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Journal of Plastic, Reconstructive & Aesthetic Surgery (2009) xx, 1e9

Adipose-derived stem cells enhance peripheral

nerve regeneration
P.G. di Summa a,b, P.J. Kingham b,c, W. Raffoul a, M. Wiberg c,
G. Terenghi b, D.F. Kalbermatten a,c,*

a
Chirurgie Plastique et Reconstructive CHUV, Université de Lausanne, Rue de Bugnon 46, 1005 Lausanne, CH, Switzerland
b
Blond McIndoe Research Laboratories. The University of Manchester, Manchester, UK
c
Departments of Surgical and Perioperative Science (Hand surgery) and Integrative Medical Biology (Anatomy), Umea
University, Umea, Sweden

Received 30 March 2009; accepted 10 September 2009

KEYWORDS Summary Traumatic injuries resulting in peripheral nerve lesions often require a graft to
Adipose-derived bridge the gap. Although autologous nerve auto-graft is still the first-choice strategy in recon-
stem cells; structions, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplan-
Peripheral nerve tation in a bioartificial conduit is an alternative strategy to create a favourable environment
regeneration; for nerve regeneration. We decided to test new fibrin nerve conduits seeded with various cell
Differentiated cells; types (primary Schwann cells and adult stem cells differentiated to a Schwann cell-like pheno-
Sciatic nerve type) for repair of sciatic nerve injury.
Two weeks after implantation, the conduits were removed and examined by immunohisto-
chemistry for axonal regeneration (evaluated by PGP 9.5 expression) and Schwann cell pres-
ence (detected by S100 expression). The results show a significant increase in axonal
regeneration in the group of fibrin seeded with Schwann cells compared with the empty fibrin
conduit. Differentiated adipose-derived stem cells also enhanced regeneration distance in
a similar manner to differentiated bone marrow mesenchymal stem cells. These observations
suggest that adipose-derived stem cells may provide an effective cell population, without the
limitations of the donor-site morbidity associated with isolation of Schwann cells, and could be
a clinically translatable route towards new methods to enhance peripheral nerve repair.
ª 2009 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by
Elsevier Ltd. All rights reserved.

Numerous surgical procedures are performed each year for
peripheral nerve repair, with a significant amount of lost
* Corresponding author. Tel.: þ41 21 314 25 06; Fax: þ41 21 314
25 31.
E-mail address: daniel.kalbermatten@bluewin.ch (D.F.
Kalbermatten).
working days with corresponding economic consequences.1
Nerve lesions without defect or with a short gap are usually
treated by end-to-end coaptation. Traumatic injuries
resulting in longer peripheral nerve lesions often require
a graft to bridge the gap. Unfortunately, full recovery may
never be achieved, particularly with extended lesions.2
Although autologous nerve autograft is still the first-choice

1748-6815/$ - see front matter ª 2009 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bjps.2009.09.012

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2 P.G. di Summa et al.
strategy in reconstructions, they have several disadvan-
tages caused by the limited availability of donor tissue,
sacrifice of functional nerve and potential formation of
neuroma.2e4
Nerve autografts have been mimicked by venous and
arterial conduit grafts, which show no functional benefits
compared with standard nerve grafts.5 Allografts have also
been tested, but they require an undesirable long-term
immunosuppressive therapy.6 Many attempts have been
made to develop conduits made of biodegradable mate-
rials, which could guide the regenerating axons without
inhibiting the process of growth and maturation.7 Recently,
we described a bioresorbable nerve constructed from fibrin
glue,8 which shows good tissue acceptance and a porous
structure that allows neurotrophic growth factors to
penetrate into the lumen of the conduit. Moreover, the
resorption of the conduit within 4 weeks avoids compres-
sion syndromes and enhances biocompatibility.9
Our aim was to optimise the fibrin conduit, previously
described, by seeding it with regenerative cells. Cell
transplantation in a bioartificial conduit is an alternative
strategy to create a favourable environment for nerve
regeneration10: in this study, we compared different cell
populations such as primary Schwann cells (SCs) and stem
cells differentiated towards to a Schwann cell phenotype.
SCs offer a highly preferred substrate for axon migration
and release of bioactive factors that further enhance
migration. They synthesise adhesion molecules (CAMs),
release growth factors (GFs) and build up basement
membranes for the support and guidance of the sprouting
axons.11,12 Despite showing regeneration enhancement,13
SCs have limited clinical applications. The culture of
a sufficient amount of SC to achieve optimal conditions for
transplantation in nerve conduits is time consuming and
requires particular care for in vitro expansion and
a constant input of GFs.14 Moreover, SCs are not easily
accessible without nerve biopsy and bear the need to
sacrifice an autologous nerve, with the related complica-
tions of anaesthesia and pain at the harvest site.4,15
Instead, the ideal transplantable cell should be easily
accessible, should proliferate rapidly in culture and should
successfully integrate into the host tissue with immunological
tolerance.16,17 The growing applicability of stem cells has
opened new frontiers to nerve regeneration: the advantage
of using these cell lines is that cell differentiation may be
stimulated by advancing axons and pluripotency, allows
multiple differentiation paths, which can create a positive
environment for axonal regeneration.1
Bone-marrow-derived mesenchymal stem cells (MSCs)
can be induced to differentiate into non-mesenchymal fates
in vitro, such as Schwann cells, improving myelin formation
and nerve regeneration in vivo after their transplantation
into different models of peripheral nerve injury.18e20
Adipose tissue is a mesodermally derived complex tissue
that, besides adipocytes and pre-adipocytes, contains
a stromal population, which includes microvascular endo-
thelial cells, smooth muscle cells, resident monocytes,
lymphocytes and stem cells.21,22 These non-adipocyte cells
are known as stromal vascular fraction (SVF) and can be
isolated by centrifugation of collagenase-digested adipose
tissue.23 In the past few years, it has been shown how
cultured SVFs give rise to multipotent precursor cell from
Please cite this article in press as: di Summa PG et al., Adipose-derived stem cells enhance peripheral nerve regeneration, J Plast
Reconstr Aesthet Surg (2009), doi:10.1016/j.bjps.2009.09.012
adipose tissue stromal cells.24e26 Although literature
terminology can be confusing,27,28 we will use the generic
nomenclature of adipose-derived stem cells (ADSCs) to
describe these cells.
As previously cited, stem and progenitor cells from adult
stromal tissue retain the capacity to differentiate towards
mesenchymal and non-mesenchymal lineages, similar to bone
marrow stromal cells,24,29 showing pluripotency above mul-
tipotency.30 Regarding neural differentiation, several studies
proved trans-differentiation of ADSC.23,24,31e33 This suggests
that adult stem cells may not be as limited by lineage as
previously thought and, in fact, manifest profound plasticity.
Recently, we showed how Schwann cell-like differenti-
ated ADSC could enhance neurite outgrowth in vitro.4 In
this current study, we have tested the ability of the
differentiated ADSC to enhance nerve regeneration in vivo.
We compared these cells with SC-differentiated MSCs and
primary adult SCs seeded in fibrin conduits.
Materials and Methods
Fibrin conduit
The fibrin conduit was prepared from two-compound fibrin
glue (Tisseel Kit VH 1.0, Baxter SA, USA). Tisseel
contains fibrinogen, 70e110 mg ml1; plasma fibronectin,
2e9 mg ml1; factor XIII, 10e50 U ml1; plasminogen,
40e120 mg ml1; aprotinin solution 3000 KIU ml1; thrombin
4 IU ml1; and calcium chloride 40 mmol l1. Fibrin glue was
dispensed in a mould prepared from silicone around
a stainless steel core and pressed into shape for 5 min
(Figure 1). This allows the generation of uniform conduits
measuring 14 mm in length, with a 2-mm lumen and 1-mm
wall thickness designed to bridge a 10-mm gap in the left
sciatic nerve of 250 g adult Sprague-Dawley rats (Janvier,
France).
Cell harvest
Schwann cells
All cells were obtained from Sprague-Dawley rats (Janvier,
France). These were euthanised by CO2 according to the
local veterinary commission in Lausanne (Switzerland) and
study was carried out in accordance with the European
Community Council directive 86/609/ECC for the care and
use of laboratory animals.
To harvest Schwann cells, sciatic nerves were exposed,
removed and kept in Dulbecco’s modified Eagle’s medium
plus glutamax (DMEM, Invitrogen, UK) containing 1% pen-
icillinestreptomycin. Nerves were then dissected in trunks,
de-sheathed and finally chopped in 1-mm segments. The
segments were then plated in a Petri dish with SC growth
medium supplemented by 14 mM forskolin and 100 ng ml1
neuregulin-1 b1 (R&D Systems, UK). The cells were incu-
bated for 2 weeks at 37  C with 5% CO2 with fresh medium
added approximately every 72 h. After these 2 weeks,
medium was aspirated and 0.125% (w/v) collagenase type
IV and 117 U mg1 dispase were added to the Petri dish.
After 24-h incubation, the cell suspension was filtered
through a 70-mm cell strainer (Falcon; BD Biosciences
Discovery Labware, Bedford, MA), centrifuged at 900 rpm
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Adipose-derived stem cells enhance peripheral nerve regeneration 3

Figure 1 Preparation of fibrin conduits.A. The fibrin conduit (Tisseel) with stainless steel wire inserted in the lumen as a spacer
while the conduit is stored before implantation.B. The fibrin conduit shows an intact lumen after removal of the stainless steel
wire.C. The fibrin conduits are stored in DMEM medium before being seeded with regenerative cells.D. A fibrin conduit implanted
in vivo to repair a rat sciatic nerve injury.

for 5 min to obtain the cell pellet. Finally, the cell pellet
was re-suspended in SC growth medium, seeded into
25-cm2 flask precoated with poly-D-lysine (Sigma, St Louis,
MO, USA) and incubated in the same conditions. The
following day, the medium was freshly changed and the
cells were left to proliferate. At confluence, the SCs were
purified by an antibody complement method to eradicate
the remaining fibroblasts.13,17,34
a 21-guage needle. The resulting cell suspension was tritu-
rated, filtered through a 70-mm Falcon filter and centrifuged
for 5 min at 600 g. The supernatant was aspirated, the cell
bolus re-suspended in mesenchymal cell growth medium and
the cells plated in 75-cm2 tissue culture flasks and incubated
in 5% CO2 at 37  C. Haematopoietic cells were eliminated by
washing daily with DMEM until all the non-adherent cells were
removed. The cells were then allowed to grow to confluence.

Adipose-derived stem cells Stem cell differentiation

Adipose-derived stem cells were harvested as described
previously.4 After euthanasia, the inguinal fat pad was care-
fully dissected and minced using a sterile razor blade. The
tissue was enzymatically digested for 2 h at 37  C using 0.15%
(w/v) collagenase type I (Invitrogen, UK). The solution was
passed through a 70-mm filter to remove undissociated tissue,
neutralised by adding modified Eagle’s medium (a-MEM;
Invitrogen, UK) containing 10% (v/v) foetal bovine serum
(FBS) and centrifuged at 800  g for 5 min. The stromal cell
pellet was re-suspended in MEM containing 10% (v/v) FBS and
1% (v/v) penicillin/streptomycin solution. The cultures were
maintained at sub-confluent levels in a 37  C incubator with
5% CO2 and passaged with trypsin/ethylenediaminetetra-
acetic acid (EDTA) (Invitrogen, UK) when required.
Bone-marrow-derived mesenchymal stem cells
MSCs were harvested from adult Sprague-Dawley rat femoral
bones34: mesenchymal cell growth medium (MEM- a; Invi-
trogen; supplemented with 10% FBS (v/v) and 1% penicilline
streptomycin) was injected through each marrow cavity using
The multi-potent nature of the cell cultures was tested by
ensuring their ability to differentiate along various lineages,
as previously described.4,34 Further, undifferentiated ADSCs
and MSCs were differentiated into an SC-like phenotype at
early passages.4,18 Growth medium was removed from sub-
confluent cultures and replaced with medium supplemented
with 1 mM b-mercaptoethanol (Sigma-Aldrich, UK) for 24 h.
The cells were then washed and fresh medium supplemented
with 35 ng ml1 all-trans-retinoic acid was added. A further
72 h later, the cells were washed and the medium replaced
with differentiation medium; cell growth medium supple-
mented with 5 ng mle1 platelet-derived growth factor (PDGF;
PeproTech Ltd., UK), 10 ng ml1 basic fibroblast growth factor
(bFGF; PeproTech Ltd., UK), 14 mM forskolin and 200 ng ml1
neuregulin-1 b1. The cells were incubated for 2 weeks under
these conditions with fresh medium added approximately
every 72 h. To confirm the effectiveness of the differentiation
of ADSC and their similarity to the Schwann cell population,
we performed cell immunostaining for typical Schwann cells

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Reconstr Aesthet Surg (2009), doi:10.1016/j.bjps.2009.09.012
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4 P.G. di Summa et al.
markers S100 and GFAP.4 Cultures of differentiated ADSC
(dADSC) and SC were compared by immunostaining: the cells
were incubated with mouse anti-glial fibrillary acidic protein
(GFAP; 1:500; LabVision, USA) and rabbit anti-S100 (1:500;
Dako, Denmark) overnight at 4  C. Phosphate-buffered solu-
tion (PBS)-washed slides were then treated with goat anti-
rabbit FITC (1:100; Vector Labs, UK) and goat anti-mouse CY3
(1:200; Amersham, UK) conjugated secondary antibodies.
Slides were examined using an Olympus IX51 inverted fluo-
rescence microscope. The dADSCs showed similar immuno-
cytochemical properties when compared with SCs (Figure 2).
Bone-marrow-derived MSCs were differentiated (dMSC) in
a similar manner as previously shown.34
Cell seeding and microsurgery
A couple of hours before implantations in rats, the cells
were trypsinised and, after centrifugation, 2  106 cells
were suspended in 50 ml of differentiation medium and
injected into the fibrin tubes. To avoid cell dispersion during
the procedure, one side of the fibrin tube was previously
capped by a fibrin glue clot. Before implantation, the fibrin
conduit was easily decapped from the fibrin clots at its ends.
The control group in the absence of cells (empty) was
similarly filled with 50 ml of differentiation medium alone.
The sciatic nerve was transected and nerve ends were fixed
to the conduit by a single epineural suture (9/0 Prolene,
Ethicon): proximal and distal nerve stumps were inserted
2 mm into the tube, thus leaving a 10-mm gap. Muscles and
fascia layers were closed with single resorbable stitches (4/
0 Softcat, Braun) and the skin by a continuous running
suture (4/0 Prolene, Ethicon). The various groups of fibrin
conduits (each one formed by six animals for a total n Z 24)
were left in place for 2 weeks and subsequently harvested
together with the proximal and distal nerve stumps.
Nerve conduit harvesting and
immunohistochemistry
Animals were sacrificed after 2 weeks by CO2 chamber
euthanasia. Harvested conduits were fixed by para-
formaldehyde (PFA) for 16 h, then rinsed 24 h at 4  C in PBS
containing 30% sucrose and 0.1% sodium azide, with PBS
changes after about 12 h to eliminate any PFA remnants.
The specimens were then embedded in OCT freezing media
(Tissue-tek, Sakura, Japan), and longitudinal cryo-sections
(14 mm) were prepared onto slides (Superfrost plus,
Menzel-Gläser, Germany).
After 3  5 min PBS washes, a casein blocking solution
was applied for 1 h before incubating slides overnight with
primary antibodies rabbit anti-S100 (1:500, Dako, UK) and
rabbit anti-PGP 9.5 (1:500, Dako, UK). The following day
after 3  5 min PBS washes, slides were incubated with
secondary antibody (1:500, Alexa Fluor goat anti-rabbit,
Invitrogen, USA) at room temperature in the dark for
40 min. After 3  5 min PBS washes, the slides were finally
mounted with Vectashield with DAPI (Vector Labs, UK) and
examined under the fluorescence microscope
(10  magnification) with the regenerating conus directed
towards the distal end.35 Axonal regeneration distance
(PGP 9.5) and S100 positive cell distribution inside the
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Reconstr Aesthet Surg (2009), doi:10.1016/j.bjps.2009.09.012
conduit were evaluated using an optical microgrid: the
length was measured from the beginning of the conduit to
the last visible sprout of the regenerating conus.
Statistical analysis
One-way analysis of variance (ANOVA) with Bonferroni
multiple comparison test was used to statistically analyse
data (GraphPad Prism). Significance was determined as
*P < 0.05, **P < 0.01 and ***P < 0.001.
Results
Cell seeding and conduit properties
Prior to in vivo experiments, we tested the fibrin conduit and
its capacity to retain cells within. We filled two different
fibrin conduits with ADSC (80  106 ml1 cell concentration),
then we fixed them at 48 h. By DAPI staining, we found a good
capacity of fibrin of retaining cells inside the lumen
(Figure 3). Fibrin conduits were easy to handle and micro-
surgical suture was uneventful. All animals survived; none of
the animals developed infections or surgical complications
except autotomy, which was present in one case out of 24.
After 2 weeks in vivo, fibrin conduits showed an intact
structure, with proximal and distal ends partially resorbed.
No signs of haematoma or infection were detected.
Axon regeneration
Sciatic nerve axonal regeneration was different depending on
the type of cells which had been seeded in the fibrin conduits.
Axonal regeneration distance was assessed proximally by PGP
9.5 immunohistochemistry (Figure 4). Conduits filled with SCs
showed the best regeneration distance (5.758  0.12 mm)
followed by fibrin containing dMSC (4.986  0.19 mm) or
dADSC (4.968  0.22 mm) (Figure 6A). All three groups with
cells resulted in better regeneration than the empty fibrin
conduit group (4.155  0.07 mm). PGP 9.5 positive axons were
associated with S100 positive cells at the proximal regener-
ating stumps (Figure 5). The distances of S100 positive cell
migration at the stumps showed a similar pattern to PGP 9.5:
the SC group (5.858  0.13 mm) followed by conduits con-
taining dMSC (5.035  0.21 mm) or dADSC (5.032  0.20 mm)
and empty fibrin conduits (4.108  0.08 mm) (Figure. 6B).
Statistical analysis showed that fibrin conduits seeded
with SC significantly improved axonal regeneration
distance, and proximal SC invasion compared with the
empty fibrin conduit (** p < 0.01). Differentiated adult stem
cells also showed a positive impact on nerve regeneration
with a significantly enhanced nerve regeneration distance
(dADSC vs. empty: p < 0.05 PGP 9.5 and p < 0.01 S100;
dMSC vs. empty: p < 0.01 for both PGP 9.5 and S100). No
significant difference was noticed between the dADSC
group and the dMSC group with either staining.
Discussion
In this study, we analysed the behaviour of fibrin conduits
seeded with different cell populations as a nerve guide to
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Adipose-derived stem cells enhance peripheral nerve regeneration 5

Figure 2 Differentiation of stem cells, dADSC and SC immunostained for S100 (green), GFAP (red) and DAPI (blue). Differenti-
ation of ADSC resulted in 40% of cells co-expressing the SC markers and with a bipolar elongated morphology. SC cultures were
routinely >97% positive for both S100 and GFAP indicating the absence of significant quantities of contaminating fibroblast cells.

Please cite this article in press as: di Summa PG et al., Adipose-derived stem cells enhance peripheral nerve regeneration, J Plast
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6 P.G. di Summa et al.

Figure 3 Analysis of cell seeding. A. Haematoxylin staining of a fibrin conduit seeded with cells in vitro and (B) shown at high
magnification.C DAPI staining of a fibrin conduit seeded with cells in vitro and (D) shown at high magnification.

repair sciatic nerve injury. We compared the regeneration
achievable with SC, dADSC, dMSC and with an empty
conduit. First, we found that all conduits seeded with cells
supported axonal regeneration without collapsing and
retained a lumen, which provides a permissive environment
for regeneration.
No signs of haematoma or infection were detected at
explantations, confirming the good tissue acceptance of
fibrin without altering the tissue environment at the
implantation side. Moreover, first signs of resorption at the
edges were noted after 2 weeks in vivo, displaying biode-
gradability of the fibrin conduit. The porous structure of
the conduit, already noted with in vitro experiments,8
seems to allow passage of neurotrophic factors into the
lumen, the surface of fibrin forms a barrier preventing
fibrous tissue to invade the lumen.36
Fibrin has been previously used as a matrix for MSC cell
suspension37 and as a new nerve guide8: our results show
that, at least in the short term, the fibrin conduit can be

Figure 5 S100 staining shows the proximal Schwann cell

Figure 4 PGP 9.5 staining shows axonal regeneration in the migration in the fibrin conduits either empty or seeded
fibrin conduits either empty or seeded with different regenera- with different regenerative cells. A. Empty fibrin conduit.
tive cells. A. Empty fibrin conduit. B. Fibrin conduit þ dADSC. B. Fibrin conduit þ dADSC. C. Fibrin conduit þ dMSC. D. Fibrin
C. Fibrin conduit þ dMSC. D. Fibrin conduit þ SC. conduit þ SC.

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Adipose-derived stem cells enhance peripheral nerve regeneration 7

Figure 6 Quantification of regeneration distances measured with (A) PGP 9.5 and (B) S100 cell staining. Graphs show
a significant improvement in axon regeneration distance in all groups (dADSC, dMSC, SC), when compared with the empty fibrin
group. Values are mean distances (mm)  SEM. (* p < 0.05; ** p< 0.01; *** p < 0.001).

optimised by cell delivery, resulting in higher peripheral
nerve regeneration.
The best regenerative values were found combining
fibrin conduits and SC. This should be expected, given the
crucial role covered by SCs in nerve regeneration and in
creating the adequate environment through which regen-
erating axons grow to reach their peripheral targets.12
Indeed, SCs were injected for the first time into a fibrin
conduit, giving encouraging results, with a better regen-
erating distance when compared statistically with the
empty fibrin conduit. These data seem to strengthen the
argument that SC can help in enhancing nerve regeneration
when seeded in a conduit.10,14
Concerning dMSC and dADSC, we also detected a statis-
tically significant improvement in nerve regeneration
distance (PGP 9.5 staining, respectively, 4.986 and 4.968
for dMSC and dADSC vs. 4.155 in the empty fibrin conduit).
From these data, we could argue that, even if these cells
still not reach the full regenerative potential of SC, they
can play an effective role in nerve regeneration. Interest-
ingly, we could find that dADSC promote regeneration in the
same manner as dMSC (difference between the two groups
p > 0.05) as we saw reporting overlapping results when we
compared the axonal regeneration distance (PGP 9.5) and
SC invasion (S100) for the two groups.
This seems to confirm the previously reported similari-
ties between these two cell populations, concerning
morphology,38 immune phenotype39 and trans-differentia-
tion capacity along mesodermal27 and non-mesodermal
lineages.26,40 This equivalence of dADSC and dMSC in
promoting peripheral nerve regeneration can be promising,
considering the already successful application of MSC
in vivo in the longer-term group.14,34,35 Stem cells could aid
regeneration by the release of soluble nerve growth factors
such as brain-derived neurotrophic factor41 or angiogenic
molecules, including vascular endothelial growth factor.42
Other studies have shown that dMSC can physically engraft
and myelineate regenerating axons in vivo20, suggesting
that multiple mechanisms may exist in stem cell therapy.
In terms of future clinical use, humans have abundant
subcutaneous fat deposits, and ADSCs can be isolated in
large quantities by conventional liposuction procedure
under local anaesthesia, thus overcoming the discomfort
and the tissue morbidity associated with bone marrow
aspiration and showing potential advantages for tissue
engineering application, compared with MSC.40,43,44
Furthermore, the frequency of MSCs in bone marrow ranges
between 1 in 25 000 to 1 in 100 000,45 whereas ADSCs
constitute a more abundant proportion, approximately 2%
of lipoaspirate cells.25,28
The promising results achieved from the combination of
regenerative cells and fibrin conduit are hopeful steps for
future clinical applications in cases of peripheral nerve
injury. Similarly, the interesting outcomes obtained from
the dADSCs, which appear comparable to the dMSC,
underline the effectiveness of this cell population and their
promising applications in nerve regeneration. A longer-term
group will be necessary to confirm the positive effects on
functional recovery and to see if dADSC will maintain this
similarity compared with dMSC.

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Acknowledgements
We thank Professor Daniel V. Egloff, director of the
department for plastic surgery CHUV for his support and
useful comments to this project and Catherine Pythoud
BSc, Jean Francois Brunet PhD for technical support. The
authors are grateful to the SwissLife Foundation, the SUVA
and the University of Lausanne FBM for their financial
support.
Conflict of Interest
None.
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Please cite this article in press as: di Summa PG et al., Adipose-derived stem cells enhance peripheral nerve regeneration, J Plast
Reconstr Aesthet Surg (2009), doi:10.1016/j.bjps.2009.09.012