Bone 70 (2015) 93–101

Contents lists available at ScienceDirect

Bone
journal homepage: www.elsevier.com/locate/bone

Review

Bone fracture healing: Cell therapy in delayed unions and nonunions

Enrique Gómez-Barrena a,⁎, Philippe Rosset b,c, Daniel Lozano d, Julien Stanovici b,c,
Christian Ermthaller e, Florian Gerbhard e
a
Dept. of Orthopaedic Surgery and Traumatology, Hospital La Paz-IdiPAZ, Universidad Autónoma de Madrid, Madrid, Spain
b
Service of Orthopaedic Surgery and Traumatology, CHU Tours, Université François-Rabelais de Tours, PRES Centre-Val de Loire Université, Tours, France
c
Inserm U957, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives (LPRO), Faculté de Médecine, Université de Nantes, France
d
Metabolic Bone Research Unit, Instituto de Investigación Sanitaria FJD, Madrid, Spain
e
Klinik für Unfallchirurgie-, Hand-, Plastische und Wiederherstellungschirurgie Zentrum für Chirurgie Universitätsklinikum Ulm, Ulm, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Bone fracture healing impairment related to mechanical problems has been largely corrected by advances in
Received 13 March 2014 fracture management. Better protocols, more strict controls of time and function, and hardware and surgical
Revised 26 July 2014 technique evolution have contributed to better prognosis, even in complex fractures. However, atrophic non-
Accepted 28 July 2014
union persists in clinical cases where, for different reasons, the osteogenic capability is impaired. When this is
Available online 2 August 2014
the case, a better understanding of the basic mechanisms under bone repair and augmentation techniques
Keywords:
may put in perspective the current possibilities and future opportunities. Among those, cell therapy particu-
Bone healing larly aims to correct this insufficient osteogenesis. However, the launching of safe and efficacious cell thera-
Nonunion pies still requires substantial amount of research, especially clinical trials. This review will envisage the
Cell therapy current clinical trials on bone healing augmentation based on cell therapy, with the experience provided by
Clinical trials the REBORNE Project, and the insight from investigator-driven clinical trials on advanced therapies towards
MSCs the future. This article is part of a Special Issue entitled Stem Cells and Bone.
© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Clinical bone healing impairment: from hypertrophic to atrophic nonunions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Clinical and imaging diagnoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Cells and molecules in bone healing after fractures and pharmacological new therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Bone healing augmentation: grafts and biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Advanced therapy (AMTP) proposed solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Safety and preclinical rationale to launch clinical trials on cell therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
On-going cell therapy clinical trials to treat long bone non unions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Conflict of interest disclosure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

Introduction

Bone fracture clinical management is oriented to obtain bone

⁎ Corresponding author at: Cirugía Ortopédica y Traumatología, Hospital La Paz,
Universidad Autónoma de Madrid, P° Castellana 261, 28046 Madrid, Spain. Fax: +34
914269774.
E-mail address: enrique.gomezbarrena@uam.es (E. Gómez-Barrena).
healing in the shortest time frame, with the best possible functional re-
covery, and with less complications. However, an overall rate of 5 to 10%
delayed union or nonunion is widely accepted as a perceived proportion

http://dx.doi.org/10.1016/j.bone.2014.07.033
8756-3282/© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
94 E. Gómez-Barrena et al. / Bone 70 (2015) 93–101
for bone healing problems, although this figure is not homogenous.
Rather, different nonunion rates are found in different types of fracture,
somewhat ranging from up to 18.5% in the tibia diaphysis [1] to 1.7% in
the femoral shaft after reamed nailing [2].
The definition of delayed union and nonunion or pseudarthrosis
certainly deserves more discussion. Those cases that correspond to a dif-
ferent healing rate than expected (slow healing rate) should be clearly
separated from those in which the bone healing is no longer expected
without treatment. A better understanding of fracture healing biology
would help in fostering preclinical studies and clinical proposals in
both of these directions: accelerating bone fracture healing in case
of slow healing rate, based on biological stimulation, and promoting
bone fracture healing in case of no healing expectations, based on
redeveloping the bone regeneration capability, whether fully lost or at
least under the required threshold to healing.
Major limb injuries related to traffic accidents and multiple trauma
are a major health issue in developed countries, resulting in long treat-
ments with substantial socioeconomic effects. But these injuries are also
severely impacting less developed countries, where secondary compli-
cations frequently generate major disabilities [3]. Long bone fractures
are difficult and slow to heal and may require months until consolida-
tion is completed. Long treatments not only associate significant loss
of working days with economic effects on the patient and the society,
but also carry the risk of nonunion and permanent disabilities related
to malunion, joint stiffness, muscular atrophy, or reflex sympathetic
dystrophy.
The ability of fractured bone to regenerate and undergo repair may
be compromised when insufficient osteogenic reaction is observed
in the fracture callus, up to developing an atrophic nonunion. Those
cases cannot be solved through a mechanical approach, as occurs with
hypertrophic nonunions. Treatment of these atrophic nonunions re-
quires some form of bone healing augmentation, providing that vascu-
larization is sufficient and confirming that infection is absent.
Conventional, standard treatment to augment bone healing is based
on bone autograft, today's most accepted gold standard. The application
of autologous cancellous and corticocancellous grafts, or larger, even
vascularized, segmental bone grafts (frequently constructed out of the
fibula) when the defect exceeds some centimeters, may permit the
most appraised personalized management to this problem. Yet this clas-
sical orthopedic approach may be not appropriate. And this happens
when the autograft strategy has already failed, when the osteogenic po-
tential of the available donor site is altered (due to cell scarcity, fibrous
tissue abundance due to previous harvesting, or other impairments), or
when the risk/benefit evaluation of the autologous bone graft obtention
is unbalanced or refused by the patient.
Fig. 1. Radiological AP and lateral views of a tibial midshaft hypertrophic nonunion.
Alternatively proposed strategies include those relying on the
osteoconductive or osteoinductive capabilities of an implanted tissue
(such as allograft or demineralized bone matrix) or a synthetic mate-
rial (such as bioceramics in different forms and compositions). Also,
different strategies have been defined to supplement potential mo-
lecular deficiency in the stimulation of local cell differentiation in
the osteoprogenitor line (such as BMP or other growth factor local
deliveries). These strategies rely on the surrounding or available cells
that might eventually produce the required local bone regeneration.
The expected fracture healing is seriously constrained in cases where
previous efforts to heal the fracture have failed. Particularly in those
cases with a supposed cell insufficiency, cell-based alternatives devel-
oped over mesenchymal stem cells (MSCs) [4] have been proposed,
and are currently under investigation and evaluation.
In this context, this review progresses from clinical concepts of bone
healing impairment to advanced therapies under trial [5]. In this jour-
ney, cellular and molecular bases of bone regeneration in fracture
healing will be considered as the foundations of so-called therapy plat-
forms [6], state of the art and recent contributions to bone induction and
augmentation will be appraised, and particular emphasis will be placed
on cell therapy proposals and current cell therapy based orthopedic
clinical trials.
Clinical bone healing impairment: from hypertrophic to
atrophic nonunions
In a normal biological environment, many skeletal fractures heal un-
eventfully in the first 6 to 8 weeks. In case of an impaired bone healing
process due to a disturbed biological or mechanical environment, or
in cases where thick cortices are involved such as in femoral and tibial
diaphysis, fractures may take a longer time to heal [7]. Per conventional
definition, if a fracture is not healed after 4 months, it can be considered
a delayed union. If no bony healing is obtained in 6 months after
the fracture, it can be clinically considered as nonunion, although the
diagnosis requires specific radiological features showing bone ending
changes.
There are two distinct variants of nonunions with opposed under-
lying pathomechanisms, namely hypertrophic and atrophic nonunions.
A hypertrophic nonunion presents with a large, vital callus, although
inefficient to regenerate bony union. On conventional radiographs,
the hypertrophic nonunion displays a large, broaden callus towards
the fracture gap, with a radiolucent area instead of bone bridging. Due
to its radiological features (Fig. 1), the hypertrophic nonunion is also
called elephant foot nonunion [8]. Its basic problem is the mechanical
disturbance of the chosen fixation technique. The most recognized
 E. Gómez-Barrena et al. / Bone 70 (2015) 93–101
etiology underlying hypertrophic nonunions is the inefficient and un-
stable fixation of the fracture allowing for multidirectional motion of
fracture fragments.
Whereas limited axial compressive movements can increase callus
formation and accelerate fracture healing [9], shear displacement has
demonstrated to hinder callus formation [10]. Up to a critical value, an
increasing interfragmentary motion leads to an increase in callus forma-
tion. Above a critical threshold, especially in combination with larger
gap sizes, interfragmentary motion leads to hypertrophic nonunions
[9,11,12]. Most frequently, the treatment of hypertrophic nonunions
is surgically oriented. Exchange of the fixation technique towards
a more stable osteosynthesis aims to restrict the fracture gap with a
limited amount of compressive forces [13,14]. Secondarily, additional
treatment by ultrasound or external shock wave therapy has also been
proposed, although definite evidence is still lacking and significant con-
troversy remains about this issue [15,16].
The pathomechanisms leading to atrophic bone nonunions are
completely different. Claimed underlying causes usually incorporate
biological impairment, sometimes in combination with mechanical fac-
tors. In most cases, atrophic nonunions are the expression of impaired
biological support for bone healing, as for damaged vascular supply,
and destruction of the periosteum and endosteum. This impairment is
frequently associated to cofactors such as polytrauma or soft tissue
damage, with detraction of surrounding tissues [17]. Consecutively,
fracture healing is impaired because of the deficiency of important me-
diators, blood supply or other indispensable biological parameters.
Mechanical reasons can also be involved in the development of atro-
phic nonunions. Excessively rigid fixation, insufficient compressive
forces, and a fracture gap too wide to allow bony bridging of the frag-
ments can also contribute. In radiological images, the atrophic nonunion
demonstrates the absence of callus tissue, the narrowing of bone ends,
and a large radiolucent zone in the fracture gap (Figs. 2 and 3). The
treatment of atrophic bone nonunion requires a surgical intervention.
The aims of this intervention are the elimination of disturbing mechan-
ical factors through modification of osteosynthesis and reduction of the
fracture gap, the increase of compressive motion between fracture frag-
ments, and the biological improvement by introduction of new cells,
vascular supply and healthy bone tissue into the fracture gap [18].
Clinical and imaging diagnoses
In the regular follow-up of patients after bone fracture, the course of
fracture consolidation is reviewed by conventional, two orthogonal pro-
jection radiographs. Therefore, the development of a nonunion can be
monitored clinically and through imaging. In case of insufficient fracture
healing, early modification of osteosynthesis like dynamization of an
Fig. 2. Radiological AP and lateral views of a femoral upper diaphysis atrophic nonunion.
95
intramedullary nail can influence the further course of healing and re-
orient a delayed union or even some nonunions towards adequate
bone consolidation. Patients with a manifest nonunion usually complain
about pain in the fracture area with, and sometimes even without,
weight bearing. The affected bone is usually sensitive upon pressure
and patients are not able to bear full weight [17].
In cases of suspected infectious genesis of nonunion with possible
additional symptoms like reddening, hyperthermia and elevated body
temperature, laboratory analysis should be obtained for infectious pa-
rameters such as white blood cell count and inflammation parame-
ters [19].
After clinical and laboratory evaluation, conventional radiographs in
two orthogonal planes represent the basic diagnostic imaging tool,
where the radiolucent gap between bone endings is associated to
closure of intramedullary canals of diaphyseal bone endings. Besides,
the basic characteristics of the nonunion (status of consolidation,
hypertrophic/atrophic nonunion, segmental bone defects) can be evalu-
ated for a more precise diagnosis.
If the amount of consolidation or the radiological signs of nonunion
do not become obvious in conventional radiographic evaluation, a com-
puter tomography (CT) of the affected region is mandatory. Three-
dimensional reconstructions and exact illustration of the fractured
region, with the amount and location of possible callus bridges, can be
evaluated through CT imaging (Fig. 3).
In some cases, especially with doubtful aseptic pathogenesis of the
non-union, additional diagnostic evaluation should be performed.
While bone scintigraphy no longer represents the state of the art diag-
nostic imaging tool, fluorodeoxyglucose positron emission computer
tomography (FDG-PET-CT) has become more and more relevant in
clinical daily routine. This imaging tool combines the exact imaging
from the CT with additional information about the metabolism of the
examined area with a high diagnostic sensitivity for a chronic osteitis.
FDG-PET-CT has been shown to be of good diagnostic accuracy in
bone pathology discrimination [20] and chronic osteomyelitis [21].
The combination of clinical examination, laboratory analysis and ra-
diological imaging by conventional radiographs, CT and possibly PET-CT
should be sufficient for a clear diagnosis. Underlying cellular and molec-
ular mechanisms are key to develop specific treatments and its review is
mandatory to further progress in clinical research strategies.
Cells and molecules in bone healing after fractures and
pharmacological new therapies
Bone healing of fractures and small bone defects is a unique and very
effective process involving complex and well-orchestrated interactions
between cells, cytokines, osteo-conductive matrix and a mechanically
96 E. Gómez-Barrena et al. / Bone 70 (2015) 93–101
Fig. 3. CT confirmation of the gap in an atrophic nonunion.
stable environment with a good blood supply, according to the
“diamond concept” [22] to generate new bone instead of a fibrous
scar, as occurs in other connective tissues.
This complex dynamic process requires the precise orchestration
of various events during overlapping stages [23] with distinctive histo-
logical characteristics, from the initial inflammatory response, the for-
mation of a cartilaginous soft callus, the formation of a bone hard
callus, and finally the bone union followed by remodeling. As is widely
accepted, this bone repair in adults recapitulates the normal develop-
ment of the skeleton during embryogenesis [24]. Moreover, the current
paradigm of bone tissue engineering also relies on biomimetics to re-
produce bone formation from development biology [25,26]. Prenatal
bone formation starts with mesenchymal cell condensation and sub-
sequent differentiation to chondrocytes (through endochondral ossifi-
cation) or, in precise cases, straight forward to osteoblasts (through
intramembranous ossification) [27]. Both processes are implicated in
the callus formation after fracture [24]. However, callus formation
in adult bone is highly influenced by factors such as inflammation,
presence of pluripotent and osteoprogenitor cells, gap distance between
bone fracture endings, and mechanical stabilization and loading. The
endochondral ossification mechanism predominates in the majority of
fracture healing cases, advancing through several phases that involve
multiple cellular and molecular events [28] in the so-called “bone
healing cascade” [29] from hematoma and inflammation to angiogene-
sis and chondrogenesis, to finally complete osteogenesis followed by
bone remodeling.
The interruption of vascular endothelium integrity is the first step
following trauma, accompanied by a disruption of the blood supply
and hematoma formation, associating the presence of necrotic material.
This facilitates a potent inflammatory response related to the pro-
duction of pro-inflammatory cytokines from aggregated platelets, as
interleukin-1 (IL-1), IL-6 or tumor necrosis factor-α, which have che-
motactic activity towards endothelial cells, fibroblasts, lymphocytes
and monocytes–macrophages [30]. Specifically, transforming growth
factor b1 (TGFb1) is a potent chemotactic stimulator of mesenchymal
stem cells that enhances osteoblast precursors and chondrocyte prolif-
eration, and may participate in recruitment of bone cells in the trauma
area [31]. In addition, TGFb1 induces the production of extracellular
bone matrix proteins such as collagen, osteopontin, and alkaline phos-
phatase [7] and regulates different cell types implicated in bone turn-
over and fracture healing [31]. Sox9, Runx2, and Osterix are three
essential transcription factors that have important roles in the cell-fate
decision process by which mesenchymal cells become chondrocytes
and osteoblasts, through activation of cell type-specific genes [32].
Macrophages can also produce and secrete these factors including
bone morphogenetic proteins (BMPs) and vascular endothelial growth
factor (VEGF), which induce angiogenesis and bone formation [33,34].
Neovascularization of damaged tissue is crucial to successful bone
healing, providing oxygen and delivering progenitor cells [35]. The
vascular endothelium lost integrity produces hypoxic conditions that
induce chondrogenesis, as occurs in the central avascular area of the
callus [36]. In this regard, VEGF is a key osteogenic and angiogenic factor
that is expressed under the control of hypoxia-inducible factor (HIF)-1α
in low oxygen tension [35]. Overexpression of HIF1α in mature osteo-
blasts, in mice with distraction osteogenesis, stimulates bone regenera-
tion indicating an angiogenic response related to new bone formation.
BMPs, parathyroid hormone (PTH)-related protein (PTHrP) and other
osteogenic factors stimulate the expression of VEGF in osteoblastic
cells [37,38]. In this reparative phase, neoangiogenesis and chondrogen-
esis predominate to bridge the gap in the fracture and complete bone
healing, but this soft callus is then replaced with a hard callus connecting
bone fragments with new bone. Osteoblasts can form woven bone rap-
idly, but it is randomly arranged and mechanically weak [28], requiring
bone remodeling by which newly formed woven bone is replaced by la-
mellae through the activity of osteoclasts and osteoblasts [39].
This cellular and molecular background justifies different strategies
to promote bone regeneration based on molecular osteoinduction. The
use of agents that increase vascularization and osteoblastic maturation
could contribute to early callus formation. In this context, PTH exerts
anabolic actions throughout cAMP–PKA pathway activation, implicating
on bone formation in vitro and in vivo [40] and interacting with impor-
tant bone local factors such as PTHrP, BMPs, Wnt-β-catenin, EGF, and
FGF [40]. The possibility of using Wnt pathway molecules as anabolic
agents in bone repair is complex because their effects depend on
the cell differentiation state [41]. In addition, this pathway is implicated
in tumoral processes. Studies with Wnt pathway antagonists such
as DKK-1, SFRP and sclerostin are in progress. Several studies demon-
strated that the use of these factors can promote bone formation in
rodent models associated with a decreased BMD and higher bone turn-
over [42,43]. Sost (sclerostin-encoding gene) is a key modulator of
bone remodeling and its expression was rapidly reduced in the callus,
indicating that this would allow osteoblasts to escape from its inhibitory
effect to promote bone repair [44].
However, translation into clinical trials is limited at this point. PTH
trials in fractures [45,46] aimed at accelerating fracture repair, particu-
larly in fractures that are seldom prone to nonunion. BMPs have been
frequently used in clinical trials with significant heterogeneity. A sys-
tematic review and metaanalysis on 11 trials observed comparable
times to fracture healing between BMP and controls and confirmed
some evidence of increased healing rates with BMP without a secondary
procedure compared with usual care control in acute tibial fractures
[47], but observed that achieving union for nonunited fractures was
similar to bone graft substitutes. Other strategies such as local applica-
tion of FGF2 were found to accelerate tibial shaft fractures [48], although
no data are available in nonunions.
Bone healing augmentation: grafts and biomaterials
Bone grafting is widely used in hospitals to repair injured, aged or
diseased skeletal tissue. In Europe, about one million patients encounter
 E. Gómez-Barrena et al. / Bone 70 (2015) 93–101
surgical bone reconstruction annually and the numbers are increasing
due to our aging population. Bone grafting intends to facilitate bone
healing through osteogenesis (i.e. bone generation) at the site of damage,
but this is only attained when augmentation includes cells capable
of forming bone. Other options to augment this bone repair include
osteoinductive (i.e. bone inducers) and osteoconductive (i.e. bone
guides) capabilities of the supplied coadjuvants to the surgical treatment.
Bone autograft is the safest and most effective grafting procedure,
since it contains a patient's own bone growing cells (to enhance osteo-
genesis) and proteins (to enhance osteoinduction), while providing a
framework for the new bone to grow into (osteoconduction). However,
bone autograft is limited in quantity (about 20 cm3) and its harvesting
(e.g. from the iliac crest) represents an additional surgical intervention,
with frequent consequences of pain and complications [49].
The next solution is allograft bone directly coming from tissue
banks (fresh-frozen) or prepared to be conserved (dried or lyophi-
lized). This solution does not contain living cells and some matrix pro-
teins are destroyed by virus-inactivation treatments and the freezing
process, thus it only guarantees osteoconductive properties. Moreover,
allograft bone may transfer disease or lead to immunological rejections
[50]. An interesting alternative is to combine allograft with MSCs
from concentrated bone marrow, as has been proposed in bone defects
after revision hip surgery [51] but also preliminarily explored in long
bone pseudarthrosis [52]. Yet the number of cells may be an issue, as
the available evidence in preclinical models recommends a high num-
ber of cells [53] and many ongoing clinical trials are thus based on
high number of MSCs that require cell expansion, as will be discussed
later.
Since both autograft and allograft have drawbacks, scientists have
long searched for biocompatible materials that could be used in place
of the transplanted bone [50,54]. Several biomaterials can be chosen
depending on the goal (mechanical strength or filling) and approach
(percutaneous or surgical). The most widely used biomaterials are
calcium-phosphate ceramics, which usually combine hydroxyapatite
and tricalcium phosphate as granules or, more rarely, sticks, and exhibit
interconnected pores each measuring 100–400 μm. These biomaterials
promote the adhesion, proliferation, and osteoblastic differentiation of
MSCs, as well as the production of the collagen matrix that subsequently
undergoes mineralization. Collagen sponges and biodegradable poly-
mers can also be used. The biomaterials must be absorbable, at a vari-
able rate depending on their anticipated biomechanical role, and must
allow the ingrowth of newly formed blood vessels from the neighboring
tissues. Good quality vascularization of the tissue in contact with the im-
plant is crucial.
Although most of the available synthetic bone substitutes possess
some of the positive properties of autograft (particularly, osteoconductive
capabilities and occasionally, osteoinductive properties), none has all
the benefits of one's own bone yet (osteogenic properties). Basically
and besides bone autografting, which is the only truly osteogenic mate-
rial, orthobiological solutions today available to surgeons include
osteoconductive and osteoinductive products, such as different prep-
arations of bone allograft (fresh-frozen or dried by lyophilization,
warranting osteoconduction), different synthetic substitutes (with
variable properties but particularly osteoconductive), and synthetic
pharmaceuticals with osteoinductive properties (such as bone morpho-
genetic proteins, BMPs). Available evidence confirms the outcome of
fractures and non-unions treated by surgical techniques augmented
by autograft [55] and by BMPs [47]; thus this information may be com-
pared to efficacy studies about other solutions.
An alternative strategy to accelerate bone healing includes the use
of degradable biomaterials in combination with osteogenic factors.
Besides the already mentioned growth factors, emerging anabolic oste-
ogenic factors are under scrutiny. This applies not only to PTH but also to
PTHrP whose C-terminal 107–111 domain (also known as osteostatin)
exhibits osteogenic features in vitro, and stimulates bone formation
in vivo [56–60]. PTHrP also conferred both osteogenic and angiogenic
97
preclinical features when coating Si-based ceramics both in vitro and
in vivo [61,62].
But besides bone grafts, substitutes and their augmentation with
growth factors and anabolic strategies, cell therapies have been pro-
posed to evolve towards new osteoinductive and osteogenic solutions
that could safely and efficaciously compete with currently available
standards.
Advanced therapy (AMTP) proposed solutions
In view of these limitations and the increasing number of bone
grafting procedures, surgeons are looking for alternatives with added
value compared to osteoconductive substitutes, such as cell therapy
and tissue engineering [63].
According to the above mentioned diamond concept [22], MSCs play
a crucial role in bone repair, and thus cell therapy can serve as an alter-
native to autologous bone grafting. A large number of osteoprogenitor
cells may be implanted at the injury site, either alone or combined
with a matrix. Autologous bone marrow (BM) is rich in growth factors
and osteoprogenitors as MSCs are present in the mononuclear cellular
fraction of the bone marrow. Bone marrow MSCs are currently the
most appropriate cells for inducing bone repair, as they have a strong
osteogenic potential and are easily obtained by culturing iliac crest aspi-
rates. Several MSC-based cell therapy modalities have been developed,
i.e., with and without cell culturing, and with or without a matrix.
The mononuclear cell fraction of the bone marrow, which contains
the MSCs, can be used directly by percutaneous injection of aspirated
BM into the injury site. To increase the number of injected mononucle-
ar cells and consequently of MSCs, it is possible to separate the mono-
nuclear cells by centrifugation and concentrate them 3-fold to 6-fold
[64] with good results in pseudarthrosis [65]. The healing rate in-
creased in proportion to the injected MSC concentration. Patients
whose fractures did not heal received fewer than 1000 MSCs per mL
and fewer than 30,000 MSCs in total, whereas those whose fractures
healed received significantly higher MSC concentrations and counts,
with a mean of 1500 MSCs per mL and 54,000 MSCs in total, in a volume
of 20 mL.
Concentrated or unconcentrated mononuclear cells can be mixed in
the operating room with a synthetic or natural osteo-conducting matrix
(e.g., allogeneic bone graft or coral) before implantation. Few published
studies assessed the combined use of concentrated or unconcentrated
BM with a biomaterial [66–68]. This method is a valid option for every-
day practice, provided that CE-marked (that is, approved for clinical use
in Europe) biomaterials are used and concentration (if used) is achieved
via an approved procedure.
Mononuclear cells may also be cultured in vitro to allow selection
and expansion of an adherent fraction corresponding to MSC. This
increases the number of MSC to millions of cells. Expanded MSCs can
be extemporaneously mixed with scaffolds during surgery (Fig. 4) so
that this composite material is used in the same way as bone grafts.
Quarto et al. [69] first reported the use of cultured BM MSCs combined
intra-operatively with hydroxyapatite blocks to fill large bone defects
(4–7 cm). They successfully treated 3 patients, with defects in the
tibia, humerus, and ulna, respectively. A subsequent study confirmed
healing of the defects after 6–7 years [70]. The expanded MSCs may
also be injected alone percutaneously in the site of fracture or osteotomy
with interesting results in two studies [71,72].
Tissue engineering combines bone marrow cells or mesenchymal
stem cells, synthetic scaffolds and molecular signals (growth or differ-
entiating factors) in order to form hybrid constructs. In a classical ap-
proach, bone tissue engineering consists of harvesting bone marrow
from a patient, isolating MSCs by their adherence to tissue culture plas-
tic, expanding and differentiating those cells in culture to a sufficient
number, and then seeding them onto a suitable synthetic scaffold to
be expanded in vitro on this scaffold during several days/weeks. This al-
lows for scaffold colonization and for cell differentiation, before grafting
98 E. Gómez-Barrena et al. / Bone 70 (2015) 93–101
Fig. 4. Cell and bioceramic intraoperative preparation before nonunion augmentation.
of this processed composite material at the affected site, prior to im-
plantation into the same patient [73].
For bone reconstruction purposes, human MSCs have been seeded
and cultured on porous calcium phosphate ceramics in osteogenic
media (dexamethasone, ascorbic acid, β-glycerophosphate). Early pro-
posals lead to clinical studies with low numbers of patients using this
approach, but the outcomes were inconsistent showing low efficacy
in bone regeneration. From these, it is clear that the strategy requires
significant tuning [74,75].
The reasons of the limited clinical success may be due to several
bottlenecks in the multidisciplinary field of bone tissue engineering,
particularly about biomaterials and cell limitations. Biomaterials used
as bone void fillers are inspired by the bone extracellular matrix
(hydroxyapatite, collagen I) but need to be colonized by cells and
vascularized in order to promote bone tissue formation and healing.
The regenerative capabilities of current biomaterials are still limited
to small bone defects. Regarding cell limitations, barriers are found in
the autologous approach, the cell selection, the association of cells and
materials, and the osteogenic differentiation of implanted cells. The au-
tologous approach for isolation and osteogenic differentiation of MSCs is
highly demanding in terms of logistics, production and safety of culture
conditions leading to a costly therapeutic procedure. The selection of a
restricted population of cells from different donors with age and genetic
diversities remains a challenge for regenerative medicine at this early
stage of research due to patient variability. The association of biomate-
rials and osteoprogenitor cells raises technical challenges (i.e. cell
sources, types, doses, timing) and regulatory issues (devices with me-
dicinal drugs) to implement clinical trials. Moreover, bone formation re-
quires different cell populations that cooperate to set up complex 3D
tissue under the guidance of biomechanical cues while vascularization
plays a major role in tissue healing. Finally, osteogenic differentiation in-
duced in vitro is not fully supported by the in vivo release of osteogenic
factors from the graft itself.
An alternative to the previous strategies is to implant the composite
material (cell + scaffold) into a heterotopic site, e.g. in a richly
vascularized muscle, to promote angiogenesis and blood vessel growth
into the construct for some weeks and then to transfer it to the affected
site with vascular anastomoses for the transferred muscle flap con-
taining the implant [76], but only anecdotical experience is available.
Most crucial in the lack of evidence on satisfactory AMTP solutions
that could be acceptable in current clinical practice is the insufficient
number of clinical trials with reasonable, standardized, and preclinically
well-supported cell products. Scientific preclinical proofs of efficacy are
frequently weak, and the proposed cell products are also difficult to re-
produce in a standardized manner, based on the provided information
in many publications, which compounds the difficulties to confirm
these products in well-designed clinical trials.
Safety and preclinical rationale to launch clinical trials on
cell therapy
Not only are complex design and management of clinical trial regu-
lation and subsequent approval applicable to cell therapy, but specific
ethical and regulatory issues are also present. Therefore, a substantial
amount of efforts are required to support clinical trial proposals on pre-
clinical strong arguments and data. In this context, cell therapy is con-
sidered an advanced therapy (AT) by the European legislation [77],
where cells or tissues are considered ‘engineered’ if they have been
subjected to substantial manipulation and are not intended to be used
for the same essential function or functions in the recipient as in the
donor. Principles applying to advanced therapies include marketing
authorization (pre-market approval), demonstration of quality, safety
and efficacy, and post-authorization vigilance. Manufacturing of these
products requires authorization by the competent authority of the
member state ensuring national traceability and pharmacovigilance re-
quirements as well as specific quality standards. The regulatory require-
ments, currently derived from the field of pharmaceutical medications,
will have to evolve in accordance with the specific characteristics of cell
therapy trials in surgery.
At present, only autologous MSCs are used for bone repair cell thera-
py. Intra-operative BM concentration in the operating room using small
centrifuges and CE-marked kits does not require authorization and is per-
formed under the responsibility of the surgeon. Safety of this procedure
has been confirmed by Hernigou on 1873 patients [78]. For the cultured
MSCs, Tarte et al. [79] found no evidence of deleterious changes or malig-
nant transformation of cultured MSCs used in two national multicentric
immune-hematology trials. However, the immunomodulating effects of
MSCs and their stromal properties (ability to maintain the survival and
growth of associated cells) warrant caution in patients treated for neo-
plastic diseases, most notably bone malignancies.
Preclinical rationale requires solid indices of feasibility and efficacy.
In this field, preclinical studies only orient towards the real feasibility
and efficacy, whose definite proof requires clinical trials. Bone research
scientists scaling their hypothesis to clinical trials on bone cell therapy
should be aware of the underlying strategy that should orient their pre-
clinical research to maximize the probabilities of satisfying regulators
and obtaining the required approvals to launch trials. Understandably,
this strategy will be modified as upcoming evidence may make some
requirements unnecessary, while other new data may recommend dif-
ferent preclinical approaches prior to clinical trials.
In this context, the REBORNE European Union FP7th large inte-
grating project (www.reborne.org) has fostered our consortium to or-
ganize the current preclinical requirements to request approval from
multinational European competent authorities. Both in vitro and animal
studies have been launched to preclinically support the derived clinical
trials. Particularly, a clinical multicentric phase I/IIa trial (EudraCT 2011-
005441-13, NCT01842477) aiming at safety and efficacy of cellular
therapy was started in May 2013 to assess the use of cultured, expanded
autologous BM cells intra-operatively loaded onto biphasic calcium-
phosphate granules as an alternative to autologous cancellous bone
grafting in patients with long bone nonunion or delayed union.
On-going cell therapy clinical trials to treat long bone non unions
The review of international clinical trial databases is the only
updated source of on-going clinical trials. Search can be performed ini-
tially through the WHO International Clinical Trials Registry Platform —
ICTRP [80]. This platform incorporates weekly updates of the European
Clinical Trials Database — EudraCT [81], the ClinicalTrials.gov database
[82], the International Standard Randomised Controlled Trial Number
Table 1
Clinical trials related with long bone fracture and mesenchymal cell therapy, cited in trial registries and completed or recruiting patients, with sufficient information about intervention.

Title Identifier Sponsor Principal Study phase Indication Intervention Status

investigator

BMAC injected
Treatment of non-union of long bone fractures by NCT01206179 Royan Institute, Mohssen Emadedin 1 Long bone nonunion BMAC injected percutaneously Completed 2011
autologous mesenchymal stem cell Tehran, Iran
Percutaneous autologous bone-marrow grafting NCT00512434 University Hospital, Philippe Rosset Open tibial fractures BMAC injected percutaneously Completed 2013
for open tibial shaft fracture (IMOCA) Tours, France
The efficacy of mesenchymal stem cells for NCT01788059 Emdadi Kamyab Mohammad 2 Nonunion femur BMAC injected percutaneously Completed 2013
stimulate the union in treatment of non-united Hospital, Khorasan, Iran Taghi Peivandi and tibia
tibial and femoral fractures

BMAC with bone substitute or DBM

Autologous implantation of mesenchymal stem NCT00250302 Hadassah Medical Meir Liebergall Distal tibia fracture BMAC + carrier Completed 2011
cells for the treatment of distal tibial fractures Organization,
Jerusalem, Israel

E. Gómez-Barrena et al. / Bone 70 (2015) 93–101

Clinical trial based on the use of mononuclear NCT01813188 FFISRM, Murcia, Spain Luis Meseguer 2 randomized Tibial pseudarthrosis BMAC + TCP + DBM Recruiting
cells from autologous bone marrow in patients vs autologous participants
with pseudoarthrosis bone graft
Evaluation the treatment of nonunion of long bone NCT01958502 Emdadi Kamyab Mohammad 2 Nonunion femur BMAC + collagenic 3-D Recruiting
fracture of lower extremities (femur and tibia) Hospital, Khorasan, Iran Taghi Peivandi and tibia scaffold with BMP-2 participants
using mononuclear stem cells from the iliac wing
within a 3-D tissue engineered scaffold
Use of adult bone marrow mononuclear cells in NCT01581892 HUCA, Oviedo, Spain Jesus Otero 1–2 Nonunion BMAC + osteogenic matrix Recruiting
patients with long bone nonunion participants

Expanded MSC alone

Treatment of atrophic nonunion by preosteoblast NCT00916981 University Hospital of Jean Philippe Hauzeur 1–2 Long bone nonunion Expanded MSC injected Completed 2012
cells Liege, Belgium percutaneously
Pivotal phase 2b/3 study on autologous osteoblastic EUCTR2011-005584-24-NL, Bone Therapeutics S.A., Stefan Desmyter 2b-3 randomized Long bone nonunion Expanded MSC injected Recruiting
cells implantation in hypotrophic non-union NCT01756326 Belgium vs autologous percutaneously (PREOB®) participants
fractures bone graft
Phase 1/2a study on allogeneic osteoblastic cells NCT02020590* Bone Therapeutics S.A., Enrico Bastianelli 1–2a Long bone nonunion Allogeneic osteoblastic cells Recruiting
implantation in delayed-union fractures Belgium injected percutaneously participants
(ALLOB®)

Expanded MSC + bone substitute

Feasibility study of Aastrom tissue repair cells to NCT00424567 Aastrom Biosciences, USA Matthew Jiménez 1-2 Long bone nonunion Cultured bone marrow Completed 2007
treat non-union fractures. tissue + bone matrix
Mesenchymal stem cells; donor and role in NCT01626625 Indonesia University Phedy Phe 1 compared Nonunion Expanded MSC + HA Recruiting
management and reconstruction of nonunion to iliac bone participants
fracture autograft
Evaluation of efficacy and safety of autologous MSCs EUCTR2011-005441-13, INSERM, France, Enrique Gómez Barrena 1–2a Long bone nonunion Expanded MSC + HA + TCP Recruiting
combined to biomaterials to enhance bone NCT01842477 (FP7 European participants
healing (OrthoCT1) Project REBORNE)

NCT: ClinicalTrials.gov; EUCTR: EudraCT; BMAC = bone marrow aspirate concentrate; DBM = demineralized bone matrix; HA = HYDROXYAPatite; TCP = tricalcium phosphate; MSC = mesenchymal stem cell.
All mentioned studies are on autologous cells except if indicated by an *.

99
100 E. Gómez-Barrena et al. / Bone 70 (2015) 93–101
Register — ISRCTN, and the Australian New Zealand Clinical Trials Regis-
try, as well as monthly updates of national clinical trial registries.
A particular distinction of European clinical trials on advanced ther-
apies is the large proportion of sponsors from academic and charitable
organizations, as seen in a recent review of 318 trials from 2004 to
2010 on 250 therapies [83]. This aspect is reinforced by the fostering
of investigator-driven clinical trials from institutions and organizations
across Europe [84], spreading the opportunities for more available clin-
ical information about the myriad possibilities that can be considered in
the cell therapy field.
Yet, many declared clinical trials in any of the available international
and national trial registries, both from academic and industrial spon-
sors, do not offer results or just provide initial information about the
research effort, and then the development of the trial and the final out-
comes are difficult to trace. This is equally confirmed in the long bone
nonunion cell therapy trials. To further illustrate the current situation,
the available on-going trials on the topic of this review are summarized
in Table 1.
Excluding trials with unknown status or not yet recruiting, 13 trials
related with long bone fracture or nonunion and mesenchymal cell
therapy were identified as they have been cited in clinical trial registries
as completed (6 of them) or recruiting patients. They may be classified
into four groups to allow for comparative analysis. The first group, with
3 trials all completed, includes the simplest technique, percutaneous
injection of bone marrow aspirate concentrate (BMAC); none of these
trials have been published yet. In the second group (4 trials), BMAC
is associated with bone substitutes or demineralized bone matrix
(DBM); results have been published about one single trial only [85],
observing a shorter time to bone union with cells than in the controls.
In the third group, 3 trials intend to test percutaneous injection of ex-
panded MSCs, but the only completed trial is not yet published. In the
fourth group, 3 trials address the association of expanded MSC and
bone matrix or substitute, but the only completed trial has not been
published yet. Needless to say that follow-up of these and other trials
on the topic will enlighten the future of the field.
A major criticism on the available trials are the underreported re-
sults, which may reflect lack of protocol adherence, patient heterogene-
ity in small unicentric trials, confounding efficacy results in part due to
patient or to protocol variability, or others. Many of these trials do not
offer sufficient information about the cell product to correlate with
the results in other trials and many are also impossible to reproduce
in other centers due to lack of transparency. However, reliability is par-
ticularly challenged by the size and design of the currently available
trials. Unless large, comparative trials with well-defined cell products
are published, evidence on this therapy will remain controversial or
even negative.
Future directions
A strong need of clinical results is required to further progress in cell
therapy. Launched trials will hopefully provide this information in the
near future. If clinical results are positive, far greater challenges may
be raised by the development of more complex tissue engineering tech-
niques, and this may allow the treatment of large bone defects and
unsolved situations [86] after appropriate in vivo models confirm the
specific solution to submit to trials. A multidisciplinary approach will
be required to improve implanted cell survival and to ensure prompt
vessel ingrowth into the biomaterial via careful selection of structure
and shape, together with addition of cytokines and growth factors.
The development of new materials and cell combinations (hydrogel-
based, bioceramic-based, or other) that could eventually craft solutions
for supplying cells and biomaterials percutaneously is expected in the
near future. The immunosuppressive properties of MSCs may allow
the transplantation of allogeneic MSCs in various orthopedic conditions,
with the establishment of cell banks for regenerative medicine. Early
trials evaluating allogeneic MSCs in delayed unions are already under
way. And last but not least, a future step that may help to further define
and spread these therapies is a careful cost–benefit assessment and
a broad economic evaluation to clarify the best indications of bone
repair cell therapy as a standard procedure, if confirmation of safety
and efficacy is clearly derived from current trials.
Conflict of interest disclosure
The authors do not have anything to disclose.
Acknowledgments
This work was supported in part by the European Commission,
Seventh Framework Programme (FP7), through the REBORNE Project,
grant agreement no. 241879.
EGB also acknowledges support from the Spanish General Direc-
torate on Scientific and Technological Research, Ministry of Economy
and Competitiveness (grant no. SAF2012-40149-C02-01).
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