The biologicalmechanisms that provide a rationale for bone grafting are osteoconduction,osteoinduction and osteogenesis.Osteoconduction occurs when the bone graftmaterialserves as a scaffold for new bone growth that is perpetuated by the native bone.Osteoinduction involves the stimulation of osteoprogenitor cells to differentiate into osteoblasts,which then begin new boneformation.Osteogenesisoccurswhenvitalosteoblastsoriginating from the bonegraftmaterialcontributetonew bonegrowthalongwiththebonegrowth generatedviatheothertwomechanisms[40].
In thermal decomposition, amorphous calcium phosphate (ACP) or calcium deficient HA,which is obtained under neutralor acid conditions,is used for preparation.In thephosphorylated process,controlby rigorousaddphosphoricacid and the correct potentialof hydrogen is used to synthesize pure TCP.Three polymorphs of TCP can be produced according to the sintering temperature: low-temperatureβ-TCP,andtwohigh-temperatureforms,α-TCP andά-TCP.The lastonehasno practicalusebecauseitonly existsattemperatures>1430℃ and reverts almost instantaneously to α-TCP upon cooling below the transition temperature.In contrast,β-TCP is stable at room temperature and transforms irreversibly to α-TCP at~1125℃,which can beretained during cooling to room temperature[41-45].Therefore,strictcontrolofthesinteringtemperatureisneeded.
Throughoutthe synthesis processes,each process to determine a valid sintering temperature.Asresults,avalid sintering temperatureofCaO andβ-TCP were95 0℃ and1050℃.
Bone graftmaterials such as β-TCP has been shown to conductosteoblastic
differentiation and proliferation of bone marrow mesenchymal stem cells [44]. Calcium phosphatematerialshavebeen used increasingly in thepast40 yearsas bone graftsubstitutes in the dentaland orthopedic fields.β-TCP powder was synthesizedbyusing naturalmaterialssuchaseggshells,cuttlefishboneandlyster shellhadalreadystudied.But,itisdifficulttoberecycledandtheseshellscontain massprotein.Contrary,abaloneshellswereeasy to recycled becauseofthe vast majority ofthem arediscarded asrubbish.And then,abaloneshellscontain very littleofprotein.Sothat,itiseasilysynthesistohighpurityofbiocompatiblesorce asCaCo3.
Inthisstudy,thesynthesisbehaviorwasdependentonthephosophoricacid,and thetemperatureforsynthesis.Theabaloneshellpowderwaseasily turnedtoCaO by sintering at 950℃ temperature. There are two major distinct phases of tricalcium phophatecrystals:α-TCP andβ-TCP.In spiteoftheirsimilarchemical composition,their differet crystallographic features result in different resorption patterns.Theα-TCP isobtainedbyheatingabove1170℃ andismoresolublethan β-TCP.In addition,β-TCP is more stable atroom temperature than α-TCP,it presentshighersolubilitythanHA and,consequently,canbedegradedfasterinthe body,allowingadesirablegradualreplacementbythenewlyformedbone[46-48].
Studiesconcerning β-TCP efficiency asabonegraftwerealready conducted.In vivostudieswith β-TCP implanted in theratfemoralcondylewereconducted by Kondo,et al.,are repoted that β-TCP has a good biocompatibility,sinc both bioresorption and boneformation started atan early phaseafterimplantation [49]. Furthermore, shiratori, et al., are report that also β-TCP an osteoconductive biomaterial, based on the histological and molecular fidings of bone tissue withdrawn from bone defects in rat femurs previously implanted with β-TCP
granules[50,51].In thiscontext,thepresentwork showed thesynthesisβ-TCP from naturalmaterialsuch asabaloneshell.A biomaterialaiming toassociatethe osteoconductivityofβ-TCP.
However,before performing in vivo studies on the impactofsuch materialin bonetissuetherapy,an in vitroevalution ofcytocompatibility should beconduted.
TheFT-IR spectraexhibitthecharacteristicbandsofCaO,CaCO3andDCP andβ -TCP,attesting theaccuracy ofthebiomaterialsynthesis[52-54].Lima,etal.,are reportthat,assessed thenumberofbiablebalb/c3T3 fibroblastsafterexposureto severalmetal-modifiedapatiteextractsfor24hrandconcludedthecellsrespondto themetalthatsubstitutesCa orphosphateionsin thecrystallatticeofHA.For thatreson,solublebiomaterialssuch asmetallicion-substituted calcium phospates mustbeevaluatedin termsoftheircytocompatibility beforeclinicaluse.TheCaO, CaCO3,DCP and β-TCP synthesized from abalone shellofanalysis thestructure andmorphology measuredby SEM andXRD todetermine.TheMTT assay result shows that β-TCP are cytocompatible. Concerning the MTT assay result, considerable celldensity was observed in the cells cultivated with effluents of controlβ-TCP andabaloneshellβ-TCP.
Comparisonofcellsurvival culturedincontrolβ-TCP andabaloneshellβ-TCP medium confirmed by DAPIstaining and CellLive& Dead assay.Assessmentof cellsurvivalin thesamplesused MG-63and hNOKscells.DAPIisafluorescent stain thatbinds strong to A-T rich regions in DNA.Itis used extensively in fluorescencemicroscopy.DAPIcan passthrough an intactcellmembranetherfore itcan be used to stain both live and fixed cells,though itpassed through the membtanelessefficientlyinlivecellsandthereforetheeffectivenessofthestainis
lower.As theresult,a majority ofMG-63 and hNOKscells wereobserved with blue-fluorescent.TheLive& Deadassay stain solutionisamixtureoftwohighly fluorescentdyesthatdifferentiallylabelliveanddeadcells.Thelivecelldyelabels intact,viable cells green.The dye is membrane permeantand non-fluorescent untilubiquitousintracellularesterasesremovetheestergroupsand renderthe molecule fluorescent.The dead celldye labels the cells with a compromised plasma membrane red.This is membrane-mpermeantand binds to DNA with highaffinity.
As shown in Fig.12a,when cells die,their nucleisize decreases,which is shownasahigherintensityofDAPIfrom deadcells.Viablecellshaveveryround andclearnuclei,whereasdeadcellshavesmaller,condensed,cutorchoppednuclei. Thecontrolβ-TCP andabaloneshellβ-TCP werethesameasthecontrolgroup.
Thelive& deadviability/cytotoxicityassayprovidedatwo-colorfluorescencecell viabilityassaybasedonthesimultaneousdeterminationofliveanddeadcellswith two probes thatmeasure the recognized parameters ofcellviability intracellular esteraseactivityandplasmamembraneintegrity.AsshowninFig.12b,theMG-63 and hNOKscellsin each groupexhibited high cellviability and survival.Mostof thecellswerealivewithalmostnodeadcellsobserved.
Atpresent,many otherstudies have confirmed thatbiocompatible β-TCP was successfully synthesizedby using naturalmaterialssuch aseggshellandcuttlefish boneandoystershell[55,56].However,many studiesusedcomplex process,high levelconcentration and severalkind ofacid to reaction.In this study,the basic raw materialwas used naturalmaterialas abalone shellforsynthesized β-TCP.
Afterwards,the abalone shellpowder was easily turned to CaO By sintering
wasused assourceofphosphorforthesynthesisprocess.Therefore,themethod ofsynthesized β-TCP from abalone shellwas only using phosphoric acid and sintering process. As the FT-IR and XRD analysis results, the materials compositionandcrystallinewereconfirmed.
Finally,thebeta tricalcium phosphate(β-TCP)synthesized by abaloneshellare expectedtoabiocompatiblematerialandmassproductmaybepossibleinthelow costandsimpleprocess.