Combined microcomputed tomography, biomechanical and histomorphometric analysis of the peri-implant bone: A pilot study in minipig model

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at

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j ou rn a l h om ep a ge :w w w . i n t l . e l s e v i e r h e a l t h . c o m / j o u r n a l s / d e m a

Combined

microcomputed

tomography,

biomechanical

and

histomorphometric

analysis

of

the

peri-implant

bone:

a

pilot

study

in

minipig

model

Matteo

Gramanzini

a,b,∗,1

_,

_Sara

_Gargiulo

a,b,1

_,

_Fernando

_Zarone

c

_,

Rosario

Megna

a

_,

_Antonio

_Apicella

d

_,

_Raffaella

_Aversa

d

_,

_Marco

_Salvatore

e

_,

Marcello

Mancini

a

_,

_Roberto

_Sorrentino

c,d

_,

_Arturo

_Brunetti

f,b

a_Institute_of_Biostructure_and_Bioimaging,_National_Research_Council,_Via_T._De_Amicis_95,₈₀₁₄₅_Naples,_Italy

b_CEINGE_scarl,_Via_G._Salvatore_486,₈₀₁₄₅_Naples,_Italy

c_Department_of_{Neurosciences,}_Reproductive_and_{Odontostomatological}_Sciences,_School_of_Medicine,_University

“FedericoII”,ViaPansini5,80131Naples,Italy

d_Department_of_Architecture_and_Industrial_Design,_Second_University_of_Naples,_Borgo_San_Lorenzo,

81031Aversa,Italy

e_IRCCS_SDN,_Via_E._Gianturco_113,₈₀₁₄₃_Naples,_Italy

f_Department_of_Advanced_Medical_Sciences,_University_“Federico_II”,_Via_Pansini_5,₈₀₁₄₅_Naples,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5October2015

Receivedinrevisedform

9March2016

Accepted22March2016

Keywords:

Dentalimplant

Insertiontorque

Resonancefrequencyanalysis

␮CT

Histomorphometry

Minipigmodel

a

b

s

t

r

a

c

t

Objectives.Topresentapracticalapproachthatcombinesbiomechanicaltests,

microcom-putedtomography(␮CT) andhistomorphometry, providingquantitativeresultsonbone

structureandmechanicalpropertiesinaminipigmodel,inordertoinvestigatethespeciﬁc

responsetoaninnovativedentalbiomaterial.

Methods.Titaniumimplantswithinnovativethree-dimensionalscaffoldswereinsertedin

thetibiasof4minipigs.Primarystabilityandosseointegrationwereinvestigatedbymeans

ofinsertiontorque(IT)values,resonancefrequencyanalysis(RFA),bone-to-implantcontact

(BIC),bonemineraldensity(BMD)andstereologicalmeasuresoftrabecularbone.

Results.AsigniﬁcantpositivecorrelationwasfoundbetweenITandRFA(r=0.980,p=0.0001).

BMDattheimplantsiteswas18%lessthanthereferencevalues(p=0.0156).Peri-implant

Tb.Thwas50%higher,whileTb.Nwas50%lowerthanthereferencezone(p<0.003)and

theywerenegativelycorrelated(r=−0.897,p=0.006).

∗ _{Corresponding}_author_at:_Institute_of_Biostructure_and_Bioimaging,_National_Research_Council,_Via_T._De_Amicis_95,₈₀₁₄₅_Naples,_Italy.

Tel.:+390812203401;fax:+390812203498.

E-mailaddresses:matteo.gramanzini@ibb.cnr.it(M.Gramanzini),sara.gargiulo@ibb.cnr.it(S.Gargiulo),fernandozarone@mac.com

(F.Zarone),rosario.megna@ibb.cnr.it(R.Megna),antonio.apicella@unina2.it(A.Apicella),raffaella.aversa@unina2.it(R.Aversa),

marsalva@unina.it(M.Salvatore),marcello.mancini@ibb.cnr.it(M.Mancini),errestino@libero.it(R.Sorrentino),brunetti@unina.it

(A.Brunetti).

1 _These_authors_contributed_equally_to_this_work.

http://dx.doi.org/10.1016/j.dental.2016.03.025

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Signiﬁcance.␮CTincreasesevaluationthroughputandoffersthepossibilityforqualitative

three-dimensionalrecordingofthebone–implantsystemaswellasfornon-destructive

eval-uationofbonearchitectureandmineraldensity,incombinationwithconventionalanalysis

methods.Theproposedmultimodalapproachallowstoimproveaccuracyand

reproducibil-ityforperi-implantbonemeasurementsandcouldsupportfutureinvestigations.

1. Introduction

Improvements in bone–implant integration are crucial in

moderndentalsurgery[1,2].Theprocessesofosseointegration

involve an initial mechanical interlocking between

alveo-lar bone and the implant body (primary implant stability)

and,lateron,abiologicalﬁxationthroughcontinuousbone

apposition(contactosteogenesis)andremodelingtowardthe

implant(secondaryimplantstability).[3].

Bioﬁdelityisaraisingtopic inmodernmedicine;sucha

concept derives from the study ofthe biomechanic of

tis-suesandmaterialsandofthebiomimeticapproachnowadays

common intissueengineering toimitate the properties of

naturaltissueswithbioinspiredmaterials. Thebone tissue

isasanaturalnanostructuredbiomaterialcontaininga

col-lagenmatrixandhydroxyapatiteﬁller[4].Tissueengineering

usingsyntheticbiomaterialsisnowadaysusedinregenerative

therapiesandimplantology,sincetheymayactasbioactive

scaffoldspromotingwoundhealingandboneformation[5].

Thephysico-chemical and structural characteristics of the

scaffoldmaterialshavebeenobservedtosigniﬁcantly

inﬂu-encetheir invivoactivity;3Dscaffoldsmay then represent

newclinicalapplicationsinbonereconstructivesurgeryand

implantology[6].Inparticular,ithasbeenshownthat

scaf-foldsmadeupofporoushydroxyapatitemaybecolonizedby

osteoblastsfavoringimplantstabilityimprovement[7].

Differentmethodsweredevelopedtoobjectivelyevaluate

implantprimarystability.Inparticular,peakinsertiontorque

(IT)andresonance frequencyanalysis(RFA)arethe

param-etersthataremostgloballyusedandacorrelationbetween

primarystabilityandimplantinsertiontorquehasbeenoften

suggestedindentalliterature[8–18].Insertiontorquevalues

werecorrelatedtohistologicalbone-to-implantcontactand

radiologicalbone density[19]. RFA isanother effectiveand

reliablemethodtoassessimplantstability,whichpositively

correlateswithITmethod[20,21].

Theuseofpatientscarenewlydevelopedimplant

materi-alsoftenrequirestheuseofpreclinicalmodelstotesttheir

biocompatibility,mechanicalstabilityandsafety.Before

clin-icaltrialsinhumans,minipigsarearecommendedasanimal

model for translational research of new dental materials,

sincetheirbonestructureissimilartothatofhumans.This

speciesisconsideredtobecloselyrepresentativeofhuman

bonetissuewithregard tomorphology[22],bone

composi-tion[23],microstructure[24]andremodelingcharacteristics

[25,26]. Moreover,the boneofminipigshows similaritiesin

mineraldensityandconcentrationwiththatofhumans[27].

However,minipigsboneshaveadensertrabecularnetwork:

meancavitypathlengthsvaluesintheadultmanaretypically

1200␮mand350␮mintheminipig[25],whilemean

trabe-cularpathlengthsinchildaretypically190␮mand280␮min

theminipig[28].Boneregenerationrateinminipigsis

com-parable to that ofhumans (namely,1.2–1.5mmper day in

minipigsand1.0–1.5mmperdayinhumans)[29],andcortical

bonemineralizationrateissimilartoman[30],although

mes-enchymalstemcellsinminipigshowedasigniﬁcantlylower

abilitythanhumanonestoformdifferentiatedandfunctional

osteoblasts[31].Basedontheaboveconsiderations,minipig

hasbeenselectedinthisstudyaslargeanimalmodelforthe

pivotalpreclinicaltestingofourinnovativebiomaterialsand

implants.Thismodelwouldbeneﬁtfrom anon-destructive

imagingmodalitysothatmechanicalandmorphological

end-pointscanmorereadilybeexaminedinthesamespecimens.

Presently, microcomputedtomography(␮CT)mayrepresent

apromisingcandidate toolforbonecharacterizationwhen

pairedtobiomechanicaltestingandstandardhistology.The

implantstabilitywhichiscriticallydependentonstructural

characteristics and bone quality ina relatively broad area

around the implant, may be correlated to bone properties

assessedby␮CT[32,33].Thistechniqueisfast,preciseand,

unlikeconventionalmicroscopicmethods,doesnotneedlong

timeforspecimenpreparationanditisnotimpairedbythe

assessmentofalimitednumberofsections.High-resolution

␮CTallowsmeasurementsoftrabecularandcorticalbone[34]

andathree-dimensional(3D)representationofbone

forma-tion attheperi-implant region[35]. Inaccordancewiththe

principles of the “reﬁnement”and “reduction” declared by

Russelland Burch in1954,␮CT allowslessuse ofsamples

necessaryforbothbiomechanicalandmorphometricstudies.

Inthepresentinvestigation,aninnovativehybrid

nanocom-positeworkingasabiomimeticscaffoldwasusedtoevaluate

theperformanceofthenovelmaterialinimprovingimplant

primarystabilityinpost-surgicalsites.

Ourinnovativebiomaterialappearsasananoporous

trans-parentglassypolymer(Fig.1B)abletoprogressivelyswellinto

ahydrogelform(Fig.1C)whenincontactwithwatersolutions

(picking upto30–50%inweightofwater),withakineticof

swellingat37◦Cofabout0.1mmperhour[36].The

geomet-ricalconfigurationofthemodifiedimplant(Fig.1A)confines

thepolymericscaffoldinanichethatallowsswelling

defor-mationonlyintheradialimplantdirection.Thisradialguided

swellingcouldbeusedtostabilizetheimplantinthesocket

while creating a biomechanically active interface for bone

growth [36]. Moreover, the nanoporositycan bemodulated

tohostcellsandgrowth factors.Inpreviousinvestigations,

the materialwas characterizedfor physical, chemical and

mechanicalproperties[37].Itshowedabioactivesurfacewith

improvedosteoblasticadhesionandproliferationanditcould

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Fig.1–(A)Implantprototype,(B)implantwithglassy

polymerscaffold,and(C)implantwithswollenscaffold.

andrapidosseointegration.Furthermore,theﬁniteelement

analysis(FEA)wasusedtoevaluatethescaffoldinteractions

betweentheswolleninterfaceandthebone[36].Theelastic

modulusofthe scaffoldsintheswollenstatewasreported

torangebetween2and10MPa,whichiscomparabletothat

ofthe periodontalligament(2–50MPa)[38–40]. Thescaffold

hadtoplaytwobiomechanicalfunctions:astructuralone,as

partoftheﬁxture,andabioactiveone,asbonegrowth

stimu-lus.Physiologically,thestressesanddeformationsinducedby

thestretchingoftheperiodontalligamentactsasa

biome-chanical stimulus that favors new bone apposition in the

tooth socket [36]. Sincethe elasticmodulus oftheswollen

scaffoldwascomparabletothatoftheperiodontalligament,

the FEAconﬁrmed thatthe swelling ofthe nanocomposite

couldactasabiomechanicalbonegrowthinput[36].

Accord-ing to Frost’slaw regarding the strain dependent adaptive

propertiesofbone[41],thestrainvaluesrecordedatthe

bone-scaffoldinterfacefellwithintheadaptedwindowandwere

compatiblewithanorganizedbonegrowth.Thepurposeof

thispilotstudywastoexaminetheutilityof␮CTforevaluating

bone–implantintegrationinaminipigmodel,asanadjunct

toconventional histomorphometryandbiomechanical

test-ing.Thepresentedapproachcanserveasamodelforfuture

investigationstostudyimplantsofdifferentcompositionor

shapewithcomplementary␮CTanalysis.Thenullhypothesis

statedthattherewasnoassociationbetweenthe

biomechan-ical,histomorphometricand ␮CTstudy variablesofcontrol

andexperimentalimplantsites.

2. Materials

and

methods

2.1. Surgicalprocedures

AllexperimentalprocedureshavecompliedwiththeItalian

D.L.no 116 of1992and associatedguidelines inthe

Euro-peanCommunitiesCouncilDirectiveofthe24thofNovember

1986(86/609/ECC). Titaniumimplants (WinsixImplant

Sys-tem,Biosaﬁn,Ancona,Italy)modiﬁedwith3Dscaffoldswere

used.

The proposed biomaterial was a hydrophilic hybrid

composite made up of nanoinclusions of hydroxyapatite

dipped in a ceramo-polymeric matrix composed by

poly-hydroxylethylmethacrylateandfumednanosilica.

Thescaffoldsweredesignedusingareverseengineering

approachandwereretainedbymodiﬁedimplantprototypes.

Standard titanium implants with a diameter of 5.2mm, a

lengthof13mmandahexagonalinternalconnectionwere

used.Animplantwasimportedinasolidmodelerandmeshed

todiscretizeitsgeometry.Then,aﬁniteelementmodelingwas

usedtomodifytheﬁxtureandcreatetheexperimental

pro-totype,whoseshapewassuitabletoincorporatethescaffold

(Fig.1A).Thecoronalpartofthescrewthreadwasremoved

soastoincorporatethescaffold,thathadalengthof5mm

andtheshapeofatruncatedconewiththemajorbase

api-cally(majorbase0.6mm,minorbase0.4mm).Thetitanium

implantprototypeshadamicro-roughsurfacethatwasacid

etched(Fig.1A).

A steel cylinderwas used as trayfor the fabricationof

the modiﬁedimplants.Adoubleimpressiontechniquewas

performed usingputtyand lightsiliconimpression

materi-als. First,the shapeofastandardunmodiﬁedimplant was

impressedinthesilicontoobtaintheexternalproﬁleshape

mold. Subsequently, the modiﬁed titanium implant

proto-types were inserted in the silicon molds by means of an

implant holder and the unpolymerized novelmaterial

liq-uidcompositeresin[36,37]wasinjectedintheemptyspace

betweentheexternalshapemoldandthemodiﬁedimplant.

The material was let to self-polymerize at room

tempera-turefor15min.Then,theimplant threadswerethoroughly

polishedbymeansofsiliconrubbers.Although,thenew

bio-materialwasdesignedasself-settingatroomtemperature,

anadditionalthermaltreatementat80◦Cwasaddedtothe

fabricationprocedures inorder tofavorcomplete

polymer-izationandstabilizationofthescaffoldsintheirhighlyrigid

glassy state (with an elastic modulus comparable to that

of the bone) [36,37] before the surgical insertion (Fig. 1B).

The scaffolded implants were then sterilized by means of

gamma-rays.Thescaffoldedimplantswere introduced

uni-cortically intothe leftand rightproximaltibialmetaphysis

of 4 male Yucatan minipigs (>18-month old, average body

weight65kg)withaninter-implantdistanceof5–9mm.The

implants were inserted ﬂush with the corticalbone, so as

the scaffoldscouldinterfacewithboththecorticaland the

spongybone(Fig.2).Thiswasmadetoevaluateifthe

differ-entelasticityofthebonytissuescouldaffecttheswellingof

thescaffolds.Theanimalswere sedatedbyan

intramuscu-larinjection(10mg/kg)ofketamine(Inoketam1000®_,_Virbac

S.r.l., Milan, Italy)and 0.5mg/kg midazolam PHG (Hospira,

LakeForest,IL,USA).Anesthesiawasinducedbymaskand,

afterendotrachealintubation,theminipigsweremaintained

inspontaneouslybreathingbyinhalationof3%isoﬂuraneand

oxigen.Thesurgicalﬁeldswereshavedanddisinfectedwith

benzoxoniumchloride(Citrosil®_,_Manetty_&_Roberts,_Milan,

Italy).Thetibiaswereexposedbyskinincisionsand

fascial-periostealﬂaps.Theimplantsiteswerepreparedusingdrills

withincreasingdiameterandatorque-controlledhandpiece

implant unit at a rotation speed of 25–30RPM, with

con-tinuous external sterilesaline irrigation to minimize bone

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Fig.2–Representativesagittal␮CTsliceshowingscaffold-implantssystemsinsertedintothetibiaofminipigsafter8

weeksofhealingtimeandthecylindricalvolumetricregionofinterest(VOI),wheremeasurementsinperi-implantbone

weremade(A),atthelevelofthetestzoneand(B),atthelevelofreferencezone.

placedineach tibia, according tolocal anatomyand bone

quality. A total of28 scaffolded ﬁxtures was inserted in 7

tibias; 1 tibia was not used because of unfavorable local

anatomy. Theinsertion torquevalues were recorded using

anelectronictorque-controlimplanthandpiece.Theimplant

positionwascheckedbymeansofradiographsand

intensiﬁ-cationofbrilliance,inordertoverifythatthescaffoldswere

inthetransitionarea betweenthe corticaland thespongy

bone. The skin and the fascia-periosteum were closed in

separatelayerswithsyntheticmonoﬁlamentnonabsorbable

polypropylenesuture(Prolene® _2-0,_Ethicon,_Somerville,_NJ,

USA) and a single absorbable suture (Vicryl® _3-0, _Ethicon,

Somerville,NJ,USA),respectively.Perioperatively,theanimals

receivedenroﬂoxacin2.5mg/kg/12h(Baytril®_,_Bayer,_Barmen,

Germany)asantibioticfor6daysandketorolac1mg/kg/24h

as anti-inﬂammatory medication for 3 days. The animals

wereinspectedaftertheﬁrstfewpostoperativedaysforsigns

ofwounddehiscenceorinfectionand,thereafter,weeklyto

assessgeneralhealth.Theanimalsdidnotshowsignsof

phys-icalimpairmentsandwereinstableconditionthroughoutthe

study.After8weeks,animalsweresacriﬁcedandspecimens

containingthe implants were ﬁxedin10% pH7.0-buffered

formalin.

2.2. Biomechanicalanalysis

Insertiontorquevalues(Ncm)wererecordedduringimplant

placement bymeans ofa torque-controlled implant

hand-piece.Moreover,onceimplant placementwascompleted, a

RFAwasperformedusingadedicatedelectronicdevice(Osstell

ISQ,OsstellAB,Göteborg,Sweden).Thevaluesofresonance

frequencywererecordedasanimplantstabilityquotient(ISQ)

inordertoallowacomparisonofprimarystabilitybetween

differentﬁxtures[42–44].

2.3. CTand3Dreconstruction

Thebonesurrounding theimplant scaffoldswasexamined

using␮CT(eXploreLocus,GEHealthcare,London,Canada).

Thedetectorofsuch␮CTsystemwasmadeupof2

compo-nents,ascintillatorandaCCDcamera,whichwereconnected

withopticalﬁbers.Thescintillatorisathinscreenofcesium

iodide,thatluminesceswhenexposedtoX-rays.Thecamera

behinditrecordstheimagethatisprojectedontothe

scin-tillator. Theprojectionissaved indigital format.Atotalof

466␮CTslices,withapixelsizeof45␮m,wereimagedatan

X-rayenergylevelof80kVpandacurrentof450␮A.Exposure

timewas100mswithatotalscanningtimeof11min.Images

werereconstructedusingamodiﬁedFeldkampalgorithmand

scaledintoHounsﬁeldunits(HU).Acalibrationstandard,

con-tainingacompartmentwith1073mg/cm3_SB3_(an_epoxy_based

bone-mimickingmaterial),wasusedforbonemineraldensity

(BMD)calibration.AlldatawereevaluatedinMicroView,2.1.2

(GEHealthcare,London,Canada).Datasetswereresampledto

reorient implantsperpendicular tothe cross-section plane.

Beforesegmentation,thresholdlevelsforbone,implantand

scaffoldweredetermined,basedonvisualinspectionofthe

completeslicesandonthegray-scalehistogram.Theupper

and lower thresholdlevels forbone, implants and scaffold

weredeterminedinall7samplesanddidnotoverlap,

allow-ingtomakeacleardistinction.Anautomaticsegmentation

technique,thatutilizestheOtsumethod,wasusedfor

calcu-latethevaluetobeusedasthethresholdtoextractthebone

fromthesurroundingstructures.Themeanswerecalculated

andusedforeachsample.Theinter-andintra-examiner

vari-abilitieswithin1standarddeviationwerecalculated.Then,a

cylindrical,volumetricregionofinterest(VOI)wasplacedin

correspondenceofthescaffoldelementoftheimplant

circum-ferentially expanded2.5mm aroundthe scaffold,including

bothcorticalandcancellousbone(Fig.2C),whichwasdeﬁned

asthe testzone (Fig. 3A). Subsequently,bone,implant and

scaffoldintheROIweredifferentiatedbasedontheir

thresh-oldlevels.Outcomevariableswere:BMD(mg/cm3)andbone

mineralconcentration(BMC,mg);bonevolumefraction(BVF),

beingthe%oftheROIwhichincludesbone;bonevolume(BV),

beingthemm3_of_bone_that_is_present_in_the_region_of_interest;

theratioofthevolumeofbonepresent(BV)tothetotalvolume

(TV)ofinterest(%);theratioofthesurface(BS)tothevolume

(BV)ofbone(mm−1);theaveragetrabecularthickness(Tb.Th,

mm);themeantrabecularnumber(Tb.N,mm−1);themean

diameter to the marrowspaces (Tb.Sp, mm).These values

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Fig.3–(A)High-resolution␮CT3-Dvolumerenderingofminipigtibiaswithimplants.(B)Representativeaxial␮CTimageof

minipigtibiaswithimplant–boneinterfacevisualization.(C)Representativephotographofminipigtibiaswithimplant

positionvisualization.(DandE)High-resolution␮CT3-Dvolumerenderingofscaffold.(F)High-resolution␮CT3-Dvolume

renderingperi-implantbone.

medianimplants,asreferencezone(Fig.3B).3-Dvolume

ren-deringimageswereperformedon27␮m␮CTdatasetusing

OsirixImagingSoftware5.8.5(Pixmeo,Bermex,Switzerland)

(Fig.2A,B,E,F).

2.4. Histologyandhistomorphometry

Theformalin-ﬁxedspecimensweretransferredto70%ethanol

solution,dehydratedinascendingconcentrationofethanolup

to100%,andtheninﬁltratedandembeddedinahydrophilic

acrylicresin (LR White, London Resin Company, Berkshire,

England).Afterpolymerization,thespecimensweresectioned

along the longitudinalaxis witha high-precision diamond

diskatabout 100␮m,providedwithacustom-built sawing

andagrindingapparatus(TTSystem,TMA2,Grottammare,

Italy).Atotalof3sectionswasobtainedforeachimplant(1

longitudinalcentralsectionand 2sections atacutting

dis-tanceof600␮m).Thesectionswere grounddowntoabout

60±10␮mandstainedwithacidfuchsineandtoluidineblue.

The investigation was conducted in a transmitted

bright-ﬁeldandcircularlypolarizedLightMicroscopeAxiolab(Zeiss

Oberchen,Germany) connected toa high-resolutiondigital

camera(FinePix S2Pro,FujiPhotoFilmCo. Ltd,Minato-Ku,

Japan).A histomorphometricsoftware packagewith image

capturing capabilities(Image-ProPlus 6.0, Media

Cybernet-icsInc, Bethesda, MD, USA)was used. Toensure accuracy,

the software was calibrated for each experimental image

usinga featurenamed“Calibration Wizard”, which creates

alinearremappingofthepixelnumbers.Theunit of

mea-surementwas the pixel. The analyzed parameter was the

bone-to-implantcontact(BIC,%),deﬁnedastheratiobetween

the lengthofthe scaffold materialsection incontact with

boneand theperimeterofthescaffoldarea,at25×

magni-ﬁcation.Ateach measurement,thesoftwarewascalibrated

usingthelengthoftheimplantasreference.Measurements

wereperformedbyasingleexperiencedandcalibrated

oper-ator.Intra-examinervariabilitywascontrolledbycarryingout

2measurements;ifthedifferencebetweenthe2valueswas

greaterthan5%,themeasuringwasrepeated.

2.5. Statisticalanalyses

The study variables were divided into 4groups asfollows:

biomechanical (IT and RFA), histomorphometric(BIC), ␮CT

of implant sites (BMD, BMC, BVF, BV, BV/TV, BS/BV, TbTh,

TbN,TbSp)and␮CTofcontrolsites(cBMD,cBMC,cBVF,cBV,

cBV/TV,cBS/BV,cTb/Th,cTbN,cTbSp).Mean,standard

devia-tion,median,minimumandmaximumvalueswerecalculated

forallthevariables.NormalitywastestedusingShapiro–Wilk

testandvariancehomogeneityusingBartlettorLevenetest.

When data were normally distributed and variance

homo-geneitywasmet,thevariableswerecomparedusingone-way

analysisofvariance(ANOVA).Incaseofviolationofnormality

orvariancehomogeneity,Kruskall–WallisorMann–Whitney

test were performed. The mean values of21 variables for

7 tibialsampleswere used forthe statistical analysis.The

Pearson correlation wasperformed forall possiblepairsof

variables,foratotalof210combinations.Thelinear

regres-sioncoefﬁcientswerecalculatedforthe12pairsofvariables

foundtobecorrelated.Moreover,IT,RFA,BIC,BMD,BMC,TbTh,

TbNandTbSpwereanalyzedforcomparisonamongimplant

sites.Samplesizerequiredtoreacha5%signiﬁcancewitha

80%powerwasestimatedforMann–Whitney,Shapiro–Wilk,

AnovaandPearsoncorrelationtests.Statisticalanalysiswas

performedusingRstatisticalsoftware(version3.1.2,R

Foun-dationforStatisticalComputing,Vienna,Austria)andthelevel

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Fig.4–Representativesagittal␮CTsliceshowing

scaffold-implantssystemsinsertedintothetibiaof

minipigsafter8weeksofhealingtime.

3. Results

Theminipigsshowedgoodgeneralehealthaftersurgeryand

signsofinfectionwerenotfoundatclinicaland␮CT

exami-nation.NoeffectsfromX-rayscatteringorringartifactswere

foundinthe␮CTimagesintheperi-implantzoneusedfor

measurements(Fig.4).Alltheimplantswereosseointegrated

and the peri-implant bone structure consisted of lamellar

bonearchitecture(Fig.5).TheITandRFAvalueswerereported

inTable1.

Accordingtotheresultsofthepresentinvestigation,the

nullhypothesiswasrejected.

Descriptivestatisticsforbiomechanical,␮CTandhistologic

variablesweresummarizedinTable2.Themeanandstandard

deviationoflower andupperthresholdgray-levelsfor

tita-niumimplants,forceramo-polymericscaffoldsandforbone

madeby2independentandcalibratedoperatorswere

summa-rizedinTable3.TheﬁrstboxofTable2showsthevaluesofthe

3seriesofrepeatedmeasuresforeachbonesample;the

sec-ondboxthoseofallsamplesforeachseriesofmeasurements;

the third onethe global thresholdvalue forimplant,

scaf-foldandbone.Theinter-andintra-examinervariabilitieswere

equivalentwithin1standarddeviation.ANOVAdidnotreveal

differencesamongtheimplantsitesforBIC(F=1.76,p=0.112)

and IT (F=2.580, p=0.058), unlike RFA (Kruskall–Wallis,

p=0.0156).For␮CTparameters,signiﬁcantdifferenceswere

foundamongtheimplant sitesforBMC(F=5.201,p=0.003)

andBMD(Kruskall–Wallis,p=0.037),unlikeforTbTh(F=2.61,

p=0.056),TbN(Kruskall–Wallis,p=0.037)andTbSp(F=1.764,

p=0.167).Forcomparisonsbetweenimplantandcontrolsites,

ANOVA did not reveal differences between the ␮CT pairs

ofvariablescBMC–BMC(F=1.26,p=0.2831)andcTbSp–TbSp

(F=8.68,p=0.0514),whereascBMD-BMDandcBV–BVwere

sig-niﬁcantlydifferent(Mann–Whitneytest:p<0.05).FiftyPearson

correlationswereobservedwithp<0.05,bothbetween

vari-ablesofthe samegroupthatbetweenvariables ofdifferent

groups.Thecorrelationmatrixofallcorrelationcoefﬁcients

betweenthesetofvariableswerereportedinTable4.

Relevant correlations were found between IT and RFA

variables (r=0.980, p=0.0001); the ␮CT pairs of variables

cBV–BMC(r=0.796,p=0.0322),cBV–BVF(r=0.823,p=0.0230),

cBV–BV(r=0.826,p=0.0220),cBV–BS/BV(r=−0.873,p=0.0103),

cBV–TbTh (r=0.830, p=0.0208), cBV–TbN (r=−0.778,

p=0.0394), cBVF–TbN (r=0.772, p=0.0419), cBV/TV–TbN

(r=−0.795, p=0.0326) and cBV/TV–BS/BV (r=−0.761,

p=0.0470);andbetweenIT–cTbSpandRFA–cTbSp(r=−0.859,

Fig.5–(A)Microphotographofalongitudinalsectionofaspecimenstainedwithfuchsinacidandtoluidineblue.(B)

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Table1–Insertiontorque(ITinNcm)andresonancefrequencyanalysis(RFAinISQ)valuesoftheexperimental scaffoldedimplants.

Minipig Tibia Measurement Implant

#1 #2 #3 #4 #1 Left IT(Ncm) 44.2 46.2 47.3 44.5 RFA(ISQ) 71 73 74 71 Right IT(Ncm) 48.1 44.6 45.1 44.4 RFA(ISQ) 75 71 72 71 #2 Left IT(Ncm) 43.4 44.7 42.9 42.5 RFA(ISQ) 70 71 70 69 Right IT(Ncm) 51.1 45.2 45.8 44.7 RFA(ISQ) 79 72 72 71

#3 Left IT(Ncm) Excludedbecauseofunfavorable

localanatomy RFA(ISQ) Right IT(Ncm) 42.0 45.6 42.9 43.1 RFA(ISQ) 69 72 70 69 #4 Left IT(Ncm) 46.2 43.7 45.2 43.8 RFA(ISQ) 73 70 72 70 Right IT(Ncm) 42.6 39.9 43.2 42.2 RFA(ISQ) 69 66 70 69

Table2–Descriptivestatisticofsampledata.

Variable Unit Mean SD Min Median Max

IT Ncm 44.39 1.63 41.90 43.83 46.70 RFA ISQ 71.11 1.71 68.50 71.25 73.50 BMD mg/cm3 _623.7 _42.1 _533.2 _631.9 _665.0 BMC mg 46.62 5.71 36.18 47.47 54.34 BVF % 0.7809 0.0639 0.6705 0.7925 0.8579 BV mm3 _74.04 _4.42 _67.47 _73.98 _79.39 BV/TV % 0.8394 0.1118 0.7332 0.8164 1.0743 BS/BV mm−1 1.641 0.182 1.451 1.634 1.891 TbTh mm 1.244 0.140 1.058 1.228 1.405 TbN mm−1 0.6392 0.0335 0.5846 0.6316 0.6866 TbSp mm 0.3311 0.0744 0.2246 0.3165 0.4351 cBMD mg/cm3 _764.0 _31.5 _719.9 _765.5 _808.9 cBMC mg 52.40 12.36 33.62 54.13 73.82 cBVF % 0.6955 0.0983 0.4933 0.7322 0.7799 cBV mm3 _63.70 _12.47 _44.50 _69.52 _73.82 cBV/TV % 0.7075 0.1009 0.5043 0.7467 0.7913 cBS/BV mm−1 3.722 1.307 1.923 4.117 5.517 cTbTh mm 0.6152 0.2675 0.3624 0.4857 1.0399 cTbN mm−1 1.274 0.355 0.761 1.391 1.617 cTbSp mm 0.2407 0.0817 0.1507 0.2474 0.3561 BIC % 76.91 4.17 69.86 77.44 82.34

p=0.0134 and r=−0.834, p=0.0196, respectively. A lack of statistical signiﬁcance was found between the values of BICand all other variables.Table 5summarized thelinear

regressioncoefﬁcientsforthepairsofvariablescorrelated.

4. Discussion

Preclinicalresearchisconsideredanessentialsteptotest

den-talmaterialsandimplantspriortotheirclinicaluse.Minipigs

are suitable models fortraslational validation due totheir

anatomicalandphysiologicalcorrespondenceswithhuman

bone[45].Itisgenerallyacceptedthatthechemicaland

phys-icalpropertiesofimplantsurfaceshaveamajorinﬂuenceon

thestructureoftheperi-implantboneandthuscanimprove

osseointegration. The scaffolds should work as a medium

that offersmechanicalproperties similartothe hostbone,

promoting cell adhesion and activity and inducing ﬁnally

bonegrowth.Thetestedinnovativenanocompositematerial

allowedtofabricatestable3Dimplantscaffoldsthatproved

tobeeasyhandlingandsuitableforthesurgicalprocedures.

Inapreviousinvestigation[36],itwasdemonstratedthatthe

volumetricexpansionofthebioactivenovelmaterialdueto

theswellingofthescaffolds,signiﬁcantlycontributedtothe

primarystabilityoftheimplants.Sinceitisknownthatthe

developmentofastablebone–implantinterfacedependson

differentfactors,itisdesirabletouseseveralmethods

capa-bletoevaluatedifferentboneproperties.Inthisperspective,

theintegrationofbiomechanicaldata,of3D␮CTinformation

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T able 3 – Descr ipti v e sta tistics for intr a-oper a tor and inter -oper a tor repr oducibility (mean ± standar d de via tion). OP 1 OP 2 Implant Scaffold Bone Implant Scaffold Bone LT h UTh LT h UTh LT h UTh LT h UTh LT h U T h LT h UTh Bone sample #1 4999.3 ± 1.2 13,873.7 ± 1.5 0.3 ± 0.6 1499.3 ± 1.2 1699.0 ± 1.0 4699.0 ± 1.0 4999.3 ± 1.2 13,873.7 ± 1.5 0.3 ± 0.6 1499.7 ± 0.6 1699.0 ± 1.0 4699.0 ± 1.0 #2 4998.6 ± 1.2 13,872.7 ± 1.2 0.0 ± 0.0 1500.0 ± 0.0 1699.3 ± 0.6 4699.3 ± 0.6 4999.3 ± 1.2 13,872.7 ± 1.2 0.3 ± 0.6 1499.0 ± 1.0 1699.3 ± 0.6 4699.3 ± 0.6 #3 5000.0 ± 0.0 13,874.7 ± 1.2 0.3 ± 0.6 1499.3 ± 0.6 1698.0 ± 0.0 4694.6 ± 0.6 5000.0 ± 0.0 13,872.7 ± 1.2 0.3 ± 0.6 1499.3 ± 0.6 1694.7 ± 6.7 4694.0 ± 1.0 #4 4999.0 ± 1.0 13,873.0 ± 1.7 0.3 ± 0.6 1499.6 ± 0.6 1698.3 ± 0.6 4693.6 ± 1.5 4999.0 ± 1.0 13,873.7 ± 1.5 0.0 ± 0.0 1499.6 ± 0.6 1695.7 ± 5.8 4694.3 ± 2.1 #5 4999.3 ± 1.2 13,873.7 ± 1.5 0.3 ± 0.6 1499.6 ± 0.6 1699.6 ± 0.6 4699.6 ± 0.6 4999.3 ± 1.2 13,873.7 ± 1.5 0.7 ± 0.6 1499.6 ± 0.6 1699.6 ± 0.6 4699.3 ± 0.6 #6 4999.3 ± 1.2 13,873.0 ± 1.7 0.3 ± 0.6 1499.0 ± 1.0 1699.0 ± 1.0 4699.3 ± 1.2 4999.3 ± 1.2 13,872.7 ± 1.2 0.3 ± 0.6 1499.0 ± 1.0 1699.0 ± 1.0 4699.0 ± 1.0 #7 4999.3 ± 1.2 13,872.0 ± 1.2 0.3 ± 0.6 1499.3 ± 0.6 1700.0 ± 0.0 4699.7 ± 0.6 4999.3 ± 1.2 13,872.0 ± 1.2 0.3 ± 0.6 14,993 ± 0.6 1700.0 ± 0.0 4699.7 ± 0.6 Re peated measur es series 1 ◦ 4999.3 ± 0.9 13,874.1 ± 1.1 0.1 ± 0.4 1499.6 ± 0.8 1698.7 ± 1.0 4698.0 ± 2.5 4999.6 ± 0.8 13,873.3 ± 1.3 0.2 ± 0.4 1499.3 ± 0.7 1698.8 ± 0.8 4697.7 ± 2.6 2 ◦ 4999.1 ± 1.0 13,872.7 ± 1.3 0.1 ± 0.4 1499.1 ± 0.7 1698.8 ± 0.7 4698.2 ± 2.6 4999.0 ± 1.1 13,873.0 ± 1.3 0.2 ± 0.4 1499.3 ± 0.5 1697.2 ± 3.8 4697.7 ± 2.4 3 ◦ 4999.4 ± 1.0 13,873.1 ± 1.5 0.6 ± 0.5 1499.7 ± 0.5 1699.8 ± 0.8 4697.4 ± 2.9 4999.4 ± 1.0 13,873.0 ± 1.3 0.7 ± 0.5 1499.5 ± 0.8 1697.7 ± 4.9 4697.2 ± 2.9 Global v alue 4999.3 ± 1.0 13,873.3 ± 1.4 0.3 ± 0.5 1499.5 ± 0.7 1699.3 ± 0.9 4697.3 ± 2.6 4999.3 ± 0.9 13,873.1 ± 1.2 0.3 ± 0.5 1499.4 ± 0.7 1698.2 ± 3.5 4698.3 ± 2.5 OP: oper ator; LTh: lo w e r HU thr eshold; UTh: upper HU thr eshold.

sampleenablestoimproveevaluationofimplantstability.

Pri-maryimplantstabilityisconsideredofparamountimportance

toachieveosseointegrationand ITandRFAvaluesare

vali-datedmechanicalparameterstoevaluatethestrenghtofthe

interactionbetweenthehostboneandimplantsurface[46].In

dentalliterature,minimumITvaluesforimmediateloading

rangedfrom32to50Ncm[8,12,14,47–51],sufﬁcientfor

retain-ingmicromovementswithinlimits[12,49].RFAhasbeenused

atearlystagesofosseointegrationorinmonitoringovertime

andincreasesduringthehealingphase[52].Theacceptable

rangeofRFA,thatindicatesstability,liesbetween55and85

ISQ[53–55].Moreover,ITandISQvalueshaveshownapositive

correlation[33,56].ThemeasurementsofITandRFArecorded

inthisstudywereintheintervalofvaluesreportedas

ade-quateinliterature.Inagreementwiththeliterature,astrong

correlationbetweenITandRFAwasfound.Secondary

stabil-ityisdeterminedbythenewboneformationandremodeling

attheimplantinterface,andgreaterbonecontactis

gener-allybelievedtoresultinbetterimplantstability[57].Inthe

examinedsamples,lamellarbonewasvisibleinintimate

con-tact withthescaffoldsurface. TheBICvaluesinthisstudy

werefoundtobeappropriatetowarrantstability,sincethey

werehigherthan50%,theminimumvalueconsidered

neces-saryforloadingandforensurelong-termsurvivalofimplants

[58].Goodprimarystabilitymayberelatedtobetterbone

tis-sueresponse.However,theformulaofhigherbiomechanical

parameterstranslatingintobetterosseointegrationmaynot

alwaysbetrue,becausethequantityandqualityofbonemay

vary signiﬁcantlyamongsubjects. DespiteITand RFAhave

beenconsidered validtoolstoforeseethebonequalityand

primarystabilityofimplantsites,theircomparisonwith

radio-logicor histomorphometrictestsgavecontradictoryresults

[59–69]. Friberg et al. [61]demonstrated that IT was

corre-lated tohistomorphometricBICandtoradiological BMDof

thepreparedsites[19,52].Otherauthors demonstratedthat

ITvalueswascorrelatedwithBMDofthereceivingbonesite,

obtainedbymeasuringCTor␮CT[9,70,71].Incontrast,ithas

beenreportedthatRFAsuffersfromalackofsensitivitytothe

qualityofsurroundingbone[72],andacorrelationwith

histo-morphometricBICwasnotfound[56,73].Thebiomechanical

propertiesoftheboneseemstoberelatedbothtoitsmineral

content and microarchitecture [74]. Moreover,bone quality

parametersassessedby␮CTinperi-implantregionsdidnot

seemlinearlycorrelatedtobiomechanicalvariables[68].The

present study failedto ﬁnda correlation betweenBIC and

biomechanicalor␮CTparameters.Nevertheless,astrong

rela-tionshipbetweenIT–cTbSpandRFA–cTbSpwasfound,where

negativevaluesofrindicatethat,increasingcTbSp,ITandRFA

decreased.Althoughhistologyisthemostcommonmethod

for analysingbone formation around implants,it alsohas

limitations.Samplepreparationistimeconsuming,mayalter

bone–implantinterfaceandalimitednumberof2Dsections

per sampleare usedfortheapproximationofmeanvalues

ofBIC.Conversely,␮CTisanon-destructive,fastandprecise

technique,thatprovidesaccuratemodelingandanalysisof3D

imagesofbonesamples,andcanevaluatebothqualitativeand

quantitativemorphometryofboneintegrationarounddental

implants[57,75].␮CTproducesvoxelsintherangeof5–50␮m,

orapproximately1.000timessmallerinvolumethanclinical

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d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 794–806

Table4–Pearsoncorrelationmatrix.

IT RFA BIC TMD TMC BVF BV BV/TV BS/BV TbTh TbN TbSp cTMD cTMC cBVF cBV cBV/TV cBS/BV cTbTh cTbN RFA 0.980* BIC −0.171 −0.286 BMD 0.321 0.384 0.108 BMC 0.226 0.281 0.123 0.928* BVF 0.310 0.352 0.199 0.899* _0.978* BV 0.036 0.094 0.206 0.772* _0.941* _0.937* BV/TV −0.093 −0.193 −0.052 −0.775* _−0.535 _−0.462 _−0.350 BS/BV −0.407 −0.467 −0.054 −0.755* _−0.857* _−0.931* _−0.857* _0.290 TbTh 0.408 0.455 0.107 0.743 0.840* _0.925* _0.838* _−0.268 _−0.996* TbN −0.520 −0.596 0.123 −0.527 −0.566 −0.688 −0.564 0.181 0.896* _−0.897* TbSp −0.242 −0.244 −0.297 −0.800* _−0.945* _−0.965* _−0.939* _0.280 _0.868* _−0.870* _0.567 cBMD −0.249 −0.094 −0.205 0.320 0.491 0.473 0.673 −0.215 −0.552 0.494 −0.480 −0.394 cBMC 0.071 0.243 −0.463 0.304 0.415 0.410 0.523 −0.221 −0.560 0.492 −0.602 −0.279 0.918* cBVF 0.301 0.406 −0.161 0.287 0.465 0.544 0.612 0.003 −0.736 0.695 −0.772* _−0.474 _0.794* _0.869* cBV 0.327 0.450 −0.081 0.717 0.796* _0.823* _0.826* _−0.452 _−0.873* _0.830* _−0.778* _−0.709 _0.782* _0.823* _0.864* cBV/TV 0.320 0.426 −0.134 0.329 0.492 0.574 0.628 −0.043 −0.761* _0.722 _−0.795* _−0.496 _0.788* _0.863* _0.998* _0.884* cBS/BV 0.294 0.146 0.139 −0.172 −0.299 −0.336 −0.503 0.140 0.505 −0.470 0.566 0.228 −0.921* _−0.840* _−0.751 _−0.663 _−0.750 cTbTh −0.453 −0.292 −0.201 0.157 0.248 0.242 0.432 −0.232 −0.362 0.322 −0.403 −0.124 0.903* _0.795* _0.593 _0.561 _0.591 _−0.968* cTbN 0.643 0.501 0.134 −0.037 −0.068 −0.050 −0.233 0.242 0.140 −0.117 0.206 −0.064 −0.719 −0.563 −0.302 −0.294 −0.302 0.854* _−0.941* cTbSp −0.858* _−0.834* _0.066 _−0.210 _−0.298 _−0.390 _−0.260 _−0.173 _0.502 _−0.488 _0.524 _0.402 _−0.032 _−0.264 _−0.587 _−0.478 _−0.589 _−0.087 _0.297 _−0.585 *_p_<_0.05.

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Table5–Linearregressioncoefﬁcientsforcorrelatedvariables(y=˛*x+ˇ). y x ˛ err.st.˛ ˇ err.st.ˇ IT RFA 0.930 0.084 −21.73 6.00 cTbSp IT −0.043 0.012 2.156 0.512 cTbSp RFA −0.040 0.012 3.070 0.837 cBV TMC 1.739 0.591 −17.39 27.74 cBV BVF 160.6 49.6 −61.74 38.84 cBV BV 2.333 0.712 −109.1 52.8 cBV BS/BV −59.77 14.92 161.8 24.6 cBV TbTh 73.82 22.18 −28.15 27.74 cBV TbN −289.7 104.6 248.9 66.9 cBVF TbN −2.267 0.834 2.144 0.534 cBV/TV TbN −2.396 0.818 2.239 0.523 cBV/TV BS/BV −0.422 0.161 1.399 0.265

using␮CT,includingteeth,boneandmaterialssuchas ceram-ics,polymersorbiomaterialscaffolds[34].Althoughformalin

ﬁxationmayalterthemechanicalpropertiesofbone,several

studiesshowedthatithasnoeffectonthemineral

composi-tionofbone[78,79].Moreover,inarecentstudyitwasreported

thatformalinﬁxationandfreezingwouldnotadverselyaffect

theviscoelasticandelasticmechanicalpropertiesofmurine

bone[80].Inthepresentinvestigation,noartifactswerefound

inthe ␮CTimages incorrespondenceofthe zonesaround

thescaffolds(Fig.4).However,itisdifﬁculttodeterminethe

preciseeffectofmaterialcompositionontheimagequality.

Nevertheless,theselimitsdonotnecessarilypreventsuitable

analysisofperi-implantboneby␮CT,evenifextensive

exper-imentationforappropriateset up shouldbeperformed for

eachequipment.Moreover,intra-andinter-examiner

repro-ducibilityandreliabilityofthresholdlevelsformethodology

validationwerecompared,withgoodresults.Therefore,this

methodcanbeproperlyused ascomplementofthe

estab-lishedgoldstandard,allowingareﬁnementintheplanning

ofexperimentswithanimalmodels,inaccordancewiththe

3Rsprinciples,andtoimprovethequalityoffuturestudies.

Themeandensityofthenewlyformedbonecanbean

indi-cationofhow the remodelingprocess has been successful

intermsofnewbonetissuedensity,similartothereference

areas.Inthepresentstudy,theentityofosseointegrationwas

visualizedandquantiﬁedby␮CT3Dreconstruction.␮CTfor

preclinicalapplicationprovideshigherspatialresolutionthan

clinicalCT,thenperi-implantbonemeasurementsat45␮m

resolutioncouldbeusedasaguidetopredictimplant

inte-gration.Intheexaminedsamples,cBVofthereferencezone

wasfoundcorrelatedwithBMC,BV,Tb.N,Tb.Thofthebone

surroundingthescaffold.Peri-implantBMDwas18%lessthan

thecontrolvalue,probablyduetothefactthatminipigswere

sacriﬁced 8weeks after implants placement. An adequate

healingperiod forosseointegration in humans is assumed

tobe12 weeks[81]andboneformationand remodelingin

minipigshavebeenestimatedtobeapproximately

compara-bletothoseofhumans[29]. Inminipigs,anhealingperiod

of4weeksisconsidered short[82]and histological

evalua-tionofbonedensitydemonstratedanincreaseofmeanvalues

overtimeat4,8and12weeksafterreceivingdifferenttypes

ofdentalimplants[83].Nevertheless,inthisstudyan

inter-val ofreference valuesformineral density ofperi-implant

bonewasdeﬁnedafterthewidelyadoptedhealingperiodof

8weeksinminipigs,corresponding tothedesirable values

ofthebiomechanicalproperties.Boneinthe vicinityofthe

implantsshowedevidenceofanincreaseof50%intrabecular

thicknessbutasimilardecreaseoftheirnumberwhen

com-paredtotheboneinthecontrolzone(p=0.0021).Itislikely

thatthesechangesintrabecularmicroarchitecture

parame-tersmaybeawayforlowdensitybonetorespondtothestress

inducedbyloading,sincebonestrengthincreases

proportion-allytothesquareofthetrabecularradius[84].Insomestudies,

bonedensityweremeasuredpreoperativelyinimplant

recip-ientsiteofhumanpatientsbyclinicalCTandwererecorded

inHounsﬁeldunits(HU)orconvertedinmg/cm3_using_a

cali-brationphantomwithknowndensityvalues[19,46,66–68].As

tobonedensityanalysis,themethodusingBMDcalibration

standardismoreaccuratelycomparedwiththemethodwith

Hounsﬁeldunits[85].Bilhanetal.[86]reportedinanexvivo

studythatHUvaluesassessedbyclinicalCTcouldbea

mis-leadingtoolforthedeterminationofbonedensityandcould

resultinanoverestimationofmicro-architecturalparameters,

while␮CT,withaspatial resolutionnotmorethan 100␮m,

wasabletoreasonablyevaluatebonequantitativelyand

qual-itativelywithagoodcorrelationbetweenhistomorphometric

andmicrotomographicdata[85].Themainlimitationofthis

proof ofconcept study was the relatively smallnumber of

samples.Thus,cautionisrequiredintheinterpretationofthe

results.Estimatedsamplesizespergrouprangedfrom11for

Pearsoncorrelationteststo26forAnova.Furtherstudiesina

largergroupofanimalsareneededtoassessthesensitivityof

thisapproachthatcombinesbiomechanicaltests,

microcom-putedtomography(␮CT)andhistomorphometrytoimprove

evaluationofimplantstability.

5. Conclusion

Inconclusion, theresultsfromthis pilotstudyshowed the

feasibility and usefulness of ␮CT imaging in the

charac-terization ofbone densityand microarchitecture topredict

structuralchangesinminipigboneundergoingnew

bioma-terialsandimplants,inacomplementarywaytothecommon

biomechanicaltesting and hystomorphometry.The

innova-tive hybrid ceramopolymeric nanocomposite was effective

infabricatingthree-dimensionalbioactivescaffolds.Further

clinicalstudiesareneededtoconﬁrmthattheexperimental

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avoidingmicromotionatthetissue/biomaterialinterface,

pro-motingacceleratedimplantosseointegrationandloading.

Conﬂict

of

interest

Theauthorsdeclarethatthereisnoconﬂictofinterest.

Author

contributions

• MatteoGramanzini: conception and design ofthe work;

acquisition,analysis and interpretation of data; imaging

datareconstructionandpost-processing;criticalrevisionof

dataandstatistics;draftingarticle.

• SaraGargiulo:conceptionanddesignofthework;

acqui-sition,analysis and interpretation of data; imaging data

reconstructionandpost-processing;criticalrevisionofdata

andstatistics;draftingarticle.

• Roberto Sorrentino: conception and design of the work;

acquisition,analysisandinterpretationofdata;critical

revi-sionofarticle.

• Rosario Megna: statistics; analysis and interpretation of

data;criticalrevisionofarticle.

• AntonioApicella:conceptionanddesignofthework;

anal-ysisandinterpretationofdata.

• RaffaellaAversa:acquisition,analysisandinterpretationof

data;criticalrevisionofarticle.

• Marco Salvatore: critical revision of article and ﬁnal

approval;analysisandinterpretationofdata.

• Marcello Mancini: critical revision of article and ﬁnal

approval;analysisandinterpretationofdata.

• FernandoZarone:conceptionanddesignofthework;

criti-calrevisionofarticleandﬁnalapproval.

• ArturoBrunetti:conceptionanddesignofthework;critical

revisionofarticleandﬁnalapproval.

Acknowledgements

TheauthorswouldliketothankProf.EnricoFeliceGherlone

andhisteamoftheUniversityVitaSaluteSanRaffaeleofMilan

(Italy)forthesupportinhistologicalandhistomorphometrical

analysesandBiosaﬁn(Ancona,Italy)forthetechnical

sup-portprovidingtheimplantprototypesoftheWinsixImplant

System.

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s

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