Content uploaded by Mehra Nazempour
Author content
All content in this area was uploaded by Mehra Nazempour on Sep 26, 2019
Content may be subject to copyright.
J Cosmet Dermatol. 2019;00:1–7. wileyonlinelibrary.com/journal/jocd
|
1
© 2019 Wiley Periodicals, Inc.
Received:24April2019
|
Revised:2 0July2019
|
Accepted:23July2019
DOI:10.1111/jocd.13109
ORIGINAL CONTRIBUTION
The effect of allogenic human Wharton's jelly stem cells
seeded onto acellular dermal matrix in healing of rat burn
wounds
Mehra Nazempour MSc1 | Davood Mehrabani PhD2,3,4,5 |
Rouhollah Mehdinavaz‐Aghdam PhD6 | Seyedeh‐Sara Hashemi PhD3 |
Amin Derakhshanfar PhD4 | Shahrokh Zare MSc2 | Mitra Zardosht MSc3 |
Javad Moayedi MSc4 | Mahjoob Vahedi DVM4
1Depar tmentofBiomedicalandTissue
Enginee ring,S ciencea ndResearch
Branch,IslamicAzadUniver sity,Tehran,Iran
2StemcellTechnologyResearch
Center,Shir azUnive rsityofMedic al
Science s,Shir az,Iran
3BurnandWoundHealingRese arch
Center,Shir azUnive rsityofMedic al
Science s,Shir az,Iran
4Compar ativean dExpe riment alMedicine
Center,Shir azUnive rsityofMedic al
Science s,Shir az,Iran
5Depar tmentofPathol ogy,Unive rsityof
Alber ta,Edmonton,Alber ta,C anada
6SchoolofMetallurgya nd
MaterialsEngine ering ,Collegeof
Enginee ring,U niversityofTehran,Tehran,
Iran
Correspondence
DavoodMehrabani,StemcellTechnolog y
ResearchCenter,Shi razUniversit yof
MedicalSciences,Shiraz,Ir an.
Email:mehrabad@sums.ac.ir
Rouholl ahMehdinavaz‐Aghdam,S chool
ofMetallurgyandMater ialsEngineering,
CollegeofEnginee ring,U niversityofTehran,
Tehran,Iran.
Email:mehdinavaz@ut.ac.ir
Funding information
Nationa lInstituteforMedicalResearch
Develop mentofIr anMinis tryofH ealth ,
TreatmentandMedic alEducation,G rant/
AwardNumber:963 474
Abstract
Background: Variousmethodswereintroducedtoovercometheautograftshortage
inburnwoundcare,includingcelltransplantationandtissueengineering.
Aims: Toevaluatethe healing effect of allogenic human Wharton's jellystem cells
(hWJSCs)seededontoacellulardermalmatrix(ADM)inratburninjuries.
Patients and Methods: Human Wharton's jell y stem cells provided f rom umbilical
cord tissue were characterized before transplantation, and the growth kinetic was
determined. Skin samples from cosmetic surgeries were used for preparation of
ADM.FortymaleSprague Dawley ratswererandomly dividedinto4 equalgroups.
Third‐degreeburnwasinducedforallanimalsbyexposingtohotwaterusinga2cm
ring for 10seconds. Group1 was burnedratsthat did not receive any treatment.
Afterburninjur y,thesecondgroupreceivedsilversulfadiazine(SSD),thethirdgroup
was treated jus t by using ADM, and the four th group received 2 × 106 hWJSC s
seededontoADM.Theanimalswereeuthanizedforhistologicevaluationafter7,14,
and21days.
Results: HumanWharton'sjelly stem cells were characterizedto be spindle shape
andpositiveforosteogenicandadipogenicinductionandformesenchymalmarkers
butlacked hematopoietic markers. Population doubling time (PDT)was 40.1 hours
withanincreasinggrowthtrenduntilday6th.Macro‐andmicroscopically,theheal‐
ingwasmildinADMgroupandmoderateinADM+hWJSCsgroupafter21days.
Conclusion: Allogenic hWJSCs seeded ontoADM improved thehealingprocessin
burn wounds d enoting to their ther apeutic and anti‐inflam matory effec ts in burn
woundsthatcanbeaddedtotheliterature.
KEY WORDS
acellulardermalmatrix,burn,healing,Wharton'sjellystemcells,wound
2
|
NAZEMPOUR Et A l.
1 | INTRODUCTION
Burnisresponsibleforabout265000globaldeathsperyearandis
stilloneofthemosttraumaticinjuriesworldwidewithpublichealth
concernleadingtosocialandeconomicconsequences.1Burninjury
ischaracterizedbydestructionofskinstructures,functions,andloss
of progenito r cell populat ions that are nec essary fo r regeneratio n
andrestorationof the skin.Burnwoundscandelayanddisruptthe
cic at rici alprocesses,andin creasethech an ce soffu nc tional an daes‐
thetic se quelae and ar e responsible f or formation of sc ar and ke‐
loidduetoscarcontractureanduncontrollableproliferationofscar
tissue.2
Therefo re, an effecti ve wound manageme nt has an import ant
roleinburntherapyfor timelyandpermanentclosure offull‐thick‐
ness burnwounds. Graftingtechniques such as split‐thicknessand
full‐thickness graftsarewelldevelopedto improvetheappearance
and function of the lesions, eventhe effectiveness of thesemeth‐
ods is limite d because of t he availabilit y of donor ski n in patients
with large total body surface area (TBSA) burns, and the fact that
burn patients may experience a significant delayinwoundclosure,
infection,scarring,anddeath.3Toovercometheautograftshortage
in burn wou nd care, var ious alternat ive methods we re introduce d
including allogeneic and synthetic skin substitutes and xenograf ts,
eventheyaretemporar yforwoundcoverageanddeliverdifferent
bio‐factorstofacilit atethe angiogenesis and granulationof wound
bedforfurthersurgery.4
Burninjurycan bedeepenedbyinfection,andtherebyincrease
thetotaltissuedamageandtheriskofcomplicationssuchashyper‐
trophicscarring.5 Soitisnecessarytodevelopadjuvanttreatments
withantimicrobialpropertiessuchassilversulfadiazine(SSD).6 SSD
hasbeen introduced asagoldstandardwithanenviable safety re‐
cord in bur n treatment with an timicrobial prop erties, but i ts side
effects suchas leukopenia werereported as disadvantages of this
medication.7 In past d ecades, cell t ransplant ation has emerge d as
anoth er po pu la roptionfo rimprovedburnwou ndhealingan dregen‐
erationofskinstructureandfunc tions.8
Thetherapeuticroleofmesenchymalstemcells(MSCs)inheal‐
ing is the topic of interest, because they have the ability to differ‐
entiate into mesodermal, ectodermal, and endodermal cells with
lowimmunogenicity and paracrine activities.8 MSCs were isolated
from varioustissuesincludingbone marrow,9adipose tissue,10 and
dentalpulp.11They are easily cultured and grown, take more time
tobecome senescent andexpressMSCmarkers,and lack hemato‐
poieticmarkers.12The transplantationofMSCsinburninjuries was
demonstrated toimprovescarqualityby earlyclosureof thelesion
andto promoteregenerationoftheskinanditsappendagesandto
attenuatetheinflammatoryprocess.13
The Whar ton's jelly‐like matrix of the umbilical cord is a rich
sourceofMSCscalledWharton'sjellystemcells(WJSCs)thathave
a high immun omodulator y activity, simil ar to bone marrow MS Cs
(BM‐MSCs).Currently,WJSCs have clinical application for a broad
scope of diseases.14 Human Wharton's jelly stem cells (hWJSCs)
haveproper tiessuchasrobustproliferationanddifferentiation,and
weak immunogenicit y,thereby posing a great potential in thefield
ofregenerativemedicine.15MSCscanbeseededontoseveralscaf‐
folds prov iding a three‐d imensional env ironment for th e MSCs to
grow and improvethe reconstruction of the injuredtissues.These
scaffoldswereshowntopossessproper ties such asporosit y,cyto‐
compatibility,biodegradability,surfacephysicochemical,andbiome‐
chanicalactivities.16
Tissue engineering by using MSCs seeded onto the scaffolds
aimstooptimizetheaestheticandfunctionalreconfigurationofthe
skin with t he potential in t reatment of bur n injuries, wou nd heal‐
ing, skin regeneration, reducing inflammatory responses, and im‐
provingsc arring.17G reat advance s in dermal sca ffolds resul ted in
introductionofacellulardermalmatrix(ADM)tobeextensivelyused
inreconstructivesurgery.18Itisapopulardermal scaffoldmaterial
commonlyusedfortreatmentofburninjuries.19
It was first introduced in 1995 in treatment of full‐thickness
burninjuries20 andlaterinotherclinicalsettingstorepairwounds.
Several surgeonshave startedcollaboration inordertoincorporate
tissueengineeringandstemcellsasanefficacioustreatmentoption
withthehope tobeimplementedwithlowrisk, lowmorbidity,and
withmorebenefitsoverconventionaltreatment.17, 19 Therearesev‐
eral challenges to the use of stemcellsalone in burninjuries to be
adminis tered topic ally or intr avenously. This s tudy was cond ucted
toevaluate the healing effect of allogenichWJSCsseeded onto an
ADMscaf foldinburninjuriesinexperimentalratmodel.
2 | SUBJECTS AND METHODS
2.1 | Animals
Forty male Sprague Dawley rats (180‐200 g) purchased from
Laboratory AnimalCenterofShirazUniversityof MedicalSciences,
Shiraz, Iranwereenrolled. The animalswerekept singlein cagesat
controlledtemperature (22.0±2.0°C) and lighting (12hours light/
darkcycles)andhadfreeaccesstofoodandwater.Ketamine(5mg/
kg;Woerden, Netherlands)andxylazine(20mg/kg;Alfaz yne)were
usedintraperi toneallytoa nesthet izetheanima lsd uringallinter ven‐
tions. Pos tburn analge sia was provided b y subcutaneo us adminis‐
tration of 0.0 5 mg/kg bupre norphine, th ree times dail y (Produlab
Pharma) until sacrifice. Throughout the experiment,any changesin
theskin, complications and infection were recorded.Therats were
euthani zed after 7, 14, and 21 days po stintervent ions. All expe ri‐
mentswereundertakenbasedonIranVeterinar yOrganizationrules
andregulationsforworkingwithlaboratoryanimals.Thisstudywas
financi ally suppor ted and ethica lly approved by Nat ional Institu te
for Medical Research Development of Iran Ministry of Health,
TreatmentandEducation(Grantno.of963474).
2.1.1 | Isolation of hWJSCs
HumanWhar ton'sjellystemcellswereprovidedfromumbilicalcord
tissuebyenzymaticdigestion asdescribedbefore.14Briefly,umbili‐
calcordsamples(n=3)wereprovidedfromhealthyvolunteersafter
|
3
NAZEMP OUR Et A l.
receiptofaconsentletter.TheywereasepticallytransferredtoBurn
andWoundHealingResearchCenterofShirazUniversityofMedical
Sciences,Shiraz,Iraninphosphatebuffersaline(PBS:Sigma‐Aldrich)
containingpenicillin, streptomycin,andamphotericin B(100U/mL,
100µg/mL,and0.25µg/mL,respectively;Invitrogen).After3times
washing with Hanks' balanced saltsolution (Invitrogen), theywere
horizontally cut into1‐cm pieces with eachpiece cut open length‐
wiseandplacedwithitsinnersurfacefacedownintoenzymaticso‐
lution consistedof collagenase typeI,collagenase typeIV(Thermo
FisherScientific),andhyaluronidase(Sigma‐Aldrich).
Theywerelatertransferredin5%CO2incubatorat37°Candsat‐
uratedhumidity for 45 minutes. The enzymaticsolution resulted in
dissolve d gelatinous mas ses of Wharto n'sj elly. It was then filt ered
th r o u gha s y r i nge o f18 ‐ gaug e n eedl e t ofu r th e r brea k u pthe r e main e d
ge l a tino u s mass e s a ndt o f a cil i t a tet h e r elea s e ofc e l lsf r o mthe m a sses.
Itwascentrifuged,and the viablecellswere plated at a densityof1
millioncells/cm2in100‐mmcellculturedishesadding90%Dulbecco's
ModifiedEagle'sMedium/F12(DMEM/F12,Invitrogen)with10%FBS
(Gibco), 1%nonessential amino acids (Invitrogen), 2 mmol/L L‐gluta‐
mine, (Invitrogen) and1%penicillin, streptomycin, andamphotericin
B. Afte r 5 days, the medium w as replaced and s ubsequent cult ure
mediawaschangedevery3day s.T headhere ntcell sweretransferred
to5%CO2incubatorat37°Candsaturatedhumidity.Cellcounting
wasconduc tedusingtrypanbluedyeandNeobarhemocytometer.
2.2 | Characterization of WJSCs
Human Wha rton's jelly s tem cells were ev aluated mor phologic ally
for being spindle shape. They were assessed for in vitro osteo‐
genic induction seeding 5 × 104WJSCsina12‐wellplatecontain‐
ingDMEM, supplementedwith 10% FBS, and osteogenicmedia of
200 μmol/L L‐ascorbicacid (Sigma),10 mmol/L glycerolphosphate
(Sigma),and100nmol/Ldexamethasone(Sigma).Themediawasre‐
placed for 3 weeks every3days. The dif ferentiation was assessed
byalizarinredstaining(Sigma)thatis boundtocalciummineralized
depositsandrevealingaredcolor.
Toevaluate in vitroadipogenicproper ty,5× 104 hWJSCswere
seeded i n a 12‐well plate in DMEM supp lemented wit h 10% FBS,
containingadipogenic medium of 1 μmol/L dexamethasone,5μg/
mLinsulin(Sigma),0.5mmol/Lisobutylmethylxanthine(Sigma),and
60 μmol/Lindomethacin(Sigma)for21days,whilethemediumwas
changed every 3 days. The cells were later stained with Oil red O
(Sigma) revealing red color droplets. Flow c ytometry was under‐
taken on 3rdpassageofhWJSCstoassess the expression of mes‐
enchymal surface markers for CD73 and CD90 and hematopoietic
surfacemarkersofCD34,andCD45(Dako,Denmark).
2.3 | Growth kinetics
Atotalof4×104hWJSCsfrompassage3seededin24‐wellculture
platesweretransferredin5%CO2incubatorat37°Cand saturated
humidity,while the media was changed every3days. Cellviabilit y
wasevaluated eachdayfor7 days using 0.4% tr ypan bluesolution
(Biowest ), a Neubauer hem ocytomet er slide and a phas e contrast
microscope. The population doubling time (PDT) was determined
by plotting the grow th curve and using the formula of PDT = T
ln2 = ln(Xe/Xb); while T was incubationtime in hours, Xb was cell
numberatthebeginningofincubationtime,andXewascellnumber
attheendoftheincubationtime.Themeannumberofcellsateach
time point wasplottedusing GraphPadPrism (GraphPad Software
Inc).Cellswerecr yop re ser vedata de nsityof 1×106c ells/mLin10 %
dimethyl sulphoxide (DMSO;MPBio USA) (V/V),50% fetalbovine
serum(V/V),and40%DMEM.
2.4 | Acellular dermal matrix (ADM) preparation
The human s kin sampl es were asepti cally pre pared from co smetic
surgeries of Depar tment of Plastic Surgery, Shiraz University of
Medica l Sciences, S hiraz, and t ransfer red to Stem Cell L aborator y
of Burn and Wound Healing Research Center af filiated to Shiraz
University of Medical Sciences. The tissue samples were kept in
PBS, whil e penicillin‐s treptomycin an d fungisone wer e added. For
washing,0.5%TritonX‐10 0and10mmol/LEDTA(Sigma)wereused
2times.Allepidermal,fattissues,andhairswereseparated,andthe
re m a i n e dtis s u e w ask e p tat −2 0 ° C u n t ilu s e . To remov e e pide r m a l t is‐
sue,1MNaCl,0.5%TritonX‐100and10mol/LEDTAsolutionwere
appliedinashakingincubatorat37°Cfor24hours.Decellularization
wasconduc tedusing0.5%sodiumdodecylsulfate(SDS),10mmol/L
HEPES, an d 10m mol/L EDTAso lutions (Sigm a)i n an incubator at
37°C for 1 ho ur.Be fore lyophiliz ation, peni cillin‐str eptomycin and
fungisonewereaddedtothesolution.
2.5 | Experimental design
FortymaleSprague Dawley 180‐200g ratswererandomly divided
into 4 equal g roups. Third‐ degree burn i nduction w as undert aken
for all anim als by exposin g to hot water using a 2 cm ring for t en
seconds.InGroup1,ratswereexposedtohotwaterfor10seconds,
without a ny treatment meas ure. The second gro up received SSD
ointment after burn induc tion, the thirdgroup wastreated just by
ADM scaffold, andt hefour thgroup underwent treatment by cell
tra nspla ntationof2×10 6hWJSC sseed edontoADMsc af fold.After
treatment,theanimalswereeuthanizedformacroscopicandhisto‐
logicevaluation.
2.6 | Histologic assessment
Histolog ic assessment of A DM was done before l yophilization by
fix ationoftiss uesam plesi n4%buf fere dfo rmali n.Theywe resubse‐
quentlyembeddedinparaffin.Tissuesections(5micronsthickness)
wereprepared and stained withhematoxylin and eosin(H&E), and
Alcian bl ue. Skin sam ples were fi xed in 10% formali n and embed‐
dedinparaffin.Specimenswerelatersectioned(5μmthickness)and
dried over night at 37°C . Sectio ns were depar affinize d, rehydrate d
withagradedethanolseries,andstained withH&Eformicroscopic
evaluation(Olympus).
4
|
NAZEMPOUR Et A l.
3 | RESULTS
3.1 | Characterization of WJSCs
Morphologically,WJSCswere adherent to the culture flasks and
werespindleshape(Figure1).Theosteogenicinductionpropert y
ofthecells was confirmed by Alizarin red staining after3weeks
dueto the presenceofcalcium depositsinredcolorintheflasks
(Figure 1). Oi l Red O staining of WJSCs af ter 21 days revealed
adipogenicinduction of the cellsinred color droplets(Figure 1).
Under fl owcytometr y, th e cells were positive fo r expression of
mesenchymalmarkersincludingCD73andCD90,andwerenega‐
tiveforexpressionof hematopoieticmarkers ofCD34andCD45
(Figure1).
3.2 | Growth kinetics
PDT of the 3rd pas sage of hWJSCs for seven days was s hown in
Figure2revealingaPDTof40.1hours.Therewasanincreasingtrend
incellmultiplicationuntilday6,andthenadecreasewasnoticed.
FIGURE 1 HumanWhar ton'sjellystemcellsindifferentpassages.A,Passage1(40×),B,Passage2(40×),C,Passage3(40×),D,Control
inpassage3(40×).E,Osteogenicdif ferentiationofhWJSCsafter21dusingAlizarinRedstainingdenotingtopresenceofcalciumdeposits
indifferentiatedcells(40×).F,AdipogenicdifferentiationofhWJSCsafter3wkusingOilRedOstaining(40×).G,FlowcytometryofhWJSCs
showingthepositiveexpressionofmesenchymalmarkers(CD73andCD90)andthenegativeexpressionofhematopoieticmarkers(CD34
andCD45)
(A) (B) (C)
(D)
(G)
(E) (F)
|
5
NAZEMP OUR Et A l.
3.3 | Histologic evaluation
Macroscopicallyingroup1 (leftuntreatedaf terburninjury),thele‐
sion size was 14.6%, d ecreased by 5 4.1%in g roup 2 (SSD group),
71.3%ingroup3(justADMscaffold),and87.6%ingroup4(hWJSCs
seededontotheADM).AsFigure3showsinADMsamples,H&E(A)
andAlcian blue(B)staining methodsconfirmed presenceofhyalu‐
ronicacid(acidicglycosaminoglycans)inthescaffolds.After3weeks
in group 1 (C), v asodilatio n, conjest ion, hemor rhage, infi ltration of
inflammatorycells,andextensivenecrosisandthelackofepidermal
layerwerenoted.
Ingroup2(D)receivingSSD,conjestion,infiltrationofinf lamma‐
torycells,andformationofgranulationtissuewerevisible.Ingroup
3(E)receivingADMalone,infiltrationoffewinflammatorycells,an‐
giogenesis, formation of granulation tissue, par tial epithelialization
revealing a m ild healing pro cess were obser ved. In group 4 usin g
WJSCsseededontoADM(F),angiogenesis,formationofgranulation
tissue, epithelialization revealing a moderate healing processwere
seen.
4 | DISCUSSION
Thefirstrepor tedapplicationofstemcelltherapyinburncare was
byShumakovetalin2003comparingallogenicandautologousBM‐
MSCswithembr yonicfibroblast sinburnwoundsof40ratsshowing
thatstemcelltransplantationcouldexpeditethewound healing.21
Their anti‐inflammatory, immunomodulatory, angiogenic, and re‐
generativeactivitiesinhealingofburnwoundsforskinandskinap‐
pendagewereillustratedlater,22whiletheycouldimprovet helevels
ofcollagendeposition,granulationtissue formationinburninjuries
too.23
Itwasshownthattheadministered dosageof MSCshasan im‐
por tantroleinef fi ca cyofce llthe ra pyofbu rnwounds.24Karim ietal
report edusing5×106adipose‐derivedstemcells(ADSCs)inhealing
ofacuteburninjuries.24Thetherapeuticefficacyofautologousand
allogenicADSCshasbeencomparedinhealingofacuteburninjuries
showing th at autologous st em cells were supe rior in wound hea l‐
ing.25Inou rs tud y,2 ×10 6all og enichWJ SC sse ede do nt oA DMinrat
burninjuriescouldacceleratewoundhealing.Ourfindingsweredif‐
ferent frompreviousrepor ts thatrevealedautologousMSCs were
superiortoallogenicstemcellsinwoundhealing.26
Inburn injuries,MSCs have been applied fromvarioussources
of autologous, allogeneic, or xenogeneic and in conjunction with
FIGURE 2 GrowthcurveandPDTofhWJSCs
FIGURE 3 HistologicevaluationofADMscaffoldbyA ,H&EandB,Alcianbluestaining.Blueregionsdenotetomatrixsecretion(Black
arrowsdenotetoglycosaminoglycans,400×).C,Group1receivednotreatmentafterburninductionshowingnecrosisandabsenceof
epidermallayerafter21d(40×).D,Group2receivedSSDafterburninductiondemonstratingnecrosis,inflammation,andformation
ofgranulationtissueafter3wk(40×).E,Group3whenjustADMwasappliedaf terburninductionrevealsangiogenesis,formationof
granulationtissue,andimprovedepithelializationleadingtoamildhealingprocessafter21d(40×).F,Group4thathWJSCswereseeded
ontoADMafterburninductiondisplayedepithelialization,angiogenesis,andformationofgranulationtissueresultinginamoderatehealing
process(40×)
(A) (B) (C)
(D) (E) (F)
6
|
NAZEMPOUR Et A l.
differentscaffolds.26Whenscaffoldswereaddedtocelltherapyin
burninjuries,afasterandbetterwoundhealingweredemonstrated.
Orbay et al s howed that ADSCs se eded onto ADM result ed in a
better implantforwoundvascularization,volumemaintenance,and
collagenquantity.27AftertopicalapplicationofBM‐MSCs together
with fibrin glue, anaccelerated burnhealing with skin appendages
wasseeninratswithscaldburns.28
In burn wou nds treated wit h ADSCs in conjun ction with atel o‐
collagen matrix with silicone membrane (ACMS), an advanced gran‐
ulationtissueandcapillaryformationwereobserved.29 Motamedet
al evaluate d regeneratio n of burn tissue us ing ADSCs see ded onto
the human a mniotic membr ane and noticed an a ccelerated woun d
healinginratswiththelowestinflammatorycellinfiltrateafter7and
14day s.30Inpor cin em ode lo fb urn injur y, BM‐ MC S sa nds ki n‐d eri ve d
keratino cytes in co njunction w ith human amni otic membra ne were
compared.Itwasshownthatthetransplantedcellscouldimproveep‐
ithelialization,angiogenesis,andthe amountsofcollagendeposition
andgranulationtissueandacceleratethehealingofthewound.31
Autologous ADSCs together with absorbable human cellular
collagenmatrixweredemonstratedto recruitendothelialcellsand
enhance formation of vascular network andimprove thetissue re‐
modeling and angiogenic dynamics, and increase the amount of
granulation tissue.32 Another study used ADSCs seeded onto a
collagen sc affold in b urn injur y and repor ted increas ed neovascu‐
larization,acceleratedmaturationof the wound,increased collagen
deposition,anddecreasedwoundbeddepthafterthreeweeks.33
Inour study,seedingofWJSCsontothe ADM scaffoldresultedin
animprovedangiogenesis, and granulation tissue formation, and de‐
creasedinflammation,necrosisandfibrosisafter21days.Similartoour
findings,an intensereductionininflammationand fibrosiswas exhib‐
it edw hen ADS C sw ere tran spl ant edi nbu rnw oun d s.34A ls oaf t ert r ans‐
plantationofMSCs, a reduction in necrosis (20%) in comparisonwith
thecontrolgroup(100%)wasdemonstratedinacuteburninjuries.35
Theanti‐inflammatoryroleofMSCsinburn skin tissues can be
explainedbydownregulationofinflammatorymarkers,upregulation
of anti‐inflammatory markers and a local increase in anti‐inflam‐
matory cytokines.36 The immunom odulatory effect of MSC s was
shown to be due to secretion of “secretomes”orextracellularves‐
icles tha t furthe r downregul ate the IL‐6 and nitric ox ide syntha se,
andi ncreasetheIL‐10andATP.37Liuetalbyintravenousapplication
ofhumanUC‐MSCson burn woundsofratsrepor tedadecreasein
inflammatorycellinfiltrates,interleukin‐1and6,andTNFalpha,and
ahigherratioofcollagentypesI/III,vascularization,neo‐angiogene‐
sis,andkeratinizationleadingtoafasterwoundhealing.38
Inourstudywh encomparingthelesi onsizein diff erentgr ou ps,
it was shown t hat in rats l eft untrea ted after bu rn injury, the l e‐
sionsizewas14.6%,thatdecreasedby 54.1% afterapplying SSD,
by71.3% afterusing ADM alone and by 87.6%when the animals
received WJSCs seeded onto the ADM. Similar to our findings
aftercreatingburninjuries,asignificantchangeinsizeoftheburn
woundwasnoted aftertransplantation ofADSCs (84.9%)in com‐
parisonwith the control group (69.7%).30Francketalreporteda
statisticallysignificantdecreaseintheburnsizewithtransplanted
ADSCs(40.4 4%)whencomparedwiththecontrolgroup(32.26%).2
InporcinemodelwithcontactburnsusingallogeneicMSCswitha
fibrinmatrix,astatisticallyreducedburnwoundsizewasnoted.39
There wereseveral limitations in our study including the use
oftheratmodel for studying human woundhealing as histologic
structuresandhealingcharacteristicsinhumanaredif ferentfrom
rats. A lso in rats, co ntraction p lays an impor tant role in woun d
healing , while in human, t he wound healing ha ppens by re‐epi‐
thelialization.Inrats,thepanniculuscarnosusplaysanimportant
role inwound contraction that was removedaf terdebridement
influenci ngo urr esult s.Thesecondlimit ationofourst udywasthe
number ofhWJSCs(2×106cells)thatwasusedthatwasinsmall
amountquantity,whilealargerquantit yof stem cellsseems nec‐
essary for celltherapyin burn patients. The third limitation was
the allogenic source of transplanted cellsthat maydecrease the
immunomodulating and anti‐inflammatory activities of hWJSCs
andthelastlimitationwastheduration ofsamplinguntil3weeks
thatmaynecessitatelongertimeintervalsupto1‐3months.
5 | CONCLUSION
Our findings showed that allogenic hWJSCs seeded onto ADM
couldimprovethehealingprocessdenotingtothetherapeuticef‐
fic ac yofWJSC sintreatme ntofac uteburni njuri es .Mo restudiesi n
smallandlargeanimalmodelswithlargerquantityofstemcellsand
duringalongertimeintervalareneededtodrawsolidconclusions.
ACKNOWLEDGMENTS
We would like to thank National Institute for Medical Research
Development of Iran Ministr y of Health, Treatment and Medical
Education(Grantno.of963474)forfinancialsupportandStemCell
TechnologyResearchCenterandtheComparativeandExperimental
Centerforproviding the neededspacesfortheexperimentsof this
stud y.
CONFLICT OF INTEREST
Theauthorshavedeclaredthatnocompetinginterestsexist.
ORCID
Davood Mehrabani https://orcid.org/0000‐0002‐5738‐1719
Seyedeh‐Sara Hashemi https://orcid.org/0000‐0002‐9395‐9573
REFERENCES
1. World Health Organization. 2016 Burns fact sheet worldwide.
2017;2:9. http://www.who.int/mediacentre/factsheets/fs365/en
Accessed6March2018.
2. FranckCL,SenegagliaAC,LeiteL,deMouraS,FranciscoNF,Ribas
FilhoJM.Influenceofadiposetissue‐derivedstemcellsontheburn
woundhealingprocess.Stem Cells Int.2019;2019:2340725.
|
7
NAZEMP OUR Et A l.
3. Rowan MP, Cancio LC , Elster EA , et al. Bur n wound healin g and
treatment:reviewandadvancements.Crit Care.2015;19:243.
4. Halim AS, Khoo TL, Yussof M. Biologic and synthetic skin substi‐
tutes:anoverview.Indian J Plast Surg.2010;4 3:S23‐S28.
5. Middelkoop E, van den Bogaerdt AJ, Lamme EN, Hoekstra MJ,
BrandsmaK,Ulrich MM.Porcine woundmodels for skin substitu‐
tionandburntreatment.Biomaterials.2004;25:1559‐1567.
6. Mohtasham Amiri Z, Tanideh N, Seddighi A, et al. The effect
of lithospermum officinale, silver sulfadiazine and alpha oint‐
ments inhealingofburn wound injuries in rat. World J Plast Surg.
2017;6:313‐318.
7. Sedighi A , Mehrabani D, Shirazi R . Histopathological evalua‐
tion of the healingef fects ofhuman amniotic membrane trans‐
planta tion in third‐ degree bur n wound injur ies. Comp Clin Pathol.
2016;25:381‐385.
8. Mehrab ani D, Rabiee M, Tamadon A , et al. The growth kinetic,
differentiation properties, karyotyping, and characterization of
adipose tissue‐derived stem cells in hamster. Comp Clin Pathol.
2016;25:1017‐1022.
9. Iravani K, Sobhanmanesh A, Ashraf MJ, Hashemi SB, Mehrabani
D,ZareS .Thehealing ef fect of conditioned mediaandbone mar‐
row‐derivedstemcellsinlaryngotrachealstenosis:acomparisonin
experimentaldogmodel.World J Plast Surg.2 017;6:190‐197.
10. Mehrabani D, Khodakaram‐Tafti A , Shater zadeh‐Yazdi H, Zamiri
B, Omidi M . Comparison of the regenerative ef fect of adipose‐
derived stem cells, fibrin glue sc affold, and autologous bone
graftin experimental mandibular defectinrabbit.Dent Traumatol.
2018;34:413‐420.
11. Mehrabani D, Mahdiyar P, Torabi K, et al. G rowth kinetics and
characterization of human dental pulp stem cells: comparison
between third molar and f irst premolar teeth. J Clin Exp D ent.
2017;9 :e172‐e177.
12. Mehrabani D, Mehrabani G, Zare S, Manafi A. Adipose‐derived
stemcells(ADSC)andaestheticsurger y:aminireview.World J Plas t
Surg.2013;2:65‐70.
13. Hu ang L, Burd A . An update review of s tem cell applic ations in
burnsandwoundcare.Indian J Plast Surg.2012;45:229‐236.
14. Ko ng CM, Lin HD, Bi swas A, B ongso A , Fong CY. Manufac turing
of human Wh arton's jelly s tem cells for cli nical use: se lection of
serumisimportant.Cytotherapy.2019;21(4):483‐ 495.[Epub ahead
ofprint].
15. HanY,SunT,HanY,etal.Humanumbilicalcordmesenchy malstem
cellsimplantationacceleratescutaneouswoundhealingindiabetic
ratsviatheWntsignalingpathway.Eur J Med Res. 2019;24:10.
16. Khodakaram‐TaftiA ,MehrabaniD,Shaterzadeh‐YazdiH, ZamiriB,
OmidiM.Tissueengineeringinmaxillarybonedefects .World J Plast
Surg.2018;7:3‐11.
17. ZhangCP,FuXB. Therapeuticpotential ofstemcellsin skin repair
andregeneration.Chin J Traumatol.2008;11:209‐221.
18. NamnoumJD.Expander/implantreconstructionwithalloderm:re‐
centexperience.Plast Reconstr Surg.2009;124:387‐394.
19. Lai C, Song G, Zhao B, et al. Preparation and charac terization
of human sc ar acellular dermal matrix. J Biomater Sci Polym Ed.
2019;30:769‐78 4.[Epubaheadofprint]).
20. Wainwright DJ. Use of an acellular allograft dermal mat rix
(AlloDerm) in the management of full‐thickness burns. Burns.
1995;21:243 ‐248.
21. Shumakov VI, Onishchenko NA, Rasulov MF, Krasheninnikov ME,
Zaiden ov VA. Mesen chymal bone m arrow stem ce lls more ef fec‐
tivelystimulateregener ationofdeepburnwoundsthanembr yonic
fibroblasts.Bull E xp Biol Med.2 00 3;136:19 2‐195.
22. Im am RA , Rizk AA . Effic acy of er ythrop oietin pre treated me sen‐
chymal stem cellsin murine burnwound healing: possible in vivo
transdifferentiationintokeratinocytes.Folia Morphol (Warsz). 2019.
[Epubaheadofprint].
23. FDA .proposedapproachtoregulationofcellularandtissue‐based
products. http://www.fda.gov/downloads/Biologi‐casBloodVa
c c i n e s / G u i d a n c e C o m p l i a n c e R e ‐ g u l a t o r y I n f o r m a t i o n / G u i d a n c e s /
Tissue/UCM‐ 062601.Accessedon26July,2011.
24. K arimi H, So udmand A , Orouj i Z, Taghiabadi E , Mousavi S J. Burn
wound healing with injection of adipose‐derived s tem cells: a
mousemodelstudy.Ann Burns Fire Disasters.2014;27:44 ‐49.
25. ChangYW,WuYC,HuangSH,WangH,KuoYR,LeeSS.Autologous
andnot allogeneic adipose‐derived stemcellsimprove acuteburn
woundhealing.PLoS One.2018;13:e0197744.
26. Fra ncis E, Kear ney L, Clove r J. The effe cts of ste m cells on bur n
wounds:areview.Int J Burns Trauma.2019;9(1):1‐12.eCollection.
27. OrbayH,Takami Y,Hyakusoku H,Mizuno H. Acellulardermal ma‐
trixseededwith adipose‐derivedstemcellsasasubcutaneousim‐
plant.Aesth Plast Surg.2011;35:756‐763.
28. YangY,ZhangW,LiY,FangG,ZhangK.Scaldedskinofrattreated
byusingfibringluecombinedwithallogeneicbonemar rowmesen‐
chymalstemcells.Ann Dermatol.2014;26:289‐295.
29. NambuM,IshiharaM,NakamuraS,etal.Enhancedhealingofmito‐
mycinC‐treatedwoundsinratsusinginbredadiposetissue‐derived
stroma l cells withi n an atelocol lagen matr ix. Wound Repair Regen.
20 07;15:50 5‐510 .
30. MotamedS, Taghiabadi E, Molaei H, et al. Cell‐based skin substi‐
tutesaccelerateregenerationofextensiveburnwoundsinrats.Am
J Surg. 20 17; 214 :762‐769.
31. YanG,SunH, WangF,et al.TopicalapplicationofhPDG F‐A‐mod‐
ified po rcine BMSC and keratinoc ytes loaded on acellul ar HAM
promote s the heali ng of combin ed radiat ion‐wound sk in injur y in
minipigs.Int J Radiat Biol.2011;87:591‐600.
32. G hieh F,J urjus R, Ib rahim A , et al. The use of s tem cells in bu rn
woundhealing:areview.Biomed Res Int. 2015;2015:684084.
33. FoubertP,BarillasS,GonzalezAD,etal.Unculturedadipose‐derived
regenerative cells (ADRCs) seeded incollagen scaffold improves
dermalregeneration,enhancingearlyvascularizationandstructural
organizationfollowingthermalburns.Burns.2015;41:1504‐1516.
34. Chen Y‐W,Scutar uTT,G hetu N, et al. Theeffects of adipose‐de‐
rivedstemcell‐differentiatedadipocytesonskinburnwoundheal‐
inginrats.J Burn Care Res.2017;38:1‐10.
35. SingerDD,SingerAJ,G ordonC,Br inkP.Theef fect sofratmesen‐
chymalstemcellsoninjuryprogressioninar atmodel.Acad Emerg
Med.2013;20:398‐402.
36. Liu P, Deng Z, Han S, et al. Tissue‐engineered skin containing
mesenchymal stem cells improves burn wounds . Artif Organs.
2008;32:925‐931.
37. Cr uz FF,Ro cco P.Stem‐ cell extr acellula r vesicles an d lung repa ir.
Stem Cell Investig. 2017;4:78.
38. LiuL ,YuY,HouY,etal.Humanumbilicalcordmesenchymals tem
cellstransplantationpromotescutaneouswoundhealingofsevere
burnedr ats.PLoS One.2014;9:e8 8348.
39. Cl over AJP, Kumar AHS, Is akson M, et al . Allogeneic me senchy‐
mals temcells, but not culturemo difiedmonocy tes, improveburn
woundhealing.Burns.2015;41:548‐557.
How to cite this article:NazempourM,MehrabaniD,
Mehdinavaz‐AghdamR ,etal.Theeffectofallogenichuman
Wharton'sjellystemcellsseededontoacellulardermal
matrixinhealingofratburnwounds.J Cosmet Dermatol.
2019;00:1–7. https://doi.or g/10.1111/jo cd .1310 9
