Supportive use of platelet-rich plasma and stromal vascular fraction for cell-assisted fat transfer of skin radiation-induced lesions in nude mice

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vascular fraction for cell-assisted fat transfer of skin

radiation-induced lesions in nude mice

Baptiste Bertrand, Julia Eraud, Melanie Velier, Cécile Cauvin, Nicolas

Macagno, Mohamed Boucekine, Cecile Philandrianos, Dominique Casanova,

Jeremy Magalon, Florence Sabatier

To cite this version:

Baptiste Bertrand, Julia Eraud, Melanie Velier, Cécile Cauvin, Nicolas Macagno, et al..

Sup-portive use of platelet-rich plasma and stromal vascular fraction for cell-assisted fat transfer

of skin radiation-induced lesions in nude mice.

Burns, Elsevier, 2020, 46 (7), pp.1641-1652.

�10.1016/j.burns.2020.04.020�. �hal-03155462�

Supportive

use

of

platelet-rich

plasma

and

stromal

vascular

fraction

for

cell-assisted

fat

transfer

of

skin

radiation-induced

lesions

in

nude

mice

Baptiste

Bertrand

a,b,

*

,

Julia

Eraud

a

,

Mélanie

Velier

b,c

,

Cécile

Cauvin

d

,

Nicolas

Macagno

e

,

Mohamed

Boucekine

f

,

Cécile

Philandrianos

a

,

Dominique

Casanova

a

,

Jeremy

Magalon

b,c

,

Florence

Sabatier

b,c

a_{DepartmentofPlasticSurgery,LaConceptionHospital,AssistancePublique HôpitauxdeMarseille,France} b_{Aix-MarseilleUniv,C2VN,INSERM,INRA,France}

c

CultureandCellTherapyLaboratory,INSERMCICBT-1409,LaConceptionHospital,AssistancePublique Hôpitaux deMarseille,France

d_{DepartmentofRadiotherapy,HopitalPrivéClairval,Marseille,France}

e_{DepartmentofPathology,laTimoneHospital,AssistancePublique HôpitauxdeMarseille,France} f_{Aix-MarseilleUniv,EA3279 PublicHealth,ChronicDiseasesandQualityofLife ResearchUnit,France}

a

b

s

t

r

a

c

t

Background: External radiotherapy has become indispensable in oncological therapies. Unfortunately,radiationisresponsibleforserioussideeffects,suchasradiodermatitis.The skinisweakenedandulcerated.Ourstudyaimedtoevaluatethesubcutaneoustransferof microfat(MF)aloneandtwomixes:MF+Platelet-richplasma(PRP)andMF+stromalvascular fraction(SVF)totreatradiation-inducedskinlesions.

Method:Wedefinedrandomlyfiveexperimentalgroupsofninemice:1healthycontrolgroup and4irradiated(60Grey)andtreatedgroups.Theskinlesionsweretreated3monthsafter irradiationbyMF,MF+PRP(50% 50%),MF+SVF(90% 10%)orRinger-lactatesubcutaneous injections.Woundhealingwasevaluatedat1,2and3monthspost-injectionandhistological woundanalysisat3months,aftereuthanasia.

Results:Alltheirradiatedmicepresentedwithwounds.Aftersham-injection,thewoundarea increasedby91.171.1%versusadecreaseof15.923.1%afterMFalone(NS),27.323.8% afterMF+SVF(NS)and76.47.7%afterMF+PRP(P=0.032).Asignificativereductionofskin thicknessinwoundperipherywasmeasuredforthethreetreatedgroupscomparedto sham-injection(P<0.05)butnotinthehealedwounds(NS).Themostimportantsubcutaneous neo-vesseldensitywasshownafterMF+SVFinjection.

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Accepted15April2020 Keywords: Radiodermitis Fattransfer Platelet-RichPlasma StromalVascularFraction WoundHealing

Abbreviations:MF,microfat;PRP,plasma-richplatelets;SVF,stromalvascularfraction;NS,notsignificant.

* Correspondingauthorat:DepartmentofPlasticSurgery LaConceptionHospital AssitancePublique-HôpitauxdeMarseille, Aix-MarseilleUniv147,BoulevardBaille,13005Marseille,France.

E-mailaddresses:Baptiste.bertrand@ap-hm.fr,Baptiste.bertrand@gmail.com(B.Bertrand),Julia.eraud@ap-hm.fr(J.Eraud),

Melanie.velier@ap-hm.fr(M.Velier),Cecile.cauvin@yahoo.fr(C.Cauvin),Nicolas.macagno@ap-hm.fr(N.Macagno),

boucekine.m@gmail.com(M.Boucekine),Cecile.philandrianos@ap-hm.fr(C.Philandrianos),Dominique.casanova@ap-hm.fr

(D.Casanova),Jeremy.magalon@ap-hm.fr(J.Magalon),Florence.sabatier@ap-hm.fr(F.Sabatier).

https://doi.org/10.1016/j.burns.2020.04.020

0305-4179/©2020ElsevierLtdandISBI.Allrightsreserved.

Available

online

at

www.sciencedirect.com

ScienceDirect

Conclusion:TheMF+PRPmixwasthemostefficientproducttoincreasehealing.TheMF+SVF mixshowedthehighestrateofneo-angiogenesisbutwasdisappointingintermsofhealing. Levelofevidence:Notgradable.

©2020ElsevierLtdandISBI.Allrightsreserved.

1.

Introduction

Externalradiotherapyhasbecomeindispensableinoncology therapy,conferringmanybenefitstopatients.Unfortunately, radiationisresponsibleforseriousandfrequentsideeffects, suchasradiodermatitis[1].Skindirectlyexposedtoradiation becomes thickened and fibrotic, with diminished micro-vascularisation[2].Afteraninitialphaseofvascular hyper-permeability,microvasculardensitydecreasesduetofibrotic and irregularcapillaries,whichareoften occluded[3]. The epidermisisweakenedandulcerationsappearafewmonths oryearsafterradiation[4].Healingoftheseulcersispoor,and thewoundsbecomechronic.Asurgicalprocedureis frequent-lynecessarytoexcisethedamagedskinandreplaceitwitha flap[5,6].

SincethefattransferprocedurewasdevelopedbySydney Colemanin1995[7],manystudieshaveshownaregenerative, as well as a volumizing, effect of the fat [8]. Injecting autologous fat under irradiated skin resulted in a pro-cicatrizingandantifibroticactioninpreclinical[9]andclinical studies [10,11]. The stromal vascular fraction (SVF) of the adipose tissue is the product remaining after enzymatic digestionofthematureadipocytes.Thiscell-basedproduct seemstobetheprincipalactorintheregenerativeeffects[12

15]ofautologousfatgrafting[16].Platelet-richplasma(PRP), aconcentrateofbloodplatelets,isanattractiveproductto promote healing of chronic wounds [17,18]. PRP delivers various growthfactors thatstimulateneoangiogenesisina paracrinemanner[19].Thesethreeautologousproducts(fat, SVFandPRP)areinteresting,bothseparatelyandtogether,for thetreatmentofskinlesionsinducedbyradiation.Nostudy has compared the action of these different regenerative productsonwoundhealinginthecontextofradiodermatitis. Theobjectiveofourstudywastoevaluateandcomparethe efficacyofmicrofat(MF)alone,PRPmixedwithMF,andSVF mixedwithMFversusacontroltreatmentinamousemodelof radiation-inducedskin-lesions.Apositiveeffectofthesenew cell-based products would demonstrate the potential for clinicalapplicationtotreatorpreventradiation-inducedskin lesions.

2.

Materials

and

methods

2.1. Animals

This experimental study was approved by the National Animal Care and Ethics Committee (#00506.02). Based on previous experimental studiesby ourstem celllaboratory

andbySultanetal.[9]andFogeronetal.[20]demonstrating thepositiveeffectoffatgraftingonradiationskindamagein murinemodels, we determined that nine mice per group wouldbesufficienttoshowasignificantdifferencebetween the various products evaluated. We couldnot predict the number of deaths a priori because our model is the first murinemodelofskinulcerationafterradiotherapy.Tolimit theeffectofmortalityonthedecreaseinpowerofthetests, wehaveplannednon-parametricteststotakeintoaccount thelackofnormalityofgroupsbyusingallthetestdata.We also included more subjects than previously published studies toprovide enough subjects forcomparison. Thus, forty-fivemaleorfemalenudeNMRI-Foxnu1nu/numice(8 weeks old, 28 g) were purchased from the January Lab (Genest-Saint-Isle,France).Themicewererandomlydivided intotwogroups:ninecontrolmiceand36miceexposedto externalradiotherapy.

2.2. Experimentalgroups

Ninemiceeachwererandomlyassignedtofiveexperimental groups(Fig.1):four treatedgroups(MFalone,MF+SVF,MF +PRPandshaminjectionseeTable1)exposedtoradiationand onehealthycontrolgroup.Themicewerehousedinindividual cagesafterradiotherapy.Treatmentwasdelayedfor3months toreproducethechronicradiodermatitiswoundsandlesions. Aftertreatment, the miceunderwent a3-month follow-up beforeeuthanasia.

2.3. Irradiation

Targetedirradiationofthedorsalcutaneousliningwascarried outsimultaneouslyformiceofthefourtreatedgroups(n=36)

aftergeneralanaesthesiawithanintraperitonealinjectionof ketamine/xylazine (200/10mg/kg). The dorsal skin was re-tractedfromtheunderlyingbonyskeleton,maintainedbytwo separatedsuturesbetweentwo20cm20cmfieldsof plex-iglass.Thetwosutureswerereproduciblyspaced3cmapart, centredonthemidlineofthebackofeachmouse,soastohold thedorsalskinofeachmouse1cmundertheplexiglassfield. Fourblocksof8-cm-thick Cerrobend(CerroMetalProducts, Bellefonte,PA,USA)wereplacedoneachedgeofthePlexiglas abovethemicetoprotectthebodyandspinefromthediffusion oftheradiation.(Fig.2).Theirradiationwasdeliveredatadose of60Gyinonetargetedfractionina20cm20cmirradiation field,attheexactsizeoftheplexiglassplates,usingalinear accelerator Elektasynergy system(ElektaBeam Modulator; ElektaOncologySystems,Crawley,UK)with6MVphotons.The acceleratorarmwassetto180,andthecollimatorwassetto0 witha prescriptionat1.5cmand askinsourcedistanceof 100cm. The flow rate was 400 EU/min. Plexiglass is an homogeneous medium. Thus the irradiation delivered on the irradiation field was identical at all points under the plexiglassplate.

2.4. Celltherapyproducts

Celltherapyproductswereobtainedfromahealthy 35-year-old woman volunteer donorfollowing provision ofwritten informed consent. The lipoaspirate residue and a blood sample were recovered during the aesthetic liposuction procedure.FatandMFwereharvestedfromthelateralflank areas, and peripheral whole blood was taken during the intervention.Thedonorhadnorelevantdiseasesandwasfree ofanydrugsknowntoaffectplateletfunctionfor7daysbefore sampling.Allcelltherapyproductsweremanufacturedina

Table1–Compositionofthevariousproductsevaluatedinthisstudy.

PRP SVF Microfat Ringer-lactate Shaminjection (n=9mice) 1cm3 MF(n= 9mice) 1cm3 MF PRP(n=9 mice) 0.5 cm3 _{0.5 cm}3 MF SVF(n= 9 mice) 0.1 cm3 _{0.9 cm}3 Characteristics andComposition

Injectedcells/mouse Viableinjectedcells/mouse CanulaSt’RIM canula14Gauges Purification Cen-trifugation 1200g 3min Platelets(106_{) 36295.6% Viability 80%}

RedBloodCells (106₎ 15 3.9% Leukocytes Including Macrophages/Monocytes Lymphocytes Neutrophils 46910(17.6%) 7488(2.8%) 33388(12.5%) 6034(2.3%) Leukocytes(106_{) 1.8} 0.5% Endothelial ProgenitorCells 106 731(40.2%) Increasefactorin platelets 2.26 Pericytes 48411(18.2%) Increasefactorin leukocytes 0.5 StromalCells 63948(24.0%)

TheEnglishinthisdocumenthasbeencheckedbyatleasttwoprofessionaleditors,bothnativespeakersofEnglish.Foracertificate,pleasesee:

ClassAmicrobiologicalsafetyfacilitylocatedintheculture and cell therapy laboratory of our university hospital and injectedonthesameday,within3hofharvest.

2.5. Microfat

TheMF(36cm3_{)wasaspiratedwitha14Gcannulafromthe}

st’RIMprocedurepack (Thiebaud Biomedical Devices, Mar-gencel, France) forMF transfer. TheMF was purified after centrifugation (1200g for 3min) with a microcentrifuge (Medilite;ThermoScientific,Waltham,MA,USA)toeliminate oilyandbloodyresidue.

2.6. Stromalvascularfraction

Fat(184cm3_{)wasaspiratedwitha12G,12-holeharvesting}

cannula (Khouri Harvester, Koume, Plantation, FL, USA) mountedona10cm3_{syringe.SVFwaspurifiedbyenzymatic}

digestionofmatureadipocytesduring45minat37 usinga manualmethodandcollagenaseattheconcentrationof0.25U/ mL (NB5, Heideberg,Germany). Total viable nucleated cell recovery and cell viability were determined using the Nucleocounter NC100 instrument (ChemoMetec,Denmark). CellularcomponentswithinisolatedSVFwereidentifiedbya flowcytometryanalysis(BeckmanNaviosinstrument)usinga panelofcellsurfacemakersinagreementwithInternational FederationforAdiposeTherapeuticsandScience(IFATS)and theInternationalSocietyforCellularTherapy(ISCT) recom-mendations[21].

2.7. Platelet-richplasma

PRP was produced according toa previously described cell therapymethodology [22,23]. Briefly,18cm3_{ofwhole blood}

was collected from the same healthy donor with 4cm3

adenosine citrate dextrose-acid solution (ACD-A, Ref. Fig.2–Experimentalirradiationprotocol.Thedorsalskinwasretractedfromtheunderlyingbonyskeleton,maintainedbytwo separatedsuturesbetweentwofieldsofPlexiglass.Thetwosutureswerereproduciblyspaced3cmapart(A),centredonthe midlineofthebackofeachmouse,soastoholdthedorsalskinofeachmouse1cmundertheplexiglassfield(B).Fourblocksof 8-cm-thickCerrobend(CerroMetalProducts,Bellefonte,PA,USA)wereplacedoneachedgeofthePlexiglasabovethemiceto protectthebodyandspinefromtheradiation.Theirradiationwasdeliveredatadoseof60Gyinonetargetedfractiononthe PlexiglasfieldbyalinearacceleratorElektasynergysystem(ElektaBeamModulator;ElektaOncologySystems,Crawley,UK) with6MVphotons.

BDB8651;Fenwal Inc.,Portland,OR,USA)tomakea22cm3

solution.Anothertube,coatedwiththeEDTAanticoagulant, was used to determine the number and concentration of platelets with an automatic cell counter (ADVIA1 2120; SiemensDiagnosticSolutions,Malvern,PA,USA).Theblood wascentrifugedat130gfor15minandthenagainat250g for15minintwo11cm3conicaltubes (NUNC1,Ref. 56423; ThermoScientific) using a microcentrifuge (Medilite1, Ref. 448; Thermo Scientific). The supernatant or platelet-poor plasma(PPP)wasremovedbygentleaspiration(approximately 1cm3_{/tube).ThePRPpelletswereresuspendedintheresidual}

PPPandpooled.Atthefinalmeasurement,weobtainedatotal of5.6cm3 concentrated PRP. A 250

m

lsample was used to determinethe finalPRPformulationwithanautomaticcell counter(ADVIA12120;SiemensDiagnosticSolutions, Erlan-gen,Germany).

2.8. Mixtures

MF,PRPandSVFwerepreparedinacelltherapylaboratory (Table1).Allproductswerepackagedundersterileconditions in1cm3_{syringesforreinjectionintothemice.Ringer'slactate}

wasusedasthemediumtoresuspendtheSVF.TheMF+SVF mixwasinourinitialhypothesisthemostefficientproduct evaluated. Thus, Ringer-Lactate, as a medium for SVF resuspension, was chosen as the placebo for the sham-injectiongroup.

2.9. Injections

Aftermixing whennecessary, the injectionprocedurewas performedblindlyundergeneralanaesthesiabyinhalationof halogenated gas (sevoflurane; Baxter France, Maurepas, France). Two diametrically opposed 18 G punctures were made5mm fromeachwound tointroducethe 21 Gblunt cannula for reinjection into the st’RIM pack (Thiebaud Biomedical Devices). A 1cm3 _{syringe of MF, MF+PRP, MF}

+SVForRinger'slactatewasinjectedusingtheconventional Coleman's procedure. The product was evenly distributed underthewoundineachmouse.

2.10. Animalexamination

Theweightofeachmouseandthesizeofthewoundswere measuredonthedaytheproductswereinjected,andevery monththereafterduringthe3-monthpost-treatmentperiod under general halogenated gas anaesthesia (sevoflurane; Baxter).Healingwasevaluatedbymeasuringstandarddigital photographs of the wounds using ImageJ 1.45s freeware (National Institutes of Health, Bethesda, MD, USA; http:// imagej.net/ImageJ); afixed landmark(millimetreruler)was includedineachphotograph[24].Thepercentageofhealing wascalculatedbyrelatingthemeasuredwoundareatothe area ofthe initialwound(100 ((wound area/initial wound area)100)).

2.11. Histologicalexamination

Blinded histological analyses were performed by a skin pathologist.Afterthemicewereeuthanised,theirskinwas

fixed in 10% formalin solution and embedded in paraffin. Then,5-

m

m-thicksectionswerestainedwithhaematoxylin and eosin, Masson's trichrome and orcein to evaluate the histopathological changes and detect collagen fibres. The sectionswereexaminedunderalightmicroscope(Axiophot; Zeiss,Oberkochen,Germany)andphotographedwithadigital camera. A semi-quantitative evaluation was performed to assesstheskinfatgrafts(0,notraceoffat;+,traceoffat;++,fat stillpresent)[22].Epidermal,dermalandtotalskinthicknesses weremeasuredquantitativelyintheskinexposedtoradiation onthewoundperipheryandinthehealedwoundarea.Skin thicknesseswerecalculatedusingGraphPadsoftware (Graph-PadInc.,LaJolla,CA,USA).Sixmeasurementsweremadeper mouse,andthemeanswerecomparedforeachexperimental condition.Asemi-quantitativeevaluationwasperformedto evaluate subcutaneous vascularisation using the following scale:0,capillariespresentinnormalnumbersandshowing normal morphology; +, increased number of congested capillaries;++,numerouscongestedcapillaries.

2.12. Statisticalanalyses

Allstatisticaldataareexpressedasmeansstandarderrorof themeans(SEM).Differencebetweengroupswereassessedby Kruskal WallisandMann WhitneyUtestsorNonparametric analysisoflongitudinaldata.AprobabilityvaluesofP<0.05 were considered significant. Bonferroni corrections were madeformultipletesting.DatawereanalysedusingnparLD packageofRsoftware(http://www.R-project.org/)andSPSS20 (IBM Corp.Released2011.IBMSPSSStatisticsforWindows, Version20.0.Armonk,NY:IBMCorp).

3.

Results

3.1. Clinicalmanifestationsoftheradiation

Irradiationinducedskinsclerosisandchronicwoundsinthe exposed dorsal skin. The irradiated dorsal skin appeared thickerandinduratedtothetouch.Alltreatmentgroupmice presented with wounds (wound area was 14415mm2 _on

average, no statistical difference between the 4 groups (P=0.563))at3monthsafterirradiationandshowedalossof bodyweightcomparedtohealthymice(irradiatedmicebody weight=29.650.41g; healthy mice body weight=31.37 0.31g,P=0.041).

3.2. Characterisationandinjectionofthedifferentcell therapyproducts

3.2.1. Stromalvascularfraction

We obtained 10.3cm3 _{of SVF from 184cm}3 _{harvested fat,}

correspondingto27.4millionofviablenucleatedcellsleading to274000viablenucleatedcellsinjected/mouse.Thecellular compositionoftheSVFisdetailedinTable1.

3.2.2. Platelet-richplasma

Weobtained5.6cm3_{PRPwithaplateletconcentrationof724G/}

l, correspondingto362millionofplatelets injected/mouse. The cellcomposition ofthe PRP isshown in Table1. This

formulation was similar to that previously reported for purifiedPRP[23,25].

3.3. Animalevaluation

Wenotedtwodeathsintheplacebogroup(at1and2months post-treatment),twodeathsintheMF+SVFgroup(at2and3 monthspost-treatment)onedeathintheMF+PRPgroup(at2 monthspost-treatment)andonedeathintheMFalonegroup (at 1 month post-treatment) during the 3 month post-treatment period. Body weight in the placebo group was 30.390.55g vs. 32.110.76g in the MF+SVF group, 31.76 0.75gintheMF+PRPgroupand31.890.53gintheMFgroup (NS,P=0.231)at3monthspost-treatment.

3.4. Woundhealingevaluation

Thewoundareainthesham-injectiongroupincreasedby91.1 71.1%onaverage( 11.029.5mm2

ofwoundareahealedon average, initial wound area: 129.227.2mm2_{) at 3 months}

post-treatment.Incontrast,thewoundsinalltreatedgroups hadpartiallyhealedat3monthsaftertreatment.Wereport that15.923.1%onaverageofthewoundareahealedintheMF group(NS)(61.042.7mm2_{ofwoundareahealedonaverage,}

initialwoundarea:147.633.0mm2_{onaverage),27.323.8%}

onaverageofthewoundareahealedintheMF+SVFgroup(NS) (39.524.2mm2 of wound area healed on average, initial woundarea: 176.527.2mm2

on average)and76.47.7%on average of the wound area healed in the MF+PRP group (P=0.032)(99.319.3mm2_{ofwoundareahealedonaverage,}

initialwoundarea:121.321.5mm2_)(Fig.3).

3.5. Histopathologicalchangesaftertreatment

Threemonthsfollowingtreatment,theregenerative epider-misofthetreatedmicewasthicker.Inparticular,thehealing marginsdisplayedmorehyperplasiacomparedtotheskinof the micethat had received sham injection. In the dermal granulationtissue,inwhichsmallclustersofadipocyteswere statedinthemicetreatedbyMF+PRP,highercellularitywas

Fig.3–Comparisonofwoundhealingfromirradiation-inducedskinulcersaftertreatmentwithasham-injection(Ringer's lactate),microfat(MF),MF+platelet-richplasma(PRP)andMF+thestromalvascularfraction(SVF)duringthe3monthfollow-up.

statedcomparedtoshaminjectedmice(Fig.4).Asignificant reduction in dermal thickness and epidermal thickness secondarytoskinsclerosisinducedbyradiationwasobserved intheperipheryofthewoundaftertreatmentwithMF,MF

+PRP, and MF+SVF compared to the placebo (P<0.05).No significantdifferencewasdetectedamongthethreegroups treated with cell-based therapyproducts. Data for dermal, epidermalandtotalskinthicknessesaftertreatmentwiththe Fig.4–Histologicalfindingsofthewounds,3monthsaftertreatmentbyMF+PRP(A)orsham-injection(Ringer'slactate)(B):the regenerativeepidermaltonguesarethicker,withmorehyperplasia,andthedermisdisplaysahighercellularityinthetreated group(A)comparedtosham-injection(B).Smallclustersofadipocytesareintegratedwithinthegranulationtissueofthewound treatedbyMF+PRP(yellowarrows).

MF+PRP,MF+SVF,MFandplacebo areshown inFig.5. No differences in scar thickness were observed between the differenttreatmentsandthesham-injection.Theappearance ofbloodvesselsandthedensityinareastreatedwithMF,MF +PRP and MF+SVF denoted an increase in vessel density, particularly in the MF+SVF group showing the greatest increaseindensityanddiameterofvessels(Fig.6).

3.6. Fatgraftviability

TheMFinjectedinthesubcutaneousplanewasstillpresentat 3monthspost-treatmentinallthreegroups.TheMFformed homogeneous, adherent and well-vascularised fat nodules underthepanniculuscarnosus(Fig.7).

4.

Discussion

Radiodermatitisischaracterisedbyfibrotictransformationof thesubcutaneoussoft-tissuesassociatedwithadecreasein cutaneousvascularisation[26,27].Immediatelyafter irradia-tion,acuteradiodermatitisisassociatedwithdermal inflam-mationandepidermalinvolvement.Thechronicityofdermal

inflammation is responsible for the fibrotic changes and epidermalthickening,whichleadtoepidermal hypovascular-isation,skinulcerationsandnecrosis[28].Medicalor acciden-tal exposure ofalarge area aswellas elevated“hotspot” radiationexposureoftheskinleadtosevereskindamages named“cutaneousradiationsyndrome”(Hopwell).Irradiation severlyimpairswoundhealing,dependingonthe radiation dose,depthofpenetrationandquality.Ourmodelisrelevant to the human accident situation of large area irradiation exposure. In this situation,the skinulceration observedis irremediablyextensive[29]asinourmurinesham-injected group.Treatmentoftheseradiation-inducedwoundspresents a challenge for clinicians.Actually, autologousfat grafting seemstobethemoreefficientproceduretoattenuatethese lesions[10,11,30]inalternativetoradicalskinexcisionanduse ofacover flap.However,furtherexperimentaland clinical studieshaveshowninterestingeffectsofusingPRPandSVFto treatthesecutaneousradiationlesions.

Ourradiation-inducedskinlesionmodelusedwasadapted frompreviousexperimentalstudies.Thaniketal.andSultan et al. described a murine model of dorsal radiodermatitis carriedoutafteraone-timedoseof50Greysand45Greysof radiationtherapyrespectivelyviaalinearaccelerator[2,9].In Fig.5–Comparisonofepidermal,dermalandtotalskinthicknessesat3monthsaftertreatmentbysham-injection(Ringer's lactate),MF,MF+PRPandMF+theSVFversusthehealthycontrolgroup.

ourstudy,aone-timedoseof60Greyswasdeliveredbylinear acceleratortoobtainalatedorsalskinnecrosis,ulcerations andwounds12weeksafterradiationwithareducedmortality. However,alimitofthisimpairedwoundhealingmodelisthe contractionofthemurinepanniculuscarnosusleadingtothe woundcontraction,absentinphysiologichumanhealing.In fact,inconventionalmurinemodelsofwoundhealing,a full-thicknesswoundisperformedwithpunchbiopsyandwound contractionisprevented withdonut-shaped silicone splint fixedaroundthewounds[30].Inourmurinewoundmodel,the irregular and unpredictable shape of the radiodermititis woundsdidnotallowtheuseofthosesplints.

Sultanetal.evaluatedpreviouslytheeffectoffatgrafting onradiationskindamagesinasimilarmurinemodel.Four weeksafterirradiation,theauthors reportedoverabundant collagen deposits resulting in dermal thickening and a decreaseinskinmicrovascularity.Fourweeksaftertreatment by fat grafting or by sham injection, they reported an

epidermalthicknessesof36.306.1

m

minthesham-grafted group versus 8.270.64

m

m in the fat-grafted group [9]. Comparatively,Thaniketal.evaluatedanovelmousemodel of cutaneous injury and reported 6 weeks after 45-Grey irradiationanincreaseofdermalthicknessandskinfibrosis

[2]. In our study, we reported 6 months after a 60-Grey irradiationthus3monthsaftertreatment,athickeningofthe epidermis(epidermalthickness=7027

m

m)andthedermis (380121

m

m) in the sham-grafted group compared to the healthycontrolgroup(epidermisthickness:221

m

m;dermis thickness: 1736

m

m) and the treated groups (epidermis thickness: 20 to 242

m

m; dermis thickness: 163 to 191 21

m

m), revealing a decrease of skin fibrosis in the 3 irradiatedandtreatedgroups.

Asubcutaneousinjectionoffatisasimpleandvalidated treatmentforirradiation-inducedcutaneouslesions.Mojallal etal.showedanincreaseinextracellularmatrix,collagenfibre and neovascularisation around the transplanted fat [31]. Fig.6–Skinvascularisationaftertreatmentbysham-injection(Ringer'slactate),MF,MF+PRPandMF+theSVF.Thenumberand diameterofdermalcapillariesincreasedaftertreatmentwithMF+SVF(left)incomparisontotheshaminjection(right).

Phulpinetal.showedimprovedskinqualityafter subcutane-ously injecting autologous fat in 11 patients with chronic radiodermatitisoftheheadandneck[11].Theyobservedan improved vascularnetworkinthetreatedskinassociated witha decreaseintheirradiatedmorphologicalpatternsandnoskin ulcerations.Inasimilarstudy,Rigottietal.showedimproved healingin10of11patientswiththoraciccutaneousulcerations secondarytoirradiationafterasubcutaneousautologousfat transfer [10]. The histological analysis showed improved neovessel formation and dermal hydration. The authors concludedthattheadipose-derivedstemcells(ASCs)contained in theSVF of the fat weretheprincipalskin-regenerating agents. TheactionofASCsinthetreatmentofradiodermatitislesions hasbeenstudiedpreviouslyinpigs[20,32].Inthesestudies, injecting autologous ASCs locally into the skin lesions de-creased the subcutaneous defects and muscle fibrosis in comparisontothecontrols.Theauthorsreportedadecrease in disorganised myofibres,associated with a decrease in the size ofthe fibroticand necroticareas,after ASCs wereinjected comparedtothecontrol.Iddinsatal.usedtheSVFfromfat,a concentrateofgrowthfactorsandnon-expandedASCs,ina clinical case report. TheSVF was used in association with 0.2cm3ofMFafter allreferencetreatmentsfailedtotreata chronic ulceration of the thumb secondary to accidental irradiation[33].Themixturewasinjectedinthesubcutaneous planeofthepulp.Thewoundhealedcompletelywithin290 days,withaprominentdecreaseinfingerparaesthesiaduetoa decreaseinsubcutaneousfibrosis.Inourexperimentalstudy, theSVFdidnotshowsuperiorefficacytotheotherproducts withrespecttoulcerhealing.However,theskintreatedwithMF +SVFshowedagreaterincreaseinvasculardensitythanthat treatedwiththeothermixtures(Fig.7).

PRPseemstobeeffectiveforhealingchronicwounds.Once activated,thePRPsecretesmorethan30pro-healinggrowth factors [34]. Fujita et al. demonstrated the efficacy of PRP injections intoskin lesionsinamurine modelofimpaired woundhealingafterirradiation[35].Theyshowedasignificant decrease in the ulcer at 15 days after the PRP injection associated with a significant increase in the number of neovesselsandcollagenformationcomparedtothecontrol. Inourstudy,injectingtheMF+PRPmixturewasmoreefficient forincreasingwoundhealingcomparedtoMFaloneorMF +SVF(Fig.4).At3monthsaftertheinjection,thehealingwas significantly better inthe MF+PRPgroup compared to the sham-injected group (P<0.001). However, we observed an importantvariationofthewoundsizeinthesham-injection group.Therandomisationofthemicedistributionensuresno selectionbias.AsreportedbyAgayetal.inaMinipigmodelof cutaneousradiationsyndrome,theskinulcerationappeared variableandextensive,principallyinthecontrolgroup[29]. Thus,microfattreatmentseemstodecreasethepost irradia-tionulcersizevariability,promotingwoundhealinginthe3 treatedgroups.

Theeffectivenessoffat,PRPandSVFhasbeenshownin severalanimalmodels,andafewclinicalcasestudies,asa treatmentforskinlesionsinducedbyradiation.Inourstudy, weassessedMFaloneandMFinassociationwithPRPorSVF. PRPandSVFarefluidproductsthatdiffuserapidly.Aprevious studybyourlaboratorydemonstratedthesuperiorityofthese productswhenusedinassociationwithMF[36].PRPandSVF seemtostayinthesubcutaneousplaneforalongerperiod, which couldexplaintheincreasedeffectiveness.Ourstudy suggeststhatMF+PRPisthemostefficientproductforwound healing,withawoundhealingrateof81%comparedto16% and21%forMFandMF+SVF,respectively.However,theinitial wound area inthe MF+PRP group (121.321.5mm2) had a tendencytobeinferiortotheMF+SVFgroup(176.527.2mm2₎

withoutsignificantdifference(P=0.434,Kruskal WallisTest), despite the initial randomisation. This tendancy could be pejorativefortheSVFeffectcomparedtothePRPeffect.This possiblebiascouldbeavoidedinafutureexperimentalstudy withadifferentdesignusingeachmouseasitsowncontrol. All threeproductsdecreasedskinsclerosissimilarly.MF wasstillpresentat3monthsafterinjectioninthethreetreated groups. The clinicalresults obtainedwithSVF were disap-pointing and were not significantly superior to MF alone. However,asuperiorrateofneoangiogenesiswasreportedin thegrouptreatedwithSVF.ThelowclinicalimpactofSVFon woundhealingcouldbeexplainedbythenon-optimalmixture of 10% SVF and 90% MF. A future study with a 50%/50% proportionofMFandSVFshouldbeperformedusingthesame radiation-inducedskin-lesionmodel.

5.

Conclusion

Inthisstudy,amixtureofMFandPRPprovedmostefficientto increasehealinginachronicimpairedwoundhealingmodel after skin radiation. MF alone and MF+SVF resulted in increasedhealingrates comparedwiththesham-injection, but not significantly. An increase in neoangiogenesis and decreaseinsubcutaneoussclerosisseemedtobetheprincipal Fig.7–Subcutaneousfatviabilityat3monthsaftertreatment

mechanism of action of these new combined cell-based therapies.

Financial

disclosure

Theauthorsreportednoconflictsofinterest.Allaspectsofthis studywerefundedbytheFondationdel’Avenir(RMA 2015-004).

Reviewers’

names

-DrBarbaraHERSANT,PlasticSurgeon,Paris,AP-HP,Hôpital deCréteil,FRANCE

- DrNathalieKERFANT,PlasticSurgeon,CHUdeBrest, FRANCE

- DrBenoitCHAPUT,PlasticSurgeon,CHUdeToulouse, FRANCE

- PrAliMOJALLAL,PlasticSurgeon,CHUdeLyon,France

Declarations

Ethicsapprovalandconsenttoparticipate.

Thisexperimental study was approved by the National AnimalCareandEthicsCommittee(#00506.02).

Consent

for

publication

Notapplicable.

Availability

of

data

and

material

Thedatasetsusedand/oranalysedduringthecurrentstudy areavailablefromthe correspondingauthoronreasonable request.

Competing

interests

Theauthorsdeclarethattheyhavenocompetinginterests.

Authors’

contributions

BB: conception, drafting, final approval, agreement to be accountableforallaspectsofthework.

JE:conception, drafting, finalapproval,agreement tobe accountableforallaspectsofthework.

MV:conception,revision,finalapproval,agreementtobe accountableforallaspectsofthework.

CC: design, revision, final approval, agreement to be accountableforallaspectsofthework.

NM:conception,revision,finalapproval,agreementtobe accountableforallaspectsofthework.

MB:conception,revision,finalapproval,agreementtobe accountableforallaspectsofthework.

MAL: drafting, revision, finalapproval, agreement tobe accountableforallaspectsofthework.

DC:conception,revision,finalapproval,agreementtobe accountableforallaspectsofthework.

JM: drafting, revision, final approval, agreement to be accountableforallaspectsofthework.

FS:conception, revision,finalapproval,agreementtobe accountableforallaspectsofthework.

Acknowledgements

WewouldliketothankDoctorLucileAndracforperforming theinitialhistologicalanalysis.Wewouldalsoliketothank ProfessorGuyMagalonandProfessorDidierCowenfortheir helpincarryingoutthisstudy.

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