Lentiviral-Mediated Integrin a5 Expression in Human Adult
Mesenchymal Stromal Cells Promotes Bone Repair
in Mouse Cranial and Long-Bone Defects
Samer Srouji,1,2Dror Ben-David,2Olivia Fromigue ´,3,4Pascal Vaudin,5,6,*
Gisela Kuhn,7Ralph Mu ¨ller,7Erella Livne,2and Pierre J. Marie3,4
Adult human mesenchymal stromal cells (hMSCs) are an important source for tissue repair in regenerative
medicine. Notably, targeted gene therapy in hMSCs to promote osteogenic differentiation may help in the
development of novel therapeutic approaches for bone repair. We recently showed that a5 integrin (ITGA5)
promotes osteoblast differentiation in bone marrow–derived hMSCs. Here, we determined whether lentiviral
(LV)-mediated expression of ITGA5 in hMSCs derived from the bone-marrow stroma of healthy individuals may
promote bone repair in vivo in two relevant critical-size bone defects in the mouse. In a first series of experiments,
control or LV-ITGA5-transduced hMSCs were seeded on collagen-based gelatin sponge and transplanted in a
cranial critical-size defect (5mm) in Nude-Foxn1nu mice. Microcomputed tomography and quantitative histo-
logical analyses after 8 weeks showed no or little de novo bone formation in defects implanted with collagen
sponge alone or with hMSCs, respectively. In contrast, implantation of collagen sponge with LV-ITGA5-
transduced hMSCs showed greater bone formation compared with control hMSCs. We also tested the bone-repair
potential of LV-mediated ITGA5 expression in hMSCs in a critical-size long-bone defect (2mm) in femur in Nude-
Foxn1nu mice. Bone remnants were stabilized with external fixation, and control or LV-ITGA5-transduced
hMSCs mixed with coral/hydroxyapatite particles were transplanted into the critical-size long-bone defect.
Histological analysis after 8 weeks showed that LV-ITGA5-transduced hMSCs implanted with particles induced
85% bone regeneration and repair. These results demonstrate that repair of critical-size mouse cranial and long-
bone defects can be induced using LV-mediated ITGA5 gene expression in hMSCs, which provides a novel gene
therapy for bone regeneration.
multiple lineages, including chondroblasts, osteoblasts, and
adipocytes (Bianco and Gehron Robey, 2000; Kassem, 2004;
Oreffo et al., 2005). In recent years, there has been a growing
interest in using bone marrow–derived MSCs, in particular
human MSCs (hMSCs), for potential therapeutic applications
in bone regeneration and repair (Prockop et al., 2003; Prockop,
(MSCs) are adherent cells that can differentiate into
2009; Augello and De Bari, 2010; Charbord, 2010). Although
adult hMSCs are considered a valuable source for bone-tissue
regeneration in human diseases, the capacity of autologous
hMSCs to differentiate into functional bone-forming osteo-
blasts remains relatively limited for bone regeneration in vivo
(Petite et al., 2000). An important issue for efficient bone re-
generation is therefore to develop targeted gene therapies
for specifically promoting hMSC osteoblast differentiation
and osteogenic potential for optimal bone repair (Gazit
et al., 1999; Franceschi, 2005; Heyde et al., 2007; Evans, 2011).
1Oral and Maxillofacial Surgery Department, Carmel Medical Center, 32000 Haifa, Israel.
2Department of Anatomy and Cell Biology, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, 32000 Haifa, Israel.
3Laboratory of Osteoblast Biology and Pathology, Inserm U606, Paris, F-75475 France.
4UMR 606, University Paris Diderot, Paris, F-75475 France.
5Inserm U966, Paris, F-75475 France.
6University of Tours, Tours, F-37032 France.
7Institute for Biomechanics, ETH Zurich, 8093 Zurich, Switzerland.
*Present address: UMR 6175, INRA, Nouzilly, 37380 France.
HUMAN GENE THERAPY 23:167–172 (February 2012)
ª Mary Ann Liebert, Inc.
Several molecular mechanisms are now known to trigger the
osteogenic differentiation of MSCs (Marie and Fromigue,
2006; Bianco et al., 2010). However, a limited number of gene-
delivery strategies were shown to promote osteogenic dif-
ferentiation in hMSCs. One widely used strategy consists of
hMSC engineered with bone morphogenetic proteins to pro-
mote bone formation (Turgeman et al., 2001; Peterson et al.,
2005; Aslan et al., 2006) with, however, variable reported ef-
fects on bone repair (Vilquin and Rosset, 2006). A distinct
strategy is to target genes that are more specifically involved
in osteogenic differentiation, such as the transcription factor
Runx2 (Lian and Stein, 2003). Indeed, Runx2 gene delivery in
MSC was found to be efficient in promoting bone regenera-
tion (Hou et al., 1999). A third possibility is to target hMSCs
using genes that can promote Runx2 expression or activity. In
this context, we recently showed that a5 integrin (ITGA5)
promotes hMSC osteogenic differentiation through activation
of signaling pathways, resulting in increased Runx2 expres-
sion and activity (Hamidouche et al., 2009; 2010). This
knowledge led us to postulate that overexpression of ITGA5
using gene delivery in hMSCs may promote the osteogenic
differentiation potential of hMSCs and bone regeneration
in vivo. We therefore investigated the capacity of lentiviral
(LV)-mediated ITGA5 overexpression in hMSCs to promote
de novo bone formation and bone repair in two relevant criti-
cal-size bone defects in the mouse.
Materials and Methods
Lentivirus production and transduction
The ITGA5 coding sequence (Giancotti and Ruoslahti, 1990)
was amplified by PCR from pcDNA3 by using the following
primers: ITGA5-ATG, 5’ CAGGGAAGAGCGGGCGCTAT
GG; and ITGA5-STOP, 5’ GGGAGTCTGAAATTGGGAG
GACTCAGG. The amplified ITGA5 coding sequence was
cloned into the pCR8/GW/TOPO TA plasmid (Invitrogen,
Carlsbad, CA) and transferred into pLentiGW vector bearing
the cytomegalovirus (CMV) promoter (Invitrogen) by in vitro
recombination. Viral production was performed using human
embryonic kidney cells HEK293T grown in Dulbecco’s mod-
ified Eagle’s medium supplemented with 10% fetal bovine
serum, 1% L-glutamine, penicillin/streptomycin (10,000U/ml
and 10,000lg/ml, respectively), and 2mM HEPES buffer
(Hamidouche et al., 2008). The day before transfection, 2·106
cells were seeded on a 175-cm2flask. LV transfer vector
(50lg), vesicular stomatitis virus glycoprotein (VSV-G) viral
envelope plasmid (pCMV-G; 10lg), and packaging construct
(pCMV DR8.2; 50lg) were mixed with water up to 800ll and
200ll of 2 M CaCl2and then added to 1ml of HEPES-buffered
saline solution 2·(5mM NaCl, 1mM KCl, 150mM Na2HPO4,
0.5mM HEPES, pH 7) and incubated for 20min at room
temperature. This DNA solution was then added dropwise
onto HEK293T cells with medium, swirled gently, and then
incubated overnight at 37?C. The following day, the trans-
fection solution was removed, and the cells were rinsed with
serum-free medium before addition of 15ml of complete me-
dium. After 24 and 48hr of incubation, the supernatants were
collected, centrifuged at 1,200rpm to remove cell debris, fil-
tered through a 0.45-lm low-protein binding filter (Corning,
Bath, UK), aliquoted, and stored at -80?C. hMSCs were de-
rived from the bone-marrow stroma of healthy individuals
after informed consent was obtained (Delorme and Charbord,
2007). For transduction, subconfluent hMSCs were incubated
with LV particles and 4lg/ml Polybrene (Sigma, St. Louis,
MO) for 48hr in complete medium. After 48hr, transduction
medium was discarded and cells were ready for experiments.
Previous data showed that all cells were transduced using this
method (Hamidouche et al., 2009), and that the expression of
the transgene was found to be optimal for increasing in vitro
osteogenesis with a two- to threefold increase in ITGA5 ex-
pression level (Hamidouche et al., 2009).
Cranial critical-size defect
Athymic Nude-Foxn1nu mice, 8 weeks old and weighing
20–25g (Harlan Laboratories, Jerusalem, Israel), were used
for the cranial critical-size defect model. All procedures in-
volving the use of animals were conducted in accordance
with the guidelines of the Institutional Animal Care and
Use Committee of the Technion, Israel. Two bilateral full-
thickness circular defects (5-mm diameter) were created with
a hand drill and trephine bit in the parietal bones of the skull
on both sides of the sagittal suture line. Care was taken not to
damage the sagittal suture or the dura mater beneath the
bone. Control or LV-ITGA5-transduced hMSCs were seeded
on collagen-based gelatin sponge (Levy Dental Co., Tel Aviv,
Israel) cut in the shape of a disc (5·1mm) and transplanted
in the defects. Defects with collagen sponge only served as a
negative control. Five replicates were made for each group
(five mice per group). After 8 weeks, mice were euthanized
and the harvested skulls underwent microcomputed to-
mography (lCT) scanning and histological analyses.
Long-bone critical-size defect
To confirm the bone-repair potential of ITGA5-expressing
hMSCs, the cells were transplanted in a mouse long-bone
critical-size defect model as previously described (Srouji
et al., 2011). In brief, athymic Nude-Foxn1nu mice, 8 weeks
old and weighing 25g (Harlan Laboratories), were used for
the experiments. Mice were anesthetized with a 0.5-ml in-
traperitoneal injection of xylazine and ketamine (1:1). Under
aseptic conditions, a longitudinal incision was made over
the lateral aspect of the thigh, and muscles were split at the
fascia lata to expose the femur proximally and distally to
the medial aspect of the femoral condyle. Four holes, two in
the distal region and two in the proximal region, were drilled
in the midshaft of the femurs. Rods were inserted manually
into the drilled holes by penetration through one lateral
cortex to the opposite cortex. The protruding ends of the rods
on the lateral and medial sides were then connected with
acrylic dental paste (Unifast Trad; GC America Inc., Alsip,
IL). Under saline irrigation, a critical-size bone defect of
2mm between the rods was created in the cylindrical mid-
shaft part of the femur using a motorized minidrill. The
muscles were opposed over the bone defect, and the wound
closed with vicryl sutures (Srouji et al., 2011). Control or LV-
ITGA5-transduced hMSCs mixed with coral/hydroxyapatite
particles were transplanted into the critical-size defect for 8
weeks. Defects with coral/hydroxyapatite particles only
served as a negative control. Five replicates were performed
for each group. At the end of the experiment, mice (five per
group) were euthanized, and the harvested legs were taken
for histological analysis for accurate investigation of bone
168SROUJI ET AL.
Upon termination of the experiment, animals were eu-
thanized, and transplanted skulls were fixed in 10% neutral
buffered formalin for lCT analysis on a 40 Imaging System 6
(Scanco Medical AG, Bassersdorf, Switzerland) operated at
an energy of 55 kVp and an intensity of 145lA with an
acquisition time of 200ms and no frame averaging. Scans
were performed in high-resolution mode resulting in a
nominal isotropic resolution of 30lm. A constrained
Gaussian filter (sigma 1.2, support 1) was used to partly
suppress the noise in the images. Mineralized bone tissue
was segmented from nonmineralized tissue using a global
thresholding procedure (21% of the maximum gray value)
(Ruegsegger et al., 1996). The defect region was then identi-
fied by a cylindrical contour, and the bone volume was cal-
culated within this fixed volume of interest (Mu ¨ller and van
Lenthe, 2006). Furthermore, we calculated the bone coverage
from a projection of the superior part of the skull in the
superior–inferior direction to create a high-resolution pseu-
doradiograph (Lutolf et al., 2003).
Histological and immunohistochemical analyses
Following lCT analysis, bone specimens were decalcified
using 10% ethylenediaminetetraacetic acid, embedded in
paraffin wax, and 6-lm-thick serial sections were stained
with hematoxylin and eosin (H&E) for general histology. The
orientation and alignment of the bones were carefully taken
into consideration during paraffin embedding in order to
clearly view the defect. Longitudinal serial sections of the
femur and anteroposterior sections of the calvaria were
stained with Masson’s Trichrome for general histology and
histomorphometry. The area of newly formed bone in the
defect was measured on H&E-stained sections by using the
ImagePro 6 image analysis software (Media Cybernetics,
Silver Spring, MD); the results are expressed as a percentage
of the area of the newly formed bone in the area of the
original critical-size defect. The results were determined
from five H&E-stained sections of each mouse (five mice per
group). Unstained paraffin sections were used for immuno-
histochemical localization of human osteocalcin using a
specific antibody (R&D Systems Inc., Minneapolis, MN) and
a mouse osteocalcin antibody (Abcam, Cambridge, UK).
The results are expressed as means–SEM. Comparisons
between data were performed using one-way ANOVA with
Bonferroni’s multiple comparison test, with p<0.05 consid-
ered as significant.
Results and Discussion
The development of efficient gene therapy that can induce
osteogenic differentiation of hMSCs is of major importance
for optimally promoting bone regeneration and repair. In
this study, we demonstrate that bone formation and repair
can be promoted in vivo by LV-mediated overexpression of
ITGA5 in hMSCs. In this context, cells were transduced with
a proviral copy number of the LV-ITGA5 transgene, giving
rise to a two- to threefold increase in the level of expression
of ITGA5 mRNA and protein (Hamidouche et al., 2009). The
beneficial effect of overexpressing ITGA5 using lentivirus in
hMSCs was first demonstrated by the improved bone for-
mation in a critical-size mouse cranial defect. In this model,
lCT analysis showed minimal de novo bone formation and
thereby no healing in control defects in the absence of hMSC
implantation after 8 weeks. We determined the effect re-
sulting from the use of LV-transduced hMSCs to overexpress
ITGA5 in bone regeneration at this time point, because ear-
lier time points may not give sufficient information on the
optimal capacity of LV-ITGA5-transduced hMSCs to fully
regenerate bone. Although some de novo bone formation was
observed after hMSC implantation, bone coverage in the
zone of interest was only *38% above controls in the empty
defect (Figs. 1 and 2). Remarkably, quantification of the de
novo bone formed showed that the implantation of eight LV-
ITGA5-transduced hMSCs showed a significantly greater
bone formation compared with control hMSCs. In this con-
dition, bone coverage was increased by *55% compared
with control defects (Fig. 1). Histological analysis confirmed
that the group with hMSCs had less and thinner newly
formed bone and less coverage of the defect compared with
the large amount of bone formed in the MSCs+vector group.
bone regeneration in a critical-size cranial defect in mice. lCT
analysis of critical-size cranial bone defect shows empty
defects (stars) in control mice receiving collagen-based gel-
atin sponge alone (A) and bone regeneration (B) after im-
plantation of hMSCs transduced with LV-ITGA5 compared
with control hMSCs. Quantification of the newly formed
bone in the critical-size defect (C) was performed in five mice
per group; the results are expressed as means–SEM
(*p<0.05 vs. collagen sponge and hMSCs, or vs. collagen
LV-mediated ITGA5 gene expression promotes
ITGA5 GENE THERAPY PROMOTES BONE REPAIR169
In the latter group, the bone defect was almost completely
filled with bone similar to normal calvarial bone (Fig. 2).
Quantitative analysis confirmed that bone volume/tissue
volume was increased significantly by 30% with hMSCs
alone and significantly more with hMSCs transduced with
LV-ITGA5 (47%, p<0.05), confirming the beneficial effect of
LV-ITGA5-transduced hMSCs on de novo bone formation.
We then performed an immunohistochemical analysis
using a specific anti-mouse or anti-human antibody against
osteocalcin, a late osteoblast marker, to differentiate between
human and mouse cells that participated in bone repair.
Immunohistochemical analysis using anti-mouse osteocalcin
antibody revealed no staining in the empty defect (Fig. 2D),
as expected, and a positive staining in the defect implanted
with hMSCs (Fig. 2F), as expected from the presence of
differentiated mouse osteoblasts in this area. Immuno-
histochemical analysis using anti-human osteocalcin showed
no staining in the empty defect (Fig. 2D), again as expected,
and a slight positive staining in the defect implanted with
hMSCs (Fig. 2E). After lentiviral transduction of ITGA5 in
hMSCs, osteocalcin staining was increased in implanted
human cells (Fig. 2G vs. E), whereas osteocalcin staining
remained unchanged in murine cells (Fig. 2H vs. F), indi-
cating that ITGA5 overexpression in hMSCs increased oste-
oblast differentiation and thereby bone formation through
activation of implanted human cells.
To confirm the interest of LV-ITGA5-transduced hMSCs
in bone repair, we investigated whether LV-mediated ex-
pression of ITGA5 may be effective in promoting osteogen-
esis in a nonunion long-bone model in vivo. To this goal, we
tested the effect of LV-mediated ITGA5 overexpression in
hMSCs implanted in a long-bone critical-size defect in mice
(Srouji et al., 2011). In this model, the implantation of coral/
hydroxyapatite particles alone induced little de novo bone
formation and bone repair (*24%), as shown by histo-
logical analysis after 8 weeks of implantation. Implantation
chemical analyses of bone regeneration
induced by LV-mediated ITGA5 gene ex-
pression in a critical-size cranial defect in
mice. Histological analysis of critical-size
cranial bone defect shows empty defects
in control mice receiving collagen-based
gelatin sponge alone (A) and marked
bone repair (C) after implantation of
hMSCs transduced with LV-ITGA5 com-
munohistochemical analysis of osteocalcin
using human and murine anti-osteocalcin
staining in empty defects in control
mice (D) and an increased number of
osteocalcin-positive human cells (G vs. E),
but not osteocalcin-positive murine cells
(H vs. F) in defect implanted with hMSCs
transduced with LV-ITGA5 (G, H) com-
pared with defect implanted with control
hMSCs (E, F).
Histological and immunohisto-
170SROUJI ET AL.
of hMSCs with coral/hydroxyapatite particles showed
moderate repair (*59%). In contrast, the implantation of
LV-ITGA5-transduced hMSCs with coral/hydroxyapatite
particles greatly (*85%) increased bone formation and bone
repair in this model (Fig. 3). In both calvaria and long-bone
models, implanted and not endogenous MSCs participated
in the bone regeneration, because in the absence of hMSCs,
bone regeneration was absent or limited (Figs. 1A, 2A, and
3A). Overall, the results indicate that bone regeneration in
both critical-size mouse cranial and long-bone defects are
improved by ITGA5 overexpression using lentivirus in
The important beneficial effects on bone formation ob-
served in vivo with LV-ITGA5-transduction likely resulted
from multiple cellular actions of ITGA5 at different steps of
bone repair. Both bone regeneration and repair require the
recruitment of osteoprogenitor cells and their subsequent
differentiation into bone-forming cells (Einhorn, 1998). Our
previous findings indicate that ITGA5 activates osteoblast
differentiation in adult hMSCs through activation of focal
adhesion kinase and ERK1/2-MAPKs, resulting in Runx2
phosphorylation and ectopic bone formation (Hamidouche
et al., 2009). We also found that hMSC osteogenic differen-
tiation can be induced through interactions between ITGA5
and growth factors (Hamidouche et al., 2010). Additionally,
there is in vitro and in vivo evidence from our laboratory that
ITGA5 acts as an antiapoptotic molecule in osteoblasts via
activation of phosphatidylinositol 3-kinase signaling (Kaa-
beche et al., 2005; Dufour et al., 2007). The positive effect of
LV-mediated overexpression of ITGA5 on cranial and long-
bone repair shown here may thus result from activation of
early stages of osteoblast differentiation in hMSCs and in-
creased survival of mature osteoblasts, resulting in an in-
creased number of osteocalcin-positive bone-forming cells, as
observed in this study.
In conclusion, the clear beneficial effects of targeted LV-
mediated gene therapy in the two distinct mouse models
reported here support the concept that ITGA5 over-
expression in hMSCs may be a valid therapeutic strategy to
promote osteogenic differentiation and bone repair in vivo.
These results suggest the potential for efficiently improving
bone formation in unhealed cranial and nonunion long-bone
defects in humans. This knowledge may serve as a basis for
designing innovative gene therapy to increase the osteogenic
capacity of autologous human bone marrow–derived MSCs
in individuals with severe bone loss caused by trauma or
The authors thank Dr. S. Kuwada (University of Utah, Salt
Lake City, UT) for the gift of the ITGA5 plasmid. This work
was supported in part by the Fondation de l’Avenir pour la
Recherche Applique ´e, Paris, France (no. ET9-521). The use of
ITGA5 agonists for applications in tissue regeneration is
covered by European patent no. 08290752.8 and U.S. patent
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Dr. Pierre J. Marie
2 rue Ambroise Pare
75475 Paris cedex 10
Received for publication April 11, 2011;
accepted after revision September 23, 2011.
Published online: September 29, 2011.
172SROUJI ET AL.