic Proteins (BMPs), angiopoietin, Platelet-

Derived Growth Factor and Insulin-like Growth Factor family members (Table 1). Here, we

will focus on the most extensively studied angiogenic growth factors VEGF, PlGF and FGF.

Vascular Endothelial Growth Factor

One of the most extensively studied angiogenic growth factors is VEGF, an endothelial cell-

specific mitogen. VEGF proteins are secreted by cells involved in skeletal development and

repair, including EC, macrophages, fibroblasts, smooth muscle cells, osteoblasts and

hypertrophic chondrocytes [13, 14]. Different from bone development, the early stage of

fracture repair is characterized by hematoma formation, which has strong proangiogenic

properties, predominantly due to the presence of VEGF [15, 16] (Figure 2). The importance

of an appropriate VEGF-mediated stimulation of angiogenesis during fracture healing is

eminent, as inhibition of VEGF activity through treatment with a soluble VEGFR or VEGF

antagonist resulted in impaired healing of femoral fractures and cortical bone defects in mice,

whereas local administration of VEGF lead to improved successful bone repair in both

models [15].

VEGF can influence bone regeneration by affecting bone cells indirectly or directly (Figure 3).

First, through its action on EC, VEGF induces the angiogenic process. Bone forming

precursor cells possibly migrate concomitantly with these blood vessels to the fracture callus,

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where they differentiate into osteoblasts. Secondly, through an angiocrine mechanism, VEGF

can stimulate EC to produce osteogenic cytokines that promote the differentiation of

progenitor cells into osteoblasts [17]. Lastly, VEGF may also directly influence osteoblast

function. In accordance, osteoblasts not only produce VEGF, they also respond to VEGF

itself, which regulates chemotaxis, proliferation and differentiation of osteoblasts [15, 18-21].

In accordance, VEGF inhibition leads to decreased differentiation of in vitro cultured primary

osteoblasts. In addition, conditional deletion of VEGFR-1 or VEGFR-2 [20-23] in

osteoprogenitors (Osterix-Cre) decreases trabecular bone mass in mice [24]. In this model,

the differentiation of osteoblasts lacking VEGFR-1 or VEGFR-2 was reduced, possibly due to

a decrease in VEGF-mediated RUNX2 induction. However, more experimental evidence is

warranted to elucidate the direct effect of VEGF signaling on osteoblast function during

development and particularly during fracture healing.

Taken together, VEGF activity is indispensable for normal angiogenesis, callus formation and

mineralization in response to injury. These findings support also the notion that VEGF

production is one of the major mechanisms by which angiogenesis and osteogenesis are

tightly coupled during bone repair.

Placental Growth Factor

PlGF, originally discovered in the human placenta, is a VEGF homolog. In contrast to VEGF

which can bind to both VEGFR-1 and VEGFR-2, PlGF binds exclusively to VEGFR-1 [25].

Although PlGF mediates physiological events during angiogenesis, inflammation and

endochondral ossification, it appears to be redundant during development. Indeed, PlGF

deficient mice are viable and show no organ abnormalities. In contrast, PlGF is

nonredundant during several pathological conditions including ischemia, tumor growth or

colitis. Several stimuli are known to induce the expression of PlGF and its receptor in disease

conditions, ranging from hypoxia, growth factors, and hormones to oncogenes.

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In accordance with this concept, VEGFR-1 and PlGF expression is increased during fracture

healing [23, 26] and Plgf inactivation in mice resulted in reduced fracture healing because of

a decrease in the inflammatory response, osteogenic differentiation and callus remodeling

[26].

Fibroblast Growth Factor

Members of the FGF family regulate proliferation, migration and differentiation of many

different cell types, including skeletal (precursor) cells. Ample in vitro and in vivo evidence

demonstrates a clear proangiogenic role of several members of the FGF family. Most

research has focused on FGF-1 and FGF-2, showing that these factors regulate several

aspects of the angiogenesis process, including endothelial cell proliferation, migration,

integrin and cadherin receptor expression, and intercellular gap-junction communication

(reviewed in [27]). Furthermore, FGF-2 can also induce angiogenesis indirectly through

VEGF/VEGFR signaling: (i) FGF-2 modulates VEGF expression in EC, (ii) VEGFR-2

antagonists inhibit not only VEGF but also FGF-2-induced angiogenesis, and (iii) FGF-2

increases the expression of FGF receptors (FGFRs) as wells as VEGFRs in EC [27].

Several FGF ligands, including FGF-2, FGF-9, and FGF-18, as well as their corresponding

receptors FGFR-1, FGFR-2 and FGFR-3, are involved in skeletal development [28-30] and

fracture repair [31]. Recently, it was suggested that FGF-9 is necessary for the establishment

of a vascular network during fracture healing [32]. However, given the strong mitogenic

character of FGFs, the effect on other cells including progenitor cells [33], chondrocytes [34],

osteoblasts [34] or osteoclasts [35] cannot be excluded. Therefore, more insight in the direct

or indirect regulation of endothelial or osteolineage cell function by FGFs during fracture

healing using cell-specific transgenic knockout models is required.

Matrix metalloproteinases

Cartilage and bone are rich in extracellular matrix and the remodeling of this matrix is critical

for development and repair. MMPs are enzymes mediating localized proteolytic modification

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of this extracellular matrix, allowing (i) cartilage and bone remodeling and (ii) release of

stored angiogenic signaling molecules and subsequent blood vessel invasion.

During skeletal development, mice lacking membrane-type 1 (MT1) MMP or MMP-9 display

growth plate abnormalities in the long bones and impaired bone formation, concomitant with

a delay in angiogenesis [36-38]. The function of MMP-9 during development was mirrored

during fracture repair. Mmp9-/- mice display delayed bone healing, linked to a defect in the

removal of matrix deposited by the hypertrophic chondrocytes and a delay in vascular

invasion. Surprisingly, this defect was not explained by a decrease in Vegf expression, nor its

receptor. In contrast, MMP-9 likely regulates the bioavailability of VEGF as exogenous

application of VEGF compensated for the lack of Mmp9 [39], although decisive proof is

lacking.

In addition, identifying other angiogenic growth factors and proteases will contribute in

establishing a more complete model of how remodeling of the extracellular matrix and

angiogenesis are linked during bone healing.

Novel insights in the stimulation of angiogenesis during bone repair

Recent research has characterized additional, novel angiogenic factors that are secreted by

bone-forming cells. Kim et al. identified DJ-1 as an angiogenic factor produced by human

MSC [40], which induces a direct angiogenic response in EC through activation of FGFR1

and regulates angiogenesis indirectly by stimulating VEGF production by osteoblasts. In

addition, DJ-1 stimulates the differentiation of MSC and exogenous administration of DJ-1

enhances bone regeneration in a rodent model of bone fracture repair.

Over the last several years, an increasing amount of evidence emerged implicating

microRNAs (miRNAs) in the control of angiogenesis at multiple levels. miRNAs are

expressed in blood vessel constituting cell types such as EC and vascular smooth muscle

cells and have key roles in both development and disease. For example, deletion of Dicer, an

enzyme required for miRNA biogenesis, results in early embryonic lethality, whereas mice

homozygous for a hypomorphic allele of Dicer survive to mid-gestation but have major

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defects in angiogenesis [41]. In addition, alterations in miRNA expression have been

observed in several pathologies linked to the vascular system, including cancer [42] and

diabetic retinopathy [43].

Besides an effect on angiogenic events, miRNAs appear also to regulate chondrocyte,

osteoblast and osteoclast differentiation [44-46], implying important roles in fracture repair.

Until very recently, the link between miRNAs and angiogenesis during bone healing

remained uncertain. Murata et al. showed that plasma levels of various miRNAs were altered

after fracture, including miRNA-16, miRNA-19b-1, miRNA25, miRNA92a, miRNA101 and

miRNA-129-5p [47]. Interestingly, inhibition of miRNA-92a using systemic and local

administration of antimir markedly enhanced angiogenesis in a murine fracture model,

leading to improved bone repair.

In summary, our knowledge on the role of angiogenic growth factors during bone healing is

progressively increasing, but it has mainly been focused on members of the VEGF and FGF

families. Endorsing insight in the regulation of angiogenesis by other factors, including novel

cytokines and miRNAs, may aid the development of new therapies promoting angiogenesis

during (compromised) fracture healing.

Oxygen as a key determinant of angiogenesis during bone healing

One of the immediate events after a fracture is the rupture of the local (micro)vasculature

resulting in the formation of a hematoma, and these two aspects lead to limited perfusion at

the fracture site and regional hypoxia [48] (Figure 2). Hypoxia Inducible Factor (HIF)

functions as the main transcription factor that regulates cellular responses to hypoxia [49].

HIF is a basic, heterodimeric, helix-loop-helix protein consisting of two subunits, HIF-α and

HIF-β. Under aerobic conditions, HIF-α is hydroxylated by prolyl hydroxylase domain proteins

(PHDs), which use oxygen and α-ketoglutarate as substrates. Hydroxylated HIF-α interacts

with the von Hippel–Lindau protein, the substrate-recognition subunit of an ubiquitin-protein

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ligase that targets HIF-α for proteasomal degradation. In response to hypoxia, the oxygen

sensing PHDs will become inactive and HIF binds to a specific hypoxia responsive element

(HRE; typical HRE sequence is ACGTG) in the promoter region of genes involved in

anaerobic metabolism (glucose transporter 1, lactate dehydrogenase A), erythropoiesis

(erythropoietin) and vascular response (VEGF, FGF-2, heme oxygenase-1, inducible nitric

oxide synthase).

The function of HIF in the regulation of angiogenesis under physiological and pathological

conditions has been well established (reviewed in [49]). Numerous studies have reported HIF

to be expressed during fracture healing [50, 51] suggesting that activation of the HIF-

signaling pathway to induce a VEGF-mediated angiogenic response might be an attractive

therapy to accelerate bone healing. Indeed, genetic overexpression of HIF-1α in mature

osteoblasts of mice was reported to improve bone formation and angiogenesis in a

distraction osteogenesis model, whereas osteoblasts lacking HIF-1α failed to bridge the

distraction gap [52]. In addition, pharmacological inhibition of the prolyl hydroxylases that

normally target HIF-1α for proteosomal degradation stimulated angiogenesis and bone

formation in a femur fracture and distraction osteogenesis model in mice [52, 53]. These

findings indicate that interfering with the hypoxia signaling pathway stimulates both

endochondral and intramembranous bone formation, accompanied by improved blood vessel

formation.

Taken together, the hypoxia signaling pathway is critical during fracture healing to regulate

the timely formation of a functional vascular network, and targeting this pathway is an

appealing strategy to improve bone healing.

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The vasculature: a source of skeletal stem cells for bone

regeneration

Given the importance of timely vascularization for successful bone healing, the question

remains how the angiogenic response exactly aids in restoring the damaged bone. One

obvious reason is the supply of oxygen, nutrients and minerals for bone formation. Recently,

the importance of blood vessels in delivering osteoprogenitors to the fracture site has also

become appreciated [54].

Several studies have shown that in bone marrow, MSC occupy a perivascular location [55-

58]. Different markers have been proposed in an attempt to identify these perivascular cells,

including CD-markers (e.g. CD44 or CD146 [56, 59]), Stro1 [60], Platelet-Derived Growth

Factor Receptor β [55], NG2 [61] and alpha Smooth Muscle Actin (αSMA) [62]. More

compelling evidence has been provided by recent studies showing that MSC in the bone

marrow of mice can be identified by their expression of a Nestin-GFP transgene and that

these cells occupy a perivascular location and express the pericyte markers NG2 and αSMA

[57, 63]. Despite the common consensus that perivascular cells play an important role during

development and in various injured tissues [64, 65], knowledge on their role during fracture

healing is rather limited. Perivascular cells in the periosteum are suggested to be a source of

osteoprogenitor cells during periosteal osteogenesis [66]. Recently, using a cell lineage

tracing approach in mice, it was found that αSMA-positive (αSMA+) perivascular cells exhibit

the ability to differentiate into mature osteoblasts in vitro and in vivo during normal bone

remodeling [67]. In addition, during fracture healing, the majority of callus cells including

chondrocytes and osteoblasts were derived from αSMA+ cells. Although the exact origin of

these αSMA+ cells remains to be determined, they presumably derive from the local

periosteal vasculature, since it has been shown that the periosteum supplies the vast

majority of the cells in the fracture callus [68]. In accordance with this notion, we have

recently shown that a subpopulation of culture-expanded murine periosteal cells localize

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perivascularly when co-transplanted in vivo with EC, indicating that these cells can

structurally support newly formed blood vessels [69].

Targeting angiogenesis to treat non-healing bone defects

The reestablishment of an adequate blood vessel system is of utmost importance for the

success of bone healing. Long-standing preclinical and clinical evidence has established a

strong correlation between the impairment of vascular function and failure of fracture healing

(delayed union or non-union) [70].

Bone tissue engineering represents an appealing strategy to improve non-healing osseous

defects. Reconstruction of damaged bone using this technique relies on the combination of a

biocompatible scaffold, osteogenic cells and osteogenic or angiogenic growth factors.

Furthermore, mechanical stabilization of the defect and an adequate host response are

critical to ensure successful bone formation [71, 72]. Despite the clear potential of

engineered constructs, translation into the clinic remains difficult. One of the major limitations

of this approach is the inability to provide sufficient blood vessel supply during the early

stages of bone regeneration, reflected by the limited survival of the osteogenic cells early

after implantation [73, 74]. Upon implantation of the construct, an angiogenic response is

elicited by inflammatory cytokines as part of the normal healing process [75].

Neovascularization of the scaffold will occur by invading blood vessels deriving from the

surrounding host vasculature. However, the slow rate of invasion of blood vessels into the

scaffold (<1 mm per day) [76] makes it an insufficient process to timely vascularize tissues of

clinically relevant size.

To overcome this problem, several methods have been proposed to improve the

vascularization of bone constructs in experimental models: the delivery of angiogenic growth

factors, the use of EC to engineer a vascular network or a hybrid approach that combines

microsurgery approaches with bone tissue engineering concepts [75] (Figure 4).

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Local delivery of angiogenic growth factors

The use of angiogenic growth factors such as VEGF is an appealing strategy to improve the

vascularization of a construct. Although a single bolus of VEGF improved revascularization in

animal models [77], this approach might have some limitations due to protein instability in

vivo and possible undesirable side effects. Indeed, careful dosing of VEGF is required, as

overexpression of this angiogenic growth factor leads to aberrant bone formation, bone

marrow fibrosis, abnormal blood vessels and extramedullary hematopoiesis [78]. Current

approaches are therefore more focusing on localized and sustained delivery of growth

factors, permitting prolonged exposure of regenerating tissues to lower doses [79, 80]. To

this end, numerous natural [81], synthetic [82] and composite [83] materials have been

proposed as delivery matrices. Alternatively, advances in gene therapy also facilitate the

controlled release of angiogenic growth factors, either by injection of (viral) vectors or by

using genetically modified implanted cells. Indeed, direct injection of a recombinant adeno-

associated virus overexpressing VEGF induced mandibular condylar growth in mice [84].

Furthermore, genetically induced expression of VEGF in MSC has been shown to improve

bone formation in critical sized defects in mice, either when cells were used alone or in

combination with different BMPs and angiopoietin-1 [85, 86].

To conclude, stimulation of an angiogenic response aids bone regeneration in different

preclinical models. However, the timing and dosing of angiogenic growth factors have to be

very tightly regulated, as severe adverse effects might occur upon overstimulation.

Vascularization by autologous endothelial cells

Regardless of the strategy used to stimulate angiogenesis, sufficient and functional

endothelial cells (EC) are as much required as the abundance of angiogenic growth factors.

Several groups are therefore exploring the addition of autologous EC to tissue engineering

implants in order to accelerate the establishment of a functional vascular network.

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Mature EC can be used for these vascularization strategies, although several drawbacks are

linked to the use of this cell population. First, the low availability and proliferation capacity

limit the upscaling of the constructs for clinical applications [87]. Second, EC derived from

different tissues are phenotypically different [88-90]. In contrast, endothelial progenitor cells

(EPC) might be a more interesting source of autologous EC. These cells are characterized

by the expression of CD133, CD34 and VEGFR-2 and can be isolated from human bone

marrow, fat tissue and peripheral blood. Although limited in number, EPC can be extensively

expanded in vitro and possess the capacity to differentiate into mature EC [91, 92]. EPC are

a heterogeneous cell population consisting of early and late-stage (or blood outgrowth

endothelial cells, BOEC) EPC [93, 94]. Whereas early-stage EPC are considered to be

hematopoietic cells, BOEC are considered to be true endothelial cells. Despite their relatively

late outgrowth from blood cultures, BOEC display enhanced proliferative properties, and

have typical endothelial features such as adherens junctions, in contrast to early-stage EPC.

BOEC, either alone or in combination with early-stage EPC, have already been used in a

variety of preclinical rodent models to restore the vasculature, including bone tissue-

engineered constructs [95, 96].

Despite the enormous angiogenic potential of these EC, a perivascular component is

required in order to engineer mature, long-lasting blood vessels. Examples of perivascular

cells that can improve the stability and functionality of blood vessels include vascular smooth

muscle cells [97] and bone marrow MSC [98].

Taken together, prevascularization strategies hold promising potential, although they are at

this stage still mostly experimental and translation to the clinic is therefore very limited.

Modulating scaffold properties and in vitro prevascularization

Scaffold properties influence the behavior of the implanted cells and it is obvious that the

porosity is important, not only for osteoblast proliferation and matrix production but also for

blood vessel ingrowth. However, conflicting data make it difficult to extrapolate in vitro

findings to the in vivo situation. In vitro, endothelial cell proliferation was enhanced in

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scaffolds with smaller pore sizes [99], whereas osteoblast proliferation was diminished [100].

In contrast, higher porosity and pore size resulted in improved vascularization as well as

bone formation in vivo [101]. A recently developed and interesting technique is the fabrication

of a vascular network within a polymer-based scaffold, for example by photolithography [102]

or 3D fiber deposition [103]. Here, 2D and 3D structures are created, serving as template to

seed EC. An alternative way to realize in vitro prevascularization is the co-culture of EC and

osteoblast cells. Several reports indicate that these co-cultures were able to form

microcapillary-like structures within the scaffold, which remained stable during culture [104].

However, only limited evidence exists whether these in vitro pre-made networks can connect

to the host vasculature when implanted in vivo [105].

Preclinical and clinical studies using angiogenic growth factors

It is evident that an adequate vascular response is needed for normal bone healing and the

concept of therapeutic angiogenesis has been appreciated for many years. As discussed in

this review, preclinical data indicate that the local delivery of angiogenic growth factors

correlates with a positive outcome on bone healing. However, rigorous phase II and phase III

clinical trials demonstrating that angiogenic agents are beneficial are largely lacking.

Several factors might contribute to the slow or unsuccessful transition of preclinical to clinical

studies. First, the differences between animal models and the patients enrolled in clinical

trials cannot be neglected. Animals used in preclinical studies are typically young and

healthy, whereas patients are usually older with multiple comorbidities. Often, vascular

function is impaired in the elderly and the reduced angiogenic response compromises bone

repair. Therefore, preclinical experiments in animal models should not only be restricted to

proof-of-principle experiments showing the potential of angiogenic therapy, but should in

addition try to mimic better the compromised human situation and develop more predictive

preclinical models (e.g. aged, atherosclerotic, or diabetic animals).

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In addition, optimal dosing, duration, and timing of proangiogenic therapy have still to be

defined. Preclinical data suggest that angiogenesis is a slow process requiring the presence

of angiogenic factors during a prolonged period [75, 76], but the dosing strategies have to be

well considered to avoid dose-related side effects.

We can conclude that extensive progress has been made proving the efficacy of the

stimulation of angiogenesis for bone repair in preclinical animal models, but that the

translation into the clinic still remains limited. More relevant animal models, as well as

innovations concerning delivery and dosing of angiogenic stimuli, cell-based constructs and

advances in biomaterial design might facilitate the development of a successful clinical

application.

Conclusions and future perspectives

Fracture healing is a complex process and blood vessel formation, controlled by locally

produced growth factors and cytokines, has to be tightly regulated and has to coincidence

with the activation, proliferation and differentiation of skeletal progenitor cells. Under optimal

conditions, these processes occur synchronously resulting in full restoration of the original

morphology and biomechanical properties of the fractured bones [106]. The vascular system

plays a key role in this process, not only by supplying oxygen and nutrients, but also by

delivering osteoprogenitors to the fracture site giving rise to bone-forming cells [54, 67].

However, these healing processes are likely too slow for the reconstruction of large bone

defects created by tumor resection, trauma or infection, where accelerated bone

regeneration may be required to prevent progression to a non-union. In addition, the

regenerative process might be disturbed in pathological conditions like avascular necrosis or

in diseases associated with impaired vascular function [107]. To circumvent these problems,

strategies might focus on novel techniques for synthesizing scaffolds that serve as a

template for vascularization including 3D fiber deposition. Still, one of the main problems in

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creating a cell-seeded construct of clinically relevant size is the lag-time in blood vessel

ingrowth, seriously hampering the survival of the implanted cells in the scaffold. Therefore,

an appealing strategy might be to precondition the osteogenic cells prior to in vivo

implantation by adapting their metabolism to the environment. This way, the implanted cells

may remain viable during the early critical time frame before blood vessels arrive, hereby

hopefully improving bone formation.

Taken together, tremendous progress has been made in unraveling the importance of

angiogenesis during bone healing, but novel therapies have to be developed that favor

vascular ingrowth and at the same time promote the survival of the implanted cells,

particularly in patients with an inadequate angiogenic host response.

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Table 1. Angiogenesis-stimulating growth factors.

Osteoblastica Angiogenicb Induction of VEGFc

Angiopoietin + [108] + [109] ND

Fibroblast Growth Factors + [110] + [32] + [111]

Bone Morphogenetic Proteins + [112] + [113] + [113]

Insulin-like Growth Factor family + [114] + [115] + [116]

Platelet-Derived Growth Factor + [117] + [118] + [119]

Transforming Growth Factor beta family + [120] + [121] + [122]

Vascular Endothelial Growth Factor + [20] + [15] NA

adirect effect on committed (pre)osteoblasts

bstimulation of blood vessel formation in vivo, direct or indirect

cinduction in vitro or in vivo

NA – not applicable, ND – not yet determined

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Figure legends

Figure 1. Physiological blood vessel formation. The first step of angiogenesis is vessel

sprouting. Angiogenic growth factors trigger endothelial cells to degrade the basement

membrane (A) and select for tip and stalk cells. Tip cells then migrate into the tissue stroma

while stalk cells proliferate (B). A lumen is formed by fusion of tip cells (C-D) and flow

initiates differential branch maturation (E).

Figure 2. The angiogenic response during normal fracture healing. Due to the rupture of

blood vessels, the fracture site becomes hypoxic. Activation of the hypoxia signaling pathway

stimulates the production of VEGF and PlGF by several cell types present at the fracture site.

In addition, early blood vessel formation supports the invasion of inflammatory cells which

actively contribute to the fracture healing process and produce proangiogenic cytokines.

During the formation of the soft and hard callus, the vascular system might also mediate

progenitor migration to the fracture site and promote bone regeneration by supplying oxygen,

nutrients and ions necessary for mineralization.

Figure 3. VEGF signaling during bone regeneration. VEGF stimulates the formation of

new blood vessel, which can bring progenitor cells for bone formation to the fracture callus.

In addition, VEGF upregulates the expression of osteogenic growth factors in endothelial

cells, mediating osteoblast differentiation. Lastly, VEGF might also directly affect osteoblast

behavior including differentiation, proliferation and chemotaxis.

Figure 4. Strategies to improve vascularization in a bone tissue-engineered construct.

The healing of large bone defects using cell-seeded constructs requires adequate

vascularization. Strategies to improve the formation of a functional vascular system include

the addition of growth factors or endothelial cells, and engineering scaffold properties.

21

Acknowledgements

This work was supported by grants from the Fund for Scientific Research Flanders (FWO;

G.0835.11 and G.0A72.13) and the KU Leuven (BOF-KU Leuven GOA project 3M120209).

SS is a fellow from the Agency for Innovation by Science and Technology in Flanders (IWT).

This work is part of Prometheus, the KU Leuven R&D division for skeletal tissue engineering,

http://www.kuleuven.be/prometheus.

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