Differentiation of Apical Bud Cells in a Newly Developed Apical Bud Transplantation Model Using

14 Pages·2016·11.33 MB·English

RESEARCHARTICLE
Differentiation of Apical Bud Cells in a Newly
Developed Apical Bud Transplantation Model
Using GFP Transgenic Mice as Donor
NaokiMaruo1☯,RyujiSakagami1*,YasunoriYoshinaga1‡,KazuhikoOkamura2‡,
YoshihikoSawa3☯
1 SectionofPeriodontology,DepartmentofOdontology,FukuokaDentalCollege,Fukuoka,Japan,
2 SectionofPathology,DepartmentofMorphologicalBiology,FukuokaDentalCollege,Fukuoka,Japan,
3 SectionofFunctionalStructure,DepartmentofMorphologicalBiology,FukuokaDentalCollege,Fukuoka,
Japan
☯Theseauthorscontributedequallytothiswork.
‡Theseauthorsalsocontributedequallytothiswork.
*[email protected]
Abstract
OPENACCESS
Rodentmandibularincisorshaveauniqueanatomicalstructurethatallowsteethtogrow
Citation:MaruoN,SakagamiR,YoshinagaY,
throughoutthelifetimeoftherodent.Thisreportpresentsanoveltransplantationtechnique
OkamuraK,SawaY(2016)DifferentiationofApical
BudCellsinaNewlyDevelopedApicalBud forstudyingtheapicalbuddifferentiationofrodentmandibularincisors.Incisalapicalend
TransplantationModelUsingGFPTransgenicMice tissuewithgreenfluorescentproteinfromtransgenicmousewastransplantedtowildtype
asDonor.PLoSONE11(3):e0150766.doi:10.1371/
mice,andthedevelopmentofthetransplantedcellswereimmunohistologicallyobserved
journal.pone.0150766
for12weeksafterthetransplantation.Resultsindicatethatthegreenfluorescentapicalend
Editor:SeungbokLee,SeoulNationalUniversity,
tissuereplacedtheoriginaltissue,andcellsfromtheapicalbuddifferentiatedandextended
REPUBLICOFKOREA
towardtheincisaledgedirection.Theimmunostainingwithpodoplaninalsoshowedthatthe
Received:December24,2015
characteristicsofthegreenfluorescenttissuewereidenticaltothoseoftheoriginal.The
Accepted:February18,2016 greenfluorescentcellswereonlyfoundinthelabialsideoftheincisorupto4weeks.After
Published:March15,2016 12weeks,however,theywerealsofoundinthelingualside.Herethegreenfluorescent
cementocyte-likecellswereonlypresentinthecementumclosetothedentinsurface.This
Copyright:©2016Maruoetal.Thisisanopen
accessarticledistributedunderthetermsofthe studysuggeststhatsomeofthecellsthatformthecellularcementumcomefromtheapical
CreativeCommonsAttributionLicense,whichpermits tissueincludingtheapicalbudinrodentincisors.
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare
credited.
DataAvailabilityStatement:Allrelevantdatanot
includedinthepaperareavailablefromFigshare
Introduction
(DOIs:https://dx.doi.org/10.6084/m9.figshare.
3079978.v2,https://dx.doi.org/10.6084/m9.figshare.
Rodentmaxillaryandmandibularincisorshaveuniqueanatomicalstructuresthatallowthe
3080431.v1,https://dx.doi.org/10.6084/m9.figshare.
teethtogrowthroughoutthelifetimeoftheanimal.Thereisenamelstructureformedonthe
3080434.v1).
labialsideandperiodontalligamentinthelingualside.Inthemostapicalpartoftheincisor,
Funding:ThisworkwassupportedbyProjectfor
thereisaspecializedepithelialcellgrouptermedanapicalbud,whichwasoriginallytermedas
controlofaging,industrytosupportprivate
alabialcervicalloop[1–3].Ithasbeenshownthatpluripotentcellsandstemcellnichesare
universitiesbuildinguptheirfoundationsofstrategic
presentintheapicalbudaswellasinnailsandhairfollicles[4,5].Theapicalbudisregardedas
research2012,theministryofJapaneseeducation,
science,sportsandculture,Japan. ananalogofthecervicalloopofmousemolarsandhumanteethinthedevelopmentalstage.
PLOSONE|DOI:10.1371/journal.pone.0150766 March15,2016 1/14
DevelopmentofanApicalBudDifferentiationModel
CompetingInterests:Theauthorshavedeclared Thistissueisthoughttobeanidealmodeltostudyepithelial-mesenchymalinteractionsand
thatnocompetinginterestsexist. celldifferentiation[4].
Transplantationandimplantationofrodentteethandtoothgermsinthemolarhaveshown
completetoothdevelopment[6],butitwasnotpossibletoidentifyanyreportonthetransplan-
tationofrodentmandibularincisorsexceptfortheSlavkin’sworkusingfertilizedchickeneggs
asrecipients[7].Simpleextractionofmousemandibularincisorsleavestheapicalsofttissuein
thebone,andweplannedsurgicallytoreplacetheapicaltissuebyafenestrationprocedureon
thebuccalsurfaceofthemandibularbone.
Transgenicmicewithgreenfluorescentprotein(GFP)wereusedforprovidingthetraceable
tissueinthisstudy.TheGFPisresponsibleforthegreenbioluminescenceofthejellyfish
Aequoreavictoria.Transgenicmicewithan‘enhanced’GFP(EGFP)cDNAunderthecontrol
ofachickenbeta-actinpromoterandcytomegalovirusenhancerarecalled‘greenmice’that
havebeenusedfortissuetransplantationanddifferentiationexperiments.Allofthetissues
fromthesetransgenicmice,withtheexceptionoferythrocytesandhair,weregreenunderexci-
tationlight[8].
Podoplaninisauniquecellsurfacemarkeroriginallyfoundinthelymphaticvessels[9].
Sawahasreportedthatthepodoplaninpositivecellsarefoundintheinnerenamelepithelium
andodontoblastsinmice[10].Combiningthefindingsfromanti-podoplaninimmnostaining
andGFPenablesustodistinguishthecellswithspecificcellcharactersfromthegreenmice.
Theprimarypurposeofthisexperimentistoestablishasurgicalapicalbudtransplantation
anddifferentiationmodelusingthegreenmiceasdonors.Theapicalbudandsurroundingtis-
suewereobtainedfromthegreenmice,andwasusedtoreplacetheoriginaltissueinthewild
typemice.Tothebestofourknowledge,thisisthefirstpaperthatpresentsthetransplantation
methodoftherodentmandibularincisor.
Thesecondarypurposeofthisexperimentistoinvestigatethesofarunprovenhypothesis
thatcementoblastsintheperiodontalligamentarederivedfromepithelialprecursorcells[11–
15].Someresearchesfailedtoshowtheevidenceoftheepithelialrootsheath(ERS)toundergo
epithelial-mesenchymaltransition(EMT)duringinitialcellularcementogenesis[16,17].
WhethertheERSisinfluencingtheresidualmesenchymalcellstodifferentiateintocemento-
blasts,orwhethersheathcellsthemselvescanbecomecementoblaststhroughEMTisstill
unknown.Thisstudywasundertakentoelucidatethecellmigrationanddifferentiationofthe
epithelialorigincellsintheapicalbudofrodentincisors.
Materials&Methods
ThisstudywascarriedoutinstrictaccordancewiththerecommendationsintheGuideforthe
CareandUseofLaboratoryAnimalsoftheNationalInstitutesofHealth.Theprotocolwas
approvedbytheCommitteeontheEthicsofAnimalExperimentsofFukuokaDentalCollege,
Fukuoka,Japan(PermitNumber:12008).Allsurgerywasperformedundersodiumpentobar-
bitalanesthesia,andalleffortsweremadetominimizesuffering.
Transplantation
Inthisexperiment,weusedatotalof10,5maleand5female,1-week-oldC57BL/6-Tg(CAG-
enhancedgreenfluorescenceprotein,EGFP)C14-Y01-FM131Osbmice(greenmice)asdonors
andatotalof10male4-week-oldC57BL/6miceasrecipients[8,18–20].Allofthetissuefrom
greenmice,withtheexceptionoferythrocytesandhair,werereportedtobegreenunderexcita-
tionlight[8].Thegreenfluorescencewasdirectlydetectedbyexposingspecimensto488nm
laserlight.
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DevelopmentofanApicalBudDifferentiationModel
Donorsite
Aftereuthanizingthegreenmicewithisoflurane,intactincisorsweredissectedfrombothsides
ofthemandible,mostofthedentalfollicletissuesurroundingthetoothapexwascarefully
removedwithtweezers,andapicaltissuecontainingtheapicalbudwasseparatedunderaste-
reomicroscopewith20-foldmagnification.Grafttissue,200μmlong,fromthetipoftheapex
wasstoredinsalinebeforethetransplantation(Fig1).
Recipientsite
Wildtypemicewereanesthetizedwith50mg/kgpentobarbital.Afterlocaladministrationof
0.2%xylocainewith1/800,000epinephrinetothemandible,a10mmlongincisionwasmade
intheskin,andthemassetermusclewascutattheleveloftheocclusalplane.Thebuccal
bonearoundthetoothapexwasfenestratedwitha#11scalpelbladeandtheapicaltissuewas
removed.
Thegreenfluorescenttissuewastransplantedtothelocationoftheremovedoriginaltissue
1
andthebonyholewascoveredwithaGore-Tex membrane.Themassetermuscleandthe
skinwererespectivelyclosedwithanabsorbablesuture.Theexperimentalmicewerereturned
totheirindividualcagesandallowedtomovefreely.Themicewerefedwithnormaldietduring
thehealingperiodandcarefullymonitoredeveryday.
Tissuepreparation
Atotalof4,4,and2recipientmiceweresacrificedat2weeks,4weeks,and12weeksafterthe
transplantation,respectively.Underadministrationof50mg/kgpentobarbital,themicewere
perfusedwithsalinefollowedby1.5%paraformaldehydephosphatebuffersolutionforfixation
[21].Nextthehemi-mandiblesofthemicewereembeddedinsupercryoembeddingmedium
(SCEM),andrapidlyfrozenusingliquidN .Sampleswerecutwithacryostat(LeicaMicrosys-
2
tems,Wetzlar,Germany)into4μmsectionsforHematoxylinandEosinstainingandfor
immunofluorescencestaining.Eachspecimenwascutwithatungstencarbidebladewithout
chemicaldecalcificationusingthefilmtransfermethod[22,23].
Immunostaining
Sectionswerefixedin4%paraformaldehydephosphatebuffersolutionfor3minutesatroom
temperature(RT),andtreatedwith1%goatserum(GS)dilutedwith10mMphosphate-buff-
eredsaline(PBS,pH7.4)for30minutesatRTandwereexposedbyaprimaryantibody:1μg/
mlofhamsteranti-mousepodoplaninIgG(AngioBioCo.,DelMar,CA,USA)for12hoursat
4°C.Aftertreatmentwiththeprimaryantibody,sectionswerereactedwithasecondantibody:
1μg/mlofAlexaFluor568-conjugatedgoatanti-hamsterIgG(MolecularProbes,Invitrogen,
Eugene,OR)inGS-PBSfor1houratRT,andthenexaminedbyfluorescencemicroscopy,BZ-
9000(KeyenceCorp.,Osaka,Japan)andlaser-scanningconfocalmicroscopy(Axiovert135M,
CarlZeiss,Jena,Germany),witha×63Plan-Apochromaticoilimmersionobjectivelens
(numericalaperture×1.4)[23].Thegreenfluorescencewasdirectlydetectedwithoutanyanti-
bodybyexposingspecimensto488nmlaserlight.Atthetimeoftheembedding,DAPI(4',
6-diamidino-2-phenylindole)wascounterstainedfordetectingregionsintheDNA.
Results
Successfultransplantationandnormaltoothgrowthwasobservedin3of4,2of4,and1of2
samplesat2,4,and12weeksaftersurgery,respectively.Oftheremaining4samples,2showed
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DevelopmentofanApicalBudDifferentiationModel
Fig1.Greenfluorescenceexpressionin1-week-oldC57BL/6-Tg(CAG-EGFP)mousemandibular
incisors.(A)MandibularincisorsoftheC57BL/6-Tg(CAG-EGFP)mouse(greenmouse,top)andthe
C57BL/6mouse(wildtype,bottom)inphasecontrastimage.Incisorsfromthegreenmiceshowedidentical
shapes,sizes,anddevelopmentasthosefromthewildtypemice.Thegrafttissuewasseparatedatthe
yellowdottedline.(B)FluorescentimageofthesameteethasA.Thegreenfluorescenceisobservedonlyin
themandibularincisorofthegreenmouse.(C)Sagittalsectionstainedwithhematoxylinandeosinofthe
greenmousemandibularincisor.Asteriskindicatestheapicalbud.Thegrafttissuewasseparatedatthe
yellowdottedline.(D)FluorescentimageofthesamesectionasC.Greenfluorescenceisobserved
throughoutthesectionwithdifferentintensities.Scalebars:200μminAandB;100μminCandD.
doi:10.1371/journal.pone.0150766.g001
ectopicgreenfluorescenttoothgrowthand2showednosignsofgreenfluorescentcellsinthe
mandible.
Frozensectionsat2weeksaftertransplantation
At2weeksaftersurgery,transplantedgreenfluorescentcellswereengraftedandhadmigrated
approximately3mmtowardtheincisaledge(Fig2).Inthelabialsideoftheincisor,therecipi-
entanddonor-derivedstructuresareseenintheincisalendandapicalendsides,respectively.
Thegreenfluorescentcellswereonlypresentatthelabialhalfofthemandibularincisor,and
greenfluorescencewasobservedinameloblasts,odontoblasts,andinthedentalpulpcells.In
thedentalpulp,thereisgreenfluorescenceinthecapillaries.Reactionproductswithanti-
podoplaninwereobservedinapicalbuds,pre-ameloblasts,odontoblasts,andthenervesheaths
(Fig2).
Inthemediummagnificationtheareaof(a)inFig2,thereisatransitionalareabetweenthe
recipienttissueandthedonor-derivedtissue(Fig3).Intheincisalendside,theoriginalrecipi-
enttissueremainedwithoutanygreenfluorescence,andintheapicalendsidethereweregreen
fluorescentameloblasts,odontoblasts,andstratumintermediumwithirregularenamel-like
hardtissuesurroundedbydentinlocatingbetweenthetwoareas.Odontoblastsanddentin
werecontinuousthroughrecipienttissuetodonor-derivedtissue.Ameloblastsandenamel
structurewereobservedbothintherecipientanddonorderivedtissue.Reactionproductswith
anti-podoplaninantibodywereobservedintheodontoblastsandtheameloblasts.Podoplanin-
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DevelopmentofanApicalBudDifferentiationModel
Fig2.Frozenundecalcifiedsagittalsectionofthemandible2weeksaftertransplantation.Thetop,
bottom,left,andrightofeachpanelshowthelingual,labial,incisalend,andapicalendsides,respectively.
(A)Inthesectionstainedwithhematoxylinandeosin,thelingualsideoftheincisorpresentsperiodontal
ligament(PDL),dentin(arrow),andodontoblasts(OB).Thelabialtissueconsistsofodontoblasts(OB),dentin
(arrow),enamel(arrowhead),andameloblasts(AB).DP,dentalpulpcells.Boxedareas(a)and(b)are
magnifiedinFigs3and4,respectively.(B)Greenfluorescentcellsarepresentatthelabialhalfofthe
mandibularincisor.Greenfluorescenceisobservedinameloblasts(AB),odontoblasts(OB),anddentalpulp
cells(DP).(C)Reactionproductswithanti-podoplaninareobservedinapicalbud(asterisk),pre-ameloblasts
(PAB),odontoblasts(OB),andnervesheaths(outlinedarrowheads).(D)MergedimageofB,C,andblue
fluorescenceofDAPI’s.Scalebars:500μm.
doi:10.1371/journal.pone.0150766.g002
positiveodontoblastswereobservedtoextendcontinuouslyfromtherecipienttissuetothe
donorderivedtissue(Fig3).
Intheapicalendoftheincisor,themergedgreenfluorescenttissueshowedacontinuous
structurewithoutanydisruptions.Thedentalpapillashowedrelativelydensecellsliningthe
innerenamelepithelium,withtheinnerenamelepitheliumextendedforwardtothepre-ame-
loblasts,whichlaterbecomeameloblasts(Fig4).Stronggreenfluorescencewasobservedinepi-
thelial-origincellsfromtheapicalbuds,withmoderategreenfluorescenceinodontoblastsand
indentalpulpcells(Fig4).Reactionproductswithanti-podoplaninwereobservedintheapical
buds,pre-ameloblasts,andodontoblasts.Theimmunoreactionwithanti-podoplaninwas
weakerinameloblaststhaninpre-ameloblasts.Podoplaninexpressionwasidentifiedindiffer-
entiatedodontoblasts,butnotinpre-odontoblasts(Fig4).
Intheobservationswithhighermagnificationat(a)ofFig4,therearepre-ameloblastswith
relativelylargenucleiin2or3layers(Fig5).Thegreenfluorescenceandpodoplaninexpres-
sionwasobservedinpre-ameloblastsandtheouterenamelepithelium.Greenfluorescencewas
alsoobservedinstellatereticulum,stratumintermedium,anddentalpulpcells(Fig5).
Intheobservationswithhighermagnificationat(b)inFig4,theodontoblastsshowedhigh
polaritywiththenucleialignedatthedentalpulpside(Fig6).Thegreenfluorescencewas
observedinodontoblastsbutitwasweakerthanthatintheameloblastsanddentalpulpcells.
Thegreenfluorescencewasnotobservedwithinthematrixofnewlyformedpre-dentin(Fig6).
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DevelopmentofanApicalBudDifferentiationModel
Fig3.Greenfluorescenceinthetransitionalareabetweenrecipienttissueanddonorderivedtissue.
Figuresaretheareaof(a)inFig2hereatmediummagnification.(A)Inthesectionstainedwithhematoxylin
andeosin,therecipienttissue(asterisk)isreplacedbythedonor-derivedtissue(doubleasterisk).
Ameloblasts(AB)andenamel(E)areobservedbothintherecipientanddonorderivedtissue.Inthe
transitionalareaenamel-likehardtissue(arrow)isfoundsurroundedbythedentin.Odontoblasts(OB)and
dentin(D)arecontinuousthroughrecipienttissuetodonorderivedtissue.SI,stratumintermedium.(B)Green
fluorescenceisobservedinodontoblasts(OB),ameloblasts(AB),andthestratumintermedium(SI)ofthe
donorderivedtissue(doubleasterisk).Thegreenfluorescenceisnotobservedintherecipienttissue
(asterisk).(C)Reactionproductswithanti-podoplaninantibodyareobservedintheodontoblasts(OB)and
ameloblasts(AB)oftherecipientandthedonorderivedtissue.(D)MergedimageofB,C,andblue
fluorescenceofDAPI’s.Scalebars:100μm.
doi:10.1371/journal.pone.0150766.g003
Podoplaninexpressionwasstrongindentinformingodontoblastsandmoderateinenamel
formingameloblasts(Fig6).
Frozensectionsat4weeksaftertransplantation
Inthefrozensectionsofthemandibleat4weeksaftertransplantation,therecipientand
donor-derivedtissuewerelocallyobservedinthelabialsideoftheincisor.Thetransplanted
greenfluorescentcellshadmigrated3–4mmfurthertowardtheincisaledge,comparedtothe
2weeksamples(Fig7).Stronggreenfluorescenceexpressionintheameloblastsandmoderate
expressionintheodontoblastswasobserved.Greenfluorescencewasnotobservedinthelin-
gualsideoftheincisor.Reactionproductswithanti-podoplaninantibodieswereobservedin
thelabialandlingualodontoblastsoftheincisorandintheameloblasts(Fig8).
Frozensectionsat12weeksaftertransplantation
Inthefrozensectionsofthemandible12weeksaftertransplantation,thedonor-derivedcells
wereobservedextendingallthewaytotheincisalend.Therewereperiodontalligament,
cementum,dentin,enamel,andameloblastsfromthelingualsidetothelabialsideofthe
incisor.Odontoblastsanddentalpulpcellswerefoundinthedentalpulpspace.Reactionprod-
uctswithanti-podoplaninwereobservedinnervesheaths,apicalbuds,odontoblasts,and
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Fig4.Greenfluorescenceandpodoplaninexpressionintheapicalendofthemandibularincisor.
Figuresaretheareaof(b)inFig2hereatmediummagnification.(A)Inthesectionstainedwithhematoxylin
andeosin,anapicalbud(asterisk),pre-ameloblasts(arrowhead),dentalpulpcells(DP),odontoblasts(OB),
andameloblasts(AB)areobserved.Boxedareas(a)and(b)aremagnifiedinFigs5and6,respectively.(B)
Greenfluorescenceisobservedintheapicalbud(asterisk),pre-ameloblasts(arrowhead),ameloblasts(AB),
odontoblasts(OB),anddentalpulpcells(DP).Greenfluorescenceisfoundinthecapillariesofthedentalpulp
(arrows)aswell.(C)Podoplaninexpressionisobservedinapicalbud(asterisk),pre-ameloblasts
(arrowhead),andodontoblasts(OB).Immunoreactionwithanti-podoplaninisweakerinameloblasts(AB)
thaninpre-ameloblasts(arrowhead),andisstrongerinodontoblasts(OB)thaninpre-odontoblasts(outlined
arrowhead).(D)MergedimageofB,C,andbluefluorescenceofDAPI’s.Scalebars:100μm.
doi:10.1371/journal.pone.0150766.g004
ameloblasts.Greenfluorescentcellswereobservedinthelingualsideaswellasinthelabial
side(Fig8B).Inthelingualside,greenfluorescentcellswereobservedonlyinthecementum
andnogreenfluorescentcellswerefoundintheperiodontalligamentspace(Fig8Eand8F).In
thehighmagnification,greenfluorescentcementocyte-likecellswithnucleiwereobserved
locatedinthecementum(Fig8G–8I).
Discussion
Toestablishanexperimentalmodelsuitableforstudyingdetailsoftoothdevelopment,wehave
focusedonthemouseincisorthatgrowsthroughoutthelifetimeofmice.Thegreenfluorescent
mandibularincisorswereobtainedfromtransgenicmice.Withoutanyprecedingtreatment,
thegreenfluorescentcellsfromdonormicewereproventobeutilizedfortracingaftertrans-
plantation[8,18–20,24,25].Thegreenfluorescencewasdirectlydetectedbyexposingspeci-
mensto488nmlaserlight.Thegreenfluorescenceintensityvariedbycelltype,i.e.,stronger
expressionintheapicalbudcellsthaninthepre-ameloblastsandpre-odontoblasts(Fig1D).It
seemedcellswithlargerproportionofnucleiincytoplasmand/orlessactinformingcells
showedlessGFPexpressionbecausethegreenmicewerewithanenhancedGFP(EGFP)
cDNAunderthecontrolofachickenbeta-actinpromoter[8].
Inthisexperiments,immunofluorescentstainingagainstpodoplaninwasperformedto
understandthecharacteristicsofthereplacedanddifferentiatedcells.Podoplaninisaunique
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Fig5.Greenfluorescenceandpodoplaninexpressioninpre-ameloblasts.Figuresaretheareaof(a)in
Fig4atahighmagnification.(A)Inthesectionstainedwithhematoxylinandeosin,dentalpapillacells(DP),
pre-ameloblasts(PAB),stratumintermedium(SI),stellatereticulum(SR),andouterenamelepithelium(OEE)
areobserved.(B)Greenfluorescenceisobservedinpre-ameloblasts(PAB),stratumintermedium(SI),
stellatereticulum(SR),outerenamelepithelium(OEE)anddentalpapillacells(DP).(C)Podoplanin
expressionisobservedinpre-ameloblasts(PAB)andouterenamelepithelium(OEE).(D)Mergedimageof
B,C,andbluefluorescenceofDAPI’s.Scalebars:10μm.
doi:10.1371/journal.pone.0150766.g005
cellsurfacemarkeroriginallyfoundinthelymphaticvessels[9].Sawahasreportedthatthe
podoplaninpositivecellsarefoundintheinnerenamelepitheliumandodontoblastsinmice
incisors[10].Combiningthefindingsfromanti-podoplaninimmnostainingandHEstaining
enabledustodistinguishthecellswithspecificcellcharacterssuchasameloblasts,odonto-
blasts,andapicalbudcells.
Auniquesurgicaltechniquewasoriginallydevelopedbyapplyingtheknowledgeoftheperi-
odontalsurgery.Thekeytothesuccessfultransplantationwasminimallytraumaticsurgeryto
therecipient.Bleedingduringthesurgerywasminimalaslongasthelargebloodvesselinthe
1
muscleandinthetoothapexwasnotdamaged.Gore-Tex membranewasusedtoprevent
1
othersofttissuefrommigratingintothetransplantationsite.Gore-Tex isasyntheticnonre-
sorbablematerialfrequentlyusedinperiodontalregenerativeprocedures.
Inthisexperiment,200-μmlongapicaltissuewasusedforthetransplantation.Dentalfolli-
clecellsfromtheextracteddonorteethweremicroscopicallyremovedasmuchaspossible
fromtheapexofthetooth(Fig1),buttherewasapossibilityofremainingtinyamountofden-
talfolliclecellsinthedonortissue.Furtherstudyisnowunderwaytoattemptthetransplanta-
tionofenzymaticallyisolatedapicalbuds.Weexpecttobeabletoobservemorespecificdetails
ofcelldevelopmentwiththisnewapproach.
TheGFPiswatersoluble[21],andallsampleshadtobepre-fixedwithparaformaldehyde
phosphatebuffersolutionatthetimeofsacrifice.Afterthetissuewasembeddedinthecom-
pound,Kawamoto’sfilmmethodwithatungstencarbidebladewasusedforthesectioningso
thatintacthardandsofttissuecouldbeobservedwithoutanydecalcificationprocess[22].
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DevelopmentofanApicalBudDifferentiationModel
Fig6.Greenfluorescenceandpodoplaninexpressionintheodontoblasts.Figuresaretheareaof(b)in
Fig4atahighmagnification.(A)Inthesectionstainedwithhematoxylinandeosin,theformationofpre-
dentin(arrow)byodontoblasts(OB)followedbytheearlystageenamel(arrowhead)productionfrom
ameloblasts(AB)isobserved.(B)Greenfluorescenceintheodontoblasts(OB)isweakerthanthatinthe
ameloblasts(AB),butwithinthedetectablelevel.Greenfluorescenceisminimalinthepre-dentin(arrow).(C)
Reactionproductswithanti-podoplaninantibodyareclearlyobservedintheodontoblasts(OB)andweakly
observedintheameloblasts(AB).(D)MergedimageofB,C,andbluefluorescenceofDAPI’s.Nucleiof
odontoblasts(OB)areonlylocatedinthepulpsideofthecells.Scalebars:10μm.
doi:10.1371/journal.pone.0150766.g006
Inthe2-weekspecimens,greenfluorescentcellsextended3mmfromtheapexofthetooth
(Fig2).Greenfluorescencewasobservedinameloblasts,odontoblasts,andthecapillariesof
thedentalpulp(Fig4).Thepre-ameloblaststhatlocatedbetweentheinnerenamelepithelium
andameloblastsshowedoneofthemostproliferatedcellareasoftheseincisors[26](Fig5).
Thepodoplaninpositivecellswerefoundintheapicalbud,pre-ameloblasts,nervesheaths,
andodontoblastsat2weeks.Allofthesecellsandstructureswereinthesamelocationsasin
thewildtypemice[9,10,27].Consideringthecharacteristicsofthepodoplaninexpressionin
thegreenfluorescentcells,theameloblasts,odontoblasts,anddentalpulpcellswereatthenor-
malstateandhaddifferentiatedfromthetransplantedapicalendtissue.Therefore,thetrans-
plantedtissuewasconsideredtobethegroupofpluripotentcellsthathadbeenengraftedand
wasdifferentiatedintherecipienttissue.Thesefindingsconcludedthatthisnovelapicalbud
differentiationmodelusingtransgenicrodentincisorwassuccessful.
Thedonorderivedgreenfluorescenttissuewasclearlyidentifiedinthelabialsideofthe
2-weeksamplesandatransitionalareawasobservedbetweenthegreenfluorescentnegative
andpositivetissue(Fig3).Intheincisalside,theoriginalrecipienttissueremainedwithoutany
greenfluorescence.Intheapicalside,thereweregreenfluorescentameloblastsandodonto-
blasts.Theameloblastswereobservedinseparatelocationsoftherecipientanddonorderived
tissue,whereasalignedpodoplanin-positiveodontoblastsappearedtobetransitioning,chang-
ingfromgreenfluorescentnegativetopositive(Fig3).
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DevelopmentofanApicalBudDifferentiationModel
Fig7.Frozenundecalcifiedsagittalsectionofamandible4weeksaftertransplantation.Thetop,
bottom,left,andrightofeachpanelshowthelingual,labial,incisalend,andapicalendsides,respectively.
(A)Inthesectionstainedwithhematoxylinandeosin,therecipienttissue(asterisk)anddonor-derivedtissue
(doubleasterisk)areobservedinthelabialsideoftheincisor.Thelingualsideoftheincisorpresents
periodontalligament(PDL),dentin(D),andodontoblasts(arrowhead).Thelabialtissueconsistsof
odontoblasts(arrowhead),dentin(D),enamel(E),andameloblasts(arrow).DP,dentalpulpcells.(B)Green
fluorescenceisobservedinodontoblasts(arrowhead)andameloblasts(arrow)ofthedonorderivedtissue
(doubleasterisk).Thegreenfluorescenceisnotobservedintherecipienttissue(asterisk).(C)Reaction
productswithanti-podoplaninantibodyareobservedinthelabialandlingualodontoblasts(arrowheads)and
intheameloblasts(arrow).Thereisacrossreactionwithanti-podoplaninattheenamel(E).(D)Merged
imageofB,C,andbluefluorescenceofDAPI’s.Scalebars:500μm.
doi:10.1371/journal.pone.0150766.g007
Inthe4-weeksamples,thegreenfluorescentcellshadmigratedfurthertowardstheincisal
edgedirection(Fig7).Thegreenfluorescentcellsthathadadvancedmostwerelocatedinthe
areabelowthe3rdmolarat2weeks,andwerepasttheareabelowthe1stmolarat4weeks
(Figs2and7),whichcouldbeexplainedbythegradualeruptionprocessoftheincisor.At4
weeks,thegreenfluorescentcellswereonlyobservedinthelabialsideoftheteethandnotin
thelingualside.
Inthe12-weektissuesamples,thecryosectionwasnotcutsatisfactorilylikethe2and
4-weeksamplesduetothehighercalcificationofthetoothandbone.Thethicknessofthehard
tissuesectionsseemednottobeuniform,butitwasstillpossibletoenabletheobservationsof
thedentalpulpandtheperiodontaltissueproperly(Fig8).
Thegreenfluorescentcellswerefoundbothinthelabialsideandinthelingualsideofthe
incisors(Fig8B).Thisconditionwasonlyobservedinthe12-weeksampleandnotinthe2or
4-weeksamples.Itmaybethat4weeksisnotlongenoughforthelabialcellstomigratetothe
lingualarea,suggestingthatthe12-weekintervalafterthetransplantationwasneededfor
determiningthecharacteristicsofthecellsmigratingtothelingualsideofthemandibular
incisor.
Inthe12-weeksamples,thelingualgreenfluorescentcellswereentrappedinsidethecemen-
tumclosetothedentin.Theylocatedonlyinthecellularcementum,andwerenotfoundinthe
areaclosetoorinsidetheperiodontalligament.Whetherthesegreenfluorescentcementocyte-
PLOSONE|DOI:10.1371/journal.pone.0150766 March15,2016 10/14