DSpace at VNU: Biomimetic scaffolds based on hydroxyapatite nanorod poly(D,L) lactic acid with their corresponding apatite-forming capability and biocompatibility for bone-tissue engineering

TheporosityoftheHAp/PDLLAscaffoldswasmeasuredusing theliquidsubstitutionmethod[35].Distilledwaterwasusedas thedisplacement liquid.In brief,a dry sample of each scaffold wasweighed and th

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jou rn a l h om ep ag e :w w w e l s e v i e r c o m / l o c a t e / c o l s u r f b

Biomimetic scaffolds based on hydroxyapatite nanorod/poly(d,l)

lactic acid with their corresponding apatite-forming capability and

biocompatibility for bone-tissue engineering

a School of Chemical Engineering, Hanoi University of Science and Technology, 1 Dai Co Viet Road, Hanoi, Viet Nam

b Research Center for Environmental Technology and Sustainable Development, Hanoi University of Science, 334 Nguyen Trai Street, Hanoi, Viet Nam

a r t i c l e i n f o

Article history:

Received 14 October 2014

Received in revised form 27 February 2015

Accepted 1 March 2015

Available online xxx

Keywords:

Biomimetic scaffolds

Poly(d,l) lactic acid

Hydroxyapatite nanorods

Apatite

Biocompatibility

Bone tissue engineering

a b s t r a c t

1 Introduction

Bone repair and regeneration have become a serious

chal-lenge in orthopedic surgery because of the increase in clinical

bonediseases(e.g.,boneinfections,bonetumors,andboneloss

throughtrauma)[1].Currenttherapiesfor bonedefectsinclude

autografts,allografts, and otherbone substitutes[2].Autografts

(bonesobtainedfromanotheranatomical sitein thesame

sub-ject)comprisethegoldstandardforthetreatmentofbonedefects,

but this surgicalmethodstill hasmajor disadvantages,suchas

thepossibility of donorsite morbidity, shortageof donor bone

supply,anatomicalandstructuralcomplications,aswellasgraft

∗ Corresponding author Tel.: +84 4 38680 110; fax: +84 4 38680 070.

E-mail address: nga.nguyenkim@hust.edu.vn (N.K Nga).

sorption[1].Allografts(bonesfromdonorsoranotherspecies)can

beusedasanalternative;however,thismethodcausesinherent problems(e.g.,diseasetransmissionandimmunogenicresponses) [3].Syntheticbonesubstitutes,whicharemostlymadeofmetalsor bioceramicsandglasses,haveosteoconductivepropertiesinstead

ofosteoinductiveproperties,thuslimitingtheiruse[4].Bone-tissue engineering(BTE)hasattractedscientificattentionforbeingmore effectivethanconventionalmethodsintermsofbonerepairand reconstruction The coreof BTE is to combinea biodegradable matrix(scaffold)andliving cellstogrowtissue invitroprior to implantationofthesubject[5].ScaffoldsforBTEshouldpossess severalproperties,suchashighporosity,amacroporousnetwork forinvitrocellmigration,adhesion,proliferation,andfurthertissue growth,biocompatibilityandbiodegradabilitytonon-toxic prod-ucts,aswellassufficientmechanicalstrength[1].Toachievethese properties,BTEscaffoldsareoftendesignedtomimicthestructural

http://dx.doi.org/10.1016/j.colsurfb.2015.03.001

0927-7765/© 2015 Elsevier B.V All rights reserved.

and biological functions of a naturally occurring extracellular

matrix(ECM)intermsofbothchemicalcompositionandphysical

structure[6,7]

Inthisregard,biodegradable polymers,suchas

poly(l-lactic-co-glycolicacid) [8],poly(␧-caprolactone)[9],poly(l-lacticacid)

[10],andpoly(d,l-lacticacid)(PDLLA)[11],havebeenprocessed

intothree-dimensional(3D)scaffolds.Thesepolymersshowgood

mechanical properties, with shapes and degradation rates that

areeasilyadjustable.Themaindisadvantageofthesepolymeric

materialsistheirpurebiocompatibility, given that theydo not

provide a favorable surface for cell attachment and

prolifera-tionbecauseof thelackof specificcell-recognizablesignals[1]

Bioactiveinorganicmaterials,suchashydroxyapatite(HAp)[12],

␤-tricalciumphosphate[13,14],andbioactiveglasses[15,16],have

beendesignedas3Dporousscaffoldsforboneregeneration.The

advantagesofbioactiveceramicsaretheirexcellent

osteoconduc-tivityandbiocompatibility;however,theirinherentbrittlenessand

lowmechanicalstrength(forporousspecimens)aremajor

disad-vantagesindeveloping3Dscaffolds,thuslimitingtheirapplications

[13,15,17]

Polymer/bioceramiccompositescaffoldsareattracting

increas-ingattentionin thefield ofbone regeneration becauseoftheir

mechanical stability and biocompatibility [18,19] Among such

scaffolds, HAp/polymer composites have received considerable

interestbecauseoftheircompositionandstructuralsimilarityto

naturalbone[20,21].Boneisacomplexcompositethatcomprises

anorganicphase(90%typeIcollagen,othernon-collagenous

pro-teins(e.g.,proteoglycans)),minoramountsoflipidsandosteogenic

factors(e.g.,bonemorphogeneticproteins),andamineralphase

[22] The mineral phase consists of one or more types of

cal-ciumphosphatescomprising65–70%of boneand embeddedin

a protein matrix [22–24].Among theCaP salts,hydroxyapatite

(Ca10(PO4)6(OH)2,HAp)isthemoststablecalciumphosphatein

bodyfluidsandisthemostsimilartothemineralpartofbone[23]

ThebiologicalHApsfoundinphysiologicalhardtissuesare

irreg-ularlyrod-likeorplate-likecrystalsofvariablelengthsandwidths

(30–45nm)withathickness ofapproximately5nm[22].These

HApsareorientedtothec-axis,whichisparalleltothecollagen

fibrils[22,25]inwhichdifferentcelltypes,includingosteoblasts,

osteoclasts,andosteocytes,reside.Asurveyoftheliteratureshows

thatbioceramicsthatmimic bonemineralinterms of

composi-tion,structure,andmorphologycanpromoteosteointegrationand

subsequentbonetissueformation[26–28]

PDLLAisanontoxic,biocompatible,andbiodegradable

mate-rialthathasbeenusedassuturesandtissue-engineeringscaffolds

[29].However, PDLLApresentsstronghydrophobicity owingto

theabsenceofhydrophilicgroupsandsuitablefunctionalgroups

(e.g., NH2, OH),whichresultsintheabsenceof

osteoconductiv-ity.Arecentstudyfocusedonfabricatingbioactivenanocomposite

PDLLA/nano-HApmembranesusinganelectrospinningmethodto

improvetheosteoconductivityofthepolymer[30].However,the

productsarecharacterizedbysmallporesizeswithamean

diam-eterof4.8␮mand arethusunsuitable foruseasBTEscaffolds,

giventhattheminimumporesizerequirementforbonescaffolds

is100␮m[31]

Severalmethodshavebeendevelopedtofabricate3Dscaffolds

Thesemethodsincludefiberbonding,freezedrying,phase

separa-tion,super-criticalfluidtechnology,solventcastingcombinedwith

particulateleaching,andmeltmolding[1].Amongthesemethods,

solventcastingcombinedwithparticulateleachinghasbeenwidely

usedforfabricating3Dscaffoldsbecauseofitssimplicityand

effi-ciency.Thismethodallowstoproducehighlyporousscaffoldswith

porosityupto93%andmeanporediameterupto500␮mby

vary-ingporogenparticlessize,andweightratioofpolymertoporogen

withouttheneedofthespecializedequipment[1,32].Asaresult,

inthisstudy,solventcastingcombinedwithparticulate-leaching

methodhasbeen usedas anefficient control tosynthesize3D HApnanorod/poly(d,l)lacticacid(HAp/PDLLA)scaffolds.The rod-shapedHApnanoparticleswiththesamesize asboneminerals weresuccessfullypreparedinpaststudies[33,34]andhavesince beenusedasan inorganicphase toincorporateinto thePDLLA matrixandtopreparebiomimeticHAp/PDLLAscaffoldsforBTE Theapatite-formingcapabilityofthescaffoldswasdeterminedby assessingtheformationofbone-likeapatiteonthesurfaceofthe scaffoldsafterimmersinginsimulatedbodyfluids(SBFs) Mean-while, biocompatibilitywas investigated in direct contact with humanosteoblastcelllineMG63throughinvitrotests

2 Experimental

2.1 Chemicals

Allreagentswereofanalyticalgradeandusedasreceived with-outfurtherpurification.Calciumchloridedihydrate(CaCl2·2H2O), sodium monophosphate dihydrate (Na2HPO4·2H2O), NaOH,

C2H5OH,1,4-dioxane,hydrochloricacid(HCl),pluronicco-polymer PEO20–PPO70–PEO20 (P123), NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, Na2SO4, Tris-hydroxymethyl aminomethane ((HOCH2)3CNH2),phosphatebufferedsaline(PBS)wereobtained from Sigma–Aldrich PDLLA was purchased from Boehringer Ingelheim (Ingelheim, Germany) Deionized water was usedto prepareallsolutionsandreagents

2.2 Scaffoldproduction

The HAp nanorods used in this study were prepared using the hydrothermal method assisted by a pluronic co-polymer PEO20–PPO70–PEO20 The preparation and characterizations of theseHApnanorodswereconductedinthesamemannerasinour previouswork[33].HApnanorod/PDLLAscaffoldswerethen pre-paredusingsolventcastingcombinedwithsalt-leachingmethod withNaClastheporogen.A7%polymersolutionwasproducedby dissolving0.455gofPDLLAin6.5mLof1,4-dioxanefor3hat32◦C

AsuspensionofC2H5OHwithvariousamountsofHApnanorods (0–30wt.% HAptoPDLLA) wasaddeddropwise into thePDLLA solution.Theresultingmixturewasvigorouslystirredona mag-neticstirrerataspeedof500rpmfor2htoachievehomogeneity Thehomogeneousmixturewasthencastintoa55mmglassPetri dishcontaining9gofNaClwithparticlesizesof300–450␮m.The sampleswerethenair-driedunderachemicalhoodfor24hand vacuum-driedforanother24htoremoveallsolvents.The result-ingscaffoldswerethenimmersedindistilledwaterat35◦Cfor3d (waterwaschanged3–4timeseachday)toleachoutthesalt.The producedHAp/PDLLAscaffoldswerefurtherair-driedand vacuum-driedandthenstoredinadesiccatoruntiluse.Finally,fourscaffolds wereobtainedaccordingtothepercentageofHApnanorods.These scaffoldswerelabeledasS1,S2,S3,andS4for0wt.%HAp,10wt.% HAp,20wt.%HAp,and30wt.%HAp,respectively

2.3 Scaffoldcharacterization

Themorphologiesandporestructuresoftheproducedscaffolds wereexaminedusingfieldemissionscanningelectronmicroscopy (FE-SEM)(Supra40,Zeiss,Germany)atlow(200×)andhigh(500×,

1000×and5000×)magnifications,aswellasopticalmicroscopy (SMZ800,Nikon,Japan).PriortoFE-SEMobservation,thedry sam-plesofthescaffoldswerecutintosmalldisks,whichweremounted

onanSEMstubandthensputter-coatedwithathinchromiumlayer (Quorumtech,Q150TESTurboChromiumSputter-Evaporator).The meanporediameterandporewallthicknessofthescaffoldswere measuredbyusingscanningelectronmicroscopic(SEM) images throughImageJsoftware.TheFouriertransforminfraredspectra

(FTIR)ofthescaffoldswererecordedonaNicolet6700

spectrom-eterusing KBrpellettechnique inthe rangeof 4000–400cm−1

witharesolution of4cm−1.Allmeasurementswereperformed

at25◦C.ThecompositionofHApnanoparticlesonthesurfaceof

HAp/PDLLAscaffoldswasexaminedthroughenergy-dispersive

X-rayspectroscopy(EDXS)(NovaNanoSEM450,FEI)

TheporosityoftheHAp/PDLLAscaffoldswasmeasuredusing

theliquidsubstitutionmethod[35].Distilledwaterwasusedas

thedisplacement liquid.In brief,a dry sample of each scaffold

wasweighed and then immersed in a graduated cylinder

con-tainingaknownvolumeV1 ofwaterandlefttostandtoenable

the water to penetrate into the pores of the scaffold sample

until no air bubbles emerged from thescaffold The total

vol-umeofwater and water-impregnated scaffold wasrecordedas

V2.Thevolumedifference(V2−V1)representsthevolumeofthe

HAp/PDLLAscaffoldskeleton.Thewater-impregnatedscaffoldwas

thenremovedfromthegraduatedcylinder,andtheresidualwater

volumewasrecordedasV3.Thetotalvolumeofthescaffoldisgiven

byV=(V2−V1)+(V1−V3)=(V2−V3).Bymeasuringtheinitialand

finalweightsWiandWfofthescaffoldsbeforeandafterimmersing

inwater,wecancalculatetheporevolumeofthescaffoldas

Wf−Wi

Theporosityofthescaffoldcanbecalculatedusingthefollowing

equation:

Porosity= (Wf−Wi)/H2 O

V2−V3

(2)

Atleastfivemeasurementswereconductedforeachscaffold,

andtheresultswereaveragedfromthesefivemeasurements

2.4 Invitromineralizationtests

Invitromineralizationtestswereconductedthroughthe

incu-bationoftheHAp/PDLLAscaffoldsinSBFs.SBFwaspreparedas

describedpreviously[36],filteredusinga0.22␮mMilliporefilter

systemtoeliminatebacterialcontamination,and thenstoredin

aplasticbottleinarefrigeratorat4◦C.Afterdisinfectionin70%

ethanolat4◦C,threesamplesofeachscaffoldwereimmersedin

30mL ofSBF solution placed in closed polyethylenecontainers

at37◦C.AfterbeingimmersedinSBFforthedesigneddays,the

sampleswereremoved,gentlyrinsedwithdeionizedwater,and

driedunderwarmflowingair.Therinsingprocesshasbeenusedto

removetheresidualionsoftheSBFsolutionsthatremainedonthe

scaffoldsamplesandcouldaffectthescaffoldstructure.All

opera-tionswereconductedinalaminarairflowhoodtoavoidbacterial

infection.Theapatite-formingcapabilityofHAp/PDLLAscaffolds

wasassessedthroughFE-SEMandEDXS

2.5 Invitrocellcultureexperiments

Humanosteoblastcells(MG63cellline,IZSLERBiobankingof

VeterinaryResources,Brescia,Italy)wereusedtoevaluate

biocom-patibilityofthepreparedHAp/PDLLAscaffolds.Cellsweregrown

inaminimumessentialmedium(Gibco)supplementedwith10%

(v/v) fetal bovine serum (Gibco), 2% (v/v) l-glutamine (Gibco),

1%(v/v)antibiotic,1%(v/v)sodiumpyruvate,and1%(v/v)

non-essentialaminoacids(Gibco)at37◦C and5%CO2.Themedium

waschangedevery2d.Theadherentcellswereallowedtoreach

confluence,thendetachedwith0.1%trypsin

ethylenediaminetet-raaceticacid, andsuspended ina freshculturemedium forthe

experiments

Priortocellseeding,thepreparedHAp/PDLLAscaffoldsamples

withadiameterof8mmweresterilizedin70%ethanolat4◦Cfor

24h,washedwithsteriledistilledwater,andimmersedinaculture

mediumfor1hbeforeseeding.Thereafter,thesampleswereplaced

on48-wellplates.20␮Lofcellsuspensionatadensityof7.5×104

cellspersamplewasseededonscaffoldsamples.Thesampleswere thenincubatedat37◦Cfor2htoallowcellattachmenttothe scaf-foldsurfaces,followedbyadding250␮Lofculturemediuminto eachwell.Cultureplateswerethentransferredtoanincubatorat

37◦Cand5%CO2.Culturetimeswere3,5,and7d,andthefresh culturemediumwaschangedevery2d

Cellviabilityandproliferationweredeterminedaftereach incu-bationtime usingalamarBlueassay.Theassayswereperformed after3,5,and7dofcellseeding,accordingtothemanufacturer’s protocol.Briefly,attheendofeachincubationtime,theculture mediumwasremovedfromthewellsandfreshculturemedium with10%(v/v)alamarBluewasaddedtoeach well.After4hof incubationat37◦C,aliquotsof100␮Lwerepipettedinto96-well plates,andfluorescencewasrecordedonamicroplatereaderatan excitationwavelengthof565nmandanemissionwavelengthof

595nm

Cell morphology, growth, and distribution were visualized afterstainingwithOregonGreen488phalloidin(Life Technolo-gies;Carlsbad,CA,USA)and4,6-diamidino-2-phenylindole(DAPI, Sigma–Aldrich).Cellswerefixedwith4%(w/v)formaldehydein PBS,permeabilizedwith0.2%TritonX inPBS,and stainedwith OregonGreen488andDAPI,accordingtothemanufacturer’s pro-tocol.AfterthreerinseswithPBS,thesampleswereexaminedusing confocallasermicroscopy

2.6 Statisticalanalysis

Resultswereaveragedandexpressedasmean±standard devi-ation.StatisticalanalysiswasperformedusingANOVA.Avalueof

P<0.05wasconsideredstatisticallysignificant

3 Results and discussion

3.1 Characterizationofthesynthesizedscaffolds

Four HAp/PDLLA scaffolds were synthesized with different weightpercentagesofHAp(onescaffoldwithoutHApfillingandthe otherthreescaffoldswithHApfilling).Thecompositionsandother characteristicsoftheproducedscaffoldsaresummarizedinTable1 Fig.I1(supportingmaterial)showdigitalcameraimagesofthe scaf-foldsamples(S3andS4scaffolds)andtheopticalimagesofatypical S3scaffold sample (Fig.I2).These scaffoldshave stableshapes, thicknessesof2mm(Fig.I1),andporousstructures(Fig.I2).Surface morphologiesandpore structureswerethen examinedthrough FE-SEMimaging.TheSEMimagesofallsynthesizedscaffoldsare presentedinFig.1andFig.I3.Observationsshowthatthesurface morphologies of thescaffoldsbecome coarseras theHAp con-tentincreases.AsmoothsurfacemorphologywasproducedforS1 PDLLAscaffolds(Fig.1aandI3A),whereascoarsesurface morpholo-gieswereobtainedfor HAp/PDLLAscaffolds.HAp nanoparticles werehomogeneouslydepositedwithintheporewallsofthe scaf-folds(Fig.1b–d) FE-SEMimageswithhighmagnifications(Fig I3B–D)indicatedthatnoaggregatesofnanoparticlesappearedon theporewallsforallcompositescaffolds.Moreover,thinporewalls

of1.48±0.28␮mwereobserved(Fig.I3B)forS2scaffoldwitha lowHAppercentageof10wt.%.AfurtherincreaseinHAp percent-ageproducedscaffoldswiththickerporewallsof1.81±0.3and 3.94±0.63␮m(Fig.I3CandD)forS3andS4scaffolds,respectively Fig.I3E(FE-SEMimageofahighermagnificationforS4scaffold) showedthattinyrod-likeHApparticleswereembeddedonthe polymerphase.AccordingtoTable1,increasingHAppercentage resultedinadecreaseinporesizesoftheproducedscaffolds Fur-thermore,statisticalanalysisandporesizedistributionofS1PDLLA

Table 1

Composition and some characteristics of HAp/PDLLA composite scaffolds.

a Actual amount of HAp in total PDLLA.

b Mean value ± standard deviation (SD); n = 30.

c Mean value ± standard deviation; n = 5.

scaffolds(Fig.I4A)demonstratethatthesescaffoldshaveporesizes,

whicharesignificantlylargerthanthoseoftheHAp/PDLLA

scaf-folds(IP<0.001)andaremainlyintherangeof100–450␮mwitha

meanporesizeof274␮m.S2andS3HAp/PDLLAscaffoldshave

meanporesizesof183and122␮m(Table1)withintherange

of100–350␮mand50–300␮m,respectively(Fig.I4BandC);

dif-ferencesintheirporesizesarestatisticallysignificant(IP<0.001)

S4HAp/PDLLAscaffoldshavethesmallestporesizeswithamean

poresizeof117␮m(Table1).However,statisticalanalysisshowed

thatporesizesofS4HAp/PDLLAscaffoldsaresignificantlysmaller

thanthoseofS1PDLLAandS2HAp/PDLLAscaffolds(IP<0.001),

butnotsignificantlysmallerthanthoseofS3HAp/PDLLAscaffolds

(P>0.05).TheobtainedresultsconfirmthatHApcontentgreatly

affectstheporestructureandoverallmorphologyofthecomposite

scaffolds.HigherHApcontentresultedintheproductionof

scaf-foldswithsmallporesizesandthickporewalls.Thepaststudies

demonstratedthatporesinscaffoldsplayanimportantroleinbone

tissueformation.Largepores(100–150and150–200␮m)showed

substantialboneingrowth,whilepores(75–100␮m)resultedin

ingrowthofunmineralizedosteoidtissue,andsmallerpores(10–44

and44–75␮m)werepenetratedonlybyfibroustissues[37,38].Our

resultsindicatedthattheproducedcompositescaffoldshavemean

poresizesof117–183␮m,whichisbelievedtobesuitableforBTE

scaffolds

To evaluate the interaction between PDLLA matrix and the

inorganicphase,FTIRspectraofnano-HAp(a),typicalHAp/PDLLA

scaffolds(bandc),andPDLLAscaffolds(d)arepresentedinFig.2A ThespectraoftheHAp/PDLLAscaffoldsdonotexhibitsignificant differences, which reveal thepresence of HAp onthe compos-itescaffolds.Infact,thestretchingandbendingvibrationsofthe

PO4 −groupsforHAparevisibleat1090,1030,953,602,561,and

469cm−1inthespectraoftheHAp/PDLLAscaffolds.However,these vibrationsaredetectedat1095,1032,955,604,563,and471cm−1

inthespectrumoftheHApsample.Astrongbandat1749cm−1 canbeobservedinthespectrumofS1PDLLAscaffold.Thisband

isassignedtothevibrationofthecarbonylgroup(C O)ofPDLLA However,thisbandshiftsto1754and1752cm−1 forS3andS4 HAp/PDLLAscaffolds,respectively.Inaddition,thecharacteristic peaksforC Hvibrationsweredetectedat2999and2951cm−1 fortheS1PDLLAscaffold, butobservedat2996and 2946cm−1 andat2998and2948cm−1 forS3andS4HAp/PDLLAscaffolds These resultsreveal that some molecularinteractions between HApnanoparticlesandPDLLAinthecompositescaffoldsmayhave occurred.Moreover,theweakpeakat3569cm−1ischaracteristic

ofthevibrationoftheOHgroupofHApnanoparticles.However, thispeakappearedatalowerregionat3501and3496cm−1forthe S3andS4scaffolds,respectively.Thisresultsuggeststhathydrogen bondwasformedbetweentheOHgroupofHApandtheC Ogroup

ofPDLLA,thusmakingtheHApnanoparticlesstableinthepolymer matrix

Thepresence ofHAp onthesurfaceof theHAp/PDLLA scaf-folds was verified by EDXS analysis The EDXS spectra and

Fig 1. FE-SEM images of scaffolds, synthesized at different percentages of HAp to PDLLA: (a) S1 PDLLA scaffold, (b) S2 HAp/PDLLA scaffold, (c) S3 HAp/PDLLA scaffold, and (d) S4 HAp/PDLLA scaffold at low magnification of (200×) The insets represent FE-SEM images at higher magnification of (500×).

Fig 2.(A) FTIR spectra of (a) nano-HAp powder, (b) S3 HAp/PDLLA scaffold, (c)

S4 HAp/PDLLA scaffold, and (d) S1 PDLLA scaffold and (B) EDXS spectrum of S4

HAp/PDLLA scaffold.

components of a typical S4HAp/PDLLA scaffold are illustrated

in Fig 2B Three elements, namely, O, P, and Ca, are the

majorconstituentsof thesynthesized HAp/PDLLAscaffold with

45.03±1.63at.%,5.64±0.47at.%,and9.58±0.52at.%,respectively

A trace of Cl (0.71±0.06at.%) was detected, which could be

attributedtoaresidueofthesynthesisreaction,andwas

incom-pletely eliminated from the scaffold sample through washing

Moreover,thepresenceofCinthescaffoldsamplescanpossiblybe

attributedtotheuseofcarbonadhesivetapetomountthesamples

orthepresenceofCinthepolymermatrix.Sincedelicateparticles

couldeasilybeaffected,thereforecarbonadhesivetapecouldaffect

thescaffoldmorphology.However,theCa/Pratiowas1.69,which

wasclosetothestoichiometricvalueof1.67.Thisresultconfirms

thattheHApnanoparticlesweresuccessfullyincorporatedintothe

scaffolds

Theporosity data of thesynthesized scaffoldsare shown in

Table1.Theexperimentalresultsindicatethattheporositychanges

linearlywiththeincreaseinHApcontentandexhibitsadownward

trendwithHAploading.WithintheHApcontentrangestudied,the

porosityvaluesvariedfrom89.39±2.86%to80.18±0.78%,which

showrelativelyhighporosityforallproducedscaffolds.Thehigh

porositylevelsofthesynthesizedscaffoldssuggestedthatthese

scaffoldswouldbebeneficialforinvitrocelladhesion,ingrowth,

andsurvival

3.2 Apatite-formingcapability

Animportantcharacteristicofthescaffoldsistheircapabilityto formanapatitelayerontheirsurfaces.Fig.3presentsthesurface morphologiesoftypicalHAp/PDLLAscaffoldsafterbeingimmersed

inSBFfor5and7d.TheFE-SEMimageofS3HAp/PDLLAscaffold withlowmagnification(Fig.3a)showsthataminerallayerwas alreadyformed.Thislayercoveredthescaffoldsurfaceafter5d

ofsoakinginSBF.Theimagewithhighermagnification(Fig.3b) confirmsthattheminerallayerconsistedofaggregatedflower-like particleswithameandiameterof1.08␮m.Theseparticleswere formedonthesurfaceandinsidethescaffoldpores.Thegrowthof theflower-likeparticleswasobservedwhenthesoakingtimewas prolongedto7d.Asshowninthelow-resolutionFE-SEMimagein Fig.3c,theparticlesgrewlarger(theirmeandiameterincreasedto 1.81␮m).TheFE-SEMimagewithahighresolution(Fig.3d) indi-catestheformationoftheflower-likeminerallayerconsistingof tinyneedle-likecrystalsonthescaffoldsurfaceafter7dofsoaking

inSBF.ThesurfacemorphologiesofS4HAp/PDLLAscaffoldafter7d

ofsoakinginSBFaresimilartothoseoftheS3HAp/PDLLAscaffold However,theintenseformationofsuchparticleswasobservedon theentiresurfaceoftheS4HAp/PDLLAscaffold(Fig.3e).The high-resolutionFE-SEMimage(Fig.3f)showedthatarose-likemineral layerwasexclusivelyproducedfortheS4HAp/PDLLAscaffold.This minerallayermorphologyistypicalforbone-likeapatite[36,39] Theobtainedresultsconfirmthecompleteformationofthe min-erallayeronthesurfacesofbothS3andS4HAp/PDLLAscaffolds after7dofsoakinginSBF.ToexploretheeffectofHAp nanopar-ticlesontheapatite-formingcapabilityofthecompositescaffolds,

invitromineralizationexperimentsofpurePDLLAandHAp/PDLLA scaffoldswith10wt.%ofHApwereexamined.Fig.I5showsthe FE-SEMimagesoftheS1PDLLAscaffoldafterbeingimmersedin SBFfor5and7dandthatofS2HAp/PDLLAscaffoldafter7d.Only severalspherical-likemineralparticlesformedonthesurfaceof theS1PDLLAscaffoldafter5dofsoakinginSBF (Fig.I5A).The number ofthemineralparticles increasedafter7 din SBF,and somebundlesofaggregatedmineral-likecrystals(Fig.I5B)were foundonthescaffoldsurfacebecauseoftheconjunctionofsuch particles,indicating poormineralization-formingcapability.The additionofHApat10wt.%tothePDLLAscaffoldalmostdidnot improvethemineralization-formingcapabilityofthecomposite scaffolds.TheFE-SEMimagein Fig.I5Cshowedthat thesevere aggregationofmineral-likecrystalswasobservedonthesurface

oftheS2HAp/PDLLAscaffoldafterbeingimmersedinSBFfor7d Meanwhile,theHAp/PDLLAscaffoldswithhighHApamounts(20 and30wt.%)alreadyshowedfullcoveragebytheminerallayerwith flower-likemorphologyontheirsurfacesafter7d,whichrevealed

asignificantlyhigherinvitromineralizationresponsethanthoseof purePDLLAscaffoldsandscaffoldswith10wt.%ofHAp Mineraliza-tioninvolvesthenucleationandgrowthofbone-likeapatiteonto biomaterials,whichisassociatedwithuptakeofcalciumand phos-phateionsfromthephysiologicalenvironment TheHAp/PDLLA scaffoldswithhighHAppercentagesprovidemorenucleationsites (Ca2+)forapatiteformationthanthesamecompositescaffoldswith lowHAppercentages(e.g.,10wt.%HAp)andaccordingly demon-stratebetterinvitromineralization.Ourresultsindicatethatthe HApcontentinthescaffoldssignificantlyaffectstheinductionof theminerallayerontheirsurfaces.HApcontentof 20–30wt.%, which isrelative toPDLLAis optimalforproducing HAp/PDLLA scaffoldswithhighinvitromineralization

Thechemical compositionof thereleasedminerallayerwas furtheranalyzedusing EDXSmethod.EDXSanalyseswere con-ductedfor thetypicalcomposite scaffoldsafterbeingsoakedin SBF.TableI1presentstheelementalcompositionsofthemineral layersreleasedonS3HAp/PDLLAscaffoldafterbeingsoakedinSBF for5and7d.Ca,P,andOarethreemainelementsfoundinthe

Fig 3. FE-SEM images of representative HAp/PDLLA composite scaffolds after immersion in SBF: (a and b) at 5 d with magnifications of (5000×) and (10,000×), and (c and d) at 7 d with magnifications of (5000×) and (50,000×) for S3 HAp/PDLLA scaffold; (e and f) at 7 d with magnifications of (5000×) and (50,000×) for S4 HAp/PDLLA scaffold.

releasedminerallayersaftersoakinginSBFfor5and7d

How-ever,NaandClweredetectedastraceelements(forthemineral

layer,releasedafter5dinSBF),whichwasattributedtotheresidue

fromthereactionsynthesis ofthescaffolds.AccordingtoTable

I1,theCa/Pratioswere1.24and1.55fortheminerallayers

pro-ducedonthescaffoldafter5and7dofsoakinginSBF,respectively

Apartfromapatitemineral,anumberofothercalciumphosphate

minerals(e.g.,amorphouscalciumphosphate,dicalciumphosphate

dihydrate,octacalciumphosphate,tricalciumphosphate,aswellas

␣-and␤-Ca3(PO4)2)canbeproducedunderthesameinvitro

con-ditionsthatformapatite.Theresultssuggestthatafter7dinSBFa

newbone-likeapatitephasewasalreadyproducedontheS3

scaf-fold,butafter5dinSBFdicalciumphosphateand/oroctacalcium

phosphateasintermediatephaseswereprobablyreleasedonthe

scaffold.Moreover,theCa/Pratiooftheproducedminerallayerwas

1.55,whichwaslowerthanthestoichiometricvalueof1.67,and

wasattributedtothecalcium-deficientcarbonatedhydroxyapatite,

whichsuggeststhattheformedapatiteiscarbonated

Table I2 compares the apatite-forming capability of the

HAp/PDLLA scaffolds as synthesized in this study with other

compositematerialsreportedinpreviousstudies.Thecomplete

formationofthebone-likeapatitelayerwasobservedafter7dof

incubationinSBFfortheHAp/PDLLAscaffoldsinthisstudy

How-ever,thesamelayerwasobtainedafteralongertime(21and14

d)forthenano-HAp/PDLLAfilms[40]andAkermanite/PDLLA scaf-folds[41],respectively.Moreover,thebone-likeapatitelayerwith flower-likemorphologywasobtainedfortheHAp/PDLLAscaffolds synthesizedinthisstudy(Fig.3 andf).Meanwhile,thebone-like apatitelayerformedonnano-HAp/PDLLAfilmsconsistedof numer-ousuniform-orbicular aggregates,and a worm-likemorphology wasproducedonAkermanite/PDLLAscaffolds,whichsignificantly differfromthosereleasedontheHAp/PDLLAscaffoldsinthisstudy Thecomparison revealedthat theHAp/PDLLAscaffolds demon-strated betterin vitro mineralization thanthe other composite materials, which could be attributed to the similarity (e.g., in morphologyandcomposition)oftheinorganiccomponentofthe scaffoldstothatofnaturalbone.AsshowninTableI2,theinorganic componentoftheHAp/PDLLAscaffoldsinthisstudyiscomposed

of rod-shapedHApparticles withdiameter rangingfrom 25to

30nmandlengthrangingfrom100to130nm,whichissimilar

tothevaluesforboneminerals.Theinorganiccomponentsofthe othermaterialsexhibitmorphologicaland compositional differ-encesfromthoseoftheHAp/PDLLAscaffoldsandboneminerals (seeTableI2).Based onthecomparisonoftheinvitro mineral-izationinthepresentandpreviousstudies,themorphologyand compositionoftheinorganicpartofthescaffoldshaveacrucial functionin theirbioactivityand in promotingbone-likeapatite formation

Fig 4.Cell viability and proliferation on the scaffolds after 3, 5, and 7 d of culture

through alamarBlue assay All data are expressed as means ± SD; n = 6 *P < 0.001

(data compared with those at longer culture time, and with S1 PDLLA scaffold),

**P < 0.01 and ***P < 0.05 (data compared with S1 PDLLA).

3.3 Invitrobiocompatibility

Cell–scaffoldinteractionsarethebasisofinitialcellattachment

andinfluencecellphenotypesandfunctions.Inthisstudy,invitro

cellculturetestswereconductedtoevaluatebiocompatibilityof

thesynthesizedscaffoldsintermsofcellviability,proliferation,and

attachment

Fig.4showsresultsofthealamarBlueassay,whichwas

per-formedafterMG63 cells culturedonthescaffoldsandthecell

cultureplates(servedasthecontrolgroup)for3,5,and7d.The

alamarBluereductionindicatedtheviabilityandproliferationof

MG63cells.Fig.4showsnosignificantdifferenceinthecell

viabil-itybetweenallscaffoldgroupsandthecontrolgroupafter3dof

culture.Statisticalanalysisalsoshowedthatdifferencesinthecell

viabilityamongallgroupswerenotsignificantafter3d(P>0.05)

AsshowninFig.4,thefluorescencevaluesforallscaffoldgroups

(athigherlevelsthan thoseof thecontrolgroups)increasedas

theculturetimeincreasedto5and7d,whichrevealedthatthe

cellviabilityofthescaffoldssignificantlyincreasedwithinthis

cul-tureduration(*P<0.001).At5d,thecellviabilityonS2HAp/PDLLA

scaffoldwassignificantlyhigherthanthatonpurePDLLAscaffold

(**P<0.01),butthecellviabilityforS3andS4HAp/PDLLAscaffolds

showednosignificantdifferencewiththatofpurePDLLAscaffold

At7d,thecellviabilityforallHAp/PDLLAscaffoldsbecame

signif-icantlyhigherthanthatofpurePDLLAscaffold(*P<0.001).TheS2

HAp/PDLLAscaffolds(with10wt.%HAp)demonstratedbettercell

viabilitythantheotherscaffoldsfordifferentculturetimes(5and

7d)(***P<0.05),whereasthecellviabilityforS3andS4scaffolds

wascomparableforalltheculturetimes(Fig.4)

Previousstudiesprovedthatsmallporesmightpreventcellular

penetrationandmigrationwithinscaffolds[31].Amongthe

scaf-foldsstudied,S1PDLLAscaffoldwascharacterizedbythelargest

poresizes,but thecellviabilityofS1PDLLAscaffoldwaslower

thanthatoftheHAp/PDLLAscaffolds.Thisresultcanbeattributed

totheabsenceoftheHApcomponentinthecompositionofS1

PDLLAscaffold Asa result,the surfaceof S1 PDLLAscaffold is

lackinginfunctionalgroupsascell-recognitionsignals(e.g.,OH

groups)anddoesnotsupportthatattachmentofmanycellstothe

PDLLAscaffold,evenifmanyMG63cellscanmigratetothe

scaf-fold.Amongthethreecompositescaffolds,S2HAp/PDLLAscaffolds

presenthighercellviabilitythantheothertwoscaffolds,which

maybeattributedtothefactthattheypossesslargerporesizes

(meanporesizeof183␮m)thanS3andS4scaffolds(meanpore sizesof122and117␮m,respectively).Thisresultprobablycaused morebeneficialcellmigrationandhighercellviabilityontheS2 scaffoldsthanontheS3andS4scaffolds.Theresultsofthe alamar-Blueassayindicatethatallthescaffoldsexhibitgoodcellviability, andtheadditionofHApnanorodstothePDLLAscaffold signifi-cantlyenhancedtheviabilityofMG63 cells.However,thehigh contentof HApinthescaffoldsmayinhibit theincrease incell viability

Confocallasermicroscopywasusedtoexaminecellattachment anddistribution.Fig.I6andFig.5showtheconfocallaserscanning microscopyimagesofMG63cellsculturedonthescaffoldsafter3,

5,and7d.ThestainingmethodsfornucleibyDAPIandforactin fila-mentsbyOregonGreen488phalloidinindicatethecellattachment andcytoskeletondistributiononthesurfacesofscaffolds.Infact, fewcellscouldbeobservedontheS1PDLLAandS2HAp/PDLLA scaffoldsat3d(Fig.I6AandB).Meanwhile,qualitativelyhigher

MG63cellswereobservedontheS3HAp/PDLLAandS4HAp/PDLLA scaffolds(Fig.I6CandD).Additionally,theanchorageofthecellular cytoskeletontothescaffoldswasobservedat3d,revealingthatcell attachmentoccurredonthesescaffoldsearlyintheculturetime.A furtherincreaseinculturetimeto5and7dresultedinasignificant increaseinthenumberofcellsforallscaffolds(Fig.5).At5d,all scaf-foldsshowedacytoskeletonarrangementofMG63cells,whichwas attachedtothescaffoldsurfaces(Fig.5a,c,eandg).Amongthem, theS2HAp/PDLLAscaffolddemonstratedauniformcytoskeleton arrangementwithahighdensityofMG63cellsinterconnecting theentirescaffoldat5d,butthecorrespondingcytoskeleton dis-tributionwasgraduallylostat7d(Fig.5d).TheS1PDLLAscaffold exhibitedarelativelyuniformcytoskeletondistributionat5d, how-ever,suchacytoskeletonarrangementintheS1scaffoldwaslost

at7d(Fig.5b).TheresultssuggestedthatHApnanorodsmayhave

aneffectonthecellattachmentinthesescaffolds.TheS1andS2 scaffoldswithsmallHAppercentages(0–10wt.%HAp)intheir com-positionprobablydidnotsupporttheformationofhighamountof stressfibersinthesescaffolds,whichmaintainactinbundlesand focalcontactsbetweenMG63cellsandthescaffoldsforalonger culturetime.Thisresultedinweakercellattachmentandgradual lossofcytoskeletonorganizationinS1andS2scaffoldsat7d.By contrast,amoreextendedcytoskeletonnetworkwasobservedin theS3HAp/PDLLA(Fig.5f)andS4HAp/PDLLAscaffolds(Fig.5h)

at7dthanat5d.TheseobservationsconfirmthathighHAp per-centages(20–30wt.%HAp)enabledS3andS4scaffoldstoproduce higheramountofstressfibers,whichledtostrongercell attach-mentandtheextendedcytoskeletonorganizationinthesescaffolds withincreasingculturetimeto7d.Moreover,awell-organized cytoskeletonnetworkwithalargenumberofadheredcellswas exclusivelyproducedintheS4HAp/PDLLAscaffoldat7d,which wasattributedtothehighestHAppercentageintheS4scaffold compositionamongthescaffoldsstudied.Theresultsrevealedthat allthescaffoldspossessgoodcelladhesion,proliferation,and distri-bution.AhighpercentageofHApinthecompositescaffoldsshould

behelpfulinsupportingcelladhesion,proliferation,and distribu-tionforlongercultureperiods

ThealamarBlueandconfocallasermicroscopyresults demon-strated that all the scaffolds showed good MG 63 cell affinity

in terms of cell viability, proliferation, and adhesion The S4 HAp/PDLLAscaffoldshavetheinorganiccomponentconsistingof HApnanorodswithameandiameterof28nmandameanlength

of120nm[33]andcomprise30wt.%oftheinorganiccomponent

tothepolymer,whicharetheclosestsimilaritytonaturalbone amongthescaffoldsstudied.Asaresult,theS4scaffoldsshowed higherbiocompatibility(bettercellattachmentandwell-organized cytoskeletonarrangement),comparedtotheotherscaffoldswith lowerHApcontentwithincreasingnumberofculturedays.The S4HAp/PDLLAscaffoldsthusare promisingcandidatesthat will

Fig 5.Confocal laser scanning microscopy images of MG 63 cells stained nuclei with DAPI (blue) and actin filaments of cytoskeleton with Oregon Green 488 phalloidin (green) after 5 and 7 d of seeding on (a and b) S1 PDLLA scaffold, (c and d) S2 HAp/PDLLA scaffold, (e and f) S3 HAp/PDLLA scaffold, and (g and h) S4 HAp/PDLLA scaffold Scale bars represent 100 ␮m (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

promoteproductionandorganizationofECMwithmineralization

andexpressionofosteopontin,collagentypeI,bonesialoprotein,

whicharetypicallyexpressedinnativeboneandwillbefurther

investigatedinthenextphaseofourwork

4 Conclusion

This study demonstrated that biomimetic 3D hydroxyap-atite nanorod/poly(d,l) lactic acid scaffolds were successfully

synthesized using solvent casting combined with salt-leaching

technique The synthesized scaffolds are porous, have

thick-nesses of 2mm, macropore networks withmean pore sizes of

117–183␮m,andhighporosity.Bothinvitromineralizationand

invitrocellculturetestsshowedthatthecompositescaffoldswith

highHApcontents,whichhaveastructurethatistheclosesttothat

ofnaturalbone,inducedtherapidformationofbone-likeapatite

aftera quick soakingtime in SBF anddemonstrated bettercell

adhesion,proliferation,and distributionwithincreasingculture

days.Withinthisstudy,itisconcludedthatHAp/PDLLAscaffolds

withhighHAppercentagesarepotentialbiomaterialstobeusedas

BTEscaffoldsforfurtherstudiesthatwillbeaimedatperforming

long-term in vitro cell culture experiments, degradation

stud-ies, and deeper characterizations (mechanical strength, surface

roughness)

Acknowledgments

ThisstudywasfundedbytheVietnamNationalFoundationfor

Scienceand TechnologyDevelopment (NAFOSTED)under Grant

number104.02-2012.42

TheauthorswouldliketothankProf.C.MigliaresiandA.Motta,

DepartmentofIndustrialEngineeringandBIOtechResearchCentre,

UniversityofTrento,Italy,forsupportingcellcultureexperiments

Appendix A Supplementary data

Supplementary data associated with this article can be

found,intheonlineversion,athttp://dx.doi.org/10.1016/j.colsurfb

2015.03.001

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