Tricalcium Phosphate Bone Substitutes

12 Pages·2017·4.53 MB·English

materials
Article
Physical and Histological Comparison of
Hydroxyapatite, Carbonate Apatite, and β-Tricalcium
Phosphate Bone Substitutes
KunioIshikawa1,*,YoujiMiyamoto2,AkiraTsuchiya1,KoichiroHayashi1,KanjiTsuru1,3and
GoOhe2
1 DepartmentofBiomaterials,FacultyofDentalSciences,KyushuUniversity,3-1-1Maidashi,Higashi-ku,
Fukuoka812-8582,Japan;[email protected](A.T.);[email protected](K.H.);
[email protected](K.T.)
2 DepartmentofOralSurgery,InstituteofBiomedicalSciences,TokushimaUniversityGraduateSchool,
3-18-15Kuramotocho,Tokushima770-8504,Japan;[email protected](Y.M.);
[email protected](G.O.)
3 SectionofBioengineering,DepartmentofDentalEngineering,FukuokaDentalCollege,
Fukuoka814-0193,Japan
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:28September2018;Accepted:11October2018;Published:16October2018
Abstract: Three commercially available artificial bone substitutes with different compositions,
hydroxyapatite(HAp;Neobone®),carbonateapatite(CO Ap;Cytrans®),andβ-tricalciumphosphate
3
(β-TCP;Cerasorb®),werecomparedwithrespecttotheirphysicalpropertiesandtissueresponse
tobone,usinghybriddogs. BothNeobone®(HAp)andCerasorb®(β-TCP)wereporous,whereas
Cytrans®(CO Ap)wasdense. Crystallitesizeandspecificsurfacearea(SSA)ofNeobone®(HAp),
3
Cytrans® (CO Ap),andCerasorb® (β-TCP)were75.4±0.9nm,30.8±0.8nm,and78.5±7.5nm,
3
and0.06m2/g,18.2m2/g,and1.0m2/g,respectively. Thesevaluesareconsistentwiththefactthat
bothNeobone®(HAp)andCerasorb®(β-TCP)aresinteredceramics,whereasCytrans®(CO Ap)is
3
fabricatedinaqueoussolution. DissolutioninpH5.3solutionmimickingHowship’slacunaewas
fastestinCO Ap(Cytrans®), whereasdissolutioninpH7.3physiologicalsolutionwasfastestin
3
β-TCP(Cerasorb®). TheseresultsindicatedthatCO Apisstableunderphysiologicalconditions
3
and is resorbed at Howship’s lacunae. Histological evaluation using hybrid dog mandible bone
defectmodelrevealedthatnewbonewasformedfromexistingbonetothecenterofthebonedefect
whenreconstructedwithCO Ap(Cytrans®)atweek4. Theamountofboneincreasedatweek12,
3
andresorptionoftheCO Ap(Cytrans®)wasconfirmed. β-TCP(Cerasorb®)showedlimitedbone
3
formationatweek4. However, alargeramountofbonewasobservedatweek12.Amongthese
threebonesubstitutes,CO Ap(Cytrans®)demonstratedthehighestlevelofnewboneformation.
3
Theseresultsindicatethepossibilitythatbonesubstituteswithcompositionssimilartothatofbone
mayhavepropertiessimilartothoseofbone.
Keywords: carbonate apatite; hydroxyapatite; β-tricalcium phosphate; artificial bone substitute;
crystallitesize;dissolutionrate;hybriddog
1. Introduction
Bone apatite is known as carbonate apatite [CO Ap: Ca (CO ) (PO ) ], which contains
3 10-a 3 b 4 6-c
6–9wt%carbonateinanapatiticstructure.However,sinteredhydroxyapatite[HAp;Ca (PO ) (OH) ]
10 4 6 2
andsinteredβ-tricalciumphosphate[β-TCP:Ca (PO ) ]havebeenusedinclinicsastypicalsynthetic
3 4 2
bonesubstitutes,whileCO Aphasnotbeenused[1–11].
3
Materials2018,11,1993;doi:10.3390/ma11101993 www.mdpi.com/journal/materials
Materials2018,11,1993 2of12
ThelackofchemicallypureartificialCO Apbonesubstituteisduetothethermaldecomposition
3
statusofCO Appowder. PreparationofchemicallypureCO Appowderispossible,however,CO Ap
3 3 3
powderbeginstodecomposeataround400◦C.Thus,CO Apblocksorgranulescannotbefabricated
3
bysinteringwithoutthermaldecompositionorthereleaseofCO . ThisledtotheinventionofHAp
2
andβ-TCP,whichdonotcontaincarbonateand,thus,canbesintered.
SinteredHApdemonstratedexcellenttissueresponseandgoodosteoconductivity. Therefore,
productionofsinteredHApprovesusefulforpatients. However,osteoconductivityofsinteredHApis
poorwhencomparedtothatofautografts. Moreover,incontrasttoautografts,whicharereplacedby
newbonethroughaboneremodelingprocess,sinteredHApwouldnotbereplacedbynewboneand
remainsatthebonedefect. IncontrasttoHAp,β-TCPisresorbedatthebonedefectand,thus,could
bereplacedbynewbone. However,thedissolutionrateofβ-TCPissometimesfasterthantherateof
boneformation,leadingtounsatisfactoryboneformation,orabonedefectfilledwithfibroustissue.
Autograft still remains the gold standard for reconstruction of bone defects for most of the
countries,eventhoughthereareseriousdrawbacks,includinginterventionathealthysitestocollect
autografts. SinceautograftexhibitspropertiessuperiortothoseofHApandβ-TCP,itisnaturalto
developartificialbonesubstituteswithcompositionssimilartothoseofboneorCO Ap.
3
Recently, CO Ap block was fabricated in aqueous solution via a dissolution–precipitation
3
reaction,usingaprecursorblock[12–24].CO Apblockupregulateddifferentiationofosteoblasticcells,
3
evenwhencomparedtoHap[25]. Further,CO ApshowedhigherosteoconductivitythanHap[12].
3
CO Apblockwasresorbedbyosteoclastssimilartobone. Thus,similartoautografts,CO Apblock
3 3
canbereplacedbynewbone[12].
Fortunately,chemicallypureCO Apgranules(Cytrans®)havebecomecommerciallyavailable.
3
SinceCO Apblockisnotfabricatedviasinteringbutthroughadissolution–precipitationreaction
3
inaqueoussolutionusingacalciteblock,thephysicalandhistologicalbehaviorsofCO Apmaybe
3
expectedtobedifferentfromthoseofsinteredHApandsinteredβ-TCP.However,nocomparisonhas
beenmadebetweenthecommerciallyavailablebonesubstitutes.
In the present study, physical and histological behaviors of artificial bone substitutes
were compared using three commercially available artificial bone substitutes, Neobone® (HAp),
Cytrans®(CO Ap),andCerasorb®(β-TCP).
3
2. MaterialsandMethods
2.1. ArtificialBoneSubstitutes
Neobone®(HAp,CoorsTec,Tokyo,Japan),Cytrans®(CO Ap,GC,Tokyo,Japan),andCerasorb®
3
(β-TCP, Hakuho, Tokyo, Japan), three artificial bone substitutes commercially available in Japan,
werechosenforthisstudy.
2.2. ScanningElectronMicroscopicObservation
Surfacemorphologyofthethreeartificialbonesubstituteswereobservedusingascanningelectron
microscope(SEM:S-3400N;HitachiHighTechnologiesCo.,Tokyo,Japan)underanacceleratingvoltage
of15kV,afterbeingcoatedwithgold–palladium.Coatingwasperformedusingamagnetronsputtering
machine(MSP-1S:VacuumDeviceCo.,Ibaraki,Japan).
2.3. CompositionalAnalysis
Compositions of the three artificial bone substitutes were analyzed using a powder X-ray
diffractometer,aFouriertransforminfrared(FT-IR)spectrometer,andelementalanalysis.
For X-ray diffraction (XRD) analysis, the samples were ground to fine powder, and the XRD
patternswererecordedusingadiffractometer(D8Advance,BrukerAXSGmbH,Karlsruhe,Germany),
generatingCuKαradiationat40kVand40mA.Sampleswerescannedfrom2θof10◦ to40◦ (whereθ
Materials2018,11,1993 3of12
istheBraggangle)inacontinuousmode. CrystallitesizewasalsocalculatedfromtheXRDpattern,
usingScherrer’sequation.
ForFT-IRanalysis,FT-IRspectraweremeasuredusingaKBrdiscmethod,withaspectrometer
(SPECTRUM2000LX;PerkinElmerCo. Ltd.,Kanagawa,Japan).
Carbonate content was measured using elemental analysis or a CHN coder (MT-6; Yanako
AnalyticalInstruments,Kyoto,Japan).
2.4. SpecificSurfaceAreaMeasurement
SpecificsurfaceareaswereevaluatedwithamultipleBrunauer–Emmett–Teller(BET)method
which evaluates nitrogen adsorption–desorption isotherms at 77 K using a BELSORP-mini II
(MicrotracBEL,Osaka,Japan).
2.5. PorosityMeasurement
Porosityofthebonesubstituteswasevaluatedusingtheformula
V
p
Porosity(%) = ×100, (1)
V +V
p s
where V and V are the pore volume and the volume of the specimens, respectively. The pore
p s
volumeofthebonesubstituteswasmeasuredusingaspecificsurfaceareaanalyzer.Thevolumeofthe
specimenswasmeasuredbythepycnometermethod,whereethanolwasusedastheimmersionliquid.
2.6. DissolutionBehavior
Dissolution behavior of the three artificial bone substitutes were measured using a method
definedinJIST0330-3;Bioceramics—Part3: Testingmethodofmeasuringdissolutionrateofcalcium
phosphate ceramics. In short, the dissolution rate of calcium phosphate ceramics was measured
byimmersingin0.08mol/LpH5.50aceticacid–sodiumacetatesolution,and0.05mol/LpH7.30
tris(hydroxymethyl)aminomethane–HClbuffersolutionat25±3◦C.Sample(100±1mg)waslifted
inthesolutionusingathreadwithcontinuousstirring,andtheCaconcentrationinthesolutionwas
measuredusingaCaionelectrode(n=5).
Accordingtothetestingmethod,aCaion-selectivecombinationelectrode(6583-10C,Horiba,Ltd.,
Kyoto,Japan)connectedtoapH/mV-meter(TypeF-72,Horiba,Ltd.,Kyoto,Japan)wasemployedin
thisstudy.
2.7. AnimalExperiments
Two adult male hybrid dogs (weight; 3.0–3.5 kg) were purchased from Kitayama Rabesu
(Nagano,Japan).AllhybriddogswerehousedintheAnimalUnitofHamriCo.,Ltd. (Ibaragi,Japan)
AnimalprotocolswerereviewedandapprovedbytheHamriAnimalStudiesCommittee(13-H065).
The animals maintained in individual cages were provided with water and animal specific
pellet-typelaboratoryanimalfood.Afterpremedicationwithdiethyletherbyinhalation,theanimals
were anesthetized with ketamine (10 mg/kg) and xylazine (3 mg/kg) via intravenous injection.
Threemonthsfollowingtheextractionofpremolarsandmolars,abonedefectof3.6mmindiameter
and8mmindepthwasmadeinalveolarboneusinganAadvatwistdrill,ϕ3.6mm(GC).Thedefect
wasreconstructedwithNeobone®(HAp),Cerasorb®(β-TCP),andCytrans®(CO Ap). Theanimals
3
weresacrificedusingintravenouspentobarbital(120mg/kg),4and12weeksaftersampleimplantation.
2.8. HistologicalAnalysis
Thespecimenswithsurroundingtissuewerecarefullyharvestedfromthemandibularboneand
fixedin10%bufferedformalinfor3days,andthendehydratedbyimmersionina70%to100%ethanol
gradientwithimmersionineachgradefor3days. Finally,thebonesampleswereembeddedinmethyl
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methacrylate. Then,sectionswerepreparedforpathologicalanalysisusingamodifiedinterlocked
Hdeiearmbroungdg,s aSwwi(tEzxearlkatn,dH)a. mThbeu rnge,wG ebrmonaen ya)r.eTah ienn ,thaell sdeecfteicotn swwase rmeestaasiunreedd wuistihnVg ilIlmanaugeev Ja vGeor1ld.5n1e r
soafntwdaorbes (eUrvSe Ndautisoinngal aInlsigtihtuttmesi corfo Hsceoaplteh,( DBeMth6e0s0d0aB, ,MLDei,c aUSMAic).r o systems, Heerbrugg, Switzerland).
ThenewboneareainthedefectwasmeasuredusingImageJver1.51software(USNationalInstitutes
2.o9f. HSteaatilstthic,aBl eAthneaslydsai,s MD,USA).
2.9.FSotra tissttaictiasltAicnala lyasniaslysis, one-way factorial analysis of variance (ANOVA) and Fisher’s least
significant difference (LSD) post hoc test were performed using Kaleida Graph 4. Values are
For statistical analysis, one-way factorial analysis of variance (ANOVA) and Fisher’s least
expressed as means ± SD. A p < 0.05 value was considered to indicate statistically significant
significantdifference(LSD)posthoctestwereperformedusingKaleidaGraph4.Valuesareexpressed
differences.
asmeans±SD.Ap<0.05valuewasconsideredtoindicatestatisticallysignificantdifferences.
3. Results
3. Results
TTyyppiciacal lSSEEMM imimaaggeess oof fththee (a(a––cc) )NNeeoobboonnee®® (H(HAApp),) ,(d(d––f)f )CCyytrtarannss®® (C(COO3AAp)p, )a,nadn d(g(–gi–)i C)Cerearsaosrobr®b ®
3
(β-TCP) are summarized in Figure 1.
(β-TCP)aresummarizedinFigure1.

FiFgiugruer e1.1 T.yTpypiciacla slcsacnannnining geleelcetcrtoronn mmicircoroscsocoppyy imimaaggees soof fNNeeoobboonnee®® (H(HAApp) )(a(–ac–)c,) ,CCyytrtaranns®s ®(C(COO3A3Ap)p )
(d(–df–)f, )a,nadn dCCerearsaosrobr®b ®(β(-βT-CTPC)P ()g(–gi)–.i ).
Both Neobone® and Cerasorb® displayed a porous structure, where Cerasorb® had much smaller
pores compared to Neobone®. By contrast, Cytrans® was dense, and had no pores in the granules.
Higher magnification showed that both Neobone® and Cerasorb® had smooth surfaces typical for
sintering. By contrast, the surface of Cytrans® was rough and consisted of small precipitated crystals.
Materials2018,11,1993 5of12
BothNeobone®andCerasorb®displayedaporousstructure,whereCerasorb®hadmuchsmaller
porescomparedtoNeobone®. Bycontrast,Cytrans® wasdense,andhadnoporesinthegranules.
HighermagnificationshowedthatbothNeobone® andCerasorb® hadsmoothsurfacestypicalfor
sMinatteerirailns g20.1B8,y 11c,o xn FtOraRs Pt,EtEhRe RsEuVrIfEaWce ofCytrans®wasroughandconsistedofsmallprecipitatedcry5s otaf l1s2.
TThhee XXRRDD ppaatttteerrnnss ooff ((aa)) NNeeoobboonnee®® ((bb)) CCyyttrraannss®®,, aanndd ((cc)) CCeerraassoorrbb®® aarree ssuummmmaarriizzeedd iinn FFiigguurree 22..
TThhee XXRRDD ppaatttteerrnn ooff tthhee ssttaannddaarrdd ((dd)) HHAApp aanndd ((ee)) ββ--TTCCPP aarree aallssoo sshhoowwnn ttoo ffaacciilliittaattee ccoommppaarriissoonn..

FFiigguurree 22.. XX--rraayy ddiiffffrraaccttiioonn ppaatttteerrnnss oof f(a(a) )NNeeoobboonne®e ®(H(HAAp)p, )(,b()b C)yCtyratrnasn®s (®CO(C3OAp3A), p(c),) (Cc)erCaesorarsbo®r (bβ®-
(TβC-TPC), P(d),)( dst)asntdanardda rHdaHp,a apn,dan (de)( set)asntdanardda rβd-TβC-TPC. P.
Asshown,bothNeobone®andCytrans®showedtypicalapatiticpatternswithnootherpeaks,
As shown, both Neobone® and Cytrans® showed typical apatitic patterns with no other peaks,
whereasCerasorb®showedanXRDpatterntypicalofβ-TCP.PeaksoftheNeobone®andCerasorb®
whereas Cerasorb® showed an XRD pattern typical of β-TCP. Peaks of the Neobone® and Cerasorb®
were sharp, indicating high crystallinity, whereas those of Cytrans® were broad, indicating
were sharp, indicating high crystallinity, whereas those of Cytrans® were broad, indicating low
lowcrystallinity.
crystallinity.
TheFT-IRspectraof(a)Neobone®(b)Cytrans®,and(c)Cerasorb®aresummarizedinFigure3.
The FT-IR spectra of (a) Neobone® (b) Cytrans®, and (c) Cerasorb® are summarized in Figure 3.
TheFT-IRspectraofstandard(d)HApand(e)β-TCParealsoshowntofacilitatecomparison.
The FT-IR spectra of standard (d) HAp and (e) β-TCP are also shown to facilitate comparison.
Both Neobone® and Cytrans® showed typical apatitic patterns, similar to the XRD pattern.
Both Neobone® and Cytrans® showed typical apatitic patterns, similar to the XRD pattern. In
In Cytrans®, an absorption peak typical for CO was observed at 1450 cm−1, 878 cm−1,
Cytrans®, an absorption peak typical for CO3 was obser3ved at 1450 cm−1, 878 cm−1, and 871 cm−1 [26,27].
aNnod a8b7s1ocrmpt−io1n[ 2p6e,2a7k] .aNssoiganbesdo rtpo tiOoHn pweaaks aosbssiegrnveedd taot O3H573w camso−1b (sνesrOvHed) aatn3d5 76338c mcm−1−1 (ν(νsLOOHH))a. nBdy
6c3o8ntcrmas−t, 1n(oν aLbOsHor)p.tBioync poenatrka asts,crniboeadb tsoo rCpOti3o wnapse oakbsaesrcvreidb eidn NtoeoCbOo3new®.a Isnostbesaedrv, aebdsoinrpNtieoonb poenaek®s.
Ianssctreibaded, atob sOoHrp twioenrep oebaskesrvaesdcr aibt e3d57t3o cOmH−1 awnedr e63o8b csmer−v1.e Cdeartas3o5r7b3® csmho−w1eadn adn6 F3T8-IcRm p−a1t.teCrenr atysporicba®l
sohf oβw-TeCdPa.n FT-IRpatterntypicalofβ-TCP.
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FFiigguurree 33..F oFuoruireire-trr-atrnasnfosrfmormin frianrferdarsepde cstproescctoropsycsoppeyc trsapoecft(raa) Nofe o(bao) nNe®eo(HboAnpe)®, (b(H)CAypt)r,a n(bs®) (CCyOtr3aAnps)®,
((cC)OC3eAraps)o, r(bc)® C(eβr-aTsCoPrb),® ((dβ)-TstCanPd),a (rdd) Hstaapnd,aanrdd H(ea)pst, aanndda (red) βst-aTnCdPa.rd β-TCP.
Crystallite sizes were calculated using Scherrer’s equation, and specific surface area (SSA),
Crystallite sizes were calculated using Scherrer’s equation, and specific surface area (SSA),
carbonatecontent,bulkdensity,andporosityaresummarized(Table1). CrystallitesizesofNeobone®
carbonate content, bulk density, and porosity are summarized (Table 1). Crystallite sizes of Neobone®
(HAp)andCerasorb® (β-TCP)wereapproximately80nm,whereasthecrystallitesizeofCytrans®
(HAp) and Cerasorb® (β-TCP) were approximately 80 nm, whereas the crystallite size of Cytrans®
(CO Ap)wasapproximately30nm,whichwassmallerthanhalfofNeobone®andNeobone®. Specific
(CO33Ap) was approximately 30 nm, which was smaller than half of Neobone® and Neobone®. Specific
surfaceareawasdifferentbytwoordersofmagnitude. SSAofNeobone®(HAp)was1.0m2/g. Onthe
surface area was different by two orders of magnitude. SSA of Neobone® (HAp) was 1.0 m2/g. On the
otherhand,SSAofCerasorb® (β-TCP)wasoneorderofmagnitudesmallerthanNeobone® (HAp)
other hand, SSA of Cerasorb® (β-TCP) was one order of magnitude smaller than Neobone® (HAp)
andwas0.06m2/g. Bycontrast,SSAofCytrans®(CO Ap)wasoneorderofmagnitudelargerthan
and was 0.06 m2/g. By contrast, SSA of Cytrans® (CO33Ap) was one order of magnitude larger than
Neobone®(HAp)andwas18.2m2/g. NocarbonatewasfoundinNeobone®(HAp)andCerasorb®
Neobone® (HAp) and was 18.2 m2/g. No carbonate was found in Neobone® (HAp) and Cerasorb® (β-
(β-TCP),whereasCytrans®(CO Ap)had11.9wt%ofcarbonatecontent. Cytrans®(CO Ap)hadthe
TCP), whereas Cytrans® (CO3A3p) had 11.9 wt % of carbonate content. Cytrans® (CO33Ap) had the
largestbulkdensityandNeobone®(HAp)hadthesmallestbulkdensity.
largest bulk density and Neobone® (HAp) had the smallest bulk density.
Table1.PhysicalpropertiesofNeobone®(HAp),Cytrans®(CO Ap),andCerasorb®(β-TCP).
Table 1. Physical properties of Neobone® (HAp), Cytrans® (CO33Ap), and Cerasorb® (β-TCP).
BBonoen eSuSbusbtistutitteu te CryCstrayllsitt(aenl Slmiitz)ee S(nizme) SS(AmS S3(m/Ag3)/g) CCOO33 C(C%oon)ntetennt (t%) BBuul(lkgk/ DcDmeenn3)ssiittyy (g/cm3P) o(r%oPs)oi(rt%oys)i ty
NNeeoobobnoen®e (®H(AHpA) p) 7755.4.4 ±± 0.90 .9 11..00 -- 0.47±0.407. ±0 20.02 85.1±85.01. ±5 0.5
CCyytrtarnans®s ®(C(OC3OApA) p) 3300.8.8 ±± 0.80 .8 1188..22 111.19.9 0.99±0.909. ±0 30.03 68.7±68.07. ±9 0.9
3
Cerasorb® (β-TCP) 78.5 ± 7.5 0.06 - 0.72 ± 0.03 76.4 ± 0.8
Cerasorb®(β-TCP) 78.5±7.5 0.06 - 0.72±0.03 76.4±0.8
As a result of bulk density and similar theoretical density, packing porosity was largest in
NeobAosnea® r(eHsuAlpt)o ffolblouwlkedd ebnys iCtyeraansodrbs®im (βil-aTrCtPh)e aonredt iCcayltrdanens®s i(tCy,Op3Aacpk)i.n g porosity was largest in
NeobTohnee ®di(sHsoAlupt)iofonl lboewheadviboyrsC oefr Naseoorbbo®n(eβ®- (THCAPp))a,n CdyCtryatnrsa®n (sC®O(C3AOp3)A, apn).d Cerasorb® (β-TCP) in pH
7.3 CTahPeOd4-isfrseoel ubtiuofnfebr eshoalvuitoiorsn,o farNee soubmonme®ar(izHeAd pi)n, CFyigtruarnes ®4. (CInO t3hAep n),eauntrdalC esoralusotirobn®, (sβim-TuClaPt)ining
ppHhy7s.i3olCogaPicOal4 c-fornedeibtiuofnfe, rCseoralusotirobn®, (aβr-eTsCuPm) smhaorwizeedd tihne Ffaigsuterset 4d.isIsnotluhteionne uratrtae lfosolllouwtieodn ,bsyi mCuytlaratinnsg®
p(ChOys3iAolpo)g aicnadl cNoenodbitoinoen®, C(HerAapso).r b®(β-TCP)showedthefastestdissolutionratefollowedbyCytrans®
(CO Ap)andNeobone®(HAp).
3
MMataetreirailasls2 2001188,1, 111,,1 x9 9F3OR PEER REVIEW 77o off1 122
Materials 2018, 11, x FOR PEER REVIEW 7 of 12

FFigiguurere4 .4.D Disissosolulutitoionnb ebhehavavioiorro foNf Neoeboobnoen®e®( H(HAApp),)C, Cytyrtarnasn®s®( C(COOA3App),),a annddC Cerearsaosorbrb®®( β(β-T-TCCPP))i nin
3
CFCaigaPPuOOre4- -f4frr.e eeDep ipsHsHo7 l7u.3.3tib obunuf ffbefererhs soaovluliuotitroi onon.f. Neobone® (HAp), Cytrans® (CO3Ap), and Cerasorb® (β-TCP) in
4
CaPO4-free pH 7.3 buffer solution.
TThheed disisssoolluuttiioonn bbeehhaavviioorrss ooff NNeeoobboonnee®® ((HHAApp)), ,CCyyttrraannss®® (C(COO3A3Ap)p, )a,nadn dCeCrearsaosrobr®b (®β-(TβC-TPC) iPn) pinH
p5H.5 5b.T5uhfbfeue rdf fisesorslosuoltuliuotintoi onan rbeae hrseuamvsuimomrasmr oiazfr eNidzee iodnb iFoningFeui®gr (euH r5eA. 5Ipn.) I,t nChetyh twreaewnasek®a ak(CcaiOdcii3cdA iscpo)sl,uo altnuiodtni oC, nse,irmsaismuolruabtlia®n t(igβn -tgThtCeh Pien)is niinds iepd Hoef
o5tf.h5te hb reuufrffufelfrefl sebooblurodtriedorne or afor ofe sostusetomecomlcalsaatrssit,z seC,dyC tiyrnat rFnaisng®su ®(rCe(O C53.O AIn3pA )t hpse)h oswhweoaewdk e atdhcietdh ifeca ssftaoeslsutte tsdiotinsds,i osslsiumotliuuoltnaio trinnatgrea ttfheoelf loionlwlsoiedwdee bodyf
btChyeeCr raeusrofafrslbeo ®rb bo(®βrd-(TβeCr- TPoC)f Poa)sntadeno dcNlNaesoetbso,ob nCoeny®te r®a(Hn(HsA®A p()Cp, )O,in3iAnstspeta)e dasd hooofwf CCedee rrtaahsseoo rrfbab®s® te((βsβt-- TTdCCisPPs)o),,l uwwtihhoiincc hhr asstheho ofwwoleeldodw fafeasdst etbesystt
dCdiseissrsoaosluloutritboio®n n(a βat-tp TpHCHP7 7).3 .3.a.n d Neobone® (HAp), instead of Cerasorb® (β-TCP), which showed fastest
dissolution at pH 7.3.

FFigiguurere5 5..D Disisssoolulutitoionnb behehavavioiroro foNf Neoeboobnoen®e®( H(HAApp),)C, Cytyrtarnasn®s®( C(COO3A3App),),a annddC Cerearsaosorbr®b®( β(β--TTCCPP))i ninp pHH
5F5.5i.g5bu burufeff ef5er. rDs osiolsulsutoitloiuontn.io. n behavior of Neobone® (HAp), Cytrans® (CO3Ap), and Cerasorb® (β-TCP) in pH
5.5 buffer solution.
TheVillanuevaGoldner-stainedhistologicalimagesofthethreeartificialbonesubstitutes,4weeks
The Villanueva Goldner-stained histological images of the three artificial bone substitutes, 4
and12weeksafterimplantation,areshowninFigures6and7.
weekTsh aen Vd i1ll2a nwueeevkas Gafoteldr nimerp-slatanitnaetido nh,i satroel oshgoicwaln i imn aFgiegsu roefs t6h ea ntdh r7e.e artificial bone substitutes, 4
weeks and 12 weeks after implantation, are shown in Figures 6 and 7.
MMateartiearlisal2s0 21081,81,1 1,11, 9x9 F3OR PEER REVIEW 8 8ofo 1f21 2
Materials 2018, 11, x FOR PEER REVIEW 8 of 12

Figure 6. Histological findings of Neobone® (HAp), Cytrans® (CO3Ap), and Cerasorb® (β-TCP)
FiFigmiugpurelraen6 .t6e.d H Hiisnisttotool oldoggoiicgca alml fiafninndddiiinbngugslsa oro fbf NoNneeeoo bdboeonfneeec®®t (a(HHt AA4 ppw)),,e CeCkyystt rraaafnntessr®® i((mCCOpOl3a3AnAptpa),t) i,oaannn d(d VCCileleraranasuoseorvbrba® ®G(β(o-βTld-CTnPCe)rP )
imismptalpianlnaitnnegtde)d.i n Gitnroetdeono d gaomrge aam,n bdaoinbndueil;ba rurelbdaor a nrbeeodan,e eof esdctteeofaeitdc4t. wAater er4ok wswasef etienkrdsi imcaafptteela ra nlitimagtnpioeladnn (ctVautiblilooanind ua(Vel voilaslatGenooubledlvanaset rsG saotralodinuninendrg ).
Gsrttheaeein naiabnrugen)a.d, Gbaonrentleeyn; fraoerrdemaa,er edba oo,nosetse;t oeroeiddid oa.nrAe trahr,e oo wssutseriofnaiddcei. c Aoaftr ertohaweli gnsn eiewndd biccuoanbteeo .i adElaiBgl,no eesxtdies octibunlbagos bitdsoaanlre oo; usNtneBdo,b tnhlaeeswats bb uaornnodeua;n nOdt,l y
fotorhmset eeaodbiudon;s Hdtea,o nHitdlAyo pfno (rtNhmeeeosdbu oornfsaetec®oe);i odCf ,o tCnhO eth3nAee pwsu (rbCfoaynctreea .onEfs B®th),;e eT xn,i esβwt-iTn bCgoPbn o(eCn. eEer;BaN,s oeBxr,bisn®te)i.nw g bboonnee;; ON,Bo, snteeowid b;oHne,;H OA, p
(Noestoebooidn;e H®),; HCA,Cp O(N3Aeopb(oCnyet®r)a; nCs, ®C)O;T3A,βp- T(CCyPtr(aCnesr®a);s oTr, bβ®-T).CP (Cerasorb®).

FiFgiugruere7 .7H. Hisitsotloolgogiciaclalfi fninddininggsso offN Neeoobboonnee®® ((HHAApp)),, CCyyttrraannss®® ((CCOO33AApp)),, aanndd CCeerraassoorrbb®® imimpplalnantetded inintot o
doFdgioggmu mraena 7dn. idHbiubislutaolralrob gboioncneaeld fdeinefefdecictntag atst 1 o12f2 wNweeeoeekbksos naaefftt®ee (rrH iimmApppll)aa, nnCttyaatttriioaonnn s((®VV (iiCllllaOann3uAueepvv)a,a aGGnodold lCdnneererar ssstoatraibnin®in iinmgg)p.) G.laGrnerteeened na irnaetraoe, a ,
bodbnooegn; emr;e ardendad riaebraue,laao, rso tbseotoeniodei d.dE.e EfBeB,c,et exaxits i1st2itni wnggeb ebokonsn eae;f;Nt NerBB i,m,n npeewlwan bbtoaontnieoe;n; OO (V,, oiolsslatteenoouiieddv;; aHH G,, oHHldAAnppe r((N Nsteeaooinbbioonnngee)®.® )G;) ;CrCe,e ,CnCO aOr3Ae3aAp, p
(Cb(yCotnyreatr;n arsne®sd)® ;)a;Tr T,eaβ, ,β- oT-TsCtCePoP(i Cd(C.e eErraBas,so eorxrbbi®s®t))i..n g bone; NB, new bone; O, osteoid; H, HAp (Neobone®); C, CO3Ap
(Cytrans®); T, β-TCP (Cerasorb®).
Materials2018,11,1993 9of12
Materials 2018, 11, x FOR PEER REVIEW 9 of 12
At 4 weeks after implantation, new bone formation was observed from existing bone to the
At 4 weeks after implantation, new bone formation was observed from existing bone to the
center of the bone defect in the images of Neobone® (HAp), Cytrans® (CO Ap), and Cerasorb®
3
(βce-nTtCerP )o(fF tihgeu rbeon6)e. dNeefwectb ionn ethwe aimsfaogrems eodf oNneothbeonsue®r f(aHceAspa)n, dCiyntsraidnes®t h(eCOpo3Areps)o, fanCde rCaseorarbso®rb(β® -(TβC-P)
TCP) (Figure 6). New bone was formed on the surfaces and inside the pores of Cerasorb® (β-TCP)
(Figure6f). Inthecentralareaofthedefect,fibroussofttissuehadinfiltratedintotheinterconnected
(Figure 6f). In the central area of the defect, fibrous soft tissue had infiltrated into the interconnected
porousstructureofNeobone®(HAp)andCerasorb®(β-TCP).ThestructureofCerasorb®(β-TCP)was
porous structure of Neobone® (HAp) and Cerasorb® (β-TCP). The structure of Cerasorb® (β-TCP) was
apparentlyresorbedandbecameobscure. ThenewboneinNeobone® (HAp)andCerasorb® (β-TCP)
apparently resorbed and became obscure. The new bone in Neobone® (HAp) and Cerasorb® (β-TCP)
(Figure6d,f)wasonlyobservedintheneighborhoodofexistingbone(Figure6a). Ontheotherhand,
(Figure 6d,f) was only observed in the neighborhood of existing bone (Figure 6a). On the other hand,
newboneformationinCytrans® (CO Ap)hadreachedmorecentralareasofthedefectscompared
new bone formation in Cytrans® (CO3A3p) had reached more central areas of the defects compared to
tothoseinNeobone®(HAp)andCerasorb®(β-TCP)(Figure6b). NewboneandCytrans®(CO Ap)
those in Neobone® (HAp) and Cerasorb® (β-TCP) (Figure 6b). New bone and Cytrans® (CO3A3p)
granuleswereindirectcontactwitheachotherwithoutanyintermediatefibroustissueinbetween
granules were in direct contact with each other without any intermediate fibrous tissue in between
(Figure 6e). Aligned cuboidal osteoblasts (arrows) were observed around the abundantly formed
(Figure 6e). Aligned cuboidal osteoblasts (arrows) were observed around the abundantly formed
osteoidonthesurfaceofthenewbone,suggestingactiveboneformationbyosteoblasts(Figure6e).
osteoid on the surface of the new bone, suggesting active bone formation by osteoblasts (Figure 6e).
SSlilgighhttc chhrroonnicici innflflaammmmaattoorryy cceellll iinnfifillttrraattiioonn-- ccoommppoosseeddl ylymmpphhooccyytteessa annddp pllaassmmaa cceellllss wwaass oobbsseerrvveedd in
tihnr ethermeea tmeraitaelrsia(Flsi g(Fuirgeu6rde– 6fd),–afn),d anthde thster osntrgoensgteisntfl ianmflammamtioantiowna wsraesc roegcnoigzneidzeind Nine Nobeoonbeo®ne(®H (AHpA)pa)n d
tahnedw theae kweesatkinesCt iynt rCayntsr®an(sC®O (C3OA3pA).pM). Moroereg rgarnanuulalatitoionnt tisisssuuee wwaass aallssoo oobbsseerrvveedd inin NNeoeboobnoen®e (®H(AHpA) p)
tthhaannC Ceerraassoorrbb®® ((ββ--TTCCPP)) aanndd CCyyttrraannss®® (C(COO3A3Ap)p. ).
AAtt 1122 wweeeekkss aafftteerr iimmppllaannttaattiioonn,, oosstteeooidid, ,wwhhicihch wwasa sobosbesrevrevde data t4 4wweeekesk sini nthteh eimiamgaeg oef of
NNeeoobboonnee®® ((HHAApp)),, CCyyttrraannss®® ((CCOO3A3App),) ,aanndd CCeerarsaosrobr®b ®(β(-βT-CTPC)P, )w,awsa gsrgardaudaullayl lryeprelapcleadce bdyb myamtuartue re
bboonnee.. TThhee aammoouunntt ooff nneeww bboonnee inin CCeerarasosorbrb® ®(β(-βT-CTPC) Pa)nadn CdyCtryatnrsa®n (sC®O(C3AOp3)A wpa)sw laarsgelar rtghearnt thhaonset hino se
inNeNoeboobnoen® e(H®A(HpA) (pF)ig(uFrieg u7dre–f7)d. T–hf)e. gTrhaenuglraatniounl atitsiosunet siseseune ast e4e wneaetk4s wwaese krespwlaacsedre bpyl aficberdoubsy tfiisbsuroeu s
taisrsouuenadr NouenodboNnee®o b(HonAep®),( CHyAtrpa)n,sC® y(tCrOan3As®p)(,C aOnd3A Cpe)r,aasnodrbC® e(βra-TsoCrPb)®, a(nβd-T thCeP i)n,falnamdmthaetoinryfl armeamctiaotno ry
rheaadct iobnechoamde bselcigohmteers laigt h1t2e rwatee1k2sw ceoemkspacroemd ptaor etdhatot taht at4 awt4eewkes.e kTsh.eTrhefeorreef,o rael,l aslhloshwoewd egdogoodo d
bbioioccoommppaattibibiilliittyya sase veivdiednecnecdebdy beyx treexmtreelmyesllyig hsltiignhflt aimnfmlaamtomryatroerayc tiroena.ctTiohne.s iTzheeo fsCizyet roafn sC®y(tCraOnsA® p)
3
g(rCaOnu3Aleps) agtr1a2nuwleese kast 1w2a wsesemkasl wlearst hsamnatllheart tahtan4 wtheaet kast. 4 weeks.
TThheea ammoouunntto offb boonnee aatt tthhee bboonnee ddeeffeecctt 44 aanndd 1122 wweeeekkss aafftteerr iimmppllaannttaattiioonn iiss ssuummmmaarriizzeedd ((FFiigguurree 8).
A8s).i nAdsi cinadteidcaitnedF iignu Freigsu6raens d6 7a,ntdh e7l,a trhgee sltarbgoenset abmonoeu anmtwouasnto bwsaesr voebdseartvbeodt hat4 bwoethek 4s wanedek1s2 awnede k1s2 in
twheeedkesfe icnt sthreec doenfsetcrtusc rteedcownsitthruCctyetdra wnsit®h (CCyOtraAnps)®. (LCaOrg3Aerpa)m. Loaurngetsr oafmboounnetws oefr eboobnsee wrveerde aotb1se2rwveede ks
3
at 12 weeks compared to those at 4 weeks, regardless of the type of bone substitute.
comparedtothoseat4weeks,regardlessofthetypeofbonesubstitute.

Figure8.Amountofnewboneformedinbonedefectareaat4weeksand12weeksafterimplantation.
Figure 8. Amount of new bone formed in bone defect area at 4 weeks and 12 weeks after implantation.
(n=2).
(n = 2).
Materials2018,11,1993 10of12
4. Discussion
In this study, three commercially available bone substitutes with different composition,
HAp (Neobone®), CO Ap (Cytrans®), and β-TCP (Cerasorb®), were compared with respect to
3
their physical character and tissue response. Sintered HAp (Neobone®), and sintered β-TCP
(Cerasorb®)showedphysicalpropertiestypicalofceramicsfabricatedbysintering. Crystallitesize
waslargeandspecificsurfaceareaswerelimited.WhenHAp(Neobone®)wascomparedwithβ-TCP
(Cerasorb®), β-TCP (Cerasorb®) had a much smaller SSA of 0.06 m2/g than the 1.0 m2/g of HAp
(Neobone®).Also,β-TCP(Cerasorb®)hadsmallerporosity,76.4±0.8%,comparedtoHAp(Neobone®),
85.1±0.5%.
These differences appear to be reasonable for obtaining proper dissolution behavior under
physiologicalandosteoclasticcondition. β-TCPisknowntohavemuchhighersolubilitythanHAp,
whichwasalsoconfirmedinthecurrentstudyusingcommerciallyavailableβ-TCP(Cerasorb®)and
HAp(Neobone®). Althoughthehighersolubilityofβ-TCPisthekeyreasonwhyβ-TCPisreplaced
bynewbone,β-TCPhasadegreeofsolubilitywhichistoohighforphysiologicalandosteoclastic
pH. Therefore, Cerasorb® (β-TCP) may be fabricated as it has very small SSA and small porosity,
whichminimizesthedissolutionrate.
CO Ap (Cytrans®) behaves differently when compared to HAp (Neobone®) and β-TCP
3
(Cerasorb®). It has a smaller crystallite size and a much higher SSA value. This is thought to be
causedbythefabricationmethod,whichisviaadissolution–precipitationreactionusingCaCO block
3
asaprecursorinaqueoussolution. Thus,crystallinityislowandcrystallitesizecalculatedfromXRD
patternissmaller(30.8±0.8nm)whencomparedtoHAp(Neobone®)orβ-TCP(Cerasorb®)with
crystallitesizesof75.4±0.9nmand78.5±7.5nm,respectively.
Dissolution of β-TCP (Cerasorb®) in pH 7.3 solutions was the fastest, even though it had the
smallest SSA (0.06 m2/g), followed by CO Ap (Cytrans®) that had the largest SSA (18.2 m2/g),
3
andHAp(Neobone®)thathadamediumSSA(1.0m2/g). Itshouldbenotedthatthedissolution
testwasdoneusingabuffersolutioncontainingnoCa-PO ,accordingtostandards. Bodyfluidwas
4
supersaturatedwithrespecttoapatite. Therefore,theleastdissolutionofHAp(Neobone®)andCO Ap
3
(Cytrans®)shouldbelowerthanthatseenintheseresults.
Interestingly,dissolutioninthepH5.5solutionwasthefastestinCO Ap(Cytrans®),followedby
3
β-TCP(Cerasorb®)andHAp(Neobone®).ThismayduetothereleaseofCO duringdissolutionin
2
pH5.5solutioninthecaseofCO Ap(Cytrans®). Althoughbehaviorofbonesubstituteatthebone
3
defectmaybemorecomplex,dissolutionbehaviorinpH5.5isthoughttoberelatedtothereplacement
bynewbone. BothCO Ap(Cytrans®)andβ-TCP(Cerasorb®)areknowntobereplacedbynewbone,
3
whereasHAp(Neobone®)isstableatthebonedefect,andwouldnotbereplacedwithbone.
Histologicalresults,summarizedinFigures6–8,revealedthatboneformationandresorptionof
bonesubstituteisdifferentaccordingtothetypeofbonesubstitute.InHAp(Neobone®),newbone
formation was limited in the neighborhood of existing bone 4 weeks after implantation, and the
amountofnewbonewasverysmall. At12weeks,theamountofbonewasalmostthesameasthat
at4weeks.
IncontrasttoHAp(Neobone®),muchlargerbonewasformedatboth4weeksand12weeks
inthecaseofCO Ap(Cytrans®). MostoftheCO Apgranuleswerepartiallyreplacedbynewbone
3 3
at 12 weeks. Cell-to-cell signaling and material-to-cell signaling may be the cause of larger new
boneformationinthecaseofCO Ap(Cytrans®).Inotherwords,osteoclastsactivatedbyresorption
3
mayreleaseclastokines,includingTRAcP,sphingosine-1-phosphate(S1P),BMP6,wingless-type10b
(Wnt10b),hepatocytegrowthfactor(HGF),collagentriplehelixrepeat-containingprotein1(CTHRC1),
where these factors activate preosteoblasts and osteoblasts [28,29]. For material-to-cell signaling,
Nagaietal. reportedthatCO Apupregulatesosteoblasticdifferentiationofthehumanbonemarrow
3
comparedtoHAp[12].
β-TCP(Cerasorb®)showedinterestingboneformation.At4weeks,almostnoboneformationwas
observed.Atthisstage,materialsremainedatthebonedefect.However,at12weeksafterimplantation,