Journal of plastic reconstructive & aesthetic surgery JPRAS 2010 di Summa (2).pdf

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

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Adipose-derived stem cells enhance peripheralnerve 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, Universite de Lausanne, Rue de Bugnon 46, 1005 Lausanne, CH, Switzerlandb Blond McIndoe Research Laboratories. The University of Manchester, Manchester, UKc Departments of Surgical and Perioperative Science (Hand surgery) and Integrative Medical Biology (Anatomy), UmeaUniversity, Umea, Sweden

Received 30 March 2009; accepted 10 September 2009

KEYWORDSAdipose-derivedstem cells;Peripheral nerveregeneration;Differentiated cells;Sciatic nerve

* Corresponding author. Tel.: þ41 2125 31.

E-mail address: daniel.kalberKalbermatten).

1748-6815/$-seefrontmatterª2009Britdoi:10.1016/j.bjps.2009.09.012

Please cite this article in press as:Reconstr Aesthet Surg (2009), doi:10

Summary Traumatic injuries resulting in peripheral nerve lesions often require a graft tobridge the gap. Although autologous nerve auto-graft is still the first-choice strategy in recon-structions, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplan-tation in a bioartificial conduit is an alternative strategy to create a favourable environmentfor nerve regeneration. We decided to test new fibrin nerve conduits seeded with various celltypes (primary Schwann cells and adult stem cells differentiated to a Schwann cell-like pheno-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 axonalregeneration in the group of fibrin seeded with Schwann cells compared with the empty fibrinconduit. Differentiated adipose-derived stem cells also enhanced regeneration distance ina similar manner to differentiated bone marrow mesenchymal stem cells. These observationssuggest that adipose-derived stem cells may provide an effective cell population, without thelimitations of the donor-site morbidity associated with isolation of Schwann cells, and could bea clinically translatable route towards new methods to enhance peripheral nerve repair.ª 2009 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published byElsevier Ltd. All rights reserved.

Numerous surgical procedures are performed each year forperipheral nerve repair, with a significant amount of lost

314 25 06; Fax: þ41 21 314

[email protected] (D.F.

ishAssociationofPlastic,Reconstruc

di Summa PG et al., Adipose-de.1016/j.bjps.2009.09.012

working days with corresponding economic consequences.1

Nerve lesions without defect or with a short gap are usuallytreated by end-to-end coaptation. Traumatic injuriesresulting in longer peripheral nerve lesions often requirea graft to bridge the gap. Unfortunately, full recovery maynever be achieved, particularly with extended lesions.2

Although autologous nerve autograft is still the first-choice

tiveandAestheticSurgeons.PublishedbyElsevierLtd.All rightsreserved.

rived stem cells enhance peripheral nerve regeneration, J Plast

mailto:[email protected]

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2 P.G. di Summa et al.

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strategy in reconstructions, they have several disadvan-tages caused by the limited availability of donor tissue,sacrifice of functional nerve and potential formation ofneuroma.2e4

Nerve autografts have been mimicked by venous andarterial conduit grafts, which show no functional benefitscompared with standard nerve grafts.5 Allografts have alsobeen tested, but they require an undesirable long-termimmunosuppressive therapy.6 Many attempts have beenmade to develop conduits made of biodegradable mate-rials, which could guide the regenerating axons withoutinhibiting the process of growth and maturation.7 Recently,we described a bioresorbable nerve constructed from fibringlue,8 which shows good tissue acceptance and a porousstructure that allows neurotrophic growth factors topenetrate into the lumen of the conduit. Moreover, theresorption of the conduit within 4 weeks avoids compres-sion syndromes and enhances biocompatibility.9

Our aim was to optimise the fibrin conduit, previouslydescribed, by seeding it with regenerative cells. Celltransplantation in a bioartificial conduit is an alternativestrategy to create a favourable environment for nerveregeneration10: in this study, we compared different cellpopulations such as primary Schwann cells (SCs) and stemcells differentiated towards to a Schwann cell phenotype.

SCs offer a highly preferred substrate for axon migrationand release of bioactive factors that further enhancemigration. They synthesise adhesion molecules (CAMs),release growth factors (GFs) and build up basementmembranes for the support and guidance of the sproutingaxons.11,12 Despite showing regeneration enhancement,13

SCs have limited clinical applications. The culture ofa sufficient amount of SC to achieve optimal conditions fortransplantation in nerve conduits is time consuming andrequires particular care for in vitro expansion anda constant input of GFs.14 Moreover, SCs are not easilyaccessible without nerve biopsy and bear the need tosacrifice 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 easilyaccessible, should proliferate rapidly in culture and shouldsuccessfully integrate into the host tissue with immunologicaltolerance.16,17 The growing applicability of stem cells hasopened new frontiers to nerve regeneration: the advantageof using these cell lines is that cell differentiation may bestimulated by advancing axons and pluripotency, allowsmultiple differentiation paths, which can create a positiveenvironment for axonal regeneration.1

Bone-marrow-derived mesenchymal stem cells (MSCs)can be induced to differentiate into non-mesenchymal fatesin vitro, such as Schwann cells, improving myelin formationand nerve regeneration in vivo after their transplantationinto different models of peripheral nerve injury.18e20

Adipose tissue is a mesodermally derived complex tissuethat, besides adipocytes and pre-adipocytes, containsa stromal population, which includes microvascular endo-thelial cells, smooth muscle cells, resident monocytes,lymphocytes and stem cells.21,22 These non-adipocyte cellsare known as stromal vascular fraction (SVF) and can beisolated by centrifugation of collagenase-digested adiposetissue.23 In the past few years, it has been shown howcultured SVFs give rise to multipotent precursor cell from

Please cite this article in press as: di Summa PG et al., Adipose-deReconstr Aesthet Surg (2009), doi:10.1016/j.bjps.2009.09.012

adipose tissue stromal cells.24e26 Although literatureterminology can be confusing,27,28 we will use the genericnomenclature of adipose-derived stem cells (ADSCs) todescribe these cells.

As previously cited, stem and progenitor cells from adultstromal tissue retain the capacity to differentiate towardsmesenchymal and non-mesenchymal lineages, similar tobonemarrow stromal cells,24,29 showing pluripotency above mul-tipotency.30 Regarding neural differentiation, several studiesproved trans-differentiation of ADSC.23,24,31e33 This suggeststhat adult stem cells may not be as limited by lineage aspreviously thought and, in fact, manifest profound plasticity.

Recently, we showed how Schwann cell-like differenti-ated ADSC could enhance neurite outgrowth in vitro.4 Inthis current study, we have tested the ability of thedifferentiated ADSC to enhance nerve regeneration in vivo.We compared these cells with SC-differentiated MSCs andprimary adult SCs seeded in fibrin conduits.

Materials and Methods

Fibrin conduit

The fibrin conduit was prepared from two-compound fibringlue (Tisseel� Kit VH 1.0, Baxter SA, USA). Tisseel�contains fibrinogen, 70e110 mg ml�1; plasma fibronectin,2e9 mg ml�1; factor XIII, 10e50 U ml�1; plasminogen,40e120 mg ml�1; aprotinin solution 3000 KIU ml�1; thrombin4 IU ml�1; and calcium chloride 40 mmol l�1. Fibrin glue wasdispensed in a mould prepared from silicone arounda stainless steel core and pressed into shape for 5 min(Figure 1). This allows the generation of uniform conduitsmeasuring 14 mm in length, with a 2-mm lumen and 1-mmwall thickness designed to bridge a 10-mm gap in the leftsciatic nerve of 250 g adult Sprague-Dawley rats (Janvier,France).

Cell harvest

Schwann cellsAll cells were obtained from Sprague-Dawley rats (Janvier,France). These were euthanised by CO2 according to thelocal veterinary commission in Lausanne (Switzerland) andstudy was carried out in accordance with the EuropeanCommunity Council directive 86/609/ECC for the care anduse of laboratory animals.

To harvest Schwann cells, sciatic nerves were exposed,removed and kept in Dulbecco’s modified Eagle’s mediumplus glutamax (DMEM, Invitrogen, UK) containing 1% pen-icillinestreptomycin. Nerves were then dissected in trunks,de-sheathed and finally chopped in 1-mm segments. Thesegments were then plated in a Petri dish with SC growthmedium supplemented by 14 mM forskolin and 100 ng ml�1

neuregulin-1 b1 (R&D Systems, UK). The cells were incu-bated for 2 weeks at 37 �C with 5% CO2 with fresh mediumadded approximately every 72 h. After these 2 weeks,medium was aspirated and 0.125% (w/v) collagenase typeIV and 117 U mg�1 dispase were added to the Petri dish.After 24-h incubation, the cell suspension was filteredthrough a 70-mm cell strainer (Falcon; BD BiosciencesDiscovery Labware, Bedford, MA), centrifuged at 900 rpm


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

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Adipose-derived stem cells enhance peripheral nerve regeneration 3

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for 5 min to obtain the cell pellet. Finally, the cell pelletwas re-suspended in SC growth medium, seeded into25-cm2 flask precoated with poly-D-lysine (Sigma, St Louis,MO, USA) and incubated in the same conditions. Thefollowing day, the medium was freshly changed and thecells were left to proliferate. At confluence, the SCs werepurified by an antibody complement method to eradicatethe remaining fibroblasts.13,17,34

Adipose-derived stem cellsAdipose-derived stem cells were harvested as describedpreviously.4 After euthanasia, the inguinal fat pad was care-fully dissected and minced using a sterile razor blade. Thetissue was enzymatically digested for 2 h at 37 �C using 0.15%(w/v) collagenase type I (Invitrogen, UK). The solution waspassed 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 cellpellet was re-suspended in MEM containing 10% (v/v) FBS and1% (v/v) penicillin/streptomycin solution. The cultures weremaintained at sub-confluent levels in a 37 �C incubator with5% CO2 and passaged with trypsin/ethylenediaminetetra-acetic acid (EDTA) (Invitrogen, UK) when required.

Bone-marrow-derived mesenchymal stem cellsMSCs were harvested from adult Sprague-Dawley rat femoralbones34: mesenchymal cell growth medium (MEM- a; Invi-trogen; supplemented with 10% FBS (v/v) and 1% penicillinestreptomycin) was injected through each marrow cavity using


a 21-guage needle. The resulting cell suspension was tritu-rated, filtered through a 70-mm Falcon filter and centrifugedfor 5 min at 600 g. The supernatant was aspirated, the cellbolus re-suspended in mesenchymal cell growth medium andthe cells plated in 75-cm2 tissue culture flasks and incubatedin 5% CO2 at 37 �C. Haematopoietic cells were eliminated bywashing daily with DMEM until all the non-adherent cells wereremoved. The cells were then allowed to grow to confluence.

Stem cell differentiation

The multi-potent nature of the cell cultures was tested byensuring their ability to differentiate along various lineages,as previously described.4,34 Further, undifferentiated ADSCsand MSCs were differentiated into an SC-like phenotype atearly passages.4,18 Growth medium was removed from sub-confluent cultures and replaced with medium supplementedwith 1 mM b-mercaptoethanol (Sigma-Aldrich, UK) for 24 h.The cells were then washed and fresh medium supplementedwith 35 ng ml�1 all-trans-retinoic acid was added. A further72 h later, the cells were washed and the medium replacedwith differentiation medium; cell growth medium supple-mented with 5 ng mle1 platelet-derived growth factor (PDGF;PeproTech Ltd., UK), 10 ng ml�1 basic fibroblast growth factor(bFGF; PeproTech Ltd., UK), 14 mM forskolin and 200 ng ml�1

neuregulin-1 b1. The cells were incubated for 2 weeks underthese conditions with fresh medium added approximatelyevery 72 h. To confirm the effectiveness of the differentiationof ADSC and their similarity to the Schwann cell population,we performed cell immunostaining for typical Schwann cells


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markers S100 and GFAP.4 Cultures of differentiated ADSC(dADSC) and SC were compared by immunostaining: the cellswere 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) ina similar manner as previously shown.34

Cell seeding and microsurgery

A couple of hours before implantations in rats, the cellswere trypsinised and, after centrifugation, 2� 106 cellswere suspended in 50 ml of differentiation medium andinjected into the fibrin tubes. To avoid cell dispersion duringthe procedure, one side of the fibrin tube was previouslycapped by a fibrin glue clot. Before implantation, the fibrinconduit was easily decapped from the fibrin clots at its ends.The control group in the absence of cells (empty) wassimilarly filled with 50 ml of differentiation medium alone.The sciatic nerve was transected and nerve ends were fixedto the conduit by a single epineural suture (9/0 Prolene,Ethicon): proximal and distal nerve stumps were inserted2 mm into the tube, thus leaving a 10-mm gap. Muscles andfascia layers were closed with single resorbable stitches (4/0 Softcat, Braun) and the skin by a continuous runningsuture (4/0 Prolene, Ethicon). The various groups of fibrinconduits (each one formed by six animals for a total n Z 24)were left in place for 2 weeks and subsequently harvestedtogether with the proximal and distal nerve stumps.

Nerve conduit harvesting andimmunohistochemistry

Animals were sacrificed after 2 weeks by CO2 chambereuthanasia. Harvested conduits were fixed by para-formaldehyde (PFA) for 16 h, then rinsed 24 h at 4 �C in PBScontaining 30% sucrose and 0.1% sodium azide, with PBSchanges 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-Glaser, Germany).

After 3� 5 min PBS washes, a casein blocking solutionwas applied for 1 h before incubating slides overnight withprimary antibodies rabbit anti-S100 (1:500, Dako, UK) andrabbit anti-PGP 9.5 (1:500, Dako, UK). The following dayafter 3� 5 min PBS washes, slides were incubated withsecondary antibody (1:500, Alexa Fluor goat anti-rabbit,Invitrogen, USA) at room temperature in the dark for40 min. After 3� 5 min PBS washes, the slides were finallymounted with Vectashield with DAPI (Vector Labs, UK) andexamined under the fluorescence microscope(10�magnification) with the regenerating conus directedtowards the distal end.35 Axonal regeneration distance(PGP 9.5) and S100 positive cell distribution inside the


conduit were evaluated using an optical microgrid: thelength was measured from the beginning of the conduit tothe last visible sprout of the regenerating conus.

Statistical analysis

One-way analysis of variance (ANOVA) with Bonferronimultiple comparison test was used to statistically analysedata (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 andits capacity to retain cells within. We filled two differentfibrin conduits with ADSC (80� 106 ml�1 cell concentration),then we fixed them at 48 h. By DAPI staining, we found a goodcapacity 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 ofthe animals developed infections or surgical complicationsexcept autotomy, which was present in one case out of 24.After 2 weeks in vivo, fibrin conduits showed an intactstructure, with proximal and distal ends partially resorbed.No signs of haematoma or infection were detected.

Axon regeneration

Sciatic nerve axonal regeneration was different depending onthe type of cells which had been seeded in the fibrin conduits.Axonal regeneration distance was assessed proximally by PGP9.5 immunohistochemistry (Figure 4). Conduits filled with SCsshowed the best regeneration distance (5.758� 0.12 mm)followed by fibrin containing dMSC (4.986� 0.19 mm) ordADSC (4.968� 0.22 mm) (Figure 6A). All three groups withcells resulted in better regeneration than the empty fibrinconduit group (4.155� 0.07 mm). PGP 9.5 positive axons wereassociated with S100 positive cells at the proximal regener-ating stumps (Figure 5). The distances of S100 positive cellmigration 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 seededwith SC significantly improved axonal regenerationdistance, and proximal SC invasion compared with theempty fibrin conduit (** p< 0.01). Differentiated adult stemcells also showed a positive impact on nerve regenerationwith 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). Nosignificant difference was noticed between the dADSCgroup and the dMSC group with either staining.

Discussion

In this study, we analysed the behaviour of fibrin conduitsseeded with different cell populations as a nerve guide to


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 wereroutinely >97% positive for both S100 and GFAP indicating the absence of significant quantities of contaminating fibroblast cells.

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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 PlastReconstr Aesthet Surg (2009), doi:10.1016/j.bjps.2009.09.012

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

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repair sciatic nerve injury. We compared the regenerationachievable with SC, dADSC, dMSC and with an emptyconduit. First, we found that all conduits seeded with cellssupported axonal regeneration without collapsing andretained a lumen, which provides a permissive environmentfor regeneration.

No signs of haematoma or infection were detected atexplantations, confirming the good tissue acceptance offibrin without altering the tissue environment at theimplantation side. Moreover, first signs of resorption at the

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


edges were noted after 2 weeks in vivo, displaying biode-gradability of the fibrin conduit. The porous structure ofthe conduit, already noted with in vitro experiments,8

seems to allow passage of neurotrophic factors into thelumen, the surface of fibrin forms a barrier preventingfibrous tissue to invade the lumen.36

Fibrin has been previously used as a matrix for MSC cellsuspension37 and as a new nerve guide8: our results showthat, at least in the short term, the fibrin conduit can be

Figure 5 S100 staining shows the proximal Schwann cellmigration in the fibrin conduits either empty or seededwith different regenerative cells. A. Empty fibrin conduit.B. Fibrin conduitþ dADSC. C. Fibrin conduitþ dMSC. D. Fibrinconduitþ SC.


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

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optimised by cell delivery, resulting in higher peripheralnerve regeneration.

The best regenerative values were found combiningfibrin conduits and SC. This should be expected, given thecrucial role covered by SCs in nerve regeneration and increating 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 fibrinconduit, giving encouraging results, with a better regen-erating distance when compared statistically with theempty fibrin conduit. These data seem to strengthen theargument that SC can help in enhancing nerve regenerationwhen seeded in a conduit.10,14

Concerning dMSC and dADSC, we also detected a statis-tically significant improvement in nerve regenerationdistance (PGP 9.5 staining, respectively, 4.986 and 4.968for dMSC and dADSC vs. 4.155 in the empty fibrin conduit).From these data, we could argue that, even if these cellsstill not reach the full regenerative potential of SC, theycan play an effective role in nerve regeneration. Interest-ingly, we could find that dADSC promote regeneration in thesame manner as dMSC (difference between the two groupsp> 0.05) as we saw reporting overlapping results when wecompared the axonal regeneration distance (PGP 9.5) andSC invasion (S100) for the two groups.

This seems to confirm the previously reported similari-ties between these two cell populations, concerningmorphology,38 immune phenotype39 and trans-differentia-tion capacity along mesodermal27 and non-mesodermallineages.26,40 This equivalence of dADSC and dMSC in


promoting peripheral nerve regeneration can be promising,considering the already successful application of MSCin vivo in the longer-term group.14,34,35 Stem cells could aidregeneration by the release of soluble nerve growth factorssuch as brain-derived neurotrophic factor41 or angiogenicmolecules, including vascular endothelial growth factor.42

Other studies have shown that dMSC can physically engraftand myelineate regenerating axons in vivo20, suggestingthat multiple mechanisms may exist in stem cell therapy.

In terms of future clinical use, humans have abundantsubcutaneous fat deposits, and ADSCs can be isolated inlarge quantities by conventional liposuction procedureunder local anaesthesia, thus overcoming the discomfortand the tissue morbidity associated with bone marrowaspiration and showing potential advantages for tissueengineering application, compared with MSC.40,43,44

Furthermore, the frequency of MSCs in bone marrow rangesbetween 1 in 25 000 to 1 in 100 000,45 whereas ADSCsconstitute a more abundant proportion, approximately 2%of lipoaspirate cells.25,28

The promising results achieved from the combination ofregenerative cells and fibrin conduit are hopeful steps forfuture clinical applications in cases of peripheral nerveinjury. Similarly, the interesting outcomes obtained fromthe dADSCs, which appear comparable to the dMSC,underline the effectiveness of this cell population and theirpromising applications in nerve regeneration. A longer-termgroup will be necessary to confirm the positive effects onfunctional recovery and to see if dADSC will maintain thissimilarity compared with dMSC.


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Acknowledgements

We thank Professor Daniel V. Egloff, director of thedepartment for plastic surgery CHUV for his support anduseful comments to this project and Catherine PythoudBSc, Jean Francois Brunet PhD for technical support. Theauthors are grateful to the SwissLife Foundation, the SUVAand the University of Lausanne FBM for their financialsupport.

Conflict of Interest

None.

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