Content uploaded by Sophie Verrier
Author content
All content in this area was uploaded by Sophie Verrier on Jul 25, 2016
Content may be subject to copyright.
87 www.ecmjournal.org
S Verrier et al. Large bone defect healing
European Cells and Materials Vol. 32 2016 (pages 87-110) ISSN 1473-2262
Abstract
Despite the high innate regenerative capacity of bone, large
osseous defects fail to heal and remain a clinical challenge.
Healing such defects requires the formation of large
amounts of bone in an environment often rendered hostile
to osteogenesis by damage to the surrounding soft tissues
and vasculature. In recent years, there have been intensive
research efforts directed towards tissue engineering
and regenerative approaches designed to overcome this
multifaceted challenge. In this paper, we describe and
critically evaluate the state-of-the-art approaches to
address the various components of this intricate problem.
The discussion includes (i) the properties of synthetic
and natural scaolds, their use in conjunction with cell
and growth factor delivery, (ii) their vascularisation, (iii)
the potential of gene therapies and (iv) the role of the
mechanical environment. In particular, we present a critical
analysis of where the eld stands, and how it can move
forward in a coordinated fashion.
Keywords: Bone, vascularisation, scaffolds, gene
therapy, stem cells, drug delivery, large bone defect, tissue
engineering, regenerative medicine, translational and
preclinical research.
*Address for correspondence:
Dr Sophie Verrier
AO Research Institute Davos
Clavadelerstrasse 8
7270 Davos Platz, Switzerland
Phone: +41 81 414 22 11
Direct: +41 81 414 2448
Fax: +41 81 414 22 88
Email: sophie.verrier@aofoundation.org
Introduction
The healing of large bone defects is a major clinical
challenge. Although bone possesses remarkable repair
and regenerative powers of its own, there are numerous
clinical conditions in which the size, location, and/or local
environment of the bone defect results in impaired healing.
Large bone defects are a problem in craniomaxillofacial
surgery, as well as in orthopaedics more generally.
Examples of large bone defects include tumour resections,
infection, fractures accompanied by substantial soft tissue
trauma, congenital deformities and segmental loss. In each
of these cases, the large volume of tissue that needs to be
replaced makes it very challenging to achieve sucient
quantity and quality of new bone formation. In addition,
the healing of larger defects is critically dependent on
the presence of an appropriate vascular supply to support
regeneration and remodelling of new bone tissue.
In clinical practice the standard treatment for large
bone defects is the use of autogenous or allogenic bone
grafting to provide an osteogenic and/or osteoconductive
stimulus, and thereby promote bone regeneration and
union. However, insucient volume of available tissue,
donor site morbidity (autogenous), inconsistent osteogenic
activity, late biomechanical failures, and the possibility of
allogenic disease transmission reduce enthusiasm for their
use. While great progress has been made with the use of
osteoconductive bone graft substitutes and distraction
osteogenesis, it is clear that complex clinical cases
where novel therapies are required still exist. Finally, the
challenging wound healing environment in which large
bone defect restoration often needs to take place, mandates
a strategy that both fills the bone gap and promotes
vascularisation and repair. Although vascularised free aps
are currently an important and successful clinical option to
address these concerns, it requires a long involved and often
risky operation, with attendant extended hospitalisation and
high cost. These challenges have motivated the eld of
TISSUE ENGINEERING AND REGENERATIVE APPROACHES TO IMPROVING
THE HEALING OF LARGE BONE DEFECTS
S. Verrier1,11*, M. Alini1,11, E. Alsberg2,11, S.R. Buchman3,11, D. Kelly4,11, M.W. Laschke5,11, M.D. Menger5,11, W.L.
Murphy6,11, J.P. Stegemann7,11, M. Schütz8,11, T. Miclau9,11, M.J. Stoddart1,11 and C. Evans10,11
1AO Research Institute Davos, Davos, Switzerland
2Departments of Biomedical Engineering and Orthopaedic Surgery, Case Western Reserve University, Cleveland,
OH, USA
3Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA
4Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland
5Institute for Clinical and Experimental Surgery, Saarland University, 66421Homburg/Saar, Germany
6Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA
7Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, USA
8Institute of Health and Biomedical Innovation and Medical Engineering Research Facility,
Queensland University of Technology, Brisbane, Australia
9Department of Orthopaedic Surgery, University of California, San Francisco, Orthopaedic Trauma Institute,
University of California, San Francisco/San Francisco General Hospital, San Francisco, CA, USA
10Rehabilitation Medicine Research Center, Mayo Clinic, 200 First Street SW, Rochester, USA
11Collaborative Research Partner Large Bone Defect Healing Program of AO Foundation, Davos, Switzerland
(All authors contributed equally to this work)
88 www.ecmjournal.org
S Verrier et al. Large bone defect healing
musculoskeletal tissue engineering to nd a solution that
will aid the surgeon and the patient in tackling some of the
most dicult and challenging reconstructive conundrums
in both orthopaedic and craniofacial surgery. The general
strategy was elegantly summarised in the Giannoudis
Diamond concept, whereby the nal outcome is dictated
by the combination of osteogenic cells, osteoconductive
scaolds, growth factors and the mechanical environment
(Giannoudis et al., 2007).
This review covers the techniques and strategies
that have been developed to address the multifaceted
challenges posed by the complicated problem of large
bone defect healing (Fig. 1). The term “large bone defect”
is used here in the sense of defects that are too large to
heal spontaneously i.e. are of critical size. The review
emphasises strategies based upon tissue engineering and
regenerative medicine, sometimes abbreviated collectively
as TERM to emphasise their overlapping nature. Unlike
fractures, critical size segmental defects have no natural
healing process and thus no native biology to model.
Rather, TERM for large bone defects engages a variety of
approaches, including scaold design and selection, drug
and morphogen delivery, cell- and gene-based therapies,
vascularisation strategies, and mechanical environments
that can be used to promote regeneration of bone. The
uniqueness of large bone defects is the size of the void that
needs to be lled and vascularised, all in the absence of
local endogenous osteogenic signals. The goal here is to
give an overview of the components that have been applied
to the problem to date, as well as to provide insight into
how these components can be combined in future more
advanced therapies.
Scaolds
Requirements for a bone regeneration scaold include
mechanical properties (e.g. desired stiffness and
compression resistance), degradability, macro- and micro-
porosity, and nanometre-scale topography. Encompassing
all of these requirements into one material or composite
is challenging and limits the number of suitable base
materials that would also have an expeditious route to
clinical application. In the following we describe some of
the materials being actively investigated for use in large
bone defects. The examples used are far from being an
exhaustive list.
Natural scaolds
Many tissue engineering strategies employ scaffold
materials to provide both mechanical support and
biological function. A logical approach to scaold design
is to mimic the materials and architectures found in native
tissues. To this end, a variety of extracellular matrix (ECM)
proteins, polysaccharides and other “naturally-derived”
materials have been used to create scaolds for bone tissue
engineering. ECM-derived scaolds have the advantage
that cells can recognise and bind to them by specic cell
surface receptors, and thereby can receive biochemical
signals directly from the scaold (Shekaran and Garcia,
2011; Siebers et al., 2005). In most cases, cells can also
degrade, synthesise, and remodel these natural matrices
in response to environmental cues (Ferreira et al., 2012).
For the repair of large bone defects, the mechanical and
space-filling attributes of the scaffold are of primary
importance. Pure naturally-derived materials, such as
collagen scaolds, typically have inferior mechanical
properties relative to both the tissues from which they are
derived and to synthetic polymer scaolds (Gibbs et al.,
2014). Accordingly, their use in large bone defects without
additional structural support is challenging. For this reason,
there is a growing interest in decellularisation of harvested
tissues for use as scaolds, in an eort to keep the native
architecture and compositional complexity intact (Cheng
et al., 2014). In addition, a variety of composite materials
that combine the desirable features of specic protein
and polysaccharide components of the ECM have been
developed and used as scaolds in bone tissue engineering
(Wang and Stegemann, 2010).
The collagen superfamily of proteins consists of over
25 molecular isoforms. The most common form is type I
collagen, which is a main structural constituent of many
Fig. 1. Large bone defect: a multifaceted challenge. Bone
healing is a complex process involving the interplay of
many factors and well-orchestrated mechanisms. Tissue
engineering approaches aim to resume the complexity
of these events by combining scaolds, cells, growth
factors and mechanical environment. The choice of cells,
their association or not with scaolds, the local delivery
of growth factors, the application mechanical stimulation
of the defect (e.g. active dynamisation and timing), but
also the patient condition and the surgical approach are
as many factors inuencing the healing outcome. Here,
we give an overview of the techniques and strategies that
have been developed in the past 10 years to address the
complex situation of large bone defects.
osteogenesis
89 www.ecmjournal.org
S Verrier et al. Large bone defect healing
tissues, and is the predominant non-mineral component
of bone. Collagen “sponges” are typically generated by
freeze-drying collagen based slurries, creating a porous
architecture (O’Brien, 2011). Such sponges are used
widely as haemostatic agents, and also have been used in
bone repair as a delivery vehicle for bone morphogenetic
proteins (BMPs) (Geiger et al., 2003; Wei et al., 2012) or
as gene delivery platforms (Curtin et al., 2012; Tierney
et al., 2013).. Their expanded use is being investigated in
preclinical studies for a variety of orthopaedic applications
(d’Aquino et al., 2009; Hosseinkhani et al., 2006). Type I
collagen has also been reconstituted into brillar form by
electrospinning (Huang et al., 2001; Matthews et al., 2002).
Eorts to further mimic the composition and function
of bone have led to collagen-ceramic composites (Wahl
and Czernuszka, 2006). Hydroxyapatite or tricalcium
phosphates are often used in this application to represent
the mineral content of bone (Gleeson et al., 2010; Zheng
et al., 2014).
Chitosan is an aminated polysaccharide that is derived
from the deacetylation of chitin, a structural component of
the exoskeleton of crustaceans and some fungi. Chitosan
can be made water soluble, and has been used in ways
similar to collagen to make sponges, meshes and scaold
materials for bone tissue engineering (Costa-Pinto et al.,
2011; Heinemann et al., 2010). In scaold form, pure
chitosan allows cell attachment and has been suggested to
be osteoinductive (Di et al., 2005). Composites of chitosan,
with other matrix components to improve its mechanical
properties, are more commonly used for orthopaedic
applications (Venkatesan et al., 2012). Blends of chitosan
and other materials have been electrospun into bre meshes
(Chen et al., 2011; Zhang et al., 2008), and composites have
been used in sponge format, including combination with
other polysaccharides (Park et al., 2013), proteins (Wang
et al., 2013), and mineral (Pighinelli and Kucharska, 2013).
The free amine groups on the chitosan molecule allow it
to be crosslinked with the same agents as used for protein
matrices (Reves et al., 2013; Wang and Stegemann, 2011),
which can increase its mechanical strength and resistance
to degradation. In addition, the positive charge on the
chitosan molecule allows the material to be used for drug
and gene delivery directly from the scaold (Cao et al.,
2012b; Goncalves et al., 2012).
Hydrogels form another class of natural polymer
scaolds. These materials are hydrated, interconnected
networks of polymer chains. An inherent advantage of such
hydrogels is that they can be delivered using minimally
invasive techniques, will ll defects of complex shapes
and can be combined with cells and/or osteoinductive
factors (Drury and Mooney, 2003). Alginate hydrogels
have been used for gene (Krebs et al., 2010) and growth
factor delivery (Kolambkar et al., 2011) and such systems
have been shown to promote functional repair of critically-
sized bone defects. Promising results have been obtained
using natural hydrogels such as brin (Chung et al., 2007;
Woodru et al., 2007) and gelatin (Yamamoto et al., 2003;
Yamamoto et al., 2006) as delivery vehicles for therapeutic
factors for bone regeneration. One concern with certain
classes of hydrogels for large bone healing is insucient
degradation of hydrogel (Rizzi et al., 2006), which may
impede vascularisation of the implant. Such problems can
potentially be overcome by modulating the hydrogel to
accelerate its rate of degradation (Alsberg et al., 2001; Jeon
et al., 2009). Hydrogels typically do not have compression-
resistant mechanical properties, but can be included within
other common orthopaedic devices (e.g. titanium cages)
and used to stimulate new bone formation.
Demineralised bone matrix (DBM) is an example of a
natural biomaterial that is commonly used clinically as a
bone graft substitute (Urist, 1965). Such grafts are typically
produced by the acid extraction of the mineral content
from allogeneic bone and contain growth factors, other
non-collagenous proteins and type I collagen (Sawkins
et al., 2013). The rigorous processing and sterilisation
that such grafts must undergo prior to implantation can
negatively impact their osteoinductive properties which
may at least partially explain the variable results seen
with DMB (Gruskin et al., 2012; Peterson et al., 2004).
To overcome such limitations, DBM can also be used as a
delivery system for novel therapeutics (Lieberman et al.,
1999). Decellularised ECM derived from other mammalian
tissues such as small intestine submucosa have been used
as biological scaolds for bone regeneration (Badylak
et al., 2009; Kim et al., 2010; Moore et al., 2004). It has
also been demonstrated that bone-like ECM synthesised
in vitro by osteoblastic cells can enhance osteogenesis of
mesenchymal stem cells (MSCs) (Datta et al., 2005), and
MSC-derived ECM enhances the retention of implanted
cells into the remodelling phase of healing, resulting in
reproducible and complete repair of critical-sized bone
defects in mice (Zeitouni et al., 2012).
Synthetic scaolds
Investigators have developed a variety of synthetic
scaolds for large bone defect healing, and a common
approach involves mimicry of some aspects of the native
bone ECM.
The catalogue of synthetic bone biomaterials used in
critical sized defects features a wide range of biominerals,
including hydroxyapatite, β-tricalcium phosphate,
amorphous calcium phosphate, calcium silicate bioactive
glasses, and biphasic calcium phosphates. The bone-like
mineral layer formed on the surface of these materials
has been shown to influence critical components of
the bone formation process, including proliferation of
bone-precursor cells (Chou et al., 2005), osteogenic
differentiation of bone-forming cells (e.g. marrow-
derived MSCs, adipose-derived MSCs, pre-osteoblasts,
and osteoblasts) (Barradas et al., 2012; Chou et al.,
2005; Murphy et al., 2005), and localised sequestering
of bone growth factors (Suarez-Gonzalez et al., 2012).
Recent studies suggest that released mineral ions (e.g.
calcium (Barradas et al., 2012), phosphate (Shih et al.,
2014), magnesium (Hussain et al., 2012; Schwartz and
Reddi, 1979), strontium (Yang et al., 2011)) may be partly
responsible for the behaviour of bone precursor cells.
These mineral ions have been associated with expansion
of bone precursor cells, osteogenic differentiation of
marrow-derived MSCs (Barradas et al., 2012; Shih et al.,
2014), and optimised non-viral transfection of multiple
bone precursors (Choi et al., 2013).
90 www.ecmjournal.org
S Verrier et al. Large bone defect healing
Common synthetic polymer materials used to form
scaolds for bone healing include poly(alpha-hydroxy
esters) (Yu et al., 2010), poly(urethanes) (Guelcher,
2008), poly(propylene fumarate) (Wang et al., 2006), and
poly(carbonates) (Kim et al., 2012; Luangphakdy et al.,
2013). Thermoplastics such as poly(L-lactide), poly(lactide-
co-glycolide), poly(ε-caprolactone), poly(propylene
fumarate, and poly(urethanes) can be readily processed to
allow for interconnected macroporosity, with control over
the pore size, structure, and interconnectivity. In addition,
these materials can be formed via diverse manufacturing
schemes, including casting, injection moulding, and 3-D
printing. All have been applied within large bone defects,
both as void ller and as an osteoconductive matrix.
Synthetic hydrogels composed of poly(ethylene
glycol), poly(propylene fumarate-co-ethylene glycol), and
hyaluronic acid have each been used for bone precursor
cell culture in vitro or to enhance critical bone defect
healing in vivo. In each case the materials can be loaded
with bone precursor cells and/or pro-osteogenic molecules
to stimulate bone formation (Cartmell, 2009; Salinas and
Anseth, 2009). One attractive feature of these hydrogels is
their ability to incorporate and deliver controllable dosages
of biologically active molecules, including cell adhesion
peptides, proteolytically-degradable peptides, ECM
proteins, and growth factors. Another feature is the ability
to form hydrogels in situ, which opens up new minimally-
invasive clinical opportunities (Behravesh et al., 2003;
Kim et al., 2009). Self-assembling hydrogels have been
designed to gelate in situ and deliver environments that
promote bone formation. For example, peptide amphiphiles
can self-assemble into nanobrous matrices that have been
used to nucleate mineral formation (Hartgerink et al.,
2001), present peptides for cell adhesion (Webber et al.,
2010), or present peptides for growth factor binding (e.g.
transforming growth factor (TGF)-β binding (Shah et al.,
2010)).
Some recent examples highlight the potential to
combine distinct and complementary synthetic materials
to create composite. Investigators have created composites
of synthetic polymers and biominerals, taking advantage
of the resultant processing benets of the polymers and the
inherent biological activity of the biominerals, resulting
in enhanced scaold compressive modulus, improved
osteoconductivity, and greater osseointegration. In one
example, 3D printing approaches have been used to create
biomineral-coated, 70 % porous poly(ε-caprolactone)
scaffolds with mechanical properties that withstand
masticatory loads in the mandible, and stimulate bone
regeneration as they degrade (Chanchareonsook et al.,
2013). Other studies demonstrated that mineral-coated
hollow tubes composed of poly(ε-caprolactone) can
stimulate bone regeneration in sheep tibia defects (Cipitria
et al., 2013) and sheep lumbar spine fusion (Yong et al.,
2014). These examples and others suggest that innovative
manufacturing of common bone biomaterials can produce
a useful toolkit for large bone defect healing.
Furthermore, while we focus this section on synthetic
materials, it is noteworthy that a subset of naturally-derived
polymers can also be synthetically modied to create
natural/synthetic hybrids that stimulate bone formation.
For example, alginate hydrogels can be modied with
peptide ligands and used to deliver bone-forming stem
cells or osteoinductive growth factors (Drury and Mooney,
2003; Lee and Mooney, 2012). Similarly, brin hydrogels
can be used as a platform to covalently link pro-osteogenic
(Arrighi et al., 2009; Schmoekel et al., 2005) or pro-
angiogenic (Ehrbar et al., 2004) growth factors, which are
subsequently delivered during new bone formation. While
these materials generally do not match the synthetic and
manufacturing adaptability of synthetic materials, they
open up the possibility of hybrid approaches that combine
the complementary advantages of synthetic and natural
components.
Synthetic scaold design involves a series of design
trade-os, which present inherent challenges for large
bone defect healing. For example, scaffolds require
optimised mechanical properties for a particular clinical
approach, but must also provide adequate porosity for
cell inltration and tissue formation and degradability
over a timeframe that scales with the timing of new bone
formation (Hollister, 2005). In addition, scaold design
parameters such as µm-scale and nm-scale geometry have
become increasingly appreciated as critical regulators of
osteogenesis. In particular, nm-scale pillars and bres have
been associated with enhanced osteogenic dierentiation
of bone-forming stem cells in vitro (Dalby et al., 2007), as
well as increased osteogenesis in vivo (Ingavle and Leach,
2013). The diversity of existing and emerging parameters
that appear to be important for large bone defect healing
will call for ecient – and perhaps high throughput –
screening strategies to identify optimal scaold materials.
It is noteworthy that one reason why scaold materials
developed to date have been composed of similar base
materials relates to the relatively complex regulatory path
for novel bone scaolding materials. Materials comprising
new combinations of clinically established base materials
typically provide a more rapid route to regulatory approval
and clinical applications. In particular, while combinations
of existing, FDA approved materials may only require
one to demonstrate substantial equivalence to an existing
“predicate device”, novel scaold materials often require
substantial preclinical studies and one or more clinical
studies prior to regulatory approval. The adaptable features
of commonly used scaold materials coupled with the
relatively complex regulatory path of novel materials limits
innovation.
Drug and growth factor delivery
A series of small molecule drugs (e.g. bisphosphonates) has
been used to treat orthopaedic diseases such as osteoporosis,
osteonecrosis, and osteolysis. However, small molecule
drugs have not been widely used in large bone defect
healing applications. This is perhaps not surprising, as these
drug classes are not typically designed to induce formation
of new bone tissue in large defects, but rather to regulate the
systemic balance between bone resorption and formation.
Instead, the focus of large bone defect healing studies
has been on local, bone stimulating molecules known to
inuence natural bone development and healing, such as
91 www.ecmjournal.org
S Verrier et al. Large bone defect healing
growth factors, hormones, cytokines, and antibodies. The
common strategy involves designing a molecule “carrier”,
which is then combined with an orthopaedic device (Sandor
et al., 2013; Warnke et al., 2004). Notable examples include
BMP-loaded collagen matrices combined with titanium
cages (Boden et al., 2000; Kanayama et al., 2006) or
other metallic hardware (Govender et al., 2002). The now
extensive clinical experience with BMPs and other bone
stimulating molecules suggests critical challenges that must
be addressed in the next generation of large bone defect
healing strategies. Here, we focus on describing critical
challenges to be addressed in next generation of drug
delivery strategies, with illustrative examples included.
First, there is a signicant pharmacokinetic challenge
in delivery of bone stimulating molecules. Recombinant
human (rh)BMP2 delivery for instance, is the most
prevalent drug delivery strategy used for bone regeneration.
While Medtronic’s rhBMP-2-releasing device Infuse™ has
achieved a great deal of clinical success in lumbar spine
fusion, and widespread use in other clinical indications, it
has also been associated with serious side eects (Fu et al.,
2013; Faundez et al., 2016). The side eects result in part
from the mg-scale quantity of rhBMP-2 delivered, which
is multiple orders of magnitude more rhBMP-2 than one
might nd in a healing bone defect. These side eects could
also signal that rhBMP-2 has a narrow therapeutic index,
which is a measure of the dierence between the clinically
eective dosage and the toxic dosage of a drug. As a result,
recent studies have focused on controlling the dosage and
release kinetics of bone stimulating molecules in order to
identify optimal pharmacokinetics for bone healing (King
and Krebsbach, 2012; Seeherman and Wozney, 2005). It is
not yet clear what combination of total dosage and release
kinetics can stimulate bone regeneration while limiting side
eects, but it is clear in pre-clinical models that sustained
release can decrease the total rhBMP-2 dosage needed to
stimulate bone regeneration (Jeon et al., 2008; Kolambkar
et al., 2011). Indeed, the signalling mechanisms activated
by bone stimulating molecules are typically not unique to
bone formation, and molecules are often selected based
on their ability to induce heterotopic bone formation.
Thus, there is a general need for systematic studies on the
inuence of localised dosage and release kinetics.
Second, there is a substantial formulation challenge
in delivery of bone stimulating molecules. Proteins with
significant tertiary structure have a strong tendency
to denature, degrade, and/or aggregate under standard
physiological conditions, resulting in rapid loss of
biological activity. For example, basic broblast growth
factor (FGF) loses biological activity within minutes in
aqueous solution in the absence of heparin (Nguyen et al.,
2013). These types of molecules also tend to have narrow
therapeutic indices, which results in a need to deliver
the molecules in a narrow dosage range. One illustrative
example is vascular endothelial growth factor (VEGF),
which has been shown to promote blood vessel sprouting
within a relatively limited dosage range in vivo (Lee et al.,
2000). The ideal approach would be capable of stabilising
bone stimulating molecules against inactivation, while
also enabling controllable release kinetics from a desirable
scaold material.
In view of these challenges, most common biomaterials
used for bone healing are plagued by critical limitations.
Elastomeric polymer networks (e.g. hydrogels) allow for
molecular transport, which can result in poor bioavailability
of a released molecule. Thermoplastics (e.g. poly(alpha-
hydroxy esters)) can be designed to encapsulate and release
molecules with controllable dosage and release kinetics,
but the biological activity of the released molecules is
often significantly compromised due to aggregation,
denaturation, and degradation (Zhu et al., 2000). Recent
studies with nano-structured materials provide promising
solutions to the current challenges. Lipid nanocapsules and
mineral capsules have been shown to maintain stability
of proteins (Giri et al., 2011). Recent studies indicate
that nano-structured biomineral coatings can uniquely
stabilise proteins against degradation, while also enabling
controllable release kinetics by coating dissolution (Ge et
al., 2012; Lu et al., 2009; Suarez-Gonzalez et al., 2012).
This direction is promising, as it may address each of the
major challenges in growth factor delivery. Further, it is
possible to design broadly adaptable biomineral coatings
for controllable delivery of peptides, proteins, DNA,
cells, and combinations thereof (Choi and Murphy, 2010;
Jongpaiboonkit et al., 2009; Lee et al., 2010a; Lee et al.,
2010b; Zhang et al., 2010a). In another approach, growth
factors have recently been stabilised by heparin-mimetic
ligands, which can be covalently linked within hydrogels
(Nguyen et al., 2013).
It is important to note that combining bone stimulating
molecules with an appropriate scaold while controlling
stability and pharmacokinetics is just one of several
inherent challenges in drug delivery. There are unique,
complex dynamics in each bone defect environment that
make it dicult to dene a consistently desirable delivery
dose and time scale. The integrity of the soft tissue
envelope, status of the periosteum, and age-dependent
abundance of bone-forming cell types are among the
variables that are not normalised across dierent patient
populations. These complexities make it dicult to arrive
at a denitive therapeutic index for scaold-based drug
delivery. In addition, gene expression analyses have shown
that over 6,500 genes are dierentially regulated during
bone healing (Rundle et al., 2006), which suggests a
molecularly complex environment in which multiple drugs
may be needed to promote optimal formation of bone and
other supportive tissue types (e.g. neural, vascular tissues),
particularly in large defects. However, the substantial
barriers to regulatory approval of devices that deliver a
single biologic suggest that carriers for multiple biologics
may not be clinically realistic in the foreseeable future. In
view of this complexity, there is a clear need to develop
adaptable scaolds that can be used to gain fundamental
insights into induced bone formation in a context that can
then be eciently translated to clinical applications.
Cell delivery
The rationale behind delivery of exogenous cells for
bone repair is that addition of appropriate cell types
may rescue or potentiate regeneration in cases where
92 www.ecmjournal.org
S Verrier et al. Large bone defect healing
the natural healing response is compromised or blocked.
Only cells can create bone, and therefore transplantation
of cells is a logical strategy to overcome recalcitrant
healing. Importantly, bone healing is a highly spatially
and temporally coordinated process, and therefore it is
dicult to recapitulate the normal cascade of events using
biomaterials or growth factors alone. Biomaterial-mediated
delivery of cells is often used to enhance the engraftment,
viability and function of the transplanted cells, and may
also be used in conjunction with bioactive factor delivery
to mimic physiological healing. Use of a scaffold or
matrix typically enhances the mechanical and space-lling
function of a transplant, and can provide instructive cues
to guide cell function and tissue regeneration. Most cell
delivery strategies focus on application of bone forming
cells, such as osteoblasts or their precursors (Lee et al.,
2009). However, more recent approaches have also targeted
concomitant modulation of other physiological processes,
such as development of a nourishing vasculature (Rao et
al., 2015) or management of the inammatory response
(Loi et al., 2016).
A variety of cell types have been used in bone
regeneration strategies. For large bone defects in particular,
the use of exogenously supplied cells may be necessary,
due to the need for regeneration of larger tissue volumes.
In these cases, scaolds to support cell delivery promote
engraftment and provide a space-lling function are often
used. The choice of preferred cell type may also depend
on the application and age of the patient and in some cases
combinations of cells can be applied (Wise et al., 2015).
Osteoblasts, the cells that secrete and assemble the ECM
of bone, have been delivered in hydrogel biomaterials to
enhance bone formation (Alsberg et al., 2001; Burdick
and Anseth, 2002); however, issues of immune rejection
would require an autologous source for these cells. The
diculties in isolating and expanding osteoblast cells are
substantial and make their use in clinics unlikely.
A variety of progenitor cell types have also been
examined as cell sources in bone tissue engineering.
MSCs are multipotent progenitors that have been
shown to dierentiate into connective tissue cell types,
including osteoblasts (Augello et al., 2010; Rosenbaum
et al., 2008), and also have been shown to be potent
sources of paracrine signalling factors (Parekkadan and
Milwid, 2010) that potentiate healing. Cell surface, or
CD (cluster of dierentiation) markers, are commonly
used to identify MSCs (reviewed in (Harichandan and
Buhring, 2011)), yet they should be used with caution.
There is increasing evidence that while able to distinguish
between mesenchymal and haematopoietic cells, they are
not able to dene characteristics of stemness (Whitney et
al., 2009). However, CD105+ and Stro1+ cells have been
proposed as clinically relevant populations. Many groups
have published changes in MSC phenotype and loss of
mutipotentiality with monolayer expansion (Bruder et al.,
1997; Ban et al., 2000; Bonab et al., 2006). One factor
which has been shown to be correlated with maintenance
of stemness is leukaemia inhibitory factor 1 (LIF1), the
expression of which decreases with monolayer expansion
and during dierentiation (Whitney et al., 2009).
When considering clinical use of cells, the complications
engendered by monolayer expansion provides a signicant
regulatory hurdle (Bara et al., 2014). This has increasingly
led to studies investigating whether freshly isolated,
minimally manipulated cells can be used for bone repair.
It has been shown that freshly isolated marrow cells can
lead to improved bone healing if more than 1,500 colony
forming units (CFU) of mesenchymal cells are applied
per cm3 of defect (Hernigou et al., 2005). Combining
this nding with intra-operative cell harvesting devices
provides a potential mechanism by which cell therapy can
be readily applied. The use of MSCs oers the possibility
of using banked cells, and it has been suggested that
allogeneic MSCs are hypoimmunogenic relative to other
cell types (Abumaree et al., 2012; Yi and Song, 2012).
MSCs from bone marrow (BMSCs) have been
investigated widely in bone tissue engineering (Youse et
al., 2016). MSCs can also be isolated from adipose tissue,
and these cells are often referred to adipose-derived stem
cells (ASCs). Obtained through subcutaneous aspiration,
adipose tissue presents advantages of easier accessibility
(Strioga et al., 2012) with minimal donor site morbidity
(Housman et al., 2002) and permits the harvest of larger
numbers of MSCs compared to other sources (Fraser et
al., 2006). The immunophenotype and other biological
characteristics of ASCs are generally similar to marrow-
derived MSCs, though there are some dierences (Pachon-
Pena et al., 2011). Indeed, according the cell isolation
procedure, a mixed population of cells containing both
stromal and endothelial progenitors can also be obtained
intraoperatively from the stromal vascular fraction of
adipose tissue. These properties make them attractive for
bone regeneration (Buschmann et al., 2012; Park et al.,
2012). Several attempts to heal large bone defects in animal
models have been made using scaolds loaded with ASCs,
but with inconsistent results. Success has been reported
for the healing of calvarial defects (Dudas et al., 2006;
Follmar et al., 2007), but large segmental defects in long
bones do not always heal in the absence of BMP-2 (Hao et
al., 2010; Li et al., 2007; Peterson et al., 2005). When the
eectiveness of ASCs and BMSCs was compared in a large
segmental defect in sheep (Niemeyer et al., 2010) healing
was greater with BMSCs. The latter have shown ecacy
in one human study (Quarto et al., 2001) and progenitor
cells obtained from periosteum were able to regenerate a
human phalanx when applied on a coral scaold (Vacanti
et al., 2001).
Totipotent cells sources, such as embryonic stem
cells (ESCs) and induced pluripotent stem cells (iPSCs),
have been less commonly explored in bone tissue
engineering. Culturing ESCs is technically challenging,
and the embryonic source is ethically controversial.
However, ESCs have recently been used to derive MSCs,
which in turn have been applied to bone regeneration
(Arpornmaeklong et al., 2009; Kuhn et al., 2014). iPSCs
are a newer potential cell source that oer the possibility
of generating pluripotent cells from reprogrammed adult
somatic cells (Ko and Im, 2014), and recently they have
been combined with scaold materials targeted at bone
regeneration (Liu et al., 2013).
93 www.ecmjournal.org
S Verrier et al. Large bone defect healing
Lately, much work has focused on co-transplantation of
multiple cell populations (i.e. osteogenic and angiogenic
cell populations) to enhance the bone regenerative
processes. Transplanting cells into large defects can create
regions that are hypoxic and low in nutrients necessary for
cell survival. To accelerate angiogenesis, cells capable of
contributing to the formation of a new vascular supply,
such as endothelial cells and endothelial progenitor cells
(EPC) can be used along with osteogenic cells (Cornejo
et al., 2012; Duttenhoefer et al., 2013; Herrmann et al.,
2015; Tavassol et al., 2010). Osteogenic and angiogenic
cells may communicate with each other to synergistically
improve both of these phenotypic processes (Dariima et
al., 2013). EPCs are progenitor cells of haematopoietic
lineage origin (Masuda and Asahara, 2003) and can be
easily isolated from peripheral blood using positive surface
marker selection such as CD133 and CD34 (Asahara et al.,
1997; Peichev et al., 2000). They show a high proliferation
rate compared to mature endothelial cells (Lin et al., 2000).
Several EPC sub-populations can be identified
in peripheral blood. Early and late EPCs (also called
OEC outgrowth endothelial cells) can be identied by
morphological characteristics (Hur et al., 2004; Lin et
al., 2000). Circulating EPCs are known to be responsible
for post-natal vasculogenesis, and are mobilised into the
blood stream from the bone marrow niche (Asahara et
al., 1999). Mobilisation is promoted by ischaemia and
certain cytokines such as granulocyte colony stimulating
factor (G-CSF). Circulating EPCs mobilised by G-CSF
have shown ecacy in healing non-unions in a rat model
(Mifune et al., 2008) and, when co-administered with
autologous bone graft, a small human clinical trial (Kuroda
et al., 2014).
Interestingly, signals produced by chondrocytes
may also promote the osteogenic response of stem cells
(Thompson et al., 2009) and may help recapitulate
endochondral ossification when transplanted with
osteoblasts (Alsberg et al., 2002).
Several modular approaches to cell delivery are also
being investigated. For example, lyophilised solid scaolds
have been developed that have shape memory properties
(Thornton et al., 2004). They may be delivered to a defect
in a compact form using a minimally invasive approach
such as through a catheter, and then a cell suspension can
be subsequently delivered to rehydrate them and drive
them to expand into a predetermined shape and volume.
Additionally, microscale constructs have been engineered,
such as hydrogel microspheres containing cells (Rao et al.,
2013) or self-assembling cell aggregates (Hildebrandt et
al., 2011; Solorio et al., 2012; Dang et al., 2016; Solorio
et al., 2015) that can similarly be injected. It is important
to recognise that in some cases cell delivery may not
even be necessary, if the scaold itself can present signals
capable of recruiting large enough numbers of endogenous
host osteogenic cells (Schantz et al., 2007) and/or anti-
inammatory or wound healing macrophages (Das et al.,
2013).
Vascularisation
A key challenge in the treatment of large bone defects is
the establishment of sucient vascularisation at the defect
site. Because the oxygen delivery required for the survival
of cells is usually limited to a diusion distance of ~ 150-
200 µm to a neighbouring microvessel (Colton, 1995),
the centre of cell seeded constructs rapidly die without
the establishment of a blood supply. Accordingly, various
vascularisation strategies have been developed in the eld
of regenerative medicine and tissue engineering (Laschke
and Menger, 2012), which may more successfully support
the treatment of large bone defects in future clinical practice
(Fig. 2). The close physical and biochemical interaction
between microvessels and bone cells is essential for bone
formation and repair (Carano and Filvaro, 2003). Many
angiogenic growth factors, such as VEGF or FGF, have
been shown to promote the dierentiation, migration and
proliferation of osteoblasts (Carano and Filvaro, 2003).
On the other hand, osteogenic factors, such as BMP-2,
stimulate the switch of endothelial cells from a quiescent
to an angiogenic phenotype (Finkenzeller et al., 2012).
An important structural determinant for adequate
vascularisation is the pore size of scaolds. It is well
recognised that the ideal pore size for the ingrowth of new
microvessels ranges between ~ 200-600 µm (Druecke et
al., 2004). In this size range, poly(lactic-co-glycolic acid)
(PLGA) scaolds for bone defect repair also display
suitable oxygen diusion, pre-osteoblast cell inltration,
proliferation and survival without losing their mechanical
strength (Amini et al., 2012). However, this does not
necessarily require that scaolds should be created with a
homogeneous pore size. In fact, sophisticated technologies
such as rapid prototyping oer the possibility to fabricate
scaolds with clearly dened porosity levels to ideally
promote individual key steps of the bone healing process.
Yang et al. developed ceramic scaolds with sub-µm
pores to improve cell/surface interactions, pores of tens
of µm to support osteoconduction, and corridors of 100-
600 µm to stimulate vascularisation (Yang et al., 2006).
Finally, the overall three-dimensional architecture of
scaolds has recently been shown to markedly aect their
vascularisation.
The vascularisation of bone defects may also be
improved by the application of compounds with pro-
angiogenic properties. Of interest, Holstein et al. reported
that systemic treatment with the glycoprotein erythropoietin
(EPO) is capable of stimulating bone formation, cell
proliferation and angiogenesis in a femoral segmental defect
model in mice (Holstein et al., 2011). Compared to this
systemic approach, the topical application of angiogenic
growth factors at the defect site is much more common.
For this purpose, the factors may be coated on the surface
of solid scaolds (Sun et al., 2011) or incorporated into
drug delivery systems such as microparticles or hydrogels
(Geuze et al., 2012; Ishida et al., 2010; Ratanavaraporn et
al., 2011). Alternatively, platelet-rich plasma (PRP) may
be applied, which represents a rich, autologous source of
various growth factors and can easily be isolated from
patients under clinical conditions (Lucarelli et al., 2005;
Jalowiec et al., 2016; Lippross et al., 2011). In general, it
94 www.ecmjournal.org
S Verrier et al. Large bone defect healing
should be noted that the ecacy of the administration of
growth factors may be markedly inuenced by their release
rates and inactivation (Geuze et al., 2012) (see section
“Drug and Growth Factors delivery”). Moreover, there is
no doubt that a combination of factors support individual
stages of bone healing and angiogenesis (Ratanavaraporn
et al., 2011; Su et al., 2013). Besides treatment with growth
factors, local delivery of miRNA is as a novel possibility
to optimise angiogenesis-osteogenesis coupling during
bone defect healing. Over-expression of miR-26a in
critical-size calvarial bone defect resulted in an improved
vascularisation and complete defect healing (Li et al.,
2013).
There are several possibilities for the generation of
microvascular networks within tissue constructs. These
include the in vitro seeding and cultivation of scaolds
with vessel-forming cell types (Koike et al., 2004; Wang et
al., 2007). However, this involves complex cell isolation,
seeding, and cultivation procedures, which may not be
realisable in clinical practice. Another common strategy to
induce vascularisation in bone defect healing is the seeding
of appropriate scaolds with dierentiated tissue-specic
cells (Cornejo et al., 2012; Tavassol et al., 2010), EPCs
(Seebach et al., 2010) or multipotent stem cells (Maraldi
et al., 2013; Zhang et al., 2010b) (see section “Cell
delivery”). By this method, the formation of new blood
vessels is primarily stimulated by hypoxia-driven cellular
release of angiogenic growth factors during engraftment
(Schumann et al., 2009). The seeded cells may additionally
be genetically modied to guarantee a more continuous
growth factor secretion at the defect site (see section “Gene
Therapy”). Promising growth factors in bone defect healing
include hypoxia-inducible factor-(HIF)-1α (Zou et al.,
2012), VEGF (Geiger et al., 2005; Li et al., 2009b), FGF-
2 (Guo et al., 2006; Qu et al., 2011), and angiopoietin-1
(Cao et al., 2012a).
An interesting study by Kasper et al. indicates that some
of the angiogenic and vasculogenic mechanisms may be
additionally regulated by mechanical loading of the cells
(Kasper et al., 2007). Using tube formation and spheroid
sprouting assays, they found a signicant enhancement
of angiogenesis by conditioned media from mechanically
stimulated compared with unstimulated MSCs. Thus,
they concluded that mechanical loading of MSCs results
in a paracrine stimulation of blood vessel formation,
most likely by the up-regulation of angiogenic growth
Fig. 2. Basic in vitro and in situ vascularisation strategies for tissue engineering constructs as outlined in the
section “Vascularisation”. In vitro vascularisation strategies focus on the modication of tissue constructs prior to
their implantation. This can be achieved by changing the chemical and structural properties of scaolds (1) or by
their biological activation with growth factor delivery systems (2), cells (3) and microvascular fragments (4). In
situ vascularisation strategies focus on the generation of preformed microvascular networks within scaolds by
implanting them in well-vascularised areas of the body (1) or by generation of an arteriovenous(AV)-loop (2). After
this prevascularisation phase, the scaolds are transferred to the nal defect site, where they rapidly establish a blood
supply by developing interconnections with the surrounding host microvasculature, i.e. inosculation (1), or by direct
surgical anastomosis of a vascular pedicle (2).
95 www.ecmjournal.org
S Verrier et al. Large bone defect healing
factors including VEGF and FGF. Another determinant
for the vascularisation potential of MSCs is their three-
dimensional arrangement. It was recently demonstrated
that polyurethane scaffolds, which are seeded with
multicellular MSC spheroids, exhibit a markedly improved
vascularisation when compared to control scaolds seeded
with an identical number of individual MSCs (Laschke et
al., 2013). Immunohistochemical analyses of the implants
revealed that this is due to an enhanced vessel-forming
capacity of the three-dimensional MSC spheroids, making
them attractive vascularisation units for future tissue
engineering applications.
Taken together, all of these studies indicate that
major progress has been made in recent years towards
establishing novel strategies to promote angiogenesis
and vasculogenesis in bone tissue engineering. However,
the basic problem of all of these strategies is the fact that
blood vessel formation is a time-consuming multi-step
process, which cannot be accelerated limitlessly. The
growth of newly developing microvessels is usually not
faster than ~5 µm/h (Utzinger et al., 2015). Accordingly,
complete vascularisation of large bone defects by ingrowth
of microvessels from the defect borders takes far too long
to guarantee cell survival at the defect site. A promising
concept to overcome this problem is the generation of
prevascularised tissue constructs that exhibit a functional
preformed microvascular network, which connects with
the surrounding microvasculature, known as inosculation
(Laschke and Menger, 2016).
Alternatively, it is possible to pre-vascularise scaolds
in situ by implanting them in well-vascularised areas of
the body to promote the ingrowth of new microvessels
(Laschke et al., 2011). Moreover, the incorporation of
an arteriovenous loop (AV)-loop, i.e. a ligated artery
and vein (Boos et al., 2013), or a vasculature bundle
(VB), i.e. a ligated artery and vein, into scaolds even
allows the in situ generation of tissue constructs with a
vascular pedicle, which can be surgically anastomosed to
the vessels of the defect site. Of interest, Wu et al. found
that the VB technique results in a better balance between
bone regeneration and scaold degradation than the AV-
loop strategy for the prevascularisation of bone constructs
consisting of β-tricalcium phosphate scaolds and BMSCs
(Wu et al., 2015).
Currently, in situ prevascularisation represents the
most promising approach to guarantee a sucient blood
supply to large bone constructs in the clinical setting. In
fact, Horch et al. recently reported the rst successful
application of the AV-loop technique in two patients
with large bone defects in the radius and tibia (Horch et
al., 2014). However, in situ prevascularisation strategies
normally bear the disadvantage that they require repetitive
surgical interventions for the implantation of scaolds
to the site of prevascularisation, and their removal for
nal transfer into a defect. To overcome this problem,
scaolds may be seeded in the future with adipose-derived
microvascular fragments (Laschke and Menger, 2015a).
These microvascular fragments are a randomised mixture
of fully functional arteriolar, capillary and venular vessel
segments with associated MSCs, which can be easily
isolated from adipose tissue by enzymatic digestion
(Laschke and Menger, 2015). After their implantation these
fragments survive and exhibit a high angiogenic activity,
forming new microvascular networks, which develop
interconnections to the microvessels of the host tissue.
Mechanical factors
Mechanical stability is known to be an important factor
for bone healing outcome. Indeed, beside the quality of
the implant, a large amount of experimental and clinical
evidence conrms that the course of fracture repair can
be inuenced by mechanical stimuli, and that controlled
instability at the fracture site (dynamisation) can deeply
affect bone regeneration. However, optimal loading
parameters to enhance fracture healing have not yet
been entirely dened. There are still many uncertainties
concerning the magnitude of the load, the loading timing
after fracture, but also the type of loading (e.g. axial,
bending). Despite the considerable attention paid to
fracture healing and, to some degree, sub-critical size
osteotomies, there is very little literature on the eects of
the mechanical environment on the healing of large bone
defects.
Mechanical stimulation of bone can be classified
according to the type of motion applied. Since Goodship
and Kenwright (Goodship and Kenwright, 1985), several
groups have shown that a cyclic, axial, compressive
displacement applied to a diaphysal fracture or osteotomy
induces higher healing by the formation of a stronger
cartilaginous callus leading to earlier bone bridging (Claes
et al., 1998; Wolf et al., 1998; Yamaji et al., 2001).
In certain studies, strains in the range of 5 % and 15 %
were shown to be benecial (Claes and Heigele, 1999;
Wolf et al., 1998; Yamaji et al., 2001). In other studies,
however, maximum strains of 7 % have been described to
be benecial to the gap bridging (Augat et al., 1998; Claes
et al., 1997; Claes et al., 1998), and larger displacement
was described as resulting in more brous tissue leading to
delayed bone union. In a nice experimental set up, Hente
(Hente R et al., 1990) looked at the eect of dened strain
on new bone formation (Fig. 3). A strain gradient from
0 % to more than 100 % was applied along a fracture gap.
Results showed that 0 % strain did not promote callus
formation, while strain from 30 % and higher induced
massive callus formation but without any evidence of
bridging. However, a strain of 5 % in this system was found
to be the most ecient to induce solid bridging of the gap.
Another parameter, which is still a point of discussion,
is the optimal initiation of stimulation. At the cellular level,
it has been shown that mesenchymal cells dierentiate
toward osteogenic or chondrogenic lineages at early stages
of the healing process, depending on the mechanical
environment (Le et al., 2001; Thompson et al., 2002).
Studies comparing timing of initiation of axial loading in
a rat osteotomy model, showed a positive eect of direct
post-surgery stimulation (Klein et al., 2003; Weaver et
al., 2010). In addition, Weaver also reported a positive
eect of a later starting point (10 d post-surgery), while
an intermediate time point (3 d) was not as benecial. A
benecial eect of a later starting point was also described
96 www.ecmjournal.org
S Verrier et al. Large bone defect healing
by others and was explained by the fact that this delay
might be favourable to the initiation of neo-vascularisation
(Claes et al., 2002; Wallace et al., 1994).
However, while several groups have studied the
eect of mechanical stimulation on small defects (1 to
2 mm osteotomy), only few studies have reported the
effect of mechanical loading on a large bone defect.
In a critical size goat femur bone defect (filled with
demineralised bone and MSCs), treated with either a
dynamic intramedullary rod or a static intramedullary
rod, an overall better neovascularisation of the implants
was seen in the dynamised cases (10 % strain) compared
to the rigid xations (Hou et al., 2010). Callus formation
and bone healing was compared in a 5 mm defect treated
with BMP-2, when the defects were stabilised with
interchangeable external xators creating low, medium
or high axial stiness (Glatt et al., 2012). Under constant
stiness, the low stiness group showed an increased
healing rate when compared to the medium or high stiness
groups. While switching at day 14 from low stiness to a
high stiness xator (reverse dynamisation), showed by
far improved bone healing compared to all other groups.
Epari et al. subsequently provided a theoretical basis for
these observations (Epari et al., 2013).
Thus a systematic comparison of the three above cited
parameters (timing, amplitude and loading type) is still
missing. Additionally, studies investigating osteosynthesis
devices designed specically for critical sized defects are
lacking. Such studies are needed to provide a clearer view
about the eects of mechanical stimulation on the healing
of large bone defects.
Additional approaches
Endochondral bone tissue engineering
In vitro bone tissue engineering strategies commonly
focus on promoting direct osteoblastic dierentiation
within cell-seeded constructs, mimicking the process of
intramembranous ossication. Such engineered tissues
often fail to promote bone regeneration following
implantation (Lyons et al., 2010), leading to increased
interest in endochondral bone tissue engineering strategies
(Thompson et al., 2014). This involves the implantation
of tissue engineered cartilage in an attempt to recapitulate
the normal long bone development process whereby
a cartilaginous template becomes hypertrophic, is
vascularised, and is ultimately replaced with bone. The
logic of this approach is that chondrocytes are better
equipped to survive within the nutrient and oxygen
deprived environments that exist within a large bone defect
(Farrell et al., 2009; Gawlitta et al., 2010). Furthermore,
hypertrophic chondrocytes progressing along the
endochondral pathway are known to release factors such
as VEGF to promote vascularisation of the implanted tissue
(Farrell et al., 2009; Gawlitta et al., 2010).
Chondrogenically primed BMSCs also have an
inherent tendency to become hypertrophic and undergo
endochondral ossication (Farrell et al., 2009; Pelttari
et al., 2006; Scotti et al., 2010; Vinardell et al., 2012b).
This has motivated the use of cartilaginous constructs
engineered using BMSC-seeded scaffolds for bone
regeneration. One of the earliest demonstrations of this
concept was reported by Huang and colleagues (Huang
et al., 2006), who found that cartilage tissue engineered
in vitro using BMSCs could be used for carpal bone
reconstruction in a rabbit model. More recent studies
have provided greater insight into the mechanisms by
which chondrogenically dierentiated BMSCs promote
bone formation in vivo. TGF-β typically used to promote
chondrogenic dierentiation of BMSCs, has been shown to
promote the expression not only of genes associated with
chondrogenesis and hypertrophy, but also the production of
factors critical to vascularisation such as VEGF and matrix
metalloproteinases (Farrell et al., 2009; Pelttari et al.,
2006). Following implantation, chondrogenically primed
Fig. 3. Eect of strain on callus formation (adapted from (Hente et al., 1990)). (a): schematic representation of the
experimental set up. A portion of bone is cyclically tilted along its gap of origin, creating a gradient of strain from
0 (tip of the fragment) to 100 % (top of the fragment). (b): X-ray imaging showing the presence of bone bridging
in the lower strain area compared to the higher strain where larger callus formation without bridging was observed.
97 www.ecmjournal.org
S Verrier et al. Large bone defect healing
MSCs have been shown to directly contribute to new bone
tissue formation, and also to facilitate the recruitment of
host cells capable of further driving osteogenesis (Farrell
et al., 2011; Pelttari et al., 2006; Scotti et al., 2010).
MSC-seeded scaolds have also been shown to promote
greater vascularisation than osteoblast-seeded scaolds
in vivo by activating an endochondral ossication process
and recruiting host-derived CD31+ endothelial cells,
followed by a second wave of host derived CD146+
cells that display characteristics of MSCs (Tortelli et al.,
2010). Chondrogenically primed MSCs can accelerate
the regeneration of critically sized bone defects in small
animal models. Cartilage grafts were used to promote
regeneration using a murine segmental tibial defect model
(Bahney et al., 2014). This study also used lineage tracing
experiments to show the regenerate was graft-derived,
suggesting the direct transformation of chondrocytes
into bone forming cells (Bahney et al., 2014). Finally, it
has recently been demonstrated that chondrogenically-
primed MSC-laden scaolds support greater repair of
critical-sized cranial defects than osteogenically stimulated
constructs (Thompson et al., 2016). Taken together, these
studies demonstrate the potential of endochondral tissue
engineering strategies for orchestrating bone regeneration.
A number of questions still have to be addressed to
fully realise the potential of engineered cartilage for large
bone defect healing. These include determining the optimal
duration of chondrogenic pre-culture for MSCs (Yang et
al., 2015), as well as the identication of factors that both
promote hypertrophy in vitro and accelerate endochondral
bone formation in vivo. MSCs implanted subcutaneously
into nude mice have been shown to form bone trabeculae
only if they have generated supporting hypertrophic tissue
structures prior to implantation (Scotti et al., 2010). More
advanced hypertrophic maturation of MSCs in vitro was
also found to promote the formation of larger bony tissues
in vivo (Scotti et al., 2010). Another challenge involves
the identification of suitable biomaterials to support
endochondral bone regeneration (Cunnie et al., 2015),
as well as engineering in vitro cultures that facilitate the
development of hypertrophic cartilage of sucient scale to
treat large bone defects. MSCs seeded onto collagen-based
scaolds and directed along an endochondral pathway in
vitro have been used to generate a scaled-up bone organ
in vivo which was found to contain a fully functional
haematopoietic compartment (Scotti et al., 2013). Synthetic
and natural polymeric scaolds (Yang et al., 2013; Yang
et al., 2015) and various hydrogels (Dickhut et al., 2008)
can also potentially be used for engineering scaled-up
endochondral bone tissue. An improved understanding of
how environmental cues (specic to a bone defect) will
regulate endochondral bone regeneration is also required.
For example, it has been shown that factors such as a
low oxygen environment (Sheehy et al., 2013) as well as
certain mechanical cues like compression (Thorpe et al.,
2013) and hydrostatic pressure (Vinardell et al., 2012a) can
suppress markers of hypertrophy in MSCs. This highlights
the need to consider the many factors that contribute to poor
outcomes in complex bone fractures and segmental defects
when designing novel endochondral bone regeneration
strategies.
Gene Therapy
Although several different osteogenic growth factors
show promise as agents of bone healing, their clinical
deployment is constrained by delivery problems. In
particular, it is not possible to deliver these proteins locally
at physiological concentrations in a sustained fashion.
With BMPs -2 and -7, this problem has been addressed
clinically by their implantation at very high doses on simple
scaolds. This provides modest clinical ecacy and, at
least in the case of BMP-2, provokes a number of adverse
events, some serious (Carragee et al., 2011; Faundez et
al., 2016) (section “Drug and Growth Factor delivery”).
Gene transfer technologies oer to solve these problems.
They also remove the concern that preparations of
recombinant proteins may contain denatured, and possibly
immunogenic, molecules. Moreover, there is evidence that
cells respond better to endogenously synthesised growth
factors than their recombinant equivalents.
The basic concept is quite simple. A gene, or more
usually a cDNA, encoding a protein of interest is delivered
by a vector to the site of an osseous defect. This protein
is synthesised locally in an endogenous, authentic fashion
for as long as the cDNA is present and expressed. By
incorporating regulatory elements, it is possible to control
both the level and duration of transgene expression.
Although most pre-clinical development has focused on
delivering cDNAs that encode secreted growth factors,
gene transfer is particularly well suited to delivering
intracellular proteins, such as transcription factors (Tu et
al., 2007), the Lim mineralisation proteins (Lattanzi et al.,
2008) and non-coding species of RNA (Levi et al., 2012).
Gene delivery can be accomplished with non-viral
and viral vectors. Although non-viral methods are less
expensive and generally considered to be safer than viral
vectors, they are also much less efficient. Transgene
expression is usually low and transient. However, the
literature contains examples demonstrating the ability to
heal osseous defects in animal models using non-viral gene
transfer methods (Kimelman-Bleich et al., 2011; Li et al.,
2009a). Nevertheless, most research involves the use of
viral vectors which, although more dicult and expensive
to prepare, are much more ecient.
Viral vectors that have been explored in the context of
bone healing are retrovirus (Rundle et al., 2008), lentivirus
(Virk et al., 2011), adenovirus (Baltzer et al., 2000), adeno-
associated virus (AAV) (Ito et al., 2005) and baculovirus
(Lin et al., 2012). Each has advantages and disadvantages
in terms of ease of preparation and use, persistence in the
host, immunogenicity, carrying capacity, serotype and so
forth. Much eort has been devoted to engineering novel
and improved versions of many of these viral vectors, so
simple descriptors are increasingly dicult.
Use of viral vectors raises issues of safety, which is a
key issue for non-lethal indications such as bone healing.
The major safety concern with retroviruses, including
lentiviruses, is insertional mutagenesis, which has led to the
development of leukaemia in human subjects in a clinical
trial for Severe Combined Immunodeciency Disease
(Hacein-Bey-Abina et al., 2003). The major safety issue
with adenovirus is the strong immune responses that it
generates; these led to gene therapy’s rst death, in 1999
98 www.ecmjournal.org
S Verrier et al. Large bone defect healing
(Raper et al., 2003). That said, the incidence of severe
adverse events in gene therapy trials has been remarkably
low and the safety issue is as much one of psychology as
biology. Ironically, given recent disclosures concerning
the severe adverse events generated by the large amounts
of BMP-2 present in Infuse®, USA (InductOs, Europe)
(Carragee et al., 2011), it is possible to argue that, in this
particular application, gene therapy may well be safer than
protein therapy.
Most investigators have used cDNAs encoding
proteins that promote the osteogenic dierentiation of
mesenchymal cells. BMP-2 or BMP-7 are popular choices,
as the recombinant proteins are already in clinical use.
There is also enthusiasm for using cDNAs encoding
angiogenic factors such as VEGF (Li et al., 2009b), because
osteogenesis is known to have an absolute requirement
for angiogenesis (see section “Vascularisation”). Other
transgenes of experimental interest include cycloxygenase,
which promotes osteogenesis via prostaglandin synthesis
(Rundle et al., 2008), and parathyroid hormone 1-34
(Bonadio et al., 1999), among others. Although the choice
of osteogenic genes is understandable, long bone fractures
mainly heal through the initial phases of cartilaginous callus
formation and subsequent endochondral ossication. In
recognition of this, there is increasing interest in promoting
the endochondral route to healing large bone defects (see
section “Endochondral bone tissue engineering”). BMP-
2 could be a useful transgene in this regard, because it
promotes both chondrogenesis (Palmer et al., 2005) and
the endochondral dierentiation of MSCs (Steinert et al.,
2009).
Regardless of the vectors and transgenes that are used,
there are two major strategies for their deployment: in vivo
and ex vivo. During in vivo delivery the vector is introduced
directly into the osseous defect. This has advantages of
simplicity, but raises safety concerns. Ex vivo delivery is
more cumbersome and expensive, but does not introduce
vector into the body and provides the opportunity to
deliver both osteoprogenitor cells and osteogenic genes
concomitantly to the defect. The considerable cost and
complexity of ex vivo gene delivery with autologous cells
can be mitigated with allogeneic, universal donor cells,
Fig. 4. Current approaches for gene delivery to osseous lesions. There are two general strategies: in vivo (right hand
side) and ex vivo (left-hand side). For in vivo gene delivery, the vector is introduced directly into the site of the
osseous lesion, either as a free suspension (top right) or incorporated into a gene-activated matrix (GAM) (bottom
right). For ex vivo delivery, vectors are not introduced directly into the defect. Instead they are used for the genetic
modication of cells, which are subsequently implanted. Traditional ex vivo methods (top left) usually involve the
establishment of cell cultures, which are genetically modied in vitro. The modied cells are then introduced into
the lesion, often after seeding onto an appropriate scaold. Expedited ex vivo methods (bottom left) avoid the need
for cell culture and scaolds by genetically modifying tissues such as marrow, muscle and fat, intraoperatively and
inserting them into the defect during a single operative session (Evans, 2010).
99 www.ecmjournal.org
S Verrier et al. Large bone defect healing
or by developing expedited protocols where autologous
cells or tissues are removed, genetically modied, and
reimplanted in a single operative session (Evans et al.,
2007).
Based upon these principles, four main approaches
have emerged (Fig. 4) for delivery of genes to osseous
lesions: in vivo gene delivery by direct injection or with
gene-activated matrices (GAMs), and ex vivo delivery
using expanded autologous cells or expedited approaches
accomplished intra-operatively.
The direct injection of adenovirus vectors encoding
BMP-2 (Baltzer et al., 2000; Betz et al., 2006) or BMP-6
(Bertone et al., 2004; Ishihara et al., 2008) can heal critical
size femoral defects in rats, rabbits and horses. However,
it is not reliably eective in all animals and generates a
strong neutralising immune reaction to the vector. These
immune reactions were sufficiently strong to prevent
ecacy in large bone defects in sheep (Egermann et al.,
2006b), unless the sheep had been treated previously
with cortisone, an immunosuppressant (Egermann et al.,
2006a). Of concern, immune reactions to human BMP-2
were generated in the sheep model, possibly reecting the
strong adjuvant properties of adenovirus.
GAMs provide an alternative approach. The original
GAM comprised a collagenous scaffold impregnated
with plasmid DNA encoding BMP-4 (Fang et al., 1996)
or the rst 34 amino acids of parathyroid hormone (PTH
1-34) (Bonadio et al., 1999), presently used as the drug
teriparatide (Forteo®) to treat osteoporosis. Impressive
data were reported in rat and canine models, but further
development was hindered by the low levels of transgene
expression. GAMs incorporating improved non-viral
(Tierney et al., 2012) or viral vectors show more ecient
gene transfer and expression in animal models.
Allograft revitalisation is an extension of the GAM
principle in which AAV vectors are coated onto allograft
bone (Ito et al., 2005). After implantation, host progenitor
cells encounter the vector and express transgenes, leading
to resorption of the allograft and its replacement with host
bone. Success has also been reported when AAV is coated
onto poly(epsilon-caprolactone) (Dupont et al., 2012).
Lieberman’s group pioneered the ex vivo approach,
successfully using an adenovirus vector encoding BMP-2
in conjunction with BMSCs (Lieberman et al., 1999). To
expedite matters, they now use buy coat cells obtained
intra-operatively from bone marrow, in conjunction with
a lentivirus vector that gives higher and more persistent
transgene expression (Virk et al., 2011). Because of
concerns about insertional mutagenesis with lentivirus, the
inclusion of a suicide gene, to be activated in the event of
malignant transformation or other severe adverse event, is
being explored (Alaee et al., 2014).
An alternative expedited, ex vivo approach makes use of
the remarkable osteogenic properties of muscle, reected
in the high incidence of heterotopic ossication of muscle
after blast injuries and joint replacement surgery, as well
as in the disease brodysplasia ossicans progressiva.
The latter occurs as a result of an activating mutation in
a BMP receptor, suggesting that a sustained BMP signal
eciently induces bone in muscle. Use of an adenovirus
encoding BMP-2 (Ad.BMP-2) provides such as signal.
Musgrave et al. showed that the intra-muscular injection
of Ad.BMP-2 induced bone in muscle (Musgrave et al.,
1999). This has been adapted in a strategy where biopsies
of autologous muscle are transduced with Ad.BMP-2 and
implanted into critical sized defects in rats (Evans et al.,
2009). Autologous fat is also eective, but less reliably
than muscle. Of note, this abbreviated ex vivo procedure
eliminated the humoral response to adenovirus (Evans et
al., 2009).
Despite a considerable literature, reviewed in references
(Evans, 2010; Evans, 2012; Pensak and Lieberman, 2013),
reporting successes in healing large bone defects in animal
models by gene therapy, it is not used clinically. There are a
number of reasons for this, including the need for studies in
large animals, which are costly and take a long time. Often,
insucient attention is paid to pharmacology, toxicology
and other important matters of this nature. Furthermore,
the scientists who undertake the pre-clinical research
are often naïve when it comes to the process of research
translation, which involves a wide spectrum of expertise,
ranging from regulatory issues, to clinical trial design,
ethics, and so forth. It is wise to involve individuals with
the necessary expertise early in the research programme to
forestall subsequent barriers to translation (Madry et al.,
2014).
Another constraint to clinical application lies in the
simple fact that we do not know how much of a given gene
product is needed at which time during the healing process
and for how long. Such information would greatly advance
the eld.
Conclusions
At a minimum, the regeneration of bone requires the
balanced contributions of scaolds, cells, morphogenetic
signals, vascularisation and mechanics. Each of these
elements is being studied intensively, and considerable
advances have been made in developing new
understandings, concepts and information. Because each
of these components has many facets, and therefore an even
greater number of permutations, there are innumerable
theoretical combinations that frustrate any straightforward
development of new, osteogenic technologies.
But it is also noteworthy that, despite decades of
research in this area using dierent combinations of the
components discussed in this review, we still lack an
approved engineered product that gives robust, reliable
results in the clinic. It is possible that, given the impossibly
large number of permutations of the base components
studied for the healing of large, osseous, segmental defects,
researchers have not yet arrived at the optimal combination.
However, it is also possible that we are missing something.
Bone, after all, normally heals by itself, whereas large
segmental defects do not. Perhaps we need to go back to
the beginning and discover why large segmental defects
in otherwise healthy individuals do not heal? Perhaps
formulating strategies based upon the way fractures heal
naturally is inappropriate?
Regardless of the technologies that actually work
reliably in advanced pre-clinical models, the clinical
100 www.ecmjournal.org
S Verrier et al. Large bone defect healing
development of such technologies is constrained by the
regulatory environment that governs their deployment,
as well as the nancial realities of health care economics.
The reality may be that without adequate stratication
and identication of patients, complex therapies may
not provide the economic benet to make them viable.
As described in this critical review, progress is occurring
on several fronts and it should be only a matter of time
before patients can benet from better ways to heal large
segmental defects. Achieving this will require interactive,
well-funded, sustained consortia including biologists,
physical scientists, clinicians, translational scientists, and
industrial partners.
Acknowledgements
The authors acknowledge the support of the AO Foundation
in establishing and maintaining their large bone defect
healing consortium.
References
Abumaree M, Al Jumah M, Pace RA, Kalionis B (2012)
Immunosuppressive properties of mesenchymal stem cells.
Stem Cell Rev 8: 375-392.
Alaee F, Sugiyama O, Virk MS, Tang H, Drissi H,
Lichtler AC, Lieberman JR (2014) Suicide gene approach
using a dual-expression lentiviral vector to enhance the
safety of ex vivo gene therapy for bone repair. Gene Ther
21: 139-147.
Alsberg E, Anderson KW, Albeiruti A, Franceschi RT,
Mooney DJ (2001) Cell-interactive alginate hydrogels for
bone tissue engineering. J Dent Res 80: 2025-2029.
Alsberg E, Anderson KW, Albeiruti A, Rowley JA,
Mooney DJ (2002) Engineering growing tissues. Proc Natl
Acad Sci U S A 99: 12025-12030.
Amini AR, Adams DJ, Laurencin CT, Nukavarapu SP
(2012) Optimally porous and biomechanically compatible
scaolds for large-area bone regeneration. Tissue Eng Part
A 18: 1376-1388.
Arpornmaeklong P, Brown SE, Wang Z, Krebsbach
PH (2009) Phenotypic characterisation, osteoblastic
dierentiation, and bone regeneration capacity of human
embryonic stem cell-derived mesenchymal stem cells.
Stem Cells Dev 18: 955-968.
Arrighi I, Mark S, Alvisi M, von Rechenber B, Hubbell
JA, Schense JC (2009) Bone healing induced by local
delivery of an engineered parathyroid hormone prodrug.
Biomaterials 30: 1763-1771.
Asahara T, Masuda H, Takahashi T, Kalka C, Pastore
C, Silver M, Kearne M, Magner M, Isner JM (1999)
Bone marrow origin of endothelial progenitor cells
responsible for postnatal vasculogenesis in physiological
and pathological neovascularization. Circ Res 85: 221-228.
Asahara T, Murohara T, Sullivan A, Silver M, van der
Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM
(1997) Isolation of putative progenitor endothelial cells
for angiogenesis. Science 275: 964-967.
Augat P, Margevicius K, Simon J, Wolf S, Suger G,
Claes L (1998) Local tissue properties in bone healing:
inuence of size and stability of the osteotomy gap. J
Orthop Res 16: 475-481.
Augello A, Kurth TB, De BC (2010) Mesenchymal
stem cells: a perspective from in vitro cultures to in vivo
migration and niches. Eur Cell Mater 20: 121-133.
Badylak SF, Freytes DO, Gilbert TW (2009)
Extracellular matrix as a biological scaold material:
Structure and function. Acta Biomater 5: 1-13.
Bahney CS, Hu DP, Taylor AJ, Ferro F, Britz HM,
Hallgrimsson B, Johnstone B, Miclau T, Marcucio RS
(2014) Stem cell-derived endochondral cartilage stimulates
bone healing by tissue transformation. J Bone Miner Res
29: 1269-1282.
Baltzer AW, Lattermann C, Whalen JD, Wooley P,
Weiss K, Grimm M, Ghivizzani SC, Robbins PD, Evans
CH (2000) Genetic enhancement of fracture repair: healing
of an experimental segmental defect by adenoviral transfer
of the BMP-2 gene. Gene Ther 7: 734-739.
Ban A, Muraglia A, Dozin B, Mastrogiacomo M,
Cancedda R, Quarto R (2000) Proliferation kinetics and
dierentiation potential of ex vivo expanded human bone
marrow stromal cells: Implications for their use in cell
therapy. Exp Hematol 28: 707-715.
Bara JJ, Richards RG, Alini M, Stoddart MJ (2014)
Concise review: Bone marrow-derived mesenchymal
stem cells change phenotype following in vitro culture:
implications for basic research and the clinic. Stem Cells
32: 1713-1723.
Barradas AM, Fernandes HA, Groen N, Chai YC,
Schrooten J, van de Peppel J, van Leeuwen JP, van
Blitterswijk CA, de BJ (2012) A calcium-induced
signaling cascade leading to osteogenic dierentiation of
human bone marrow-derived mesenchymal stromal cells.
Biomaterials 33: 3205-3215.
Behravesh E, Zygourakis K, Mikos AG (2003)
Adhesion and migration of marrow-derived osteoblasts on
injectable in situ crosslinkable poly(propylene fumarate-
co-ethylene glycol)-based hydrogels with a covalently
linked RGDS peptide. J Biomed Mater Res A 65: 260-270.
Bertone AL, Pittman DD, Bouxsein ML, Li J, Clancy
B, Seeherman HJ (2004) Adenoviral-mediated transfer of
human BMP-6 gene accelerates healing in a rabbit ulnar
osteotomy model. J Orthop Res 22: 1261-1270.
Betz OB, Betz VM, Nazarian A, Pilapil CG, Vrahas
MS, Bouxsein ML, Gerstenfeld LC, Einhorn TA, Evans
CH (2006) Direct percutaneous gene delivery to enhance
healing of segmental bone defects. J Bone Joint Surg Am
88: 355-365.
Boden SD, Zdeblick TA, Sandhu HS, Heim SE (2000)
The use of rhBMP-2 in interbody fusion cages. Denitive
evidence of osteoinduction in humans: a preliminary report.
Spine (Phila Pa 1976) 25: 376-381.
Bonab MM, Alimoghaddam K, Talebian F, Ghaari SH,
Ghavamzadeh A, Nikbin B (2006) Aging of mesenchymal
stem cell in vitro. BMC Cell Biol 7: 14.
Bonadio J, Smiley E, Patil P, Goldstein S (1999)
Localized, direct plasmid gene delivery in vivo: prolonged
therapy results in reproducible tissue regeneration. Nat
Med 5: 753-759.
101 www.ecmjournal.org
S Verrier et al. Large bone defect healing
Boos AM, Loew JS, Weigand A, Deschler G, Klumpp
D, Arkudas A, Bleizier O, Gulle H, Kneser U, Horch RE,
Beier JP (2013) Engineering axially vascularized bone in
the sheep arteriovenous-loop model. J Tissue Eng Regen
Med 7: 654-664.
Bruder SP, Jaiswal N, Haynesworth SE (1997) Growth
kinetics, self-renewal, and the osteogenic potential of
puried human mesenchymal stem cells during extensive
subcultivation and following cryopreservation. J Cell
Biochem 64: 278-294.
Burdick JA, Anseth KS (2002) Photoencapsulation of
osteoblasts in injectable RGD-modied PEG hydrogels
for bone tissue engineering. Biomaterials 23: 4315-4323.
Buschmann J, Harter L, Gao S, Hemmi S, Welti M,
Hild N, Schneider OD, Stark WJ, Lindenblatt N, Werner
CM, Wanner GA, Calcagni M (2012) Tissue engineered
bone grafts based on biomimetic nanocomposite PLGA/
amorphous calcium phosphate scaold and human adipose-
derived stem cells. Injury 43: 1689-1697.
Cao L, Liu X, Liu S, Jiang Y, Zhang X, Zhang C, Zeng
B (2012a) Experimental repair of segmental bone defects
in rabbits by angiopoietin-1 gene transfected MSCs seeded
on porous beta-TCP scaolds. J Biomed Mater Res B Appl
Biomater 100: 1229-1236.
Cao X, Deng W, Wei Y, Yang Y, Su W, Wei Y, Xu X,
Yu J (2012b) Incorporating pTGF-beta1/calcium phosphate
nanoparticles with bronectin into 3-dimensional collagen/
chitosan scaolds: ecient, sustained gene delivery to stem
cells for chondrogenic dierentiation. Eur Cell Mater 23:
81-93.
Carano RA, Filvaro EH (2003) Angiogenesis and
bone repair. Drug Discov Today 8: 980-989.
Carragee EJ, Mitsunaga KA, Hurwitz EL, Scuderi
GJ (2011) Retrograde ejaculation after anterior lumbar
interbody fusion using rhBMP-2: a cohort controlled study.
Spine J 11: 511-516.
Cartmell S (2009) Controlled release scaolds for bone
tissue engineering. J Pharm Sci 98: 430-441.
Chanchareonsook N, Tideman H, Feinberg SE,
Hollister SJ, Jongpaiboonkit L, Kin L, Jansen JA (2013)
Subcutaneous tissue response to titanium, poly(epsilon-
caprolactone), and carbonate-substituted hydroxyapatite-
coated poly(epsilon-caprolactone) plates: a rabbit study. J
Biomed Mater Res A 101: 2258-2266.
Chen L, Zhu C, Fan D, Liu B, Ma X, Duan Z, Zhou
Y (2011) A human-like collagen/chitosan electrospun
nanobrous scaold from aqueous solution: electrospun
mechanism and biocompatibility. J Biomed Mater Res A
99: 395-409.
Cheng CW, Solorio LD, Alsberg E (2014) Decellularized
tissue and cell-derived extracellular matrices as scaolds
for orthopaedic tissue engineering. Biotechnol Adv 32:
462-484.
Choi S, Murphy WL (2010) Sustained plasmid DNA
release from dissolving mineral coatings. Acta Biomater
6: 3426-3435.
Choi S, Yu X, Jongpaiboonkit L, Hollister SJ, Murphy
WL (2013) Inorganic coatings for optimized non-viral
transfection of stem cells. Sci Rep 3: 1567.
Chou YF, Huang W, Dunn JC, Miller TA, Wu BM
(2005) The effect of biomimetic apatite structure on
osteoblast viability, proliferation, and gene expression.
Biomaterials 26: 285-295.
Chung YI, Ahn KM, Jeon SH, Lee SY, Lee JH, Tae G
(2007) Enhanced bone regeneration with BMP-2 loaded
functional nanoparticle-hydrogel complex. J Control
Release 121: 91-99.
Cipitria A, Reichert JC, Epari DR, Saifzadeh S, Berner
A, Schell H, Mehta M, Schuetz MA, Duda GN, Hutmacher
DW (2013) Polycaprolactone scaffold and reduced
rhBMP-7 dose for the regeneration of critical-sized defects
in sheep tibiae. Biomaterials 34: 9960-9968.
Claes L, Augat P, Suger G, Wilke HJ (1997) Inuence
of size and stability of the osteotomy gap on the success
of fracture healing. J Orthop Res 15: 577-584.
Claes L, Eckert-Hubner K, Augat P (2002) The eect
of mechanical stability on local vascularization and tissue
dierentiation in callus healing. J Orthop Res 20: 1099-
1105.
Claes LE, Heigele CA (1999) Magnitudes of local stress
and strain along bony surfaces predict the course and type
of fracture healing. J Biomech 32: 255-266.
Claes LE, Heigele CA, Neidlinger-Wilke C, Kaspar
D, Seidl W, Margevicius KJ, Augat P (1998) Eects of
mechanical factors on the fracture healing process. Clin
Orthop Relat Res 355 Suppl: S132-S147.
Colton CK (1995) Implantable biohybrid articial
organs. Cell Transplant 4: 415-436.
Cornejo A, Sahar DE, Stephenson SM, Chang S,
Nguyen S, Guda T, Wenke JC, Vasquez A, Michalek JE,
Sharma R, Krishnegowda NK, Wang HT (2012) Eect of
adipose tissue-derived osteogenic and endothelial cells on
bone allograft osteogenesis and vascularization in critical-
sized calvarial defects. Tissue Eng Part A 18: 1552-1561.
Costa-Pinto AR, Reis RL, Neves NM (2011) Scaolds
based bone tissue engineering: the role of chitosan. Tissue
Eng Part B Rev 17: 331-347.
Cunnie GM, Vinardell T, Thompson EM, Daly AC,
Matsiko A, O’Brien FJ, Kelly DJ (2015) Chondrogenically
primed mesenchymal stem cells-seeded alginate hydrogels
promote early bone formation in critically-sized defects.
European Polymer Journal 72: 464-472.
Curtin CM, Cunniffe GM, Lyons FG, Bessho K,
Dickson GR, Duy GP, O’Brien FJ (2012) Innovative
collagen nano-hydroxyapatite scaolds oer a highly
ecient non-viral gene delivery platform for stem cell-
mediated bone formation. Adv Mater 24: 749-754.
d’Aquino R, De Rosa A, Lanza V, Tirino V, Laino L,
Graziano A, Desiderio V, Laino G, Papaccio G (2009)
Human mandible bone defect repair by the grafting of
dental pulp stem/progenitor cells and collagen sponge
biocomplexes. Eur Cell Mater 18: 75-83.
Dalby MJ, Gadegaard N, Tare R, Andar A, Riehle MO,
Herzyk P, Wilkinson CD, Oreo RO (2007) The control of
human mesenchymal cell dierentiation using nanoscale
symmetry and disorder. Nat Mater 6: 997-1003.
Dang PN, Dwivedi N, Phillips LM, Yu X, Herberg
S, Bowerman C, Solorio LD, Murphy WL, Alsberg E
(2016) Controlled dual growth factor delivery from
microparticles incorporated within human bone marrow-
derived mesenchymal stem cell aggregates for enhanced
102 www.ecmjournal.org
S Verrier et al. Large bone defect healing
bone tissue engineering via endochondral ossication.
Stem Cells Transl Med 5: 206-217.
Dariima T, Jin GZ, Lee EJ, Wall IB, Kim HW (2013)
Cooperation between osteoblastic cells and endothelial
cells enhances their phenotypic responses and improves
osteoblast function. Biotechnol Lett 35: 1135-1143.
Das A, Segar CE, Hughley BB, Bowers DT, Botchwey
EA (2013) The promotion of mandibular defect healing
by the targeting of S1P receptors and the recruitment of
alternatively activated macrophages. Biomaterials 34:
9853-9862.
Datta N, Holtorf HL, Sikavitsas VI, Jansen JA, Mikos
AG (2005) Eect of bone extracellular matrix synthesized
in vitro on the osteoblastic dierentiation of marrow
stromal cells. Biomaterials 26: 971-977.
Di Martino A, Sittinger M, Risbud MV (2005)
Chitosan: a versatile biopolymer for orthopaedic tissue-
engineering. Biomaterials 26: 5983-5990.
Dickhut A, Gottwald E, Steck E, Heisel C, Richter
W (2008) Chondrogenesis of mesenchymal stem cells in
gel-like biomaterials in vitro and in vivo. Front Biosci 13:
4517-4528.
Druecke D, Langer S, Lamme E, Pieper J, Ugarkovic
M, Steinau HU, Homann HH (2004) Neovascularization of
poly(ether ester) block-copolymer scaolds in vivo: long-
term investigations using intravital uorescent microscopy.
J Biomed Mater Res A 68: 10-18.
Drury JL, Mooney DJ (2003) Hydrogels for tissue
engineering: scaold design variables and applications.
Biomaterials 24: 4337-4351.
Dudas JR, Marra KG, Cooper GM, Penascino VM,
Mooney MP, Jiang S, Rubin JP, Losee JE (2006) The
osteogenic potential of adipose-derived stem cells for the
repair of rabbit calvarial defects. Ann Plast Surg 56: 543-
548.
Dupont KM, Boerckel JD, Stevens HY, Diab T,
Kolambkar YM, Takahata M, Schwarz EM, Guldberg RE
(2012) Synthetic scaold coating with adeno-associated
virus encoding BMP2 to promote endogenous bone repair.
Cell Tissue Res 347: 575-588.
Duttenhoefer F, Lara de Freitas R, Meury T, Loibl M,
Benneker LM, Richards RG, Alini M, Verrier S (2013) 3D
scaolds co-seeded with human endothelial progenitor and
mesenchymal stem cells: evidence of prevascularisation
within 7 days. Eur Cell Mater 26: 49-64.
Egermann M, Baltzer AW, Adamaszek S, Evans C,
Robbins P, Schneider E, Lill CA (2006a) Direct adenoviral
transfer of bone morphogenetic protein-2 cDNA enhances
fracture healing in osteoporotic sheep. Hum Gene Ther 17:
507-517.
Egermann M, Lill CA, Griesbeck K, Evans CH,
Robbins PD, Schneider E, Baltzer AW (2006b) Eect of
BMP-2 gene transfer on bone healing in sheep. Gene Ther
13: 1290-1299.
Ehrbar M, Djonov VG, Schnell C, Tschanz SA,
Martiny-Baron G, Schenk U, Wood J, Burri PH, Hubbell
JA, Zisch AH (2004) Cell-demanded liberation of
VEGF121 from brin implants induces local and controlled
blood vessel growth. Circ Res 94: 1124-1132.
Epari DR, Wehner T, Ignatius A, Schuetz MA, Claes
LE (2013) A case for optimising fracture healing through
inverse dynamization. Med Hypotheses 81: 225-227.
Evans CH (2010) Gene therapy for bone healing. Expert
Rev Mol Med 12: e18.
Evans CH (2012) Gene delivery to bone. Adv Drug
Deliv Rev 64: 1331-1340.
Evans CH, Liu FJ, Glatt V, Hoyland JA, Kirker-Head
C, Walsh A, Betz O, Wells JW, Betz V, Porter RM, Saad
FA, Gerstenfeld LC, Einhorn TA, Harris MB, Vrahas MS
(2009) Use of genetically modied muscle and fat grafts
to repair defects in bone and cartilage. Eur Cell Mater 18:
96-111.
Evans CH, Palmer GD, Pascher A, Porter R, Kwong
FN, Gouze E, Gouze JN, Liu F, Steinert A, Betz O, Betz V,
Vrahas M, Ghivizzani SC (2007) Facilitated endogenous
repair: making tissue engineering simple, practical, and
economical. Tissue Eng 13: 1987-1993.
Fang J, Zhu YY, Smiley E, Bonadio J, Rouleau JP,
Goldstein SA, McCauley LK, Davidson BL, Roessler
BJ (1996) Stimulation of new bone formation by direct
transfer of osteogenic plasmid genes. Proc Natl Acad Sci
U S A 93: 5753-5758.
Farrell E, Both SK, Odorfer KI, Koevoet W, Kops N,
O’Brien FJ, Baatenburg de Jong RJ, Verhaar JA, Cuijpers
V, Jansen J, Erben RG, van Osch GJ (2011) In-vivo
generation of bone via endochondral ossication by in-vitro
chondrogenic priming of adult human and rat mesenchymal
stem cells. BMC Musculoskelet Disord 12: 31.
Farrell E, van der Jagt OP, Koevoet W, Kops N, van
Manen CJ, Hellingman CA, Jahr H, O’Brien FJ, Verhaar
JA, Weinans H, van Osch GJ (2009) Chondrogenic priming
of human bone marrow stromal cells: a better route to bone
repair? Tissue Eng Part C Methods 15: 285-295.
Faundez A, Tournier C, Garcia M, Aunoble S, Le
Huec JC (2016) Bone morphogenetic protein use in spine
surgery-complications and outcomes: a systematic review.
Int Orthop 40: 1309-1319.
Ferreira AM, Gentile P, Chiono V, Ciardelli G (2012)
Collagen for bone tissue regeneration. Acta Biomater 8:
3191-3200.
Finkenzeller G, Hager S, Stark GB (2012) Eects of
bone morphogenetic protein 2 on human umbilical vein
endothelial cells. Microvasc Res 84: 81-85.
Follmar KE, Prichard HL, Decroos FC, Wang HT,
Levin LS, Klitzman B, Olbrich KC, Erdmann D (2007)
Combined bone allograft and adipose-derived stem cell
autograft in a rabbit model. Ann Plast Surg 58: 561-565.
Fraser JK, Wulur I, Alfonso Z, Hedrick MH (2006)
Fat tissue: an underappreciated source of stem cells for
biotechnology. Trends Biotechnol 24: 150-154.
Fu R, Selph S, McDonagh M, Peterson K, Tiwari A,
Chou R, Helfand M (2013) Eectiveness and harms of
recombinant human bone morphogenetic protein-2 in spine
fusion: a systematic review and meta-analysis. Ann Intern
Med 158: 890-902.
Gawlitta D, Farrell E, Malda J, Creemers LB, Alblas
J, Dhert WJ (2010) Modulating endochondral ossication
of multipotent stromal cells for bone regeneration. Tissue
Eng Part B Rev 16: 385-395.
103 www.ecmjournal.org
S Verrier et al. Large bone defect healing
Ge J, Lei J, Zare RN (2012) Protein-inorganic hybrid
nanoowers. Nat Nanotechnol 7: 428-432.
Geiger F, Bertram H, Berger I, Lorenz H, Wall O,
Eckhardt C, Simank HG, Richter W (2005) Vascular
endothelial growth factor gene-activated matrix (VEGF165-
GAM) enhances osteogenesis and angiogenesis in large
segmental bone defects. J Bone Miner Res 20: 2028-2035.
Geiger M, Li RH, Friess W (2003) Collagen sponges
for bone regeneration with rhBMP-2. Adv Drug Deliv Rev
55: 1613-1629.
Geuze RE, Theyse LF, Kempen DH, Hazewinkel
HA, Kraak HY, Oner FC, Dhert WJ, Alblas J (2012) A
dierential eect of bone morphogenetic protein-2 and
vascular endothelial growth factor release timing on
osteogenesis at ectopic and orthotopic sites in a large-
animal model. Tissue Eng Part A 18: 2052-2062.
Giannoudis PV, Einhorn TA, Marsh D (2007) Fracture
healing: the diamond concept. Injury 38 Suppl 4: S3-S6.
Gibbs DM, Black CR, Dawson JI, Oreo RO (2014)
A review of hydrogel use in fracture healing and bone
regeneration. J Tissue Eng Regen Med 10: 187-198.
Giri J, Li WJ, Tuan RS, Cicerone MT (2011)
Stabilization of proteins by nanoencapsulation in sugar-
glass for tissue engineering and drug delivery applications.
Adv Mater 23: 4861-4867.
Glatt V, Miller M, Ivkovic A, Liu F, Parry N, Grin
D, Vrahas M, Evans C (2012) Improved healing of large
segmental defects in the rat femur by reverse dynamization
in the presence of bone morphogenetic protein-2. J Bone
Joint Surg Am 94: 2063-2073.
Gleeson JP, Plunkett NA, O’Brien FJ (2010) Addition
of hydroxyapatite improves stiness, interconnectivity
and osteogenic potential of a highly porous collagen-based
scaold for bone tissue regeneration. Eur Cell Mater 20:
218-230.
Goncalves RM, Antunes JC, Barbosa MA (2012)
Mesenchymal stem cell recruitment by stromal derived
factor-1-delivery systems based on chitosan/poly(gamma-
glutamic acid) polyelectrolyte complexes. Eur Cell Mater
23: 249-260.
Goodship AE, Kenwright J (1985) The inuence of
induced micromovement upon the healing of experimental
tibial fractures. J Bone Joint Surg Br 67: 650-655.
Govender S, Csimma C, Genant HK, Valentin-Opran
A, Amit Y, Arbel R, Aro H, Atar D, Bishay M, Borner
MG, Chiron P, Choong P, Cinats J, Courtenay B, Feibel
R, Geulette B, Gravel C, Haas N, Raschke M, Hammacher
E, van der Velde D, Hardy P, Holt M, Josten C, Ketterl
RL, Lindeque B, Lob G, Mathevon H, McCoy G, Marsh
D, Miller R, Munting E, Oevre S, Nordsletten L, Patel A,
Pohl A, Rennie W, Reynders P, Rommens PM, Rondia J,
Rossouw WC, Daneel PJ, Ru S, Ruter A, Santavirta S,
Schildhauer TA, Gekle C, Schnettler R, Segal D, Seiler H,
Snowdowne RB, Stapert J, Taglang G, Verdonk R, Vogels
L, Weckbach A, Wentzensen A, Wisniewski T (2002)
Recombinant human bone morphogenetic protein-2 for
treatment of open tibial fractures: a prospective, controlled,
randomized study of four hundred and fty patients. J Bone
Joint Surg Am 84: 2123-2134.
Gruskin E, Doll BA, Futrell FW, Schmitz JP, Hollinger
JO (2012) Demineralized bone matrix in bone repair:
history and use. Adv Drug Deliv Rev 64: 1063-1077.
Guelcher SA (2008) Biodegradable polyurethanes:
synthesis and applications in regenerative medicine. Tissue
Eng Part B Rev 14: 3-17.
Guo X, Zheng Q, Kulbatski I, Yuan Q, Yang S, Shao Z,
Wang H, Xiao B, Pan Z, Tang S (2006) Bone regeneration
with active angiogenesis by basic broblast growth factor
gene transfected mesenchymal stem cells seeded on porous
beta-TCP ceramic scaolds. Biomed Mater 1: 93-99.
Hacein-Bey-Abina S, Von Kalle C, Schmidt M,
McCormack MP, Wulffraat N, Leboulch P, Lim A,
Osborne CS, Pawliuk R, Morillon E, Sorensen R, Forster
A, Fraser P, Cohen JI, de Saint Basile G, Alexander I,
Wintergerst U, Frebourg T, Aurias A, Stoppa-Lyonnet
D, Romana S, Radford-Weiss I, Gross F, Valensi F,
Delabesse E, Macintyre E, Sigaux F, Soulier J, Leiva LE,
Wissler M, Prinz C, Rabbitts TH, Le Deist F, Fischer A,
Cavazzana-Calvo M (2003) LMO2-associated clonal T
cell proliferation in two patients after gene therapy for
SCID-X1. Science 302: 415-419.
Hao W, Dong J, Jiang M, Wu J, Cui F, Zhou D (2010)
Enhanced bone formation in large segmental radial defects
by combining adipose-derived stem cells expressing bone
morphogenetic protein 2 with nHA/RHLC/PLA scaold.
Int Orthop 34: 1341-1349.
Harichandan A, Buhring HJ (2011) Prospective
isolation of human MSC. Best Pract Res Clin Haematol
24: 25-36.
Hartgerink JD, Beniash E, Stupp SI (2001) Self-
assembly and mineralization of peptide-amphiphile
nanobers. Science 294: 1684-1688.
Heinemann C, Heinemann S, Bernhardt A, Lode A,
Worch H, Hanke T (2010) In vitro osteoclastogenesis on
textile chitosan scaold. Eur Cell Mater 19: 96-106.
Hente R, Cheal E, Perren S (1990) Tissue response
to controlled interfragmentary strain gradients in fracture
healing. In: 2nd Conference of the International Society
for Fracture Repair. ed. E.Y.S Chao, Mayo Clinic, 6-8
September, p148.
Hernigou P, Poignard A, Beaujean F, Rouard H (2005)
Percutaneous autologous bone-marrow grafting for
nonunions. Inuence of the number and concentration of
progenitor cells. J Bone Joint Surg Am 87: 1430-1437.
Herrmann M, Laschke MW, Alini M, Scherberich A,
Verrier S (2015) Vascularization, survival and functionality
of tissue engineered constructs. In: Tissue Engineering,
2nd Edition. Elsevier, Chapter 14, p471-496.
Hildebrandt C, Buth H, Thielecke H (2011) A scaold-
free in vitro model for osteogenesis of human mesenchymal
stem cells. Tissue Cell 43: 91-100.
Hollister SJ (2005) Porous scaold design for tissue
engineering. Nat Mater 4: 518-524.
Holstein JH, Orth M, Scheuer C, Tami A, Becker SC,
Garcia P, Histing T, Morsdorf P, Klein M, Pohlemann
T, Menger MD (2011) Erythropoietin stimulates bone
formation, cell proliferation, and angiogenesis in a femoral
segmental defect model in mice. Bone 49: 1037-1045.
104 www.ecmjournal.org
S Verrier et al. Large bone defect healing
Horch RE, Beier JP, Kneser U, Arkudas A (2014)
Successful human long-term application of in situ bone
tissue engineering. J Cell Mol Med 18: 1478-1485.
Hosseinkhani H, Hosseinkhani M, Tian F, Kobayashi H,
Tabata Y (2006) Ectopic bone formation in collagen sponge
self-assembled peptide-amphiphile nanofibers hybrid
scaold in a perfusion culture bioreactor. Biomaterials 27:
5089-5098.
Hou T, Li Q, Luo F, Xu J, Xie Z, Wu X, Zhu C (2010)
Controlled dynamization to enhance reconstruction
capacity of tissue-engineered bone in healing critically
sized bone defects: an in vivo study in goats. Tissue Eng
Part A 16: 201-212.
Housman TS, Lawrence N, Mellen BG, George MN,
Filippo JS, Cerveny KA, DeMarco M, Feldman SR,
Fleischer AB (2002) The safety of liposuction: results of
a national survey. Dermatol Surg 28: 971-978.
Huang JI, Durbhakula MM, Angele P, Johnstone
B, Yoo JU (2006) Lunate arthroplasty with autologous
mesenchymal stem cells in a rabbit model. J Bone Joint
Surg Am 88: 744-752.
Huang L, Nagapudi K, Apkarian RP, Chaikof EL
(2001) Engineered collagen-PEO nanobers and fabrics.
J Biomater Sci Polym Ed 12: 979-993.
Hur J, Yoon CH, Kim HS, Choi JH, Kang HJ, Hwang
KK, Oh BH, Lee MM, Park YB (2004) Characterization of
two types of endothelial progenitor cells and their dierent
contributions to neovasculogenesis. Arterioscler Thromb
Vasc Biol 24: 288-293.
Hussain A, Bessho K, Takahashi K, Tabata Y (2012)
Magnesium calcium phosphate as a novel component
enhances mechanical/physical properties of gelatin
scaold and osteogenic dierentiation of bone marrow
mesenchymal stem cells. Tissue Eng Part A 18: 768-774.
Ingavle GC, Leach JK (2013) Advancements in
electrospinning of polymeric nanobrous scaolds for
tissue engineering. Tissue Eng Part B Rev 20: 277-293.
Ishida K, Matsumoto T, Sasaki K, Mifune Y, Tei K,
Kubo S, Matsushita T, Takayama K, Akisue T, Tabata
Y, Kurosaka M, Kuroda R (2010) Bone regeneration
properties of granulocyte colony-stimulating factor via
neovascularization and osteogenesis. Tissue Eng Part A
16: 3271-3284.
Ishihara A, Shields KM, Litsky AS, Mattoon JS,
Weisbrode SE, Bartlett JS, Bertone AL (2008) Osteogenic
gene regulation and relative acceleration of healing by
adenoviral-mediated transfer of human BMP-2 or -6 in
equine osteotomy and ostectomy models. J Orthop Res
26: 764-771.
Ito H, Koefoed M, Tiyapatanaputi P, Gromov K, Goater
JJ, Carmouche J, Zhang X, Rubery PT, Rabinowitz J,
Samulski RJ, Nakamura T, Soballe K, O’Keefe RJ, Boyce
BF, Schwarz EM (2005) Remodeling of cortical bone
allografts mediated by adherent rAAV-RANKL and VEGF
gene therapy. Nat Med 11: 291-297.
Jalowiec JM, D’Este M, Bara JJ, Denom J, Menzel
U, Alini M, Verrier S, Herrmann M (2016) An in vitro
investigation of platelet-rich plasma-gel as a cell and
growth factor delivery vehicle for tissue engineering.
Tissue Eng Part C Methods 22: 49-58.
Jeon O, Bouhadir KH, Mansour JM, Alsberg
E (2009) Photocrosslinked alginate hydrogels with
tunable biodegradation rates and mechanical properties.
Biomaterials 30: 2724-2734.
Jeon O, Song SJ, Yang HS, Bhang SH, Kang SW, Sung
MA, Lee JH, Kim BS (2008) Long-term delivery enhances
in vivo osteogenic efficacy of bone morphogenetic
protein-2 compared to short-term delivery. Biochem
Biophys Res Commun 369: 774-780.
Jongpaiboonkit L, Franklin-Ford T, Murphy WL (2009)
Growth of hydroxyapatite coatings on biodegradable
polymer microspheres. ACS Appl Mater Interfaces 1:
1504-1511.
Kanayama M, Hashimoto T, Shigenobu K, Yamane S,
Bauer TW, Togawa D (2006) A prospective randomized
study of posterolateral lumbar fusion using osteogenic
protein-1 (OP-1) versus local autograft with ceramic bone
substitute: emphasis of surgical exploration and histologic
assessment. Spine (Phila Pa 1976) 31: 1067-1074.
Kasper G, Dankert N, Tuischer J, Hoeft M, Gaber
T, Glaeser JD, Zander D, Tschirschmann M, Thompson
M, Matziolis G, Duda GN (2007) Mesenchymal stem
cells regulate angiogenesis according to their mechanical
environment. Stem Cells 25: 903-910.
Kim HK, Shim WS, Kim SE, Lee KH, Kang E, Kim JH,
Kim K, Kwon IC, Lee DS (2009) Injectable in situ-forming
pH/thermo-sensitive hydrogel for bone tissue engineering.
Tissue Eng Part A 15: 923-933.
Kim J, Magno MH, Waters H, Doll BA, McBride S,
Alvarez P, Darr A, Vasanji A, Kohn J, Hollinger JO (2012)
Bone regeneration in a rabbit critical-sized calvarial model
using tyrosine-derived polycarbonate scaolds. Tissue Eng
Part A 18: 1132-1139.
Kim KS, Lee JY, Kang YM, Kim ES, Kim GH, Rhee
SD, Cheon HG, Kim JH, Min BH, Lee HB, Kim MS (2010)
Small intestine submucosa sponge for in vivo support of
tissue-engineered bone formation in the presence of rat
bone marrow stem cells. Biomaterials 31: 1104-1113.
Kimelman-Bleich N, Pelled G, Zilberman Y, Kallai I,
Mizrahi O, Tawackoli W, Gazit Z, Gazit D (2011) Targeted
gene-and-host progenitor cell therapy for nonunion bone
fracture repair. Mol Ther 19: 53-59.
King WJ, Krebsbach PH (2012) Growth factor delivery:
how surface interactions modulate release in vitro and in
vivo. Adv Drug Deliv Rev 64: 1239-1256.
Klein P, Schell H, Streitparth F, Heller M, Kassi JP,
Kandziora F, Bragulla H, Haas NP, Duda GN (2003) The
initial phase of fracture healing is specically sensitive to
mechanical conditions. J Orthop Res 21: 662-669.
Ko JY, Im GI (2014) Chondrogenic and osteogenic
induction from iPS cells. Methods Mol Biol 1357: 441-450.
Koike N, Fukumura D, Gralla O, Au P, Schechner JS,
Jain RK (2004) Tissue engineering: creation of long-lasting
blood vessels. Nature 428: 138-139.
Kolambkar YM, Boerckel JD, Dupont KM, Bajin M,
Huebsch N, Mooney DJ, Hutmacher DW, Guldberg RE
(2011) Spatiotemporal delivery of bone morphogenetic
protein enhances functional repair of segmental bone
defects. Bone 49: 485-492.
Krebs MD, Salter E, Chen E, Sutter KA, Alsberg
E (2010) Calcium phosphate-DNA nanoparticle gene
105 www.ecmjournal.org
S Verrier et al. Large bone defect healing
delivery from alginate hydrogels induces in vivo
osteogenesis. J Biomed Mater Res A 92: 1131-1138.
Kuhn LT, Liu Y, Boyd NL, Dennis JE, Jiang X, Xin X,
Charles LF, Wang L, Aguila HL, Rowe DW, Lichtler AC,
Goldberg AJ (2014) Developmental-like bone regeneration
by human embryonic stem cell-derived mesenchymal cells.
Tissue Eng Part A 20: 365-377.
Kuroda R, Matsumoto T, Niikura T, Kawakami Y, Fukui
T, Lee SY, Mifune Y, Kawamata S, Fukushima M, Asahara
T, Kawamoto A, Kurosaka M (2014) Local transplantation
of granulocyte colony stimulating factor-mobilized CD34+
cells for patients with femoral and tibial nonunion: pilot
clinical trial. Stem Cells Transl Med 3: 128-134.
Laschke MW, Menger MD (2012) Vascularization in
tissue engineering: angiogenesis versus inosculation. Eur
Surg Res 48: 85-92.
Laschke MW, Menger MD (2015) Adipose tissue-
derived microvascular fragments: natural vascularization
units for regenerative medicine. Trends Biotechnol 33:
442-448.
Laschke MW, Menger MD (2016) Prevascularization in
tissue engineering: Current concepts and future directions.
Biotechnol Adv 34: 112-121.
Laschke MW, Mussawy H, Schuler S, Kazakov A,
Rücker M, Eglin D, Alini M, Menger MD (2011) Short-
term cultivation of in situ prevascularized tissue constructs
accelerates inosculation of their preformed microvascular
networks after implantation into the host tissue. Tissue Eng
Part A 17: 841-853.
Laschke MW, Schank TE, Scheuer C, Kleer S, Schuler
S, Metzger W, Eglin D, Alini M, Menger MD (2013) Three-
dimensional spheroids of adipose-derived mesenchymal
stem cells are potent initiators of blood vessel formation
in porous polyurethane scaolds. Acta Biomater 9: 6876-
6884.
Lattanzi W, Parrilla C, Fetoni A, Logroscino G, Straface
G, Pecorini G, Stigliano E, Tampieri A, Bedini R, Pecci
R, Michetti F, Gambotto A, Robbins PD, Pola E (2008)
Ex vivo-transduced autologous skin broblasts expressing
human Lim mineralization protein-3 eciently form new
bone in animal models. Gene Ther 15: 1330-1343.
Le AX, Miclau T, Hu D, Helms JA (2001) Molecular
aspects of healing in stabilized and non-stabilized fractures.
J Orthop Res 19: 78-84.
Lee JS, Lee JS, Murphy WL (2010a) Modular peptides
promote human mesenchymal stem cell dierentiation on
biomaterial surfaces. Acta Biomater 6: 21-28.
Lee JS, Wagoner Johnson AJ, Murphy WL (2010b)
A modular, hydroxyapatite-binding version of vascular
endothelial growth factor. Adv Mater 22: 5494-5498.
Lee K, Chan CK, Patil N, Goodman SB (2009) Cell
therapy for bone regeneration – bench to bedside. J Biomed
Mater Res B Appl Biomater 89: 252-263.
Lee KY, Mooney DJ (2012) Alginate: properties and
biomedical applications. Prog Polym Sci 37: 106-126.
Lee RJ, Springer ML, Blanco-Bose WE, Shaw R, Ursell
PC, Blau HM (2000) VEGF gene delivery to myocardium:
deleterious eects of unregulated expression. Circulation
102: 898-901.
Levi B, Nelson ER, Hyun JS, Glotzbach JP, Li S,
Nauta A, Montoro DT, Lee M, Commons GC, Hu S, Wu
JC, Gurtner GC, Longaker MT (2012) Enhancement of
human adipose-derived stromal cell angiogenesis through
knockdown of a BMP-2 inhibitor. Plast Reconstr Surg 129:
53-66.
Li BC, Zhang JJ, Xu C, Zhang LC, Kang JY, Zhao
H (2009a) Treatment of rabbit femoral defect by rearm
with BMP-4 gene combined with TGF-beta1. J Trauma
66: 450-456.
Li H, Dai K, Tang T, Zhang X, Yan M, Lou J (2007)
Bone regeneration by implantation of adipose-derived
stromal cells expressing BMP-2. Biochem Biophys Res
Commun 356: 836-842.
Li R, Stewart DJ, von Schroeder HP, Mackinnon ES,
Schemitsch EH (2009b) Eect of cell-based VEGF gene
therapy on healing of a segmental bone defect. J Orthop
Res 27: 8-14.
Li Y, Fan L, Liu S, Liu W, Zhang H, Zhou T, Wu D,
Yang P, Shen L, Chen J, Jin Y (2013) The promotion
of bone regeneration through positive regulation of
angiogenic-osteogenic coupling using microRNA-26a.
Biomaterials 34: 5048-5058.
Lieberman JR, Daluiski A, Stevenson S, Wu L,
McAllister P, Lee YP, Kabo JM, Finerman GA, Berk AJ,
Witte ON (1999) The eect of regional gene therapy with
bone morphogenetic protein-2-producing bone-marrow
cells on the repair of segmental femoral defects in rats. J
Bone Joint Surg Am 81: 905-917.
Lin CY, Chang YH, Kao CY, Lu CH, Sung LY, Yen
TC, Lin KJ, Hu YC (2012) Augmented healing of critical-
size calvarial defects by baculovirus-engineered MSCs
that persistently express growth factors. Biomaterials 33:
3682-3692.
Lin Y, Weisdorf DJ, Solovey A, Hebbel RP (2000)
Origins of circulating endothelial cells and endothelial
outgrowth from blood. J Clin Invest 105: 71-77.
Lippross S, Loibl M, Hoppe S, Meury T, Benneker L,
Alini M, Verrier S (2011) Platelet released growth factors
boost expansion of bone marrow derived CD34(+) and
CD133(+) endothelial progenitor cells for autologous
grafting. Platelets 22: 422-432.
Liu J, Chen W, Zhao Z, Xu HH (2013) Reprogramming
of mesenchymal stem cells derived from iPSCs seeded
on biofunctionalized calcium phosphate scaold for bone
engineering. Biomaterials 34: 7862-7872.
Loi F, Cordova LA, Zhang R, Pajarinen J, Lin
TH, Goodman SB, Yao Z (2016) The effects of
immunomodulation by macrophage subsets on osteogenesis
in vitro. Stem Cell Res Ther 7: 15.
Lu Y, Markel MD, Nemke B, Lee JS, Graf BK, Murphy
WL (2009) Inuence of hydroxyapatite-coated and growth
factor-releasing interference screws on tendon-bone
healing in an ovine model. Arthroscopy 25: 1427-1434.
Luangphakdy V, Walker E, Shinohara K, Pan H,
Heeran T, Bauer TW, Stockdale L, Saini S, Dadsetan M,
Runge MB, Vasanji A, Grith L, Yaszemski M, Muschler
GF (2013) Evaluation of osteoconductive scaolds in the
canine femoral multi-defect model. Tissue Eng Part A 19:
634-648.
Lucarelli E, Fini M, Beccheroni A, Giavaresi G, Di
Bella C, Aldini NN, Guzzardella G, Martini L, Cenacchi
A, Di Maggio N, Sangiorgi L, Fornasari PM, Mercuri
106 www.ecmjournal.org
S Verrier et al. Large bone defect healing
M, Giardino R, Donati D (2005) Stromal stem cells and
platelet-rich plasma improve bone allograft integration.
Clin Orthop Relat Res 62-68.
Lyons FG, Al-Munajjed AA, Kieran SM, Toner ME,
Murphy CM, Duy GP, O’Brien FJ (2010) The healing
of bony defects by cell-free collagen-based scaffolds
compared to stem cell-seeded tissue engineered constructs.
Biomaterials 31: 9232-9243.
Madry H, Alini M, Stoddart MJ, Evans C, Miclau T,
Steiner S (2014) Barriers and strategies for the clinical
translation of advanced orthopaedic tissue engineering
protocols. Eur Cell Mater 27: 17-21.
Maraldi T, Riccio M, Pisciotta A, Zavatti M, Carnevale
G, Beretti F, La Sala GB, Motta A, De PA (2013) Human
amniotic uid-derived and dental pulp-derived stem cells
seeded into collagen scaold repair critical-size bone
defects promoting vascularization. Stem Cell Res Ther 4:
53.
Masuda H, Asahara T (2003) Post-natal endothelial
progenitor cells for neovascularization in tissue
regeneration. Cardiovasc Res 58: 390-398.
Matthews JA, Wnek GE, Simpson DG, Bowlin
GL (2002) Electrospinning of collagen nanofibers.
Biomacromolecules 3: 232-238.
Mifune Y, Matsumoto T, Kawamoto A, Kuroda R, Shoji
T, Iwasaki H, Kwon SM, Miwa M, Kurosaka M, Asahara
T (2008) Local delivery of granulocyte colony stimulating
factor-mobilized CD34-positive progenitor cells using
bioscaold for modality of unhealing bone fracture. Stem
Cells 26: 1395-1405.
Moore DC, Pedrozo HA, Crisco JJ III, Ehrlich MG
(2004) Preformed grafts of porcine small intestine
submucosa (SIS) for bridging segmental bone defects. J
Biomed Mater Res A 69: 259-266.
Murphy WL, Hsiong S, Richardson TP, Simmons CA,
Mooney DJ (2005) Eects of a bone-like mineral lm on
phenotype of adult human mesenchymal stem cells in vitro.
Biomaterials 26: 303-310.
Musgrave DS, Bosch P, Ghivizzani S, Robbins PD,
Evans CH, Huard J (1999) Adenovirus-mediated direct
gene therapy with bone morphogenetic protein-2 produces
bone. Bone 24: 541-547.
Nguyen TH, Kim SH, Decker CG, Wong DY, Loo
JA, Maynard HD (2013) A heparin-mimicking polymer
conjugate stabilizes basic broblast growth factor. Nat
Chem 5: 221-227.
Niemeyer P, Fechner K, Milz S, Richter W, Suedkamp
NP, Mehlhorn AT, Pearce S, Kasten P (2010) Comparison
of mesenchymal stem cells from bone marrow and adipose
tissue for bone regeneration in a critical size defect of
the sheep tibia and the inuence of platelet-rich plasma.
Biomaterials 31: 3572-3579.
O’Brien FJ (2011) Biomaterials & scaolds for tissue
engineering. Materials today 14: 88-95.
Pachon-Pena G, Yu G, Tucker A, Wu X, Vendrell J,
Bunnell BA, Gimble JM (2011) Stromal stem cells from
adipose tissue and bone marrow of age-matched female
donors display distinct immunophenotypic proles. J Cell
Physiol 226: 843-851.
Palmer GD, Steinert A, Pascher A, Gouze E, Gouze JN,
Betz O, Johnstone B, Evans CH, Ghivizzani SC (2005)
Gene-induced chondrogenesis of primary mesenchymal
stem cells in vitro. Mol Ther 12: 219-228.
Parekkadan B, Milwid JM (2010) Mesenchymal stem
cells as therapeutics. Annu Rev Biomed Eng 12: 87-117.
Park BH, Zhou L, Jang KY, Park HS, Lim JM, Yoon SJ,
Lee SY, Kim JR (2012) Enhancement of tibial regeneration
in a rat model by adipose-derived stromal cells in a PLGA
scaold. Bone 51: 313-323.
Park H, Choi B, Nguyen J, Fan J, Sha S, Klokkevold
P, Lee M (2013) Anionic carbohydrate-containing chitosan
scaolds for bone regeneration. Carbohydr Polym 97: 587-
596.
Peichev M, Naiyer AJ, Pereira D, Zhu Z, Lane WJ,
Williams M, Oz MC, Hicklin DJ, Witte L, Moore MA,
Rai S (2000) Expression of VEGFR-2 and AC133 by
circulating human CD34(+) cells identies a population
of functional endothelial precursors. Blood 95: 952-958.
Pelttari K, Winter A, Steck E, Goetzke K, Hennig T,
Ochs BG, Aigner T, Richter W (2006) Premature induction
of hypertrophy during in vitro chondrogenesis of human
mesenchymal stem cells correlates with calcication and
vascular invasion after ectopic transplantation in SCID
mice. Arthritis Rheum 54: 3254-3266.
Pensak MJ, Lieberman JR (2013) Gene therapy for
bone regeneration. Curr Pharm Des 19: 3466-3473.
Peterson B, Whang PG, Iglesias R, Wang JC,
Lieberman JR (2004) Osteoinductivity of commercially
available demineralized bone matrix. Preparations in a
spine fusion model. J Bone Joint Surg Am 86-A: 2243-
2250.
Peterson B, Zhang J, Iglesias R, Kabo M, Hedrick M,
Benhaim P, Lieberman JR (2005) Healing of critically sized
femoral defects, using genetically modied mesenchymal
stem cells from human adipose tissue. Tissue Eng 11: 120-
129.
Pighinelli L, Kucharska M (2013) Chitosan-
hydroxyapatite composites. Carbohydr Polym 93: 256-262.
Qu D, Li J, Li Y, Gao Y, Zuo Y, Hsu Y, Hu J (2011)
Angiogenesis and osteogenesis enhanced by bFGF ex vivo
gene therapy for bone tissue engineering in reconstruction
of calvarial defects. J Biomed Mater Res A 96: 543-551.
Quarto R, Mastrogiacomo M, Cancedda R, Kutepov
SM, Mukhachev V, Lavroukov A, Kon E, Marcacci
M (2001) Repair of large bone defects with the use of
autologous bone marrow stromal cells. N Engl J Med 344:
385-386.
Rao RR, Peterson AW, Stegemann JP (2013) Winner
for outstanding research in the Ph.D. category for the
2013 Society for Biomaterials meeting and exposition,
April 10-13, 2013, Boston, Massachusetts: Osteogenic
dierentiation of adipose-derived and marrow-derived
mesenchymal stem cells in modular protein/ceramic
microbeads. J Biomed Mater Res A 101: 1531-1538.
Rao RR, Vigen ML, Peterson AW, Caldwell DJ, Putnam
AJ, Stegemann JP (2015) Dual-phase osteogenic and
vasculogenic engineered tissue for bone formation. Tissue
Eng Part A 21: 530-540.
Raper SE, Chirmule N, Lee FS, Wivel NA, Bagg
A, Gao GP, Wilson JM, Batshaw ML (2003) Fatal
systemic inammatory response syndrome in a ornithine
107 www.ecmjournal.org
S Verrier et al. Large bone defect healing
transcarbamylase decient patient following adenoviral
gene transfer. Mol Genet Metab 80: 148-158.
Ratanavaraporn J, Furuya H, Kohara H, Tabata Y
(2011) Synergistic eects of the dual release of stromal
cell-derived factor-1 and bone morphogenetic protein-2
from hydrogels on bone regeneration. Biomaterials 32:
2797-2811.
Reves BT, Bumgardner JD, Haggard WO (2013)
Fabrication of crosslinked carboxymethylchitosan
microspheres and their incorporation into composite
scaolds for enhanced bone regeneration. J Biomed Mater
Res B Appl Biomater 101: 630-639.
Rizzi SC, Ehrbar M, Halstenberg S, Raeber GP,
Schmoekel HG, Hagenmuller H, Muller R, Weber
FE, Hubbell JA (2006) Recombinant protein-co-PEG
networks as cell-adhesive and proteolytically degradable
hydrogel matrixes. Part II: biofunctional characteristics.
Biomacromolecules 7: 3019-3029.
Rosenbaum AJ, Grande DA, Dines JS (2008) The use
of mesenchymal stem cells in tissue engineering: A global
assessment. Organogenesis 4: 23-27.
Rundle CH, Strong DD, Chen ST, Linkhart TA, Sheng
MH, Wergedal JE, Lau KH, Baylink DJ (2008) Retroviral-
based gene therapy with cyclooxygenase-2 promotes the
union of bony callus tissues and accelerates fracture healing
in the rat. J Gene Med 10: 229-241.
Rundle CH, Wang H, Yu H, Chadwick RB, Davis EI,
Wergedal JE, Lau KH, Mohan S, Ryaby JT, Baylink DJ
(2006) Microarray analysis of gene expression during the
inammation and endochondral bone formation stages of
rat femur fracture repair. Bone 38: 521-529.
Salinas CN, Anseth KS (2009) Mesenchymal stem cells
for craniofacial tissue regeneration: designing hydrogel
delivery vehicles. J Dent Res 88: 681-692.
Sandor GK, Tuovinen VJ, Wolff J, Patrikoski M,
Jokinen J, Nieminen E, Mannerstrom B, Lappalainen OP,
Seppanen R, Miettinen S (2013) Adipose stem cell tissue-
engineered construct used to treat large anterior mandibular
defect: a case report and review of the clinical application
of good manufacturing practice-level adipose stem cells
for bone regeneration. J Oral Maxillofac Surg 71: 938-950.
Sawkins MJ, Bowen W, Dhadda P, Markides H, Sidney
LE, Taylor AJ, Rose FR, Badylak SF, Shakeshe KM,
White LJ (2013) Hydrogels derived from demineralized
and decellularized bone extracellular matrix. Acta
Biomater 9: 7865-7873.
Schantz JT, Chim H, Whiteman M (2007) Cell guidance
in tissue engineering: SDF-1 mediates site-directed homing
of mesenchymal stem cells within three-dimensional
polycaprolactone scaolds. Tissue Eng 13: 2615-2624.
Schmoekel HG, Weber FE, Schense JC, Gratz KW,
Schawalder P, Hubbell JA (2005) Bone repair with a form
of BMP-2 engineered for incorporation into brin cell
ingrowth matrices. Biotechnol Bioeng 89: 253-262.
Schumann P, Tavassol F, Lindhorst D, Stuehmer C,
Bormann KH, Kampmann A, Mulhaupt R, Laschke MW,
Menger MD, Gellrich NC, Rucker M (2009) Consequences
of seeded cell type on vascularization of tissue engineering
constructs in vivo. Microvasc Res 78: 180-190.
Schwartz R, Reddi AH (1979) Inuence of magnesium
depletion on matrix-induced endochondral bone formation.
Calcif Tissue Int 29: 15-20.
Scotti C, Piccinini E, Takizawa H, Todorov A, Bourgine
P, Papadimitropoulos A, Barbero A, Manz MG, Martin I
(2013) Engineering of a functional bone organ through
endochondral ossication. Proc Natl Acad Sci U S A 110:
3997-4002.
Scotti C, Tonnarelli B, Papadimitropoulos A,
Scherberich A, Schaeren S, Schauerte A, Lopez-Rios
J, Zeller R, Barbero A, Martin I (2010) Recapitulation
of endochondral bone formation using human adult
mesenchymal stem cells as a paradigm for developmental
engineering. Proc Natl Acad Sci U S A 107: 7251-7256.
Seebach C, Henrich D, Kahling C, Wilhelm K, Tami
AE, Alini M, Marzi I (2010) Endothelial progenitor
cells and mesenchymal stem cells seeded onto beta-TCP
granules enhance early vascularization and bone healing
in a critical-sized bone defect in rats. Tissue Eng Part A
16: 1961-1970.
Seeherman H, Wozney JM (2005) Delivery of bone
morphogenetic proteins for orthopedic tissue regeneration.
Cytokine Growth Factor Rev 16: 329-345.
Shah RN, Shah NA, Del Rosario Lim MM, Hsieh C,
Nuber G, Stupp SI (2010) Supramolecular design of self-
assembling nanobers for cartilage regeneration. Proc Natl
Acad Sci U S A 107: 3293-3298.
Sheehy EJ, Vinardell T, Buckley CT, Kelly DJ (2013)
Engineering osteochondral constructs through spatial
regulation of endochondral ossication. Acta Biomater 9:
5484-5492.
Shekaran A, Garcia AJ (2011) Extracellular matrix-
mimetic adhesive biomaterials for bone repair. J Biomed
Mater Res A 96: 261-272.
Shih YR, Hwang Y, Phadke A, Kang H, Hwang NS,
Caro EJ, Nguyen S, Siu M, Theodorakis EA, Gianneschi
NC, Vecchio KS, Chien S, Lee OK, Varghese S (2014)
Calcium phosphate-bearing matrices induce osteogenic
dierentiation of stem cells through adenosine signaling.
Proc Natl Acad Sci U S A 111: 990-995.
Siebers MC, ter Brugge PJ, Walboomers XF, Jansen JA
(2005) Integrins as linker proteins between osteoblasts and
bone replacing materials. A critical review. Biomaterials
26: 137-146.
Solorio LD, Dhami CD, Dang PN, Vieregge EL, Alsberg
E (2012) Spatiotemporal regulation of chondrogenic
dierentiation with controlled delivery of transforming
growth factor-beta1 from gelatin microspheres in
mesenchymal stem cell aggregates. Stem Cells Transl Med
1: 632-639.
Solorio LD, Phillips LM, McMillan A, Cheng CW,
Dang PN, Samorezov JE, Yu X, Murphy WL, Alsberg E
(2015) Spatially organized dierentiation of mesenchymal
stem cells within biphasic microparticle-incorporated high
cell density osteochondral tissues. Adv Healthc Mater 4:
2306-2313.
Steinert AF, Proffen B, Kunz M, Hendrich C,
Ghivizzani SC, Noth U, Rethwilm A, Eulert J, Evans
CH (2009) Hypertrophy is induced during the in vitro
108 www.ecmjournal.org
S Verrier et al. Large bone defect healing
chondrogenic differentiation of human mesenchymal
stem cells by bone morphogenetic protein-2 and bone
morphogenetic protein-4 gene transfer. Arthritis Res Ther
11: R148.
Strioga M, Viswanathan S, Darinskas A, Slaby O,
Michalek J (2012) Same or not the same? Comparison
of adipose tissue-derived versus bone marrow-derived
mesenchymal stem and stromal cells. Stem Cells Dev 21:
2724-2752.
Su J, Xu H, Sun J, Gong X, Zhao H (2013) Dual delivery
of BMP-2 and bFGF from a New nano-composite scaold,
loaded with vascular stents for large-size mandibular defect
regeneration. Int J Mol Sci 14: 12714-12728.
Suarez-Gonzalez D, Barnhart K, Migneco F, Flanagan
C, Hollister SJ, Murphy WL (2012) Controllable mineral
coatings on PCL scaolds as carriers for growth factor
release. Biomaterials 33: 713-721.
Sun XJ, Peng W, Yang ZL, Ren ML, Zhang SC, Zhang
WG, Zhang LY, Xiao K, Wang ZG, Zhang B, Wang J (2011)
Heparin-chitosan-coated acellular bone matrix enhances
perfusion of blood and vascularization in bone tissue
engineering scaolds. Tissue Eng Part A 17: 2369-2378.
Tavassol F, Schumann P, Lindhorst D, Sinikovic B, Voss
A, von See C, Kampmann A, Bormann KH, Carvalho C,
Mulhaupt R, Harder Y, Laschke MW, Menger MD, Gellrich
NC, Rücker M (2010) Accelerated angiogenic host tissue
response to poly(L-lactide-co-glycolide) scaffolds by
vitalization with osteoblast-like cells. Tissue Eng Part A
16: 2265-2279.
Thompson AD, Betz MW, Yoon DM, Fisher JP (2009)
Osteogenic dierentiation of bone marrow stromal cells
induced by coculture with chondrocytes encapsulated in
three-dimensional matrices. Tissue Eng Part A 15: 1181-
1190.
Thompson EM, Matsiko A, Farrell E, Kelly DJ, O’Brien
FJ (2014) Recapitulating endochondral ossication: a
promising route to in vivo bone regeneration. J Tissue Eng
Regen Med 9: 889-902.
Thompson EM, Matsiko A, Kelly DJ, Gleeson J,
O’Brien FJ (2016) An endochondral ossication-based
approach to bone repair: Chondrogenically-primed MSC-
laden scaolds support greater repair of critical-sized
cranial defects than osteogenically stimulated constructs
in vivo. Tissue Eng Part A 22: 556-567.
Thompson Z, Miclau T, Hu D, Helms JA (2002) A
model for intramembranous ossication during fracture
healing. J Orthop Res 20: 1091-1098.
Thornton AJ, Alsberg E, Albertelli M, Mooney DJ
(2004) Shape-dening scaolds for minimally invasive
tissue engineering. Transplantation 77: 1798-1803.
Thorpe SD, Nagel T, Carroll SF, Kelly DJ (2013)
Modulating gradients in regulatory signals within
mesenchymal stem cell seeded hydrogels: a novel strategy
to engineer zonal articular cartilage. PLoS One 8: e60764.
Tierney EG, Duy GP, Cryan SA, Curtin CM, O’Brien
FJ (2013) Non-viral gene-activated matrices: next
generation constructs for bone repair. Organogenesis 9:
22-28.
Tierney EG, Duy GP, Hibbitts AJ, Cryan SA, O’Brien
FJ (2012) The development of non-viral gene-activated
matrices for bone regeneration using polyethyleneimine
(PEI) and collagen-based scaolds. J Control Release 158:
304-311.
Tortelli F, Tasso R, Loiacono F, Cancedda R (2010) The
development of tissue-engineered bone of dierent origin
through endochondral and intramembranous ossication
following the implantation of mesenchymal stem cells and
osteoblasts in a murine model. Biomaterials 31: 242-249.
Tu Q, Valverde P, Li S, Zhang J, Yang P, Chen J
(2007) Osterix overexpression in mesenchymal stem
cells stimulates healing of critical-sized defects in murine
calvarial bone. Tissue Eng 13: 2431-2440.
Urist MR (1965) Bone: formation by autoinduction.
Science 150: 893-899.
Utzinger U, Baggett B, Weiss JA, Hoying JB, Edgar
LT (2015) Large-scale time series microscopy of neovessel
growth during angiogenesis. Angiogenesis 18: 219-232.
Vacanti CA, Bonassar LJ, Vacanti MP, Shuebarger
J (2001) Replacement of an avulsed phalanx with tissue-
engineered bone. N Engl J Med 344: 1511-1514.
Venkatesan J, Pallela R, Bhatnagar I, Kim SK (2012)
Chitosan-amylopectin/hydroxyapatite and chitosan-
chondroitin sulphate/hydroxyapatite composite scaolds
for bone tissue engineering. Int J Biol Macromol 51: 1033-
1042.
Vinardell T, Rolfe RA, Buckley CT, Meyer EG,
Ahearne M, Murphy P, Kelly DJ (2012a) Hydrostatic
pressure acts to stabilise a chondrogenic phenotype in
porcine joint tissue derived stem cells. Eur Cell Mater 23:
121-132.
Vinardell T, Sheehy EJ, Buckley CT, Kelly DJ (2012b)
A comparison of the functionality and in vivo phenotypic
stability of cartilaginous tissues engineered from dierent
stem cell sources. Tissue Eng Part A 18: 1161-1170.
Virk MS, Sugiyama O, Park SH, Gambhir SS, Adams
DJ, Drissi H, Lieberman JR (2011) “Same day” ex-vivo
regional gene therapy: a novel strategy to enhance bone
repair. Mol Ther 19: 960-968.
Wahl DA, Czernuszka JT (2006) Collagen-
hydroxyapatite composites for hard tissue repair. Eur
Cell Mater 11: 43-56.
Wallace AL, Draper ER, Strachan RK, McCarthy
ID, Hughes SP (1994) The vascular response to fracture
micromovement. Clin Orthop Relat Res 281-290.
Wang L, Rao RR, Stegemann JP (2013) Delivery of
mesenchymal stem cells in chitosan/collagen microbeads
for orthopedic tissue repair. Cells Tissues Organs 197:
333-343.
Wang L, Stegemann JP (2010) Thermogelling chitosan
and collagen composite hydrogels initiated with beta-
glycerophosphate for bone tissue engineering. Biomaterials
31: 3976-3985.
Wang L, Stegemann JP (2011) Glyoxal crosslinking
of cell-seeded chitosan/collagen hydrogels for bone
regeneration. Acta Biomater 7: 2410-2417.
Wang S, Lu L, Yaszemski MJ (2006) Bone-tissue-
engineering material poly(propylene fumarate): correlation
between molecular weight, chain dimensions, and physical
properties. Biomacromolecules 7: 1976-1982.
Wang ZZ, Au P, Chen T, Shao Y, Daheron LM, Bai H,
Arzigian M, Fukumura D, Jain RK, Scadden DT (2007)
Endothelial cells derived from human embryonic stem
109 www.ecmjournal.org
S Verrier et al. Large bone defect healing
cells form durable blood vessels in vivo. Nat Biotechnol
25: 317-318.
Warnke PH, Springer IN, Wiltfang J, Acil Y, Eunger
H, Wehmoller M, Russo PA, Bolte H, Sherry E, Behrens
E, Terheyden H (2004) Growth and transplantation of
a custom vascularised bone graft in a man. Lancet 364:
766-770.
Weaver AS, Su YP, Begun DL, Miller JD, Alford AI,
Goldstein SA (2010) The eects of axial displacement on
fracture callus morphology and MSC homing depend on
the timing of application. Bone 47: 41-48.
Webber MJ, Tongers J, Renault MA, Roncalli JG,
Losordo DW, Stupp SI (2010) Development of bioactive
peptide amphiphiles for therapeutic cell delivery. Acta
Biomater 6: 3-11.
Wei S, Cai X, Huang J, Xu F, Liu X, Wang Q (2012)
Recombinant human BMP-2 for the treatment of open
tibial fractures. Orthopedics 35: e847-e854.
Whitney MJ, Lee A, Ylostalo J, Zeitouni S, Tucker A,
Gregory CA (2009) Leukemia inhibitory factor secretion is
a predictor and indicator of early progenitor status in adult
bone marrow stromal cells. Tissue Eng Part A 15: 33-44.
Wise JK, Alford AI, Goldstein SA, Stegemann JP
(2015) Synergistic enhancement of ectopic bone formation
by supplementation of freshly isolated marrow cells with
puried MSC in collagen-chitosan hydrogel microbeads.
Connect Tissue Res 4: 1-9.
Wolf S, Janousek A, Pfeil J, Veith W, Haas F, Duda
G, Claes L (1998) The eects of external mechanical
stimulation on the healing of diaphyseal osteotomies xed
by exible external xation. Clin Biomech (Bristol, Avon)
13: 359-364.
Woodruff MA, Rath SN, Susanto E, Haupt LM,
Hutmacher DW, Nurcombe V, Cool SM (2007) Sustained
release and osteogenic potential of heparan sulfate-doped
brin glue scaolds within a rat cranial model. J Mol Histol
38: 425-433.
Wu X, Wang Q, Kang N, Wu J, Gu C, Bi J, Lv T,
Xie F, Hu J, Liu X, Cao Y, Xiao R (2015) The eects of
dierent vascular carrier patterns on the angiogenesis and
osteogenesis of BMSC-TCP-based tissue-engineered bone
in beagle dogs. J Tissue Eng Regen Med doi: 10.1002/
term.2076.
Yamaji T, Ando K, Wolf S, Augat P, Claes L (2001) The
eect of micromovement on callus formation. J Orthop Sci
6: 571-575.
Yamamoto M, Takahashi Y, Tabata Y (2003) Controlled
release by biodegradable hydrogels enhances the
ectopic bone formation of bone morphogenetic protein.
Biomaterials 24: 4375-4383.
Yamamoto M, Takahashi Y, Tabata Y (2006) Enhanced
bone regeneration at a segmental bone defect by
controlled release of bone morphogenetic protein-2 from
a biodegradable hydrogel. Tissue Eng 12: 1305-1311.
Yang F, Yang D, Tu J, Zheng Q, Cai L, Wang L
(2011) Strontium enhances osteogenic dierentiation of
mesenchymal stem cells and in vivo bone formation by
activating Wnt/catenin signaling. Stem Cells 29: 981-991.
Yang H, Yang S, Chi X, Evans JR (2006) Fine ceramic
lattices prepared by extrusion freeforming. J Biomed Mater
Res B Appl Biomater 79: 116-121.
Yang W, Both SK, van Osch GJ, Wang Y, Jansen JA,
Yang F (2015) Eects of in vitro chondrogenic priming
time of bone-marrow-derived mesenchymal stromal cells
on in vivo endochondral bone formation. Acta Biomater
13: 254-265.
Yang W, Yang F, Wang Y, Both SK, Jansen JA (2013)
In vivo bone generation via the endochondral pathway on
three-dimensional electrospun bers. Acta Biomater 9:
4505-4512.
Yi T, Song SU (2012) Immunomodulatory properties of
mesenchymal stem cells and their therapeutic applications.
Arch Pharm Res 35: 213-221.
Yong MR, Saifzadeh S, Askin GN, Labrom RD,
Hutmacher DW, Adam CJ (2014) Biological performance
of a polycaprolactone-based scaold plus recombinant
human morphogenetic protein-2 (rhBMP-2) in an ovine
thoracic interbody fusion model. Eur Spine J 23: 650-657.
Youse AM, James PF, Akbarzadeh R, Subramanian
A, Flavin C, Oudadesse H (2016) Prospect of stem cells
in bone tissue engineering: a review. Stem Cells Int 2016:
6180487.
Yu NY, Schindeler A, Little DG, Ruys AJ (2010)
Biodegradable poly(alpha-hydroxy acid) polymer scaolds
for bone tissue engineering. J Biomed Mater Res B Appl
Biomater 93: 285-295.
Zeitouni S, Krause U, Clough BH, Halderman H,
Falster A, Blalock DT, Chaput CD, Sampson HW, Gregory
CA (2012) Human mesenchymal stem cell-derived
matrices for enhanced osteoregeneration. Sci Transl Med
4: 132ra55.
Zhang H, Migneco F, Lin CY, Hollister SJ (2010a)
Chemically-conjugated bone morphogenetic protein-2 on
three-dimensional polycaprolactone scaolds stimulates
osteogenic activity in bone marrow stromal cells. Tissue
Eng Part A 16: 3441-3448.
Zhang Y, Venugopal JR, El-Turki A, Ramakrishna S, Su
B, Lim CT (2008) Electrospun biomimetic nanocomposite
nanobers of hydroxyapatite/chitosan for bone tissue
engineering. Biomaterials 29: 4314-4322.
Zhang ZY, Teoh SH, Chong MS, Lee ES, Tan LG,
Mattar CN, Fisk NM, Choolani M, Chan J (2010b) Neo-
vascularization and bone formation mediated by fetal
mesenchymal stem cell tissue-engineered bone grafts in
critical-size femoral defects. Biomaterials 31: 608-620.
Zheng H, Bai Y, Shih MS, Homann C, Peters F,
Waldner C, Hubner WD (2014) Eect of a beta-TCP
collagen composite bone substitute on healing of drilled
bone voids in the distal femoral condyle of rabbits. J
Biomed Mater Res B Appl Biomater 102: 376-383.
Zhu G, Mallery SR, Schwendeman SP (2000)
Stabilization of proteins encapsulated in injectable poly
(lactide- co-glycolide). Nat Biotechnol 18: 52-57.
Zou D, Zhang Z, He J, Zhang K, Ye D, Han W, Zhou
J, Wang Y, Li Q, Liu X, Zhang X, Wang S, Hu J, Zhu C,
Zhang W, zhou Y, Fu H, Huang Y, Jiang X (2012) Blood
vessel formation in the tissue-engineered bone with the
constitutively active form of HIF-1alpha mediated BMSCs.
Biomaterials 33: 2097-2108.
110 www.ecmjournal.org
S Verrier et al. Large bone defect healing
Discussion with Reviewers
P. Habibovic: In the Conclusion section, the authors state:
“At a minimum, the regeneration of bone requires the
balanced contributions of scaolds, cells, morphogenetic
signals, vascularisation and mechanics”. I would like
to challenge this statement by stating that a therapy
encompassing all these component will never reach the
clinic, because of the high cost and complex regulations.
Could the authors respond to this statement?
Authors: The reviewer identies an important issue that
is touched upon in the paper, but not explored in detail:
how to make TERM (Tissue Engineering and Regenerative
Medicine) aordable. This will probably require expedited
approaches that do not use expanded, autologous cells
but harness intrinsic, biological processes. Greater
investigation of simple rehabilitation techniques could also
pay dividends. Investigators need to bear in mind cost, as
well as science, when developing technologies.
I. Martin: In the conclusions, it is mentioned that “perhaps,
we need to go back to the beginning and discover why large
segmental defects in otherwise healthy individuals do not
heal”. Could you further elaborate your recommendation on
the approaches to be followed or nature of the parameters
to be investigated to bring forward our fundamental
knowledge of the biological processes which need to be
better controlled?
Authors: The early biological responses to an osseous
injury seem important determinants of whether and, if
so, how, a defect will heal (Glatt et al., eCM 2012; Kolar
al, Tissue Engineering Part B. 2010). This suggests that
study of the early biology of a segmental defect would be
protable. Useful comparisons could be made between
a critical sized defect in the presence or absence of an
osteogenic growth factor, or between a large osseous in
a bone that does not heal (e.g. femur) and one where a
similar sized defect spontaneously heals (e.g. rib). Pre-
paradigmatic observations in such systems promise to
generate experimentally testable hypotheses.
Editor’s note: The Scientic Editor responsible for this
paper was Joost de Bruijn.
