Human Periapical Cysts-Mesenchymal Stem Cells Cultured with Allogenic Human Serum are a “clinical-grade” construct alternative to bovine fetal serum and indicated in the regeneration of endo-periodontal tissues

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ORIGINAL

ARTICLE/ARTICOLO

ORIGINALE

358 CONGRESSO

NAZIONALE

BOLOGNA

2017

—

VINCITORE

PREMIO

RICCARDO

GARBEROGLIO

Human

Periapical

Cysts-Mesenchymal

Stem

Cells

Cultured

with

Allogenic

Human

Serum

are

a

‘‘clinical-grade’’

construct

alternative

to

bovine

fetal

serum

and

indicated

in

the

regeneration

of

endo-periodontal

tissues

Le

human

periapical

cysts-mesenchymal

stem

cells

coltivate

con

siero

umano

allogenico

costituiscono

un

costrutto

‘‘clinical-grade’’

alternativo

al

siero

fetale

bovino

ed

indicato

nella

rigenerazione

dei

tessuti

endo-parodontali

Marco

Tatullo

a,

*

,

Massimo

Marrelli

a

,

Francesca

Palmieri

a

,

Carlo

Rengo

b

,

Francesco

Paduano

a

,

Gianrico

Spagnuolo

b

a

TecnologicaResearchInstitute,StemCellUnit,Crotone,Italy

b_Department_of_{Neurosciences,} _Reproductive_and _{Odontostomatological}_Sciences,_University_of_Naples_{‘‘Federico}_II’’,

Naples,Italy

Received17November2017;accepted20March2018 Availableonline6April2018

KEYWORDS

Regenerativemedicine;

Stemcells;

Osteogenesis;

Humanperiapical

cyst-MSCs;

Translationalresearch.

Abstract

Aim: Ourresearchinvestigatedtheuseofhumanserum(HS)asasafeandclinical-gradeculture medium,usinganewcell-model:hPCy-MSCs.Thisarticleisaimedtoconcretelyapplicatethe concept of ‘‘waste-based regenerative dentistry’’to translateitin future endo-periodontal applications.

Methodology: HPCy-MSCswereculturedin2differentmediums,bothcontaininga-MEM:the1st with10%FBS(Controlgroup),andthe2ndwith10%humanserum(Testgroup).

PeerreviewunderresponsibilityofSocieta` ItalianadiEndodonzia.

* Correspondingauthorat:TecnologicaResearchInstitute,StreetEnricoFermi,88900Crotone,Italy.Tel./fax:+390962930414. E-mail:marco.tatullo@tecnologicasrl.com(M.Tatullo).

Availableonlineatwww.sciencedirect.com

ScienceDirect

jo ur na l h o m ep a ge : w w w.e ls e v i er.c o m / lo c at e /gi e

https://doi.org/10.1016/j.gien.2018.03.003

1121-4171/ß2018Societa` ItalianadiEndodonzia.ProductionandhostingbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Introduction

Thediscoveryofhumanperiapicalcysts-mesenchymalstem

cells(hPCy-MSCs)has,forthefirsttime,introduceddentistry

intheconceptof‘‘regenerativewastemedicine’’,the

regen-erative medicine obtainable from the reuse of biological

wastetoexploitittheclinicalpotential.HPCy-MSCsshowed

excellentability to differentiatebetweenosteogenic

phe-notypesandsurprisinglytowardthenervoustissue.1—3

Stem cell regeneration is, however, limited in clinical

practicefortheuseoffetalbovine serum(FBS),usedas a

nutritional supplementinculturemedia. Infact, its

xeno-genicorigindoesnotmakeFBSsecure inpatients

applica-tions.DespiteseveralMSCs from different sources,4_and _a

number of scaffold5—12 have been already described and

investigatedintheliterature,themainlimitationtoclinical

applicationsseemstobethestandardizationofclinical-grade

procedures,toovercomeimmunologicalandbiological

con-cernings.

In the light of the studies in the literature,13—16 our

researchanalyzedforthefirsttimehumanserum(HS)asa

safeand‘‘clinical-grade’’alternativeinregenerative

med-icine,usinganewcellularmodel:hPCy-MSCs,opening

inno-vative scenarios on the concept of ‘‘waste regenerative

dentistry’’infutureendo-periodontalapplications.

Materials

and

methods

HPCy-MSCswereobtainedasdescribedinliterature1,3 _and

culturedinamediumcontaininga-MEMwith10%FBS(Gibco)

intheControlgroup,andwith10%humanserum(HS,Sigma)

inthetestgroup.TheHSusedinthisstudy(ABplasma)isused

asbythesupplierprotocol.

PAROLE

CHIAVE

Medicinarigenerativa;

Cellulestaminali;

Osteogenesi;

Humanperiapical

cyst-MSCs;

Ricercatraslazionale.

Cellproliferation and stemness assays, geneexpression, immunophenotypic analysis and osteogenicdifferentiationwereperformedtoverifyourhypothesis.cDNAsampleswere ampli-fiedwithqPCR.

Experimentswereperformedintriplicateandanalysedwithstatisticalsoftware.

Results: ThehPCy-MSCscultivatedinamediumwithHSweremorphologicallysimilartothose cultivatedwithFBS,andshowedasignificantlyhigherproliferationrate.VonKossa’sstaining revealedthatosteoblastsfromhPCy-MSCsinHSimplementedwithosteogenicinductionfactors, showedabetterosteogenicactivity,alsoconfirmedbyasignificantupregulationofosteopotin (OPN)andmatrixextracellularphosphoglycoprotein(MEPE).

Conclusions: HPCy-MSCscultivatedinHSshowedphenotypicstabilityandaclearregenerative binding,thus,suggestingthesetwocomponentsasaclinically-gradeconstructforfuture endo-periodontaltherapies.

ß2018Societa` ItalianadiEndodonzia.ProductionandhostingbyElsevierB.V.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/ 4.0/).

Riassunto

Obiettivi: Lanostraricercahaanalizzatol’utilizzodelsieroumano(HS)comemezzodicoltura sicuroe‘‘clinical-grade’’,perusoclinico,utilizzandounnuovomodellocellulare:lehPC-MSCs. Questoarticolohaloscopodiapplicareconcretamenteilconcettodi‘‘odontoiatriarigenerativa basatasuirifiutibiologici’’,alfineditradurloinfutureapplicazioniendo-periodontali. Materialiemetodi: LeHPCy-MSCssonostatecoltivatein2mezzidicolturadiversi,entrambi contenentia-MEM:ilprimocon10%diFBS(gruppodicontrollo)eilsecondoconil10%disiero umano(gruppoditest).

Sonostatieseguitisaggidiproliferazionecellulareedistaminalita`,espressionegenica,analisi immunofenotipicaedifferenziamentoosteogenicoperverificarelanostraipotesidipartenza. CampionidicDNAsonostatiamplificaticonqPCR.

Gliesperimentisonostatieseguitiintriplicatoeanalizzaticonsoftwarestatistici. Risultati: LehPC-MSCcoltivatein unterrenoconHSeranomorfologicamentesimiliaquelle coltivate con FBS e mostravano un tasso di proliferazione significativamente piu` alto. La colorazionediVonKossaharivelatochegliosteoblastidahPC-MSCcoltivateinHSimplementato confattoridiinduzioneosteogenicahannomostratounamiglioreattivita` osteogenica, confer-mataanchedaunasignificativaup-regolazionediosteopotina(OPN)efosfoglicoproteinadella matriceextracellulare(MEPE).

Conclusioni: Le HPCy-MSC coltivate in HS hanno mostrato stabilita` fenotipica e un chiaro atteggiamentorigenerativo, suggerendoquindiquestoprotocollocome unapproccio clinica-mentevalidoperlefutureterapieendo-periodontali.

ß2018Societa` ItalianadiEndodonzia.ProductionandhostingbyElsevierB.V.Cetarticleest publie´ enOpenAccesssouslicenceCCBY-NC-ND( http://creativecommons.org/licenses/by-nc-nd/4.0/)

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Cellproliferationtests,stemcellassays,geneexpression,

immunophenotypic analysis and osteogenic differentiation

testswereperformedwithquantitativeanalysistoverifyour

hypothesis.CDNA samples wereamplifiedwith qPCRusing

sequencesofprimers.

Experimentswereperformedintriplicate,anddata

eval-uatedwithGraphPadPrismsoftware(statisticallysignificant

differenceswith:*P<0.05;**P<0.01;P<0.001;N.S.=not

significant).

Results

The hPCy-MSCscultivated ina mediumwithHS were

mor-phologically similarto those cultivated withFBS(Fig. 1A);

however,hPCy-MSCsshowedasignificantlyhigher

prolifera-tionrate,fromday6(Fig.1B).Underbothculturing

condi-tions, theamount of vitalhPCy-MSCs was about thesame

(>95%,Fig.1C).Theexpressionofthemainstemnessgenes

hasbeenverified(Fig.1D).

Figure1 Proliferation,vitality,and expressionofstemmolecularmarkersin hPCy-MSCsgrowninHSorFBS.(A)Characteristic morphologyofhPCy-MSCscultivatedin10%HSor10%FBS(days1—13).(B)CellgrowthassayofhPCy-MSCsculturedin10%HSor10%FBS. *P<0.05. (C)Cellviabilityof hPCy-MSCscultured in10%HS or10%FBS. (D)Expression of mRNAfor thegeneticindicationsof staminality:Klf4,OCT4,Sox-2,Nanog,c-mycandtheCD146pericitarymarkerinhPCy-MSCsculturedin10%HSor10%FBSanalyzedby qRT-PCR.*P<0.05,**P<0.01,***P<0.001,N.S.,notsignificant.FBS,fetalbovineserum;HS,humanserum.

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FlowcytometryofphenotypicmarkersofhPCy-MSCs

cul-tivated in HS and FBS (Fig. 2A) did not show statistically

significantdifferences(Fig.2B).

Von Kossa’s staining revealed that hPCy-MSCs induced

towardstheosteogenicphenotypeinHS,showedagreater

differentiationthanthosecultivatedinFBS(Fig.3AandB).

Theseobservationswereconfirmedbyasignificant

upregula-tionofosteopotin(OPN)andmatrixextracellular

phospho-glycoprotein (MEPE)in hPCy-MSCs cultivated inosteogenic

andbasalmediacontainingHS(Fig.3C).

Discussion

Patients treated with FBS-grown cells are exposed to a

concreteriskofimmunogenicityandtheriskoftransmission

Figure2 ExpressionofsurfacestemmarkersinhPCy-MSCscultivatedinHSorFBS.(A)Expressionofsurfacestemmarkers:CD13, CD29,CD44,CD73,CD90,CD105,CD146andCD45inhPCy-MSCscultivatedin10%HScomparedwiththosegrownin10%FBS.Thecontrol oftheisotypeisindicatedbytheemptyhistogram,whereasthecoloringof theantibodiesisindicatedbyasolidhistogram.(B) PercentagedataofsurfacestemmarkersinhPCy-MSCscultivatedin10%HScomparedwiththosegrownin10%FBS.(C)Geometricmean fluorescenceintensityofsurfacestemmarkersinhPCy-MSCscultivatedin10%HScomparedwiththosegrownin10%FBS.FBS,fetal bovineserum;HS,humanserum.

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ofprion,bacterialorviralpathologies,makingFBSdangerous

foradvancedcelltherapy.13

For more than 30 years thescientific community has

requested the use of alternative supplements to FBS14

andin2011theEuropeanCommission,withthetechnical

note EMA/410/01, suggested that animal medicines be

avoided.

Recently,someauthorshavestudiedHSasanalternative

toFBSon stemcellmodelsofdentaloriginthatare

alter-native to our15,16; however, the plasticity of hPCy-MSCs

makes them ideal for regeneration to bone-like and

neu-ron-liketissues.

Conclusions

HPCy-MSCscultivated inHSshowedexcellent skillstowards

boneregeneration,alsoavoidingtherisksderivedfromtheuse

ofFBS,thus,suggestingthesetwocomponentsasa

clinically-gradeconstructforfutureendo-periodontaltherapies.

Clinical

relevance

Human periapical cysts-mesenchymal stem cells cultured

with allogenic humanserum are clinically usable, without

Figure3 OsteogenicdifferentiationpotentialofhPCy-MSCscultivatedinHSorFBS.(A)OsteogenicdifferentiationofhPCy-MSCs, growninHS,andcomparedwiththosegrowninFBS,coloredwithredAlizarin.(B)Quantificationbyredbloodalizarinetestforthe productionofmineralized matrixinhPCy-MSCscultivatedin HScomparedwiththosegrownin FBS.*P<0.05.(C)Expressionof osteogenic markers mRNA: OSP and MEPE in hPCy-MSCs cultivated in HS compared tothose cultured in FBS after 21 days of differentiation (OS). FBS, fetal bovine serum; HS, human serum; CTR, control; OS, osteogenic differentiation. *P<0.05, ***P<0.001,N.S.,notsignificant.

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tousealternativexenogeneicserum.This‘‘clinical-grade’’

procedure,together withthegreat plasticityofhPCy-MSCs

andtherecentexperimentalresultsreportingtheirabilityto

differentiatetowardsbothbone-like,tooth-likeand

neuron-liketissues,makestheconstructmadefromsuchanewMSCs

and the ‘‘safe’’culture medium an implantable biological

devicegreatlyindicatedintheregenerationof

endo-period-ontaltissues.

Conflict

of

interest

Wedisclosenoconflictofinterest.

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