DSpace at VNU: Surfactant-assisted size control of hydroxyapatite nanorods for bone tissue engineering

A study hassuggested that betterosteoconductivity can be achieved if the synthetic HAp resembles bone minerals in composition,size,andmorphology[29].Inapreviousstudy,we successfullyprepa

Colloids and Surfaces B: Biointerfaces xxx (2013) xxx– xxx

ContentslistsavailableatScienceDirect

j o ur na l h o me p a g 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

Surfactant-assisted size control of hydroxyapatite nanorods for bone

tissue engineering

Claudio Migliaresid

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

b National Institute of Hygiene and Epidemiology, 1 Yersin Street, Hanoi, Viet Nam

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

d Department of Industrial Engineering and BIOtech Research Centre, University of Trento, Via Mesiano, 77, I-38123 Trento, Italy

a r t i c l e i n f o

Article history:

Received 29 July 2013

Received in revised form 26 October 2013

Accepted 2 November 2013

Available online xxx

Keywords:

Hydroxyapatite nanorods

Pluronic

SBF

Bioactivity

Bone tissue engineering

a b s t r a c t

Thisstudypresentsthephysicochemicalcharacterizationofthepluronicsurfactant-assistedsizecontrol

ofhydroxyapatite(HAp)nanorodsforbonetissueengineering(BTE).Rod-shapedHApnanoparticles weresynthesizedviaasimpleroutebyhydrothermal treatmentandwiththeassistanceofthe tri-blockco-polymerPEO20-PPO70-PEO20(P123).Thefilmsofpoly(d,l)lacticacid(PDLLA)wereprepared

asasubstratetospreadsynthesized HApnanorods.Powder X-raydiffraction(XRD),fieldelectron scanningmicroscopy, Fourier transform infrared spectroscopy, nitrogenadsorptionisotherms, and energy-dispersiveX-rayspectroscopywereusedtocharacterizethestructureandcompositionofthe HApsamples.Resultsshowedthatregularrod-shapedHApnanoparticles(withameanlengthof120nm andameanwidthof28nm)weresuccessfullyproduced.Moreover,synthesizedHApnanorodsrevealed therapidformationofbone-likeapatitewithadistinctivemorphology,similartoflower-likeapatite;the formationwasobservedasearlyas7daysafterincubationinstimulatedbodyfluids.Thisstudyisa posi-tiveadditiontotheongoingresearchonthepreparationofHApnanostructurestowardthedevelopment

ofbiocompatiblecompositescaffoldsforBTEapplications

© 2013 Elsevier B.V All rights reserved

1 Introduction

Bone tissue engineering (BTE) has attracted considerable

scientificattentioninthefieldsofnanomedicineand

biotechnol-ogybecauseit offersamore promisingmethodforbone repair

and regeneration than traditional methods (e.g., allografts and

autografts) [1] BTE uses scaffolding materials as template for

cell interactions and formation of extracellular matrix, thereby

providingstructuralsupporttothenewlyformedtissue[2].Till

date,anumberofbiomaterialshavebeeninvestigatedforpossible

use in BTE scaffolds, and these biomaterials can be classified

into the following categories according to their composition:

biodegradable polymers[3–9], bioactiveinorganics [10,11], and

polymer/bioactive ceramic composites [12–20] Calcium

phos-phates withCa/P ratiosof 1.5–2.0 and belonging to thegroup

of bioactive inorganics have three main compounds namely,

tetraphosphate Ca4P2O9, hydroxyapatite Ca10(PO4)6(OH)2, and

tricalcium phosphate Ca3(PO4)2 Among them, hydroxyapatite

(HAp)isthermodynamically themoststablecalciumphosphate

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

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

in physiological environments [10] and a major component of bonesandteeth.HAphasbeenwidelystudiedaloneorasfillerin polymericcompositesforBTEapplicationsbecauseofitschemical similaritytoinorganiccomponentsofbonematrix,strongaffinity forhosthardtissues,andosteoconductivity[6].Anumberof stud-ieshavefocusedoninvestigatingtheformationofnanoscaleHAp foradditionalapplications,suchasproducingrestorablescaffolds thatcanbereplacedbyendogenoushardtissuesovertime[21] Several wet chemistry methods (e.g., co-precipitation, hydrothermal, and ultrasonic) assisted by organic molecules (e.g., sodium dodecylbenzene sulfonate, sodium dodecyl sulfate [22], block copolymer poly(lactide-co-glycolide)-block-monomethoxy (polyethylene glycol) and polyvinyl pyrrolidone (PVP) [23], cetyltrimethylammonium bromide (CTAB) [24], polyvinyl alcohol [25], and polyethylene glycol 400/CTAB [26], CTAB/n-pentanol/n-hexane/water[27], double-hydrophilic block copolymer poly-(vinylpyrrolidone)-b-poly(vinylpyrrolidone-alt-maleic anhydride)-b-poly-(vinylpyrrolidone) [28]) have been studied to produce rod-shaped (or needle-like) HAp nanopar-ticles A study hassuggested that betterosteoconductivity can

be achieved if the synthetic HAp resembles bone minerals in composition,size,andmorphology[29].Inapreviousstudy,we successfullypreparedHApnanorodsviahydrothermaltechnique 0927-7765/$ – see front matter © 2013 Elsevier B.V All rights reserved.

http://dx.doi.org/10.1016/j.colsurfb.2013.11.001

lengthof85–439nm)[24].Surfactants (e.g.,CTAB) areefficient

chemicals in modifying the desired size and shape of

nano-structuresbecause of theirself-assembly intorod-like micelles

or lamellar structures at high concentrations to improve their

properties [24,30] As reported, the wide size distributions of

syntheticHApcanreducethemechanicalpropertiesofscaffolds

[31] Despite recent advances in the synthesis of HAp for BTE

applications,theproductsstill facemajordrawbacksrelated to

bioactivities,fracturetoughness,andothermechanicalproperties

[21]

Inthisstudy,wefocusonthesynthesisandcharacterization

ofHApnanoparticlesthatresemblebonemineralswithnarrow

sizedistributionstodevelopbiocompatiblecompositescaffoldsfor

BTEapplications.Rod-shapedHApnanoparticlesweresynthesized

bytriblockco-polymerPEO20-PPO70-PEO20(P123)tocontroltheir

sizes.P123isanon-ionicchemicalthathasbeenusedtoassistthe

synthesisofrod-likenanostructures[32].Apossiblemechanismfor

theformationofrod-shapedHApnanoparticleswasalsoproposed

toidentifytheeffectofP123ontheformationofHApnanorods.The

filmsofpoly(d,l)lacticacid(PDLLA)werepreparedassubstrates

tospreadHApnanorodsandtesttheirbioactivity.Bioactivitywas

determinedbyassessingtheformationofapatiteonthesurfaceof

HAp/PDLLAfilmsuponincubationinstimulatedbodyfluids(SBF)

after1,3,and7days

2 Experimental

2.1 Chemicals

Allreagentswereofanalyticalgradeandusedasreceived

with-outfurtherpurification.Calciumchloridedihydrate(CaCl2·2H2O),

sodium monophosphate dihydrate (Na2HPO4·2H2O), NaOH,

C2H5OH,CHCl3,hydrochloricacid(HCl),thepluronicco-polymer

PEO20-PPO70-PEO20 (P123), NaCl, NaHCO3, KCl, K2HPO4·3H2O,

MgCl2·6H2O, Na2SO4, and tris-hydroxymethyl aminomethane

((HOCH2)3CNH2)wereobtainedfromSigma–Aldrich.PDLLAwas

purchased from Boehringer Ingelheim (Ingelheim, Germany)

Buffer solutions (pH 4, 7, and 9) were purchased from Merck

Deionizedwaterwasusedtoprepareallsolutionsandreagents

2.2 SynthesisandcharacterizationofHApnanoparticles

For a typicalexperiment and in theabsence of P123,2.53g

CaCl2·2H2Owasdissolvedin86mLdeionizedwaterandbrought

toa pHrangingfrom9.5to11 byaddinga 0.4weightpercent

(wt%)NaOHsolution.0.2MNa2HPO4wasaddedintothesolution

bydropstoaCa/Pmolarratioof1.67.Theresultingmixturewas

vigorouslystirredat40◦Cfor4htoformwhitesuspension.The

suspensionwasthentransferredtoa200mLteflon-linedstainless

steelautoclave,andthesuspensionwasmaintainedat180◦Cfor

24h Whiteprecipitatewascollectedandwashedseveraltimes

withdeionizedwaterbeforebeingdriedat60◦Covernightand,

finally,calcinatedat500◦Cfor1h.Inatypicalreactioninthe

pres-enceofP123,amixtureofaspecificamountofP123(either1,2,or

3g)and2.53gCaCl2·2H2Oweredissolvedin86mLdeionizedwater

bystirringfor30mintoproducecleargel.Thefollowingstepsare

thesameasthoseforthesynthesiswithoutP123.Finally,aseries

ofsampleswasproducedanddenoted0P123,1P123,2P123,and

3P123basedontheamountofP123used

PowderX-raydiffractionpatternsofsynthesizedsampleswere

recordedonaSiemensD5005diffractometerthroughCuK␣

radi-ation (=0.15406nm) Powder morphology was examined by

fieldemission scanningmicroscopy(S4800,Hitachi, Japan).The

scanning electronmicroscopic (SEM) images Fourier transform infrared spectra (FTIR) were recorded ona Nicolet 6700 spec-trometerbyKBrpellettechniqueintherangeof4000–400cm−1 witha resolutionof 4cm−1.Allmeasurementswereconducted

at room temperature The chemical composition of HAp parti-cleswasdeterminedwithaJEOLJED-2300AnalysisStationwitha ZAF(atomicnumberabsorptionfluorescence)quantitativeanalysis program Nitrogen adsorption–desorptionisotherms were mea-suredat−196◦CwithaMicromeriticsASAP2020apparatus.The

totalsurfaceareaswerecalculatedbytheBrunauer–Emmett–Teller (BET)method,whereasporesizedistributionsweredeterminedby theBarrett–Joyner–Halenda(BJH)method

2.3 ProductionandcharacterizationofHAp/PDLLAfilms ThepreparationofHAp/PDLLAfilmswasdescribedinour pre-viousreport[24].To preparePDLLAfilms, we cast0.3g PDLLA dissolvedin3mLCHCl3 ona55mmaluminum plate.Thefilms wereair-driedunderachemicalhoodfor3handvacuum-dried for24htoremoveallsolvent.AsuspensionofC2H5OHwith30mg

ofHApnanorodswasdispersedontheresultingPDLLAfilms.After beingplacedat55–60◦Cfor2h,thecompositefilmsofHAp/PDLLA werevacuum-driedforanother3handstoredinadesiccatoruntil used.ThreedifferentsamplesofHApnanorods(0P123,1P123,and 2P123)wereused,which resultedinthreedifferentHAp/PDLLA filmsdenotedasF0P123,F1P123,andF2P123.The morpholo-giesofresultingfilmsandthepresenceofHApnanorodsonthe substratewerecheckedbyFE-SEMimagingatlowandhigh mag-nifications

2.4 Invitrobioactivitytests SBFwithionconcentrationssimilartothoseinhumanblood plasmawaspreparedaccordingtothemethodreportedinother studies[33]andfilteredbya0.22␮mMilliporefiltersystemto eliminatebacterialcontamination.TheSBFwaspreservedina plas-ticbottle ina refrigeratorat 4◦C Priortoinvitroexperiments, allfilmsofHAp/PDLLAwerecutintodiskswith5mmdiameter, sterilizedin70%ethanolat4◦Covernight,anddriedundera lami-narairflowhood.Twospecimensforeachkindoffilm(F0P123,

F1P123, and F2P123) were soaked in 10mL of SBF in closed polyethylenecontainersat37◦C TheSBFsolutionwasreplaced every2.5 days.After1,3,and7daysof soaking,thespecimens wereremovedfromtheSBF,rinsedwithdeionizedwater,anddried

inwarmflowingair.Alloperationswereconductedinalaminar airflowhoodtoavoidmicroorganismcontamination.Theapatite mineralizationofHAp/PDLLAfilmswasassessedthroughFE-SEM andenergy-dispersiveX-rayspectroscopy(EDXS)

3 Results and discussion

3.1 CharacterizationofHApnanoparticles FoursamplesweresynthesizedwithdifferentP123 concentra-tions(one,non-assisted-P123andtheotherthree,assisted-P123 samples).Themaincharacteristicsofrepresentativesamplesare summarizedinTable1.TheXRDpatternsofrepresentative sam-plesarepresentedinFig.1.Thediffractionpeakscanbeindexedto thehexagonallatticeofHAp(JCPDSNo.9-432),whereasthe char-acteristicpeaksareat(002),(102),(210),(211),(112),(300), (202),and (301) No characteristic peak of other phases (e.g., tricalciumphosphateandtetracalciumphosphate)wasobserved, whichwouldconfirmthatapureHApphasewasproducedforall samples.ThemorphologyoftheHApsampleswasthenidentified

byFE-SEMimages.Rod-shaped particleswithasmooth surface

N.K Nga et al / Colloids and Surfaces B: Biointerfacesxxx (2013) xxx– xxx 3

Table 1

Characteristics of representative HAp samples.

SD: standard deviation; n = 50.

Fig 1.XRD patterns of HAp samples at different concentrations of P123: (a) 0P123,

(b) 1P123, and (c) 2P123 calcinated at 500◦C for 1 h.

wereproducedforthesynthesizedsamples,independentofP123 concentration(Fig.2andFig.S1,Supportingmaterial).According

toFig.2aandTable1,largeandirregularrod-shapedHApparticles (meanwidthof103nmandmeanlengthof585nm)wereobtained fornon-assistedP123samples(0P123samples).Obtainedassisted P123sampleswithincreasingP123concentrationscanresultina significantdecreaseinthesizeofHApnanoparticles.1P123 sam-ple(Fig.2b)showsconsiderablysmallerrod-shapedparticlesthan 0P123samples;themeandiameteris74nmandthemeanlength

is383nm.Furthermore,theFE-SEMimage(Fig.2c)andparticle sizedistributionsof2P123demonstratethatthesesampleshave thesmallestand mostuniformrod-shapedparticles(theirsizes aremainlyintherangeof25–30nminwidthand100nm–130nm

inlength)amongtheassistedP123samples.However,afurther increaseofP123concentrationproducedlargerandirregular rod-shapedHAp.AsshowninFig.S1,irregularHApparticleswithmean widthof82nmandmeanlengthof365nmwereformedfor3P123 samples.Theseobservationsconfirmthatthesynthesisinthe pres-enceofP123doesnotaffectthefinalmorphologyofnanorodsbut significantlyaffectstheirsizes.2gofP123shouldbeoptimalforthe

Fig 2.FE-SEM images of representative HAp samples synthesized at different concentrations of P123: (a) 0P123, (b) 1P123, and (c) 2P123 calcinated at 500 ◦ C for 1 h The inset shows particle size distribution of 2P123.

Fig 3.(A) FTIR spectra of representative HAp samples synthesized at different

con-centrations of P123: (a) 0P123, (b) 1P123, and (c) 2P123 calcinated at 500◦C for 1 h.

(B) EDXS spectrum of an HAp sample (2P123).

productionofthemostuniformrod-shapedHApwithsizessimilar

tothoseofnaturalHAp.Table1presentsCa/Pratiosforthe

rep-resentativeHApsampleswithanupwardtendencybyincreasing

theP123concentration.Inouropinion,P123concentrationcould

notaffectthechemicalcompositionofHAp(e.g.,Ca/Pratio)because

P123wasusedastemplatetocontrolthesizesofHApnanorods,and

P123waseasilyremovedthroughsolventextraction/calcination

Ca/PratiosshowninTable1aretheaveragevaluesofthree

mea-surements Thetheoretical Ca/P ratiosfor all thesamples(e.g.,

0P123,1P123,and2P123) are1.67 However,theexperimental

Ca/Pvaluesof0P123,1P123,and2P123are1.55,1.61,and1.66,

respectively

Fig.3ApresentstheFTIRspectraofrepresentativeHAp

sam-plespreparedwithdifferentconcentrationsofP123.Thespectra

ofsamplesdonotshowsignificantdifferences,whichrevealthe

presence of characteristic groups onHAp samples Indeed, the

mainvibrationsof PO4 − groups forHAp samplesaredetected

at 1095cm−1, 1032cm−1, 960cm−1, 605cm−1, 567cm−1, and

474cm−1 Thepeaks at 1095cm−1 and 1032cm−1 characterize

adegenerateasymmetric-stretchingvibration mode,␯3,ofP O

bonds,whereastheweakpeakat960cm−1isassignedtoa

non-degenerate symmetric-stretching mode, ␯1 [24] The bands at

605cm−1and567cm−1areattributedtoadoubledegenerate

bend-ingmode, ␯4, of O P O bonds,and thebandat 474cm−1 is a

symmetricalbendingmode,␯2.Apartfromthesecharacteristics,

thebandat 3570cm−1 canbeassigned tothestretching

vibra-tionof OH groups of HAp [34], and the broad stretching peak

Fig 4. (A) Nitrogen adsorption–desorption isotherms of HAp samples synthesized

at different concentrations of P123: (a) 0P123, (b) 1P123, and (c) 2P123 (B) Pore size distributions of HAp samples.

at3433cm−1,aswellasthebendingpeakat1636cm−1,canbe assignedtoadsorbedwater

TheelementalcompositionoftheHApsampleswasdetermined

byEDXSanalysis.TheEDXSspectrumandcomponentsfora rep-resentativesample(sample2P123) arepresentedinFig.3Band TableS1.The resultsrevealedthat elementsO,P,and Cawere themajorconstituentsoftheHApsampleswith44.17±4.52wt%, 16.14±1.23wt%,and34.64±3.05wt%,respectively.AtraceofCl wasdetected,whichcouldbeattributedtoaresidueofthesynthesis reaction,andincompletelyremovedfromthesamplebywashing

Inaddition,thepresenceofCinHApsamplesispossiblyduetothe useofcarbontapetomountthesamples.Moreover,theCa/Pratios

oftheHApsamples(Table1)rangefrom1.55to1.66,whichisclose

tothestoichiometricvalueof1.67.Therefore,stoichiometricHAp wassuccessfullyproducedwiththesesamples

The surface characteristics of HAp samples were continu-ouslystudiedbyN2adsorption–desorptionmeasurements.TheN2 adsorption–desorptionisothermsoftheHApsamplesareshown

inFig.4A.AllsamplesexhibitatypeIIIisothermwithanH3 hys-teresisloopintheP/Po(0.9–1)[35],whichsuggesttheformationof slit-shapedporesassecondaryporesintheaggregatesof nanoparti-cleswithlargepores[30,36].Poresizedistributionsofthesamples werecalculatedfromdesorptioncurvesthroughtheBJHmethod (Fig.4B).Thesurfaceareas,porevolumes,andporesizesarelisted

in Table 2 These HAp samples do not demonstrate significant discrepanciesintheirBETsurfaceareaswithvaryingP123 con-centrations.TheBETsurfaceareasofallsamplesare14–27m2g−1,

N.K Nga et al / Colloids and Surfaces B: Biointerfacesxxx (2013) xxx– xxx 5

Table 2

Surface characteristics of representative HAp samples.

Sample codes S BETa(m 2 g−1) V BJHb(cm 3 g−1) D pc(nm)

a BET surface area.

b Total pore volume determined using desorption branch of the isotherms.

c Peak pore sizes from the pore size distributions.

butthesamplesdifferinthestructuresoftheirpores.Amongthree

samplesstudied,sample2P123hasnarrowporesizedistribution

withamaximumpeakof12nm,whereassample0P123showsa

disorderedporesizedistributionwiththreemaximumpeaksof

approximately3,10,and31nm.Sample1P123hasabroadporesize

distribution,whichrangesfrom20nmto80nmandconcentrated

atapproximately42nm

3.2 PossiblemechanismforHApparticleformation

ApossiblemechanismfortheformationofHApnanorodsmay

beproposedbasedontheresultsobtained(Fig.5).WithoutP123

(Fig.5A)andathighpH,thereactionbetweenthesolutionsofCaCl2

andNa2HPO4yieldsHApviathefollowingreaction:

10Ca2++6PO4 −+2OH−→ Ca10(PO4)6(OH)2

TheresultingHApwashydrothermallytreatedat180◦C,calcinated

at500◦C,andproduced largeirregularlyrod-shapedHAp

parti-cles(withmeanwidthof103nmandmeanlengthof585nm)as

showninFig.2aandTable1.Withouthydrothermaltreatmentafter

calcinationat500◦C,theaggregatedsphericalHApparticleswith

averagediameters of350nmwereobtainedinsteadof the

rod-shapedparticles(Fig.S2).Theseresultsconfirmthathydrothermal

treatmentat180◦CprovidesenergyfortheHApnucleitogrow

andproduceelongatedHApcrystals.Thiscrystalgrowthispossibly

duetotheintrinsiccrystalhabitcausedbythedifferenceinlattice

energybetweenthedifferentcrystalplanesofHAp[37].Without

anycontrol,theobtainedHApcrystalsareextremelylongandhave

irregularsize

Theoretically, P123, a non-ionic surfactant can be

self-assembledintopolymericmicelleswithvariousmorphologies(e.g.,

spherical,cylindrical, or lamellarstructures) athigh

concentra-tionsabovethecriticalmicelleconcentration[36].Thesizeand

shapeofmicellesdeterminethemorphologyof thesynthesized

HApparticles.Inaqueoussolution,P123formsnano-sized

cylin-dricalmicellesthatfunctionas“nanoreactors”.Ca2+canpreferably

bindfirstwiththehydrophilicheadgroupsontheinnersurface

oftheresultingmicellesthroughhydrogenbondsandthen,with

PO4 −/OH−toformtheP123-HApcomplexthatprovidesthe

nec-essarytopologythatinturnsdirectsmineralgrowthandleadsto

theformationofelongatedHApnanocrystals(Fig.5B).Atlow

con-centrationsofP123(e.g.,1P123samples),anumberofHApnuclei

thatareunboundtomicelles,latergrowuncontrollablytoproduce

irregularlyshapedandlargeHApcrystals.Meanwhile,thesizesof

micellesincreaseastheP123concentrationincreases.Theincrease

inthesizeofmicellesresultsinanincreaseinthesizesofHAp

par-ticles.ThemostuniformHApparticlesthatresembleboneminerals

wereobtainedwiththesuitableconcentrationof2g

3.3 InvitrobioactivityofsynthesizedHAp/PDLLAfilmsinSBF

Anessentialcharacteristicofbiomaterialsistheirabilitytobind

tolivingbonebyformingabone-likeapatitelayeronitssurface

bothinvitroandinvivo.Thepowderformof theHApparticles

is unfavorablewhen workingwithSBF Thus, thebioactivityof

theHApsamples wasevaluatedthroughinvitro testsby soak-ingfilmsofHAp/PDLLAinSBF.Fig.S3representstheSEMimages

ofHAp/PDLLAcompositefilmswithlowandhighmagnifications beforeimmersionintoSBF.Generally,thesurfacesofthe compos-itefilmsarerough.Theimageswithhighmagnificationconfirma fullcoverofrod-shapedHApparticleswithsmoothmorphologyon thesurfaceofthesubstrate

Theformationofabone-likeapatitelayerontheHAp/PDLLA filmswasinvestigatedthroughFE-SEMmeasurementsafter1,3, and7daysofsoakinginSBF.Fig.S4showsrepresentativeFE-SEM imagesofF0P123filmsaftersoakinginSBFfor1and7daysand thatofF1P123filmsaftersoakinginSBFfor3and7days Com-paredwithfilmsexaminedbeforesoaking,thesecompositefilms didnotshowsignificantchangesintheirmorphologiesduringthe initialstageofsoaking(1–3days).However,afewbundlesofHAp particleswerefoundbecauseoftheaggregationofsuchparticles

onthesurfaceofthecompositefilmsaftertheinitial(1–3days) immersioninSBF(e.g.,after1dayfortheF0P123filmandafter

3daysfortheF1P123film),asshowninFig.S4(a)and(d).After

7daysofsoakinginSBF,bothcompositesamplesexhibited mor-phologicalchanges,whicharedifferentfromthosebeforesoaking

Asshowninhigh-resolutionFE-SEMimagesinFig.S4(c)and(f), theirmorphologieswererougherwithscantdepositionof needle-likemineralcrystals(160nminlengthand20nmindiameterfor

F0P123;120nminlengthand20nmindiameterforF1P123).Such

anobservationrevealedtheinductionofamineralphaseonthe sur-faceofthesefilmsafter7daysinSBF.FE-SEMimagesofF2P123 filmsafter1,3,and7daysoftreatmentinSBFarepresentedin Fig.6.DuringincubationinSBF,themorphologyofF2P123 com-positefilmsdrasticallychangedwiththeappearanceofabundant deposits.Inparticular,theintenseformationofsphericalmineral particles(meandiameterof2␮m)composedoftinyneedle-like crystalswas already observed after1 dayof immersion in SBF (Fig.6aandb).Thesametendencywasseenafter3days(Fig.6c andd),butthesizeofthesphericalparticleswithneedle-like crys-tallitesincreasedwithlongersoakingtime(theirmeandiameter increasedto3␮m).Thereafter(beyond3days),themineralphase waslargelyspreadonthesurfaceofF2P123films.FE-SEMimages withdifferentmagnifications(Fig.6 andf)revealthatacoral-like minerallayerofnumeroustinyneedle-likecrystalswasdeposited

ontheentiresurfaceofF2P123filmsafter7daysinSBF.FE-SEM imageswithhigherresolution(Fig.6 andh)indicatetheformation

ofaflower-likeminerallayercomposedoftinyneedle-like crystal-litesontheentiresurfaceofF2P123.Thisinterestingmorphology

oftheminerallayeristypicalinbone-likeapatite[33].Thegiven resultsrevealthecompleteformationoftheminerallayeronthe surfaceofF2P123evenforshortsoakingperiods,whichindicates itshighestinvitrobioactiveresponseamongthethreecomposite filmsstudied.Asreportedinpaststudies,mineralizationinvolves thenucleationandgrowthofbone-likeapatiteontobiomaterialsin SBF[38],whichismainlyassociatedwiththeuptakeofcalciumand phosphateionsfromtheSBFsolution.Thecompositefilm(F2P123) composedofHApnanoparticles(2P123)withthesmallestsizes andthehighestBETsurfaceareaamongthestudiedHApsamples (Tables1and2)certainlyresultedinitshighuptakeforcalciumand phosphateions,therebyexhibitingthehighestbioactivity Conse-quently,thecompositefilms(F0P123andF1P123)consistedof theHApnanoparticleswithlargedimensionsandlowBETsurface areas(bothagainsttheiruptakecapacityofcalciumandphosphate ions)showconsiderablylowerbioactivity

TheCa/Pratioisakeycharacteristicusedtoidentifythemineral phaseamongbiologicallyrelevantminerals.Tofurtheranalyzethe chemicalcompositionofthereleasedminerallayer,EDXSanalysis wasconductedforthetypicalcompositefilmsafterSBFtreatment Theresultsindicatedthatthemainelements(i.e.,Ca,P,and O) werefoundintheminerallayerandCwasalsodetectedasatrace

Fig 5. Possible mechanism for the formation of (A) HAp nanoparticles in the absence of P123 and (B) HAp nanorods in the presence of P123.

element(notshown).TableS2presentstheCa/Pratiosofthe

com-positefilmsafter7daysof soakinginSBF fortheF0P123film

andafter1,3,and7daysforF2P123films.AsshowninTableS2,

theCa/PratioforF0P123after7daysoftreatmentis1.55,which

revealstheformationofanewapatitephaseonthesurfaceofthe

F0P123inSBF.However,theintensityofthecarbonpeakonEDXS

patternsofF0P123beforeandaftertreatmentinSBFincreased,

therebysuggestingthattheapatiteproducedwascarbonated.For

F2P123 films, the Ca/P ratio showed an increasingtrend with

increasingsoakingtime;theCa/Pratiowas1.36,1.47,and1.57for1,

3,and7daysofSBF,respectively.Asshownintheliterature,a

num-berofothercalciumphosphateminerals(e.g.,amorphouscalcium

phosphateCax(PO4)y·zH2O(ACP),dicalciumphosphatedihydrate

CaHPO4·2H2O(DCPD),octacalciumphosphateCa8H2(PO4)6·5H2O

(OCP),tricalciumphosphate␣-and␤-Ca3·(PO4)2 (TCP),and

Mg-substituted tricalcium phosphate (Ca,Mg)3(PO4)2·(Mg-TCP)) can

alsobeproducedunderconditionssimilartothosethatformapatite

in vitro These minerals can be transformed from one type to

anotherdependingonthepHandcompositionofthebiological

micro-environmentandcanbedistinguishedthroughtheirCa/P

ratiothroughEDXS[37].Therefore,basedonEDXSanalysesand

Ca/Pratios(TableS2),OCPandTCPwereprobablyreleasedonthe

surfacesofF2P123filmsafter1and3daysofsoaking,respectively

Thehighestvalueof1.57canbeassignedtoabone-likeapatitelayer producedinF2P123after7days.Consideringtheincreaseinthe intensityofthecarbonpeakforEDXSpatternsbeforeandafter7 daysofimmersioninSBFforF2P123,wefindthatthecarbonated apatitelayerwasalreadyproducedonthesurfaceofF2P123after

7daysofimmersioninSBF

TableS3comparestheinvitrobioactivityofHApnanorodsas preparedinthisstudywiththosereportedinpreviousstudies[39] Theformationofnewbone-likemineralwasalreadydetectedafter

1dayofincubationinSBFforthepreparedHApnanorodsinthis study;itwasdetectedonlyafter45daysforHApnanorods,HAp nanospheres,andHApfibrousmicroparticlesinpaststudies[39] Moreover,afteraquickperiodofincubationinSBF(7days),ahighly interestinganddistinctivemorphologyofbone-likeapatite resem-blingtheflower-likeapatitewasexclusivelyobservedfortheHAp nanorodspreparedinthisstudy(Fig.6 andh).Theinvitro bioactiv-ityofthepreparedHApnanorodsinthisstudythatismoreefficient thantheotherHApparticlesshouldbeattributedtotheirclose morphology(e.g.,sizeandshape)tonaturalHAp

AsshowninTableS3,theHApnanoparticlespreparedinthis studyhaveameanwidthof28nmandameanlengthof120nm, whichisextremelysimilartothoseofboneminerals.Theother HApparticlespossessmorphologiesthatareconsiderablydifferent

N.K Nga et al / Colloids and Surfaces B: Biointerfacesxxx (2013) xxx– xxx 7

Fig 6.SEM images of HAp/PDLLA composite film (F 2P123) after soaking in SBF for (a and b) 1 day, (c and d) 3 days and (e–h) 7 days.

fromthoseofthepreparedHApnanorodsandthemineralpartof

bone.Forinstance,thebestHApparticlesinthepreviousstudyhave

sphericalandnear-sphericalshape(approximately50nminsize)

orextremelyshortnanorods(averagewidthof60nmandaverage

lengthof110nm)[39].Basedonthecomparisonoftheresultsofthe

presentandpreviousstudy,thesizeandshapeofHApparticleshave

acrucialfunctionintheirbioactivityandbiocompatibilityin

pro-motingthebone-likeapatitephase.Themorphologicalsimilarityof

syntheticHApparticlescanfacilitateboththeirinvitroandinvivo bioactivity

4 Conclusion

Thisstudydemonstratesthatrod-shapedhydroxyapatite par-ticles resembling bone minerals are easily synthesized with controllablesizes witha suitable concentrationof thepluronic

andmeanlengthof120nm),Ca/Pof1.66,andstoichiometricHAp

InvitrobioactivitytestsshowedthattheHApnanorodsinducerapid

formationofbone-like apatiteafteranextremely shortsoaking

timeinSBF,whichmakesthemexcellentcandidatesforbonerepair

andreconstruction.Thisstudycanprovideanefficientprotocolfor

thecontrolledsynthesisofHApnanorodssimilartoboneminerals

inviewofdevelopingbiocompatiblecompositescaffoldsforbone

tissueengineering

Acknowledgments

Thisstudy wasfunded by theVietnamNational Foundation

forScienceandTechnologyDevelopment(NAFOSTED)undergrant

number104.02-2012.42

TheauthorswouldliketothankDr.Anne-LiseHaenni,

Insti-tutJaquesMonodInstitute/CNRS–UniversitéParisDiderot–Paris

7, France, for English correctionand helpfulcomments onthe

manuscript

Appendix A Supplementary data

Supplementary data associated with this article can be

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

2013.11.001

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