Exploring the thumbprints of Ag-hydroxyapatite composite as a surface coating bone material for the implants

Exploring the thumbprints of Ag-hydroxyapatite composite as a surface coating bone material

for the implants

Lett, J. Anita; Sagadevan, Suresh; Paiman, Suriati; Mohammad, Faruq; Schirhagl, Romana;

Leonard, Estelle; Alshahateet, Solhe F.; Oh, Won-Chun

Published in:

Journal of Materials Research and Technology

DOI:

10.1016/j.jmrt.2020.09.037

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Citation for published version (APA):

Lett, J. A., Sagadevan, S., Paiman, S., Mohammad, F., Schirhagl, R., Leonard, E., Alshahateet, S. F., &

Oh, W-C. (2020). Exploring the thumbprints of Ag-hydroxyapatite composite as a surface coating bone

material for the implants. Journal of Materials Research and Technology, 9(6), 12824-12833.

https://doi.org/10.1016/j.jmrt.2020.09.037

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Original

Article

Exploring

the

thumbprints

of

Ag-hydroxyapatite

composite

as

a

surface

coating

bone

material

for

the

implants

J.

Anita

Lett

_,

_Suresh

_Sagadevan

_,

_Suriati

_Paiman

_,

_Faruq

_Mohammad

_,

Romana

Schirhagl

_,

_Estelle

_Léonard

_,

_Solhe

_F.

_Alshahateet

_,

_Won-Chun

_Oh

b_{Nanotechnology&CatalysisResearchCentre,UniversityofMalaya,KualaLumpur50603,Malaysia} c_{DepartmentofPhysics,FacultyofScience,UniversitiPutraMalaysia,43400Serdang,Selangor,Malaysia}

d_{SurfactantResearchChair,DepartmentofChemistry,CollegeofScience,KingSaudUniversity,P.O.Box2455,Riyadh,KingdomofSaudi}

Arabia11451

e_{GroningenUniversity,UniversityMedicalCenterGroningen,AntoniusDeusinglaan1,9713AWGroningen,Netherlands} f_{ESCOM,UTC,EATIMR4297,1alléeduRéseauJean-MarieBuckmaster,60200Compiègne,France}

g_{DepartmentofChemistry,MutahUniversity,P.O.BOX7,Mutah,61710Karak,Jordan}

h_{DepartmentofAdvancedMaterialsScienceandEngineering,HanseoUniversity,Seosan-si,Chungnam356-706,RepublicofKorea}

a

r

t

i

c

l

e

i

n

f

o

Received30June2020 Accepted7September2020 Availableonline21September2020

Keywords:

Polylacticacidscaffolds Silverdopedhydroxyapatite Fuseddepositionmethod Antibacterialstudies Hemocompatibility Hardnessstudy

a

b

s

t

r

a

c

t

Polylacticacid(PLA),althoughhasmanyinterestingphysicochemicalcharacteristics,the stronghydrophobicityandalackofantibacterialactivityrestrictingitswidespread appli-cationinthemedicalsector.InaviewofaddressingsomeofthelimitationsofPLA,the currentstudyaimedtotesttheantibacterialefficacyofactivemetal-dopedbioceramic/PLA compositeformedbythefuseddepositionmanufacturing(FDM)technique.Forthetesting, wepreparedpolyvinylalcohol(PVA)boundsilver-hydroxyapatite(Ag-HAp)compositeand furtherappliedasalow-temperaturecoatingontothePLAscaffolddesignedforthe appro-priatecelldevelopment,differentiation,andbio-mineralestablishment.Fromtheanalysis, werevealedthatthelargersurfaceareaofthree-dimensional(3D)printedcomposite mate-rialhavingthematrixporositymakesitaperfectbiocompatiblematerialwithnolosstoits mechanicalpotency.TheHAp/PLAandAg-HAp/PLAcompositesweretestedforthe hemo-compatibility,andantibacterialactivity(gram-positiveandgram-negativebacteria).Further, themechanicalpropertyoftheAg-HAp/PLAscaffoldwastested.Theresultsdemonstrated thattheAg-HAp/PLAcompositeoffersthebiocompatibilityandantibacterialabilityand thereforecanserveasthepotentialboneimplantmaterial.

©2020TheAuthor(s).PublishedbyElsevierB.V.Thisisanopenaccessarticleunderthe CCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthors.}

E-mails:anitalett2014@gmail.com(J.A.Lett),drsureshnano@gmail.com(S.Sagadevan),wcoh@hanseo.ac.kr(W.Oh). https://doi.org/10.1016/j.jmrt.2020.09.037

2238-7854/©2020The Author(s). PublishedbyElsevier B.V.Thisis anopen accessarticleunderthe CC BY-NC-NDlicense (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

1.

Introduction

Additive manufacturing (AM), commonly known as three-dimensional (3D) printing offers a range of fascinating pathwaysforthefabricationofcompositematerialsoverother monolithicprototyping.Thus,bymakinguseofthisAM tech-nique,several biomaterialsare being fabricated like tissue engineering[1,2],softcomposites[3],ceramics[4],magnetic materials[5],anddirectbiologicalmaterialprintings[6].Based ontheapplication,thistechniquecanparticularlybehelpful inthebiomedicalsectorfortheregenerationofdefectivebone tissuescaused becauseofaccidentsorsurgicalamputation oftumors[7].Sincetheusageofsyntheticmaterialsforthe developmentofboneimplantshelpstoreconstructthebone tissueswithstrongstructuralsupportandnolosstothe inter-ferencesofbiologicaltissue[8,9].Toserveforsuchbehavior, thebiodegradablematricesareindicatedtoprovide momen-taryscaffoldswithinwhichthebonetissuescanregenerate. Amongmanydifferentpolymers,polylacticacid(PLA)offers excellentbiocompatibility,biodegradability,non-toxicity,and mechanicalstrengthandhencefindsitsusageinmany differ-entclinicalapplicationslikedrugdeliverysystems,surgical stitches,medicalimplantabledevicesetc.[10].

Fuseddeposition manufacturing (FDM) isthe most fre-quentlyusedpracticefortheconstructionofpureplasticparts withlowcosts,negligiblewastes,andsimplicitywith mate-rial switching [11,12]. PLA isone of the most encouraging syntheticpolymers thatcan be usedas asource biomate-rialintheFDMtechniqueandisduetothefactthatitcan undergo degradation in the aqueous medium and is bio-compatibleinthebiologicalsurrounding[12].Ingeneral,the mechanicalpropertiesofpurePLAscaffoldsarecomparable with the natural bone and so, it can be used for cranio-facialbonerestorationand cranial defects[10]. Conversely, thePLA hasstronghydrophobicitythat limitsitsextensive application in medicine and so there is astrong need for theidealbioimplantablescaffoldmaterialthatcanfacilitate the growth and activity of cells like cell adhesion, prolif-eration, and functional differentiation [13]. Besides that, it mustbebioactiveorbioresorbablewithadequate mechani-calpropertytoendurestressstatesthatmightappearatthe implantedsite.Inthatway,theimplantablescaffoldcanact asextracellularmatrix(ECM)fortissuerejuvenation.Inrecent worksbyAlvarez-Barretoetal.[14]fabricated Arg––Gly–Asp-modifiedPLAscaffoldstoregulatethecellcharacteristicsand progresstissuefunctioningforabetterandwell-organized tis-sueengineeringapproaches.ThedepositedCaPceramicover PLAnanofibrousscaffoldswascomparabletotheconstituents ofnaturalbone,butnotabletoachievethedesired osteoin-duction[15].Bearinginmindthatthemainconstituentsof natural bone are collagen fibers and hydroxyapatite (HAp) crystals,andinthatway,thenano-HAphasexcellent prop-ertiessuchasbiocompatibilityandbioactivitythatarecrucial forcelldevelopment[9].Consequently,theinorganic/organic compositematerialsencloseagreaterpotentialforbone tis-sueengineeringapplications.Numerousscaffoldshavebeen fabricatedbydirectlymixingthecompositeactiveadditives butleadstouncontrolledhomogeneityinthepolymermatrix [12].

Inourdaytodaylifedealswithvariousviruses,fungi,and bacteriaandthus,themanufacturedHApimplantable mate-rialshouldnotallowanyofthesemicroorganismstogrowon itssurface.ThesyntheticHApdoesnotexhibitany antibac-terialpropertyandthusitbecomesimportanttomodifyits surface with activebacteriostaticelements like Ag,Sr, Zn, Ce,Ti,etc[16,17].Asanexample,thepresenceofAgmetal withactiveantibacterialpropertycaneasilydamagethe bac-terialcellwall,aswellasbindtotheDNAandRNAofbacteria andtherebypreventingthegrowththatfinallyresultsin bac-terial annihilation [18]. Besides, the radius of Ag+ _atomis

0.126nmwhichisalmostthesameastheradiusofCa2+atom (0.100nm),thereforeundersomeconditions,theAgionscan bereplacedbytheCaionsintheHApcrystallattice. There-fore,toadvancethe biomedicalandclinicaluses ofHAp,it isveryessentialtoincorporateAgionsintotheHAp’scrystal latticeandinthatview,someoftheresearchershavefocused ondevelopingHAp-basedcompositecoatings[19,20].

BytakingadvantageofthebioceramicpropertiesofHApto serveasanaturalboneandAg’santibacterialproperties,we haveincorporatedAg+_{ionsintotheHApcrystalstoform}

Ag-HApandfurthersurfacemodifiedwithPLA scaffoldsusing PVA(polyvinylalcohol)tofinallygenerateAg-HAp/PLA scaf-fold. Thehighlightsofthe study reportedhere includethe fabricationofrequiredinterconnectedporousPLA scaffolds bytheFDMtechniqueusingappropriateCADdesign.Thus, thepreparedHAP/PLA,Ag-HAp/PLAcompositesandscaffold werecharacterizedforthemicro-structure,crystallinity, ele-mentalcomposition,mechanicalproperties,biocompatibility, andantibacterialefficacy.

2.

Materials

and

methods

2.1. 3DprintingofPLAframeworks

TheCADmodelsforthePLAscaffoldsweredesignedusing CatiadesignsoftwareandfabricatedbyaCrealityEnder-3 pro-HighPrecision3DprinterbyShapetool3DTechnologyFSD machine.BeforetheFDMprocess,theSTL(Standard Tessella-tionLanguage)filecreatedbytheCADsoftwareisfragmented into thin horizontalslicesand the widthofeach layercan bedrivenaspertherequirement[21].Fig.1showstheFDM printer,printingprocess,andtheformedmaterial.

2.2. SynthesisofAg-dopedHAp

For the formationofAg-Happarticles, theprecursors used includetheanalyticalreagentgradecalciumnitrate tetrahy-drate; Ca(NO3)2·4H2O, ammonium dihydrogen phosphate;

(NH4)H2PO4, and silver nitrate; AgNO3 (Merck, Mumbai,

Maharashtra, India). During the synthesis, we first formed nano-HAp bysimplydissolvingthe calciumand phosphate solutionsabovepH10toundergotheprecipitationprocess.In thisprocess,thecommonpracticerequirestheuseofa stoi-chiometricratioofCa:Ptobe1.67andinvolvesthedropwise additionofonereagenttotheotherwithcontinuousstirring underaninertatmosphere.Thisresultsinthegenerationofa suspensionunderatmospherictemperature,whichon wash-ing,drying,andsmashingformsthenano-powderofHAp.

Fig.1–SchematicrepresentationoftheFDMprintingprocessfortheformationofPLAporousscaffolds.

For the synthesis of Ag-doped HAp, 0.45M of Ca(NO3)2·4H2O along with 0.05M of AgNO3 was first

dis-solved in distilled water and adjusted to a total volume of 250mL and kept in a conical flask. Similarly, a 250mL volumeof0.3Mof(NH4)H2PO4waspreparedseparatelyusing

distilled water and transferred toa burette. On mixing of bothsolutionsataflowrateof3mL/min,roomtemperature, andstirringfor3hresultsintheformationofamilkywhite solution(havingthestoichiometricratioof1.67).Thewhole process pH was maintained at 11 (>10) by the dropwise addition of ammonia to Ca(NO3)2·4H2O+AgNO3 solution.

Thewhite colouredsolutionwaskeptatroomtemperature overnight,followed by washing thrice with distilled water, subjectedtocalcinationat100◦Cfor3h,andfinallysintered at500◦CtoobtainAg-HApcomposite.

2.3. IncorporationofAg-HApcompositeontothe3D PLAframeworks

For the surface modification ofAg-HAp composite at low-temperatureconditions,wehaveusedPLAscaffoldsandfor effectivebonding,polyvinylalcohol(PVA)wasemployedasthe bindingagent.Forthesurfacemodificationreaction,about1g ofPVA(analyticalgrade)wasmixedto50mLofdistilledwater, stirredusingamagneticstirrerat50◦Ctoformahomogenous solution. Now,the Ag-HAp compositedispersedinethanol was added to the PVA solution, whilestirring for another 2h,andaftertheperiod,theFDMprintedPLAscaffoldswere immersedandkeptinthePVAsolutionfor24h.Attheendof theincubationperiod,boththepartswereremoved,allowedto drip-dry,andthenplacedinanairdryermaintainedat50◦Cfor oneweektoobtainthefinalproductofAg-HAp/PLAscaffold.

3.

Characteristic

techniques

3.1. Morphologicalandstructuralcharacterization Themorphologicalorganizationandsurfacenatureof sam-pleswereobservedusingthefieldemissionscanningelectron microscopy (FESEM; JMS7500F; JEOL, Tokyo, Japan) and high-resolution transmission electron microscopy (HRTEM: FEI-TECNAI G2-200kV TWIN). The energy-dispersive X-ray (EDX:X-Max,USA)analysiswitharegularunit(Oxford

Instru-ments,UK)connectedtotheFESEMinstrumentwasemployed fortheelementalmappingandcompositionofelementsinthe composite.Forthesampling,thetestingmaterialswerefirst dispersedinethanol,sonicated,andthenplacedadropof sus-pensionontothecleanedaluminumfoil,driedatatmospheric air,andwassputter-coatedwithgold(EdwardsSputterCoater S150B,London,UK).Similarly, thepowder X-raydiffraction (XRD)analysis(Bruker-D8powderdiffractometer,BrukerAXS GmbH,Karlsruhe,Germany),Fouriertransforminfrared(FTIR) spectroscopy (from Agilent Cary 630, Agilent Technologies, SantaClara,CA,USA),andX-rayphotoelectronspectroscopy (XPS;SSX-100spectrometer)havingmonochromatizedX-ray beamAlK␣radiation(operatingat1486.6eV)wereemployed fortheothercharacterizations.

3.2. Biocompatibilitystudies

3.2.1. MeasurementofFBSadsorptionontotheHAp samples

ThebiocompatibilityofHApandAg-HAp/PLAcompositewas assessedbytheproteinadsorptionassayandforthe analy-sis, thetesting pellets(atadimension10×1mmdia) were engrossedintrisbufferatpH7.4fora20hperiod.Then,the scaffoldswereincubatedinMinimumEssentialMedium (␣-MEM)containing10%FetalBovineSerum(FBS;syntheticcell culturemedia)for4h.Attheendoftheincubationperiod, thescaffoldswerefirstrinsedthoroughlywithanisotonictris buffer solution, and thenthe surfacewasexposed toa2% sodiumdodecyl sulfate(SDS)solutionfor24htoelude any adsorbedproteins.Thetotalcapacityofadsorbedproteinwas investigatedusingtheBCAproteinassayequipmenthaving themicroplatereader(MolecularDevicesLLC,Sunnyvale,CA, USA).

3.2.2. Hemolyticstudies

ThehemocompatibilityofHApandAg-HAp/PLAcomposite wasexamined aspertheearlierproceduredescribed byLv et al. [22]. Briefly, about 1.25mLof sterile saline was first combinedwith1mgoftestingsample(HAporAg-HAp/PLA composite powder)andinaseparatetube, 4mLvolumeof blood collected from the healthy human was dilutedwith thatof5mLsterilesalinesolution.Fromthis,20␮Lofdiluted blood solutionwascollectedandmixedwiththatofa test-ingsamplecontainingtubeandfurtherincubatedat37◦Cfor

30minandthenall thetubeswere incubatedfor60minat 37◦Cinashakingwaterbath.Thereleaseofhemoglobinwas determined aftercentrifugationbyphotometricanalysis of thesupernatantat540nmusingtheUVspectrophotometer (ShimadzuUV2450)andfurtherthehemolyticrate(HR)was calculatedfromtheEq.(1),

HR= [(Dt−Dnc)/(Dpc−Dnc)]×100% (1)

whereDt,Dnc,andDpccorrespondstotheabsorbancesofthe

testingsample,negativecontrol,andpositivecontrol, respec-tively.

3.3. Antibacterialstudies

Forthetesting,thesamplesweremadeintopelletsof10mm diameterand2mmthickness.Toaccomplishthestudy, ster-ilePetri-dishes(90mmdiameter)comprisingsterilenutrient agarmedium(15mL)weretaken.After24hofbacterial cul-ture,thejustpreparedbacterialinoculumswerespreadover theentiresurfaceofagarmediumthriceusingasterile cot-tonswab,tomakesurethatthebacterialcultureisthoroughly andevenlydistributedonthesurfaceofplates.Thetest sam-pleswereboredinthemediumineachplateandtheplates werepreservedatnormalroomtemperaturefor45minand furtherincubatedat37◦Cfor24h.Finally,thediameterofthe zoneofinhibition(mm)wasmeasuredat3-equidistantplaces takenfromthecenteroftheinhibitionzone,andtheaverage ofthesethreevalueswastakenasthefinal.Allprocedures wereinco-operatedintriplicate.

3.4. Hardnessstudy

For this, the compressive tests were carried out on a 10×10mmdimensionofPLA-coated HApand Ag-HAp/PLA scaffold by making use of the Universal testing machine (INSTRON5566,Germany)withamaximumcompressionload cellof10kNandacross-headspeedof1.0mm/min.

4.

Results

and

discussion

ThemineralizedAg-HAp/PLAporous scaffoldsinthis work usedalow-temperatureprocess tomodifythe PLAsurface ascomparedtotheplasma-sprayedcoatingthatrequires ele-vated temperatures. The use ofmineralized Ag-HAp along withPVA asa biocompatible binder onthe surface ofPLA porous scaffolds under physiological conditions in a more rapidand simplemethodtoemerge exquisitecrystal mor-phologyforcellgrowth.Fig.2showsthecomparisonofthe powderXRDpatternofpureHApandAg-HApcomposite,and fromthefigure,theremarkablepeaksidentifiedatthe2␪of 31.74,32.18, and32.89canbeindexed tothe HApmaterial and alsomatcheswell withthe JCPDScard #89-6495. The veryfeeblepeakforsilverassociatedat2 of38.1 and44.2 (JCPDScard No.4–783) are alsofound. Thesharp peaks of theHApsampleindicateitshighlycrystallinenature(5.16%); whereas,theAg-dopedsilverHAppeaksarebroadsignifying thechange(decrease)incrystallitesize(34.8nm)andhence thecrystallinity(1.95%)ofthedopedsample[23].Theaddition

Fig.2–ComparisonofthepowderXRDpatternofHApand Ag-HApnanopowder.

Table1–Comparisonofthecrystallinitydataforthe

HapbeforeandafterdopingwithAgmetal.

Sample Thecrystallitesize(nm) Crystallinity(%)

HAp 71 5.16

Ag-HAp 34.8 1.95

ofsilvernitrate(0.05Mofsilvernitratesolutionsintocalcium nitratesolution)resultsinthereplacementofCainHApwith AgionsandthusleadstothedistortionofHAplatticeresulting intheformationofamorphousnature.Weobserved signifi-cantlynodifferencefromtheXRDpattern,anditcoulddue tothepreferredorientationofdopedelements(Ag) concen-trationaslow[24];thetracequantityofmetalnitridedopant couldnotbeabletoproduceaconsiderableimpactintheXRD pattern.Sincethedopantconcentrationislow,theimpactof thepeaksintheXRDpatternisveryfeebleandhardlydetected duetothelowerinstrumentaldetectionlimit(Table1).

TheanalysisofcompositesbytheXPStechniqueis com-monly used for the surface analysis and to detect trace elements(excepthydrogen)alongwiththeiroxidationstates inanunknownmaterial.Fig.3representstheXPSsurveyand the Agelemental spectrum ofthe Ag-HAp composite.The surveyspectrumpredictsthepresenceofmajorconstituents fortheelementsofCa,P,Ag,C,and OofAg-HAp compos-ite.Fromthespectra,weobservedthepeaksatthebinding energypositionsof348eVand351eVthatcanbeattributedto theCa2p3/2andCa2p1/2,respectively[25].Similarly,theother

peaksobservedcorrespondingtothebindingenergiesof190 eV,531eV,and532.2eVcorrespondstoP2s,O1selementsof theoxygenatomsassociatedwiththephosphategroupand adsorbedwaterrespectivelyinAg-HAp [26]. Theminuscule peakfoundobviouslyat368.2and374.3eVcorrespondstoAg 3d3/2andAg3d5/2agreeswiththeliterature[23].Althoughthe

concentrationofdopantissmall,thischaracterizationgives additionalevidenceforthesuccessfuldopingofAg+_intothe

HAplattices.

Figs. 4 and 5 show the SEM, TEM, elemental mapping, EDX,andSAEDpatternofthepureHAppowderandAg-HAp

Fig.3–XPSspectrumofAg-HApcompositeandAg’selementalspectrum.

compositerespectively.TheSEMimageprovidesinformation abouttheparticlesizeandtypicalshapefortheas-prepared samples(Fig.4).Itcanbeobservedfromthefiguresofboththe samples,dopedaswellaspureHApexhibitsagglomeration, andcanbeseenthatthedopingofAg+ _metalhas

consider-ableinfluenceonthemorphologyofHAppowder.Wenoted fromthe morphologythatthe HApsamplespreparedfrom theAg-dopinghavemuchsmallerparticlesizeasagainstthe pureHApones.Further,theEDXspectraandtheelemental mappingconfirmsfortheuniformdistributionofAginthe

Ag-HApsamplealongwithelementsofCa,P,andO,andthereby confirmingforthesuccessfulformationofAg-HApcomposite. TheTEMmicrographofpureHAp(Fig.4)showstheparticles ofnon-uniformsizedistributionwithelongatedrod-like mor-phologywithasphericallumpofaround50nmsize,whilethe Ag-HApparticles(Fig.5)exhibitsimplesmallrod-shaped mor-phology. Thelengthofthe nanorodsvariedfrom20−30nm andthediameterwasbetween5−10nmandthedoped Ag-HApsampleisamorphousasseenintheSAEDpatternand the brightspotsdiffusetoformrings.Inoneofthesimilar

Fig.5–SEM,elementalmapping,EDX,TEM,andSAEDpatternofsynthesizedAg-HApnanopowder.

study,Chungetal.(2006)reportedforthepresenceof crys-tallinephaseofAg2OalongwiththeHApandnitratephases

andisstronglyinfluencedbyAg’sdegreeofsubstitutionandin this,theformationofAg2Onucleididnotseemtobedetected

[26].

Fig. 6 displays the stereo zoom optical images ofpure HApandAg-HAp/PLAscaffoldalong withitsEDX andSEM at different magnifications. From the figure (Fig. 6e), the scaffold-coatedHApsampleseemstobeexhibitingthehighly open-porousstructure,wherethe largeporesare intercon-nectedwitheachother.Thesizeofmacroporesdependson thePLAframework,anditiseasytocustomizethescaffolds ofdesiredshapeandsizeasitisparticularlyrequiredforsome needs.Besides,theEDXprovidedelementalanalysis(Fig.6b,f) indicatesthepresenceofallelementslikeCa,P,C,O,andan additionalelementAgforthedopedHApandthereby confirm-ingforthesuccessfulformationofAg-HAp/PLAscaffold.Also, theSEMprovidedmorphologies(Fig.6g–h)showedthatallthe coatingsarecompact,even,andcontainaggregatedgranules atthesurface,ascomparedagainstthepureHAp(Fig.6c–d). TheformationofsuchporousscaffoldsbytheHApcomposite isthemostpromisingmaterialinparticularforbonedefects, repair,andtissueregeneration.Someoftheearlierresearches onscaffoldmaterialshaveconfirmedthesamethattheporous structuresareessentialcriteriafortheconductionofessential nutrientstohelpwiththetissuegrowth[27].

Itiswellrecognizedthatuponimplantation,the adsorp-tion ofessentialnutrients and proteinstakes place onthe biomaterial’sexteriorsurfaceandthisoccursbythe regula-tionofdiversebiologicalproceedingslikebiocompatibility,cell adhesion,immuneresponses,andassociatedblood coagula-tion [28]. Sincethe proteinadsorptionis avibrant process

and is also strongly influenced by the nature of the pro-teinlayersavailableatthesurfaceofbiomaterial,inaddition tothetopographical/textural/pore-sizecharacteristicsofthe biomaterial. The mentioned biomaterial characteristics, in general,decideswhichproteinsarecapabletobindoverthe material[29].Italsoassiststherestriction/adsorptionof cer-tainproteinswithinthedeformitiesofmaterial’ssurfaceand observedthatthetypeofadsorbedproteinatthesurfaceis simplyaffectedbythecharacteristicsoftheunderlying mate-rial,forexample,theporousscaffold’sphysicalandchemical propertiesrestrictitsadsorptioncapacity[30].Takinginto con-sideration these facts, the ␣-MEM with 10% FBS was used to assess the adsorptioncapacityofprotein ontothe pure HApandAg-HAp/PLAcomposites.AsshowninFig.7,thetotal amountofproteinthatgotadsorbedontothesurfacesofthe twomaterialsaredifferentandisattributedtothechanges inthemorphologiesandconstituents.TheAg-HAp/PLA sam-plesupportedthesignificantlyhighproteinadsorptionthan thatofcorrespondingpureHApduetotheavailabilityof addi-tionalAgconstituentand PLAscaffolds thatinfluencesthe crystallinityandporosity.Also,thesurfaceoftheAg-HAp/PLA sampleisunevenandimpregnatedwiththeactiveelement thatsupportsmoreproteinadsorption.However,weseethat the pure HAp materialhas protein adsorption capacity to some extent,but this can bedraggedtoa significant level bymeansofincorporatingtheAgmetalandwithscaffolds, andtherebyshiftingtothepromisinglevels.Furtherstudies havetobeperformedtoenlightenitsbiocompatibilityasa promisingscaffoldingbiomaterial.

Hemolysisisavitalaspecttoevaluatethe biocompatibil-ity ofmaterialand the hemolyticassaysare carriedout to investigate the interaction ofnanoparticles withthe outer

Fig.6–(a,e)Opticalimages,(b,f)EDXspectrums,SEMimagesat100␮m(c,g),and5␮m(d,h)ofpureHApandAg-HAp/PLA scaffoldrespectively.

Fig.7–FBSadsorptionontothesurfacesofpureHApand Ag-HAp/PLAcomposite.

membranesoftheredbloodcellbyaccessingthedischarged hemoglobin [31]. Since the HAp particles interrelate with manycopiouscellularsystemsinourbody,whilstafewof these interactions can perhapslead to defective cells and kindleplateletactivation,coagulation,andthrombus devel-opment[32,33].Fig.8showsthehemolyticassaycomparison ofHApandAg-HAp/PLAcomposite.AspertheASTMF 756-00guidelines,anysamplewithhemolysisratelessthan2% isconsideredtobenon-hemolytic,andbasedonthis,bothof ourHApcontainingsamplesareconsideredtobethesuitable materialsforbiomedicalapplications. Fromthe testing,we foundthatthehemolysisrateofAg-HAp/PLAcompositefound tobe0.8%greaterthanpureHAp,andstillbeconsideredas non-hemolyticasbothofthesample’svaluesarelessthan2%. Itcanalsobeseenthatthehemolysisratioisstronglyaffected

Fig.8–HemolyticassaycomparisonofpureHApand Ag-HAp/PLAcomposite.

bytheconcentrationofdopantanditsdegreeofcrystallinity, withthe latterhavingamoredominatingrole towardsthe hemolysis[34].

The antimicrobial activity tests were conducted for the pure HAp and Ag-HAp/PLAcomposite against the bacteria E. coli (gram-negative) and S. aureus(gram-positive), where theanalysisofresultsindicatedtheAg-dopedcompositehas higherinhibitionofbacterialgrowthasagainsttheun-doped ones.Also,withintheAg-dopedcomposite,E.colidisplayed more zone ofinhibition (3mm) in comparisonwith gram-positivebacteria(2mm)andthereby signifyingthestronger abilityofS.aureusbacteriatofightagainstthetoxicresponses ofthecomposite(Fig.9).Also,theobservationofno antibacte-rialactivityforthepureHApsampleandisduetotheabsence ofstrongerantibacterialagentAginthesample.However,the observeddifferenceofactivityfortheAg-HAp/PLAcomposite amongE.coliandS.aureusmaybeduetothedifferencesin thecellwallresponsesagainsttheAgmetal.SinceE.colihasa

Fig.9–AntibacterialassayofpureHApandAg-HAp/PLAcompositeagainstGram-positivebacteria(S.aureus)and Gram-negative(E.coli).

comparativelythinnercellwall(itsassociatedcharacteristics) andtheantioxidantenzymes(catalase)ofS.aureusprovided thesensitivityandstrongeroxidantresistance(respectively) againstthetestedAg-HAp/PLAcomposite[19].

Boneformsthe skeletonsystemofthebody andsoany basic biomechanical requirements of the implanted scaf-foldingbiomaterialincludeadequate bending,compression strength,appropriatetoequalizethestrengthofaparticular implantsite.Themechanicalcharacteristicsofnaturalboneto alargerdegreeofextentfluctuateconcerningage.Numerous researchershavereportedthatthereactionofthehosttothe implantedmaterialismainlydrivenbythemechanicalaswell asbiologicalcharacteristics[35].Researchershaveconducted studiesbycombiningpolymerswithceramicstoovercomethe restrictionsofpolymerstoimproveitshydrophobicbehavior aswellascell adhesionproperties.Inthat view,the incor-poration of PLA polymer for the implant applications has theadditionaladvantageofdecomposingthepolymeritself intocarbon dioxide,water,etcandinthatway,thereisno needtoremovefrom thebody [36]. Thestress-strain anal-ysisofthe fabricated PLAscaffold (Fig. 10) byFDMproved

Fig.10–Thestress-strainanalysisofAg-HAp/PLAscaffold.

tohaveabendingstrengthofapproximately125MPa, com-parable to the strength of cortical bone and so, it can be used for the load-bearing applications. The coating of the scaffold withbiocompatible materialimproves its biocom-patibility as demonstrated byhemolytic assay and protein adsorptionstudies.Therefore,thecustom-madescaffoldsto meettherequirementsofindividualswithdesiredshapescan bepreparedbytheFDMtechniquewithceramicpolymer com-positecoatingtomeetthestrengthcompatiblewiththatof corticalbone.

5.

Conclusion

Thismanuscriptemphasizesasimpleandeffectivewayto amendthesyntheticbio-inertPLApolymerscaffold(prepared byFDMtechnique)toabiocompatiblescaffoldbysuccessfully coatingthesurfacebyAg-HAp/PLAscaffoldbysimpledip coat-ing.TheXRDdiffractogramrevealsthatthereisanincrease incrystallinitywithareductionincrystallitesizeasHApis dopedwithsilver.TheXPSstudiesonAg-HApconfirmedthe incorporationofAgonthecrystalstructureHAp,eventhough theconcentrationofAgdidnotproducevisiblediffractgram producedbyXRD.Thesuccessfulformationofthe compos-itewasconfirmedbytheEDXanalysis,whilethemorphology studies providedthe shapesandsizesoftheparticles.The antibacterialactivityofthecompositepowdershowedgreater inhibitionefficiencytowardsE.colibetterthanS.aureus organ-isms.TheinvitrohemocompatibilitytestoftheAg-HAp/PLA compositeshowedlessthan2%ofhemolyticactivityand con-sideredtobegoodcompatibilitywithhumanblood.Theinvitro proteinadsorptiontestoftheAg-HAp/PLAcompositeshowed moderateadsorptionofFBSonthesurfacenecessaryfor vari-ousnutrientstransport.Finally,thehardnesstestconfirmsthe scaffoldbendingstrengthiscompatiblewiththecorticalbone. Overall,basedonthecumulativeanalysisofinvitro biologi-calandhardnesstests,itcanbesuggestedthatthefabricated nanocompositecoatedAg-HAp/PLAscaffoldmay serveasa potentialpolymericbiomaterialforbonetissueregeneration applications.

Conflict

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

This work was financially supported by the University of MalayaResearchGrant(RU001-2019,RU001-2020). TheKing SaudUniversityauthorisgratefultotheDeanshipof Scien-tificResearch,KingSaudUniversityforfundingthroughthe ViceDeanshipofScientificResearchChairs..

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