Behavior of macrophage and osteoblast cell lines in contact with the β-TCP biomaterial (beta-tricalcium phosphate)

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ORIGINAL ARTICLE

Behavior of macrophage and osteoblast cell lines in contact with the ␤ -TCP biomaterial (beta-tricalcium phosphate)

Comportement de lignées cellulaires de macrophages et d’ostéoblastes en contact avec le biomatériau ␤ -TCP (phosphate bêta tricalcique)

B. Arbez , H. Libouban

^∗

GEROMGroupeétudesremodelageosseuxetbiomatériaux,IRIS-IBSinstitutdebiologieensanté, universitéd’Angers,CHUd’Angers,49933Angerscedex,France

Availableonline19September2017

KEYWORDS

␤-TCP;

Macrophages;

Osteoblasts;

Osteoconduction;

Resorption

Summary Beta-tricalcium phosphate (␤-TCP) is a synthetic ceramic used for filling bone defects.Itisagoodalternativetoautologousgraftssinceitisbiocompatible,resorbableand osteoconductive.Previousinvivostudieshaveshownthatmacrophagesareoneofthefirstcells comingincontactwiththebiomaterialfollowedbyosteoclastsandosteoblaststhatwillelab- oratenewbonepackets.Studieshavefocusedonosteoclastmorphologyandveryfewofthem haveinvestigatedtheroleofmacrophages.Theaimsofthisstudyweretocharacterize(i)the biomaterialsurface;(ii)theinvitrobehaviorofmacrophages(J774.2andRaw264.7cells)using thedescriptionofcellmorphologybyscanningelectronmicroscopy(SEM)at7and14days;(iii) thebehaviorofosteoblasts(SaOs-2andMC3T3-E1cells)seededatthesurfaceofthebiomate- rial24,48and72hoursbySEMandconfocalmicroscopy.CellproliferationwasanalyzedbyMTT assays.Viabilityandaffinityofthemacrophagesfor␤-TCPwerefoundsignificantlyincreased after7and14d.MC3T3-E1cellswereanchoredandstretchedontothe␤-TCPsurfaceasearlyas 24hwithahighproliferationrate(+190%)whencomparedtothesurfaceofawellplate.SaOs- 2exhibitedthesamemorphologicalprofileat72h.Proliferationbecamesignificantlyhigher comparedtotheplasticsurfaceatonly72h(+129%).Thisstudyemphasisestheimportanceof choiceofthecelllineusedinexploringtheosteoconductiveandosteoinductivepropertiesofa biomaterial.Additionalstudiesareneededtoanalyzedifferentiationofmacrophagesintogiant multinucleatedcellsandhowthebiomaterialsurfaceinfluencesosteoblastdifferentiation.

∗Correspondingauthor.GEROM—LHEAIRIS-IBS,CHUd’Angers49933,Cedex-FRANCE.

E-mailaddress:helene.libouban@univ-angers.fr(H.Libouban).

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Morphologyofmacrophagesandosteoblastson␤-TCP 155

MOTSCLÉS

␤-TCP;

Ostéoconduction; Résorption; Macrophages; Ostéoblastes

Résumé Lebêta-tricalciumphosphate(␤-TCP)estunecéramiquesynthétiqueutiliséepour le comblement de defects osseux. Étant biocompatible, résorbable et ostéoconducteur, il représenteunebonnealternativeauxgreffesautologues.Desprécédentesétudesinvivoont montré quelesmacrophages étaient l’undes premiers typescellulairesen contactavec le biomatériauavecdescellulesmésenchymateursetdescapillaires.Ilsontsuivisparlesostéo- clastespuislesostéoblastesapposentdelamatriceosseuse.Lesétudessesontcentréessur lacaractérisationdesostéoclastesetlamorphologiedesmacrophagesaététrèspeuétudiée.

Lesobjectifsdecetteétudeontété(i)decaractériserlasurfacedubiomatériau;(ii)lamor- phologie invitrodesmacrophages déposéssurla biomatériaux(lignéesJ774.2etRaw264.7) parmicroscopieélectroniqueàbalayage(MEB)à7et14jours;(iii)d’analyserlecomporte- ment cellulairede2lignéesostéoblastiques(SaOs-2etMC3T3-E1)enMEBetenmicroscopie confocaleà24,48et72haprèsensemencementLaproliférationaétéanalyséeparuntestau MTT.Lesrésultatsontmontréunebonnesurvieetunebonneafﬁnitédesmacrophagessurle

␤-TCPà7et14jours.LescellulesMC3T3-E1ontprésentéunaspectaplatiettrèsétiréàla surfacedu␤-TCPdès24havecuneproliférationplusélevée(+190%)parrapportcelleobtenue surunesurfaceplastique.LescellulesSaOs-2ontmontrélemêmeproﬁlmorphologiqueà72h.

La proliférationestdevenuesigniﬁcativement plusélevéeparrapportàlaproliférationsur unesurfaceplastiqueà72h(+129%).L’étudemetenévidencel’importanceduchoixdela lignéecellulairedansl’étudedespropriétésinductivesetostéconductivesd’unbiomatériau.

Desétudessupplémentairessontnécessairesaﬁndemieuxappréhenderlesmécanismesimpli- quantladifférenciationdesmacrophagesencellulesgéantesmultinucléesainsiquel’inﬂuence dubiomatériausurladifférenciationostéoblastique.

Introduction

Beta-tricalcium phosphate (␤-TCP) is a synthetic ceramic thatbelongstothecalciumorthophosphatefamily.Itschem- icalcomposition(␤-Ca3(PO4)2),closetothemineralphase of bone, allows it to be used as a bone substitute for ﬁlling defects in neurosurgery, maxillofacial, reconstruc- tive, orthopedics and spinal surgeries. TCP are known to be biocompatible since almost a century. In 1920, Albee andMorrison havereportedfor theﬁrsttimethe usecal- ciumorthophosphateasbonegraftintherabbitradius[1].

Noadversereaction,inﬂammationortoxicsymptomswere observed.OsteogenesiswasstimulatedbyTCPleadingtoa fasterbonehealing.Theauthorsconcludedthatthismate- rialwassuitableforclinicalapplicationsandcouldbeused in further studies on human subjects. Studies on ␤-TCP increased in the70s, showing thebioresorption ability of

␤-TCPwiththeﬁrsthistologicobservationsin1971:biore- sorptionoccurredsimultaneouslywiththeappositionofnew bone packets after local recruitment of osteoblasts [2].

Reliable methods of ␤-TCP production were subsequently proposedthatleadtothecommercializationofthematerial inthe80s[3,4].␤-TCPis nowrecognizedasosteoconduc- tiveasitprovidesaresorbabletemplatefortheformation ofnewbone[5].Histologicalstudiesshowamarkedapposi- tionoflamellarbonedirectlyincontactwith␤-TCPwithina periodof6to24months[6,7].Inaratmodel,resorptionof

␤-TCPgranulesoccurs2weeksafterimplantationassociated withnewboneformationinside␤-TCPporesafter5weeks [2].In a rabbit model, newbone trabeculae invading the graftedbiomaterialswereevidencedasearlyas8daysby microcomputedtomography(microCT)[8].Boneformation occurring directly onto a biomaterialsurface necessitates differenttypesofcells:recruitmentofosteoprogenitorcells fromsurroundingmesenchymalcells,adhesionofosteogenic

cellsfollowedbysurvival,proliferationanddifferentiation [9].

However,thedegradationmechanismsof␤-TCPremain unclear. Degradation of calcium/phosphate biomaterials in the body is composed of two stages: cellular resorption and the dissolution of the material [10,11]. Ca/P biomaterials can be eroded, phagocytized or degraded by pH modiﬁcations caused by osteoclasts which lead to thedemineralizationof thematerial. Besides osteoclasts, macrophages(ortheirderivedgiantcellsformedbyfusion) areinvolved atan earlystageofresorption[12,13].Some studies on␤-TCP granules grafted in oral surgery suggest thatresorption of the biomaterialmay happenby phagocytosis with macrophages together with osteoclasts once newbone trabeculae are formed [6,14]. A double mech- anismofcellulardegradation of␤-TCP byosteoclasts and macrophagesismostprobable.

Surfacetopographyandporosity ofimplantsandgrafts canalsoinﬂuence bioresorption and thebehavior of cells coming in direct contact with the materials [10,15—17].

Interactions of osteoblasts and macrophages with ␤-TCP surfaceremainsunclear.Theaimofthestudywastochar- acterize: (i) the surface of plates made with ␤-TCP; (ii) themorphologyofmacrophagesseededontotheplatesby scanning electron microscopy (SEM); (iii) the behavior of osteoblast-likecellsseededontheseplatesbySEM,confocal microscopyandproliferationassay.

Material And methods

Characterizationandpreparationof␤-TCP

␤-TCPsamples

Platesof3D-printed ␤-TCP (Sinus-Up^TM)were obtain from Kasios (Kasios, L’Union, France). Sinus-Up^TM plates are

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preparedbyrapid prototyping byusingelementary ␤-TCP powder in hydroxypropyl-methylcellulose with water as binder,platesarethensubsequently sinteredathigh tem- perature and the hydroxypropylmeythylcellulose is burnt offduringsintering.Sinus-Up^TMarecommerciallyavailable andsold for sinus ﬂoor elevation.Sinus-Up^TM have a cen- tralmacroporousarea(whichwasdiscardinthisstudy)and ﬂatlateralsideswhichwerecutinplatesforexperimental purposes(0.8cmbyside).

ScanningElectronMicroscopy(SEM)

Surface morphology of the ␤-TCP plateswas analyzed by SEMonaJEOL6301F(JEOLParis,France).Allsampleswere coatedwith a20nmlayer of platinumby sputtering with a high vacuum coater (Leica EM ECA600, Leica, France).

Imageswerecapturedinthesecondaryelectronmodewith anaccelerationtensionof3kV.

Energy-DispersiveX-raySpectroscopy(EDS)

Anelementalanalysiswasperformedonthe␤-TCPplatesby energy-dispersiveX-rayspectroscopy(EDS)onaZeiss,EVO LS10SEM.Thesampleswerenotcarbonorgoldcoated.The workingpressurewas50Paandtheaccelerationtensionwas 5kV.

Ramanspectroscopy

The chemicalspectrum of the␤-TCP plateswasanalyzed byRamanspectroscopyonaSenterramicroscopewithOPUS 5.5software(Brukeroptic,Ettlingen).Theexcitationlaser wavelengthwas532nmwithanexcitationpowerof25mW and3—5cm⁻¹ resolution.The ﬁnalspectrumwasobtained byaveragingﬁvescansof20seceach.Aconcaverubberband baselinecorrectionwasapplied(11iterations,64points).

Cellculturereagentsandpreparation

All cell culture consumables were obtained from GIBCO (ThermofisherScientific,Illkirch,France).Fourculturecell lines were used: two monocyte/macrophage cell lines J774.2(European CollectionofAuthenticatedCell Culture ECACC #85011428, Salibury, UK) and Raw264.7 (American TypeCultureCollection ATCC#TIB-71,Molsheim, France), Human SaOs-2 osteoblast-like cells (ATCC #HTB-85) and pre-osteoblastcellline MC3T3-E1 subclone4 (ATCC #CRL- 2593).J774.2,Raw264.7andMC3T3-E1cellswerecultured in␣-MEM(Minimum EssentialMedium, alphaModification) andSaOs-2 cells wereculturedin DMEM(Dulbecco’s Mod- ified Eagle Medium). For all cultures, the medium was supplementedwith10%heat-inactivated fetalcalfserum, 100IU/ml penicillinand100␮g/mlstreptomycin. Medium wasreplacedevery2—3daysandtheculturesweremain- tained in humidified atmosphere of 5% CO2 at 37^◦C. At 80%confluence,MC3T3-E1andSaOs-2cellsweredetached using trypsin-EDTA (trypsin/ethylenediamine tetraacetic acid)andJ774.1/Raw264.7cellswereharvestedbyscrap- ping.

Priortocellseeding,␤-TCPplatesweresterilizedin70%

ethanolduring 24hoursand dried for an hour. They were subsequentlyimmergedduringanightinthemedia(␣-MEM supplemented with 10% fetal calf serum) to remove any

traceofethanolandtoallowproteinsfromthemediumto adhereontothe␤-TCPsurface.

Macrophagecultureandseedingon␤-TCP

J774.2 and Raw264.7 cell lines were seeded onto ␤-TCP plates at a density of 3.10⁴ cells/cm² and cultured dur- ing7 and14days(two samples/time). ␤-TCP plateswere immerged in 1mL of medium in a 24-well plate and macrophages were seeded in a homogenous way in the mediumabovethesamples.Attimeofseeding,themedium wassupplementedwith25ng/mLMacrophageColonyStim- ulatingfactor(M-CSF,Biotechnebrand,R&Dsystems,Lille, France).PreparationofcellsforSEMobservationwasthen done(seebelow).

Osteoblastscultureandseedingon␤-TCP

MC3T3-E1andSaOs-22osteoblastcellswereseededonto␤- TCPplatesin24wellplatesatadensityof210⁴ cell/cm² andculturedduring24,48and72h.The␤-TCPplateswere immerged in 1mL of culture medium and the cells were seeded in a homogenous way in the medium above the samples. Experiments for analysis of cell spreading, cell morphologyandproliferationweredoneinduplicateateach timepoint.

Cellspreadinganalyzedbyconfocalmicroscopy

Cellswerefixedin4% paraformaldehydefor20minutesat 4^◦C.Theywererinsed3times5mininPBSandstainedwith 2␮g/mL 4,6-diamidino-2-phenylindole (DAPI, Sigma, Saint Quentin-Fallavier,France)for2minatroomtemperaturein thedark.Afterrinsing6timesinPBSfor 5mineach,cells werelabeledwith6.6␮MAlexaFluor488-conjugatedphal- loidin(ThermofisherScientificIllkirch,France)for45minat roomtemperature inthedarkandrinsedin PBS(6times, 5min each)anddistilledwater(6times5min).The␤-TCP plateswithlabeledcellsweremountedbetweenglassslides with30%glycerol.LabeledcellswereobservedonaLeica TCSSP8laser-scanningconfocalmicroscope(LeicaMicrosys- tems, Heidelberg, Germany) with a HXC PL APO 63XCS2 oil immersion objective(N.A. 1.40). Excitationand emis- sionwavelengthsweresetat405nmforDAPIlabellingand 488nmforphalloidinlabelling.

Some slides were counterstained with xylenol orange 0.5mg(Sigma)for10minindistilledwaterafterthedouble labellingphalloidin/DAPItolabelthe␤-TCPsurface, CellproliferationbyMTTassay

The numberof totaland viablecells onthe surfaceof ␤- TCP plates was measured with a colorimetric MTT assay and compared with that of cells cultured directly onto the well surface. MTT assay is based on the reduction oftheyellowtetrazoliumsaltMTT(3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide, Sigma-Aldrich) by themitochondrialsuccinatedeshydrogenase.Ateachtime point,␤-TCPplatesweretransferredinanew24wellsplate;

cellswereincubatedwithMTTat0.5mg/mLfor2hoursina humidiﬁedatmosphereof5%CO2at37^◦C.MTTwasreduced to purpleformazan crystals which were then dissolved in 500␮Lacidiﬁedisopropanolperwell.Eachsupernatantwas

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Morphologyofmacrophagesandosteoblastson␤-TCP 157 transferred in 96 well plates for absorbance reading at

570nmwithaspectrophotometricplatereader(SpectraMax M2,MolecularDevices,Sunnyvale,CA)becauseabsorbance isproportionaltothenumberofviablecells.Numberofcells wasdeterminedusingastandardcurvewitharangeofeach celltypeconcentrationbetween0and10cells/cm²andthey werereportedtotheseeded surfacearea(wellsurfaceor

␤-TCPplatesurface).Priortocellculture,the␤-TCPplates wereimagedwitha numericradiographyequipment(Fax- itronX-RayLX-60,Edimex,France)andthesurfacearea(in cm²)wasmeasuredusingtheImageJsoftware1.45.

SEMofcellsseededon␤-TCPplates

Sampleswererinsedwithcacodylatebuffer(37^◦C,pH7.4) andﬁxedduringonenightat4^◦Cwithglutaraldehyde(2.5%

in cacodylate buffer 0.2M). Samples were subsequently postﬁxedwithosmium tetroxide(1%in distilledwaterfor 1hour). Theyweredehydratedwitha gradientof ethanol anddesiccatedwithhexamethyldisilazaneandexaminedas abovedescribed.

Statisticalanalysis

Statisticalanalysiswasperformedwiththestatisticalsoft- ware MedCalc version 8.2.10 (Ostend, Belgium). All data werereportedasmean±standarderrorofthemean(SEM).

Statistical signiﬁcance between groups for MTT test was determinedby anon-parametricKruskal-Wallis analysisof varianceandcomparisonbetweengroupswasdeterminedby post-hoctest.Adifferencewasconsideredsigniﬁcantwhen P<0.05.

Results

␤-TCPcharacterization

SEM

Theraw surfaceof the␤-TCPsamplesappearsonFig.1A.

Althoughthesurfaceofthebiomaterialseemedratherﬂat macroscopically,theSEManalysisrevealedinfact arough surfacewithvalleysandhills.Athighermagniﬁcations,the material surface showed a polycrystalline pavement with different polygonalcrystallites separated by grain bound- aries.Defectlineswerealsopresentonthesurfaceofthe material.Somecrystallitesshowedahexagonalpatternat thesurfaceof thepolygonalpavement(Fig.1B). Amicro- porosity was evidenced at the surface of the biomaterial betweenthesinteredgrains,itwasalsovisibleoffractured sections(datanotshown).

Ramanspectroscopy

TheRamanspectrumofthe␤-TCPplatesappearsonFig.2 forawavenumberrangingfrom50to1550cm⁻¹.Thelabeled peaks are characteristic of the internal vibration of the PO43 tetrahedricgroupsofthe␤-TCPmolecule.Thesym- metric stretching (␯1) of P O bonds of the tetrahedron correspondstothepeakswiththehighestintensityataround 950cm⁻¹and970cm⁻¹.Theasymmetricstretching(␯3)has a lower intensity and is located in the 1015—1090cm⁻¹

Figure1 (A)SEMimageofthesurfaceofa␤-TCPsintered platefromaSinusUp^TMshowinggrainboundaries(whitearrows) anddefectlines(blackarrows).Notethepresenceofamicrop- orositybetweenthesinteredgrains.(B)SEMimageofthe␤-TCP surfaceshowingshearbandswithahexagonalpattern.

108910471016971949

612549

442407

200 400 600 800 1000 1200 1400

Wavenumber (cm-1)

PO43-

ν 2 PO43- ν 4

PO43-

ν 1

PO43-

ν 3

Raman intensity (arbitrary units)

Figure2 Ramanspectrumofthe␤-TCPplate.

range.Theother vibrationalmodes(␯2and␯4)correspond toO P Obendingdeformationsofthe tetrahedron.They arerespectivelylocatedat407and548cm⁻¹.

Monocyte/macrophagecellsmorphologybySEM After7and14days,RAW264.7andJ774.2cellshavesur- vivedon␤-TCPsurface.At7daysofculture(Fig.3A),the majority of Raw264.7 cells had a round shape. After 14 days,someRAWcellsappearedﬂattenedwhensomeothers hadmaintaineda roundshape(Fig.3B). These cells have

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Figure3 RAW264.7cellsafter(A)7daysand(B)14daysofculture.At14days,somecellshadanelongatedshape(whitearrows) andsomeothershadaroundshape(blackarrows).HighermagniﬁcationofaRAW264.7cellon␤-TCPafter(C)7daysand(D)14 daysofculture.

emittedlongfilopodiathatanchorthemontothe␤-TCPsur- face(Fig.3D).Thenumberoffilopodiaincreasedbetween7 days(Fig.3C)to14days;eveniftheycannotbecountedon sucharoughmaterial,thiscorrespondstoafirmeranchor capacityofthecellstothebiomaterial.

At 7 and 14 days, the osteoblastic J774 cells seeded onthe ␤-TCP had a round shape(Fig. 4). They appeared anchoredtothesurfacewithlessﬁlopodiathanRAW264.7 cellsinthesameconditions;cytoplasmicveil-likestructures wereobservedontheirsurface(Fig.4C-D).Nodifferencein morphologybetweenthetwotimesofculturewasobserved.

MorphologicalaspectofSaOs-2andMC3T3-E1cells on␤-TCP

SEM

SEManalysisofSaOs-2andMC3T3-E1cellsincontactwith␤- TCPat24,48and72happearsonFig.5.At24h,SaOs-2cells hadaroundshape;at48h,theyflattenedandstretchedon the␤-TCPpavement(Fig.5A).Cellsexhibitedpseudopodia allowingadirectanchoragetothebiomaterialsurface.At 72h,SaOs-2cellsappearedflatandmorestretchedthanat 48h.Cellsexhibitedlongcytoplasmicextensions(pseudopo- diawithsomefilopodia)thatallowedthemtobeanchored ontheroughsurfaceofthe␤-TCP(Fig.5B).At72h,SaOs-2 cellsshowedamorphologicaladaptationtothereliefmade ofvalleysandhills.

At 24h, MC3T3-E1 cells were flat and affixed onto the ␤-TCP surface (Fig. 5C). Cells were anchored by both filopodia andlargerpseudopodia. At 48h,cells had

proliferated andformed adenselayer. At72h,theywere ﬂat,withanenlargedsurfaceandhadestablishednumerous contactbetween eachother; thus amonolayer ofMC3T3- E1 cellscovered almostall thesurfaceof thebiomaterial (Fig.5D).

Confocalmicrocopy

Fig. 6 shows confocal images obtained after a double labellingDAPI/phalloidinofSaOs-2andMC3T3cells.At24h aftercellsseeding,phalloidinlabellingshowedthatSaOs-2 cellswereattachedonthe␤-TCPsurfacebutappearedless spreadthanMC3T3-E1cells(Fig.6A-C).The actinnetwork appearedclearlymuchmoredevelopedinthecytoplasmof MC3T3-E1cellscomparedtoSaOs-2cells.Asearlyas24h, MC3T3-E1cellsappearedincontactandoverlapped;confo- calimagesevidenced cytoplasmicextensions thatinteract withneighboringcells(Fig.6C).At48h,theactincytoskele- ton of SaOs-2 cells remained poorly developed and these cells were not well spread. On the contrary, MC3T3-E1 cellsappearedwellspreadandcytoplasmicextensionswere observed.Confocalobservationatasmallermagniﬁcation, clearlyshowedadenselayerof MC3T3-cellsonthe␤-TCP surface(thatappearedinredafterxylenolorangecounter- staining)(Fig.7).

At 72h, SaOs-2 cells formed a dense layer on the ␤- TCPsurfaceandtheirmorphologicalaspectclearlyshowed improvement by exhibiting round shaped nuclei, a developed actin network andcytoplasmic extensions (Fig.6B).

MC3T3-E1cellscoveredalmostthe␤-TCPsurfaceandthese

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Figure4 J774cellson␤-TCPafter(A)7daysand(B)14daysofculture.HighermagniﬁcationofJ774cellson␤-TCPsurfaceafter (C)7daysand(D)14daysofculture.

Figure5 SEMobservationsofSaOs-2cellsbehaviorincontactwith␤-TCPplatesat48h(A)and72h(B);MC3T3-E1cellsbehavior incontactwith␤-TCPplatesat24h(C)and72h(D).

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Figure6 ConfocalmicroscopyobservationsofSaOs-2cellsadhesion(A-B)andMC3T3-E1cellsadhesion(C-D)on␤-TCPsurfaceat 24and72h.TheactinﬁlamentsarestainedingreenwithphalloidinandthenucleiarestainedinbluewithDAPI.

cellsappearedwellspreadwithlongcytoplasmicextensions (Fig.6D).

Proliferationofosteoblast-likecellson␤-TCP Proliferationkineticsof SaOs-2andMC3T3-E1cellsonthe

␤-TCP surface at 24, 48 and 72hours appears on Fig. 8.

The numberof SaOs-2 cells significantlyincreased at 48h (P<0.05 vs 24h) and at 72h (P<0.05 vs 48h). At 24 and 48h SaOs-2 proliferationwasnot significantly higherthan ontheplasticsurface.At72hproliferationofSaOs-2cells onthebiomaterialhadincreasedby129%comparedtocon- trolconditionsontheplasticsurface(P<0.05).Thenumber ofMC3T3-E1cellson␤-TCPincreasedfrom24to72hbutit becamestatisticallysignificantonlyat72hvs48h(P<0.05).

Proliferationonthe␤-TCP surfacewassigniﬁcantlyhigher when compared to controls on the plastic surface at each timepoint at 24, 48 and 72h (resp. +190%, +177%, +181%).

Discussion

Thesurfaceofthe␤-TCPplatesobservedbySEMpresented threemain characteristics: micropores, apolygonalpave- ment ofpolycrystalline tessels limitedby grainjoints and defectlines.ThedefectlinesobservedonourSEMimages, represent shear bands, a characteristicsurface defect on ceramics.Duringsinteringathightemperature,themotion of atoms allows the material to form crystallites. When forming,thesecrystallitesaresubmittedtohighshearstress thatleadstotheformationofstructuraldefectscalleddis- locations; they are 2D linear plastic deformations of the crystal. Under the shear stress, they have the ability to moveoverthecrystalliteleadingtotheformationofdefect lines, alsocalled shear bands.Some shear bandsshowan hexagonal pattern characteristic from the hexagonal lat- ticeofthe␤-TCP structure[18].Thesespeciﬁcitiesofthe surface topographyis of the upmost importanceand may inﬂuence adhesion of macrophages andexpression of dif- ferentcytokines[19].Ithasbeenshownthatthechemical

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Figure7 ConfocalmicrocopyobservationofMC3T3-E1cells cultures 48h on ␤-TCP surface stained in red with xylenol orange.Theactinﬁlamentsarestainedingreenwithphalloidin andthenucleiarestainedinbluewithDAPI.

24 48 72

0 10000 20000 30000 40000

Number of cells

24 48 72 hours

SaOS-2 MC3T3-E1

a a a

* *

#

Figure8 ProliferationofSaOs-2andMC3T3-E1cellcultured directlyon24wellplatesϒandon␤-TCPplates ,expressed innumberofcellspercm²at24,48and72hours.^aP<0.05vs plasticsurface;^*P<0.05vs24hon␤-TCPsurface;^#P<0.05vs 48hon␤-TCPsurface.

androughnesssurfaceof␤-TCPfavoredtheadhesionprocess osteoblastcellsinvitro[20].

The present study focused on cells morphology which havedevelopedinvitroon␤-TCP.Severalstudieshavebeen done usingmacrophages culturedon ␤-TCP [10,16,17]. In most ofthem, macrophageswere culturedin presenceof receptoractivatorofnuclearfactorkappa-Bligand(RANK-L) toformosteoclast-likecellsbutnoneofthemhavefocused onthemacrophagemorphology.Inapreviousstudy,wehave foundbyatime-lapsinvitrostudythatmacrophageswere abletoresorb␤-TCPgranulesandthatosteoblast-likecells couldclimbatthesurfaceofthebiomaterial[21].

The two mouse monocyte/macrophage cell lines used in this study were cultured with M-CSF. This cytokine is known to regulate and control the survival, proliferation anddifferentiationofphagocyticmacrophagesfromundif- ferentiatedprecursors[22,23].AddingM-CSFinthecultures allowscellstodifferentiateintofullymaturemacrophages.

RAW264.7andJ774cellssurvivedbetween7and14days.

Theyhadcytoplasmic veil-likeexpansions ontheirsurface characteristicofhealthymacrophages.Thecellspresented pseudopodiaandnumerousfilopodiathatanchoredthemat thesurfaceofthe␤-TCP.Previousworkonrabbitbonebiop- siesshowedthatcellularresorptionof␤-TCPoccurredintwo steps[24].GiantnucleatedTRAcP-negativecellsfirstcolo- nizedthesurfaceofthebiomaterialfrom7to14days.These cellscontainedagreatamountofmineralcrystalsfromthe calcium-phosphatematerial inside theirvacuoles suggest- ingdegradation by phagocytosis. As new bone is formed, multinucleated TRAcPpositive cells witha ruffled border (characteristicofosteoclasts)areevidencedonthesurface ofCa/Pceramics[24].Thenumberofosteoclastsincreases upontime.So,adoublepopulation ofmultinucleatedcell isresponsibleforthecellularresorptionofceramics:giant TRAcP-negativecellsthaterodethebiomaterialandosteo- claststhat resorbthe biomaterialand remodelthe newly formedbone[24,25].

Besidesosteoclasts,macrophagescouldalsobeinvolved at an early stage of biomaterial resorption. In a series of 14 patients that had sinus lift augmentation in oral surgerywith␤-TCPgranules,TRAcP-positivemultinucleated cellswereobservedincontactwithgranules[6].However, slightlyTRAcP-positivecells (characteristicofmacrophage activation) were also observed with ␤-TCP grains inside their cytoplasm after phagocytosis. Similar ﬁndings were also reported by others [8,14,26,27]. This suggests that resorption happens by phagocytosis due to macrophages togetherwithosteoclasts.Theearlyvascularizationaround thegrafted␤-TCPparticlesallowsinsitumigrationofpre- cursorcells,macrophagesandosteoprogenitors[28].Inour study,nogiantmultinucleatedcellswereobservedmeaning thatmacrophagesdidnot fuseintogiant cells in vitro.In thefuture,itcouldbeinterestingtoanalyzetheexpression ofTRAcPbymacrophagesinpresenceof␤-TCPandhowit variesovertime.

Itisadmittedthattheresorptionincaseofabiodegrad- ablematerialoccurssimultaneouslywithappositionofnew bonepackets afterrecruitmentsofosteoblasts. Numerous studies have focused onthe osteoconductive characteristics of a biomaterial in culture using an osteoblast cell line and/or bone marrow stroma cells (BMSC)[20,29,30].

The choice of a cell type in an in vitro study is of the upmostimportance.BMSCsareinterestingbecausetheycan differentiate intoosteoblasts. Indeed,such an osteogenic differentiationincontactwithabiomaterialcanreﬂectits osteoinductivepotential[30].Inthepresent study,SaOs-2 are mature osteoblast derived from a human osteosar- comaastheyexpressalkalinephosphatase[31].Incontrast, MC3T3-E1cellsarepre-osteoblastsastheydonotexpress- ing alkaline phosphatase in the absence of ascorbic acid and␤-glycerophosphate[31].MC3T3-E1havebeen shown tobe the most appropriate model in biomaterial studies [31]. CultureofMC3T3-E1oncalciumphosphate ceramics inducesalkalinephosphatasegeneexpressionafter14days

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ofculturewithoutanymediumsupplementation[30].Inour studyMC3T3-E1cellsadheredandspreadoutonthe␤-TCP surfacemorerapidlythanSaOs-2cells.At72h,thetwocell linesoccupiedmostofthesurfaceandexhibitedadeveloped cytoskeleton with a marked actin network [32]. Interac- tionbetweencellsandthebiomaterialsurfaceiscrucialto induceproliferation,followedbydifferentiation.Ourresults showedaninﬂuenceofthe␤-TCPsurfaceoncellprolifera- tionasearlyas24hwhichcouldbecorrelatedwitharapid adhesionprocessat24h. Interactionwithanextracellular matrix or a biomaterial involves is mediated by integrins thatinteractedwiththematrix. Anotherstudy hasshown thatspreadingofSaOs-2osteoblasticcellsoccurredwithin 1dayon␤-TCP(asalsofoundhere)andthatfocaladhesion areobservedat4days[20].Inourstudy,cytoplasmicexten- sionswereobservedafter48hoursallowingaﬁrmanchorage ofthecellsontothebiomaterialsurface.

In conclusion, the present study emphasises the importanceofthechoiceofacelllineinexploringtheosteo- conductiveandosteoinductivepropertiesofabiomaterial.

Additional studies are needed to better understand the resorptionprocessinvolvingdifferentiation ofmacrophage intogiantmultinucleatedcells.Thetopographical,chemical andphysicochemicalcharacteristicsof␤-TCPmayaccount for itsexcellentcapacityof inducinga regenerativebone formationassociatedwithprogressiveresorptionofthebio- material.

Disclosure of interest

B.A.receivedaPhDscholarshipfromKasiosSAS.

Acknowledgments

This work was made possible by grants, from ANR, pro- gramLabCom‘‘NextBone’’.SEMandconfocalanalysiswere performed at Service Commun d’Imagerie et d’Analyses Microscopiques(SCIAM),Université d’Angers, thanks toR.

PerrotandR.Mallet.ManythanksforKasiosSAS,18,chemin delaViolette31240L’UNION—FranceforprovidingtheSinus- Lift^TMdevices.

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