Bone Regenerative Medicine - Classic Options, Novel Strategies, and Future Directions

11/10/2016
Boneregenerativemedicine:classicoptions,novelstrategies,andfuturedirections
JOrthopSurgRes.20149:18.
PMCID:PMC3995444
Publishedonline2014Mar17.doi:10.1186/1749799X918
Boneregenerativemedicine:classicoptions,novelstrategies,andfuture
directions
AhmadOryan, 1SoodehAlidadi, 1AliMoshiri, 2,3andNicolaMaffulli4,5
1
DepartmentofPathology,SchoolofVeterinaryMedicine,ShirazUniversity,Shiraz71345,Iran
2
DivisionofSurgeryandRadiology,DepartmentofClinicalSciences,SchoolofVeterinaryMedicine,ShirazUniversity,Shiraz71345,Iran
3
DepartmentofTissueEngineeringandRegenerativeMedicine,ReproductiveBiotechnologyResearchCenter,AvicennaResearchInstitute,ACECR,
Tehran3197619751,Iran
4
DepartmentofMusculoskeletalDisorders,SchoolofMedicineandSurgery,UniversityofSalerno,Salerno84084,Italy
5
CentreforSportsandExerciseMedicine,QueenMaryUniversityofLondon,BartsandtheLondonSchoolofMedicineandDentistry,MileEndHospital,
275BancroftRoad,LondonE14DG,UK
Correspondingauthor.
AhmadOryan:oryan@shirazu.ac.irSoodehAlidadi:s_alidadi@shirazu.ac.irAliMoshiri:dr.ali.moshiri@gmail.comNicolaMaffulli:n.maffulli@qmul.ac.uk
Received2013Sep10Accepted2014Feb20.
Copyright2014Oryanetal.licenseeBioMedCentralLtd.
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Abstract
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Thisreviewanalyzestheliteratureofbonegraftsandintroducestissueengineeringasastrategyinthisfieldof
orthopedicsurgery.Weevaluatedarticlesconcerningbonegraftsanalyzedcharacteristics,advantages,and
limitationsofthegraftsandprovidedexplanationsaboutbonetissueengineeringtechnologies.Manybonegrafting
materialsareavailabletoenhancebonehealingandregeneration,fromboneautograftstograftsubstitutestheycan
beusedaloneorincombination.Autograftsarethegoldstandardforthispurpose,sincetheyprovideosteogenic
cells,osteoinductivegrowthfactors,andanosteoconductivescaffold,allessentialfornewbonegrowth.Autografts
carrythelimitationsofmorbidityattheharvestingsiteandlimitedavailability.Allograftsandxenograftscarrythe
riskofdiseasetransmissionandrejection.Tissueengineeringisanewanddevelopingoptionthathadbeen
introducedtoreducelimitationsofbonegraftsandimprovethehealingprocessesofthebonefracturesanddefects.
Thecombineduseofscaffolds,healingpromotingfactors,togetherwithgenetherapy,and,morerecently,three
dimensionalprintingoftissueengineeredconstructsmayopennewinsightsinthenearfuture.
Keywords:Bonegraft,Tissueengineering,Regenerativemedicine,Threedimensionalprinting,Orthopedic
research
Introduction
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Unlikeothertissues,thebonecanregenerateandrepairitself:inmanyinstances,boneinjuriesandfracturesheal
withoutscarformation[1,2].Nevertheless,inpathologicalfracturesorlargeandmassivebonedefects,bone
healingandrepairfail.Insufficientbloodsupply,infectionoftheboneorthesurroundingtissues,andsystemic
diseasescannegativelyinfluencebonehealing,resultingindelayedunionsornonunions[36].Boneisthesecond
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3995444/
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mostcommonlytransplantedtissueafterblood[2,7].Abonegraftisdefinedasanimplantedmaterialthatpromotes
bonehealingaloneorincombinationwithothermaterial(s)[7],throughosteogenesis,osteoinduction,and
osteoconduction[8],incombinationoralone.
Theselectionofanidealbonegraftreliesonseveralfactorssuchastissueviability,defectsize,graftsize,shapeand
volume,biomechanicalcharacteristics,grafthandling,cost,ethicalissues,biologicalcharacteristics,andassociated
complications[9].Thematerialsusedinbonegraftingcanbedividedintoseveralmajorcategories,including
autografts,allografts,andxenografts.Syntheticandbiologicallybased,tissueengineeredbiomaterialsand
combinationsofthesesubstitutesareotheroptions[10].Eachoftheseoptionshasadvantagesanddisadvantages.
Allograftsandxenograftshaveosteoinductiveandosteoconductivecharacteristicsbutlacktheosteogenic
propertiesofautografts[911].Autograftsarethegoldstandardinreconstructingsmallbonedefectsandhave
strongosteogeniccharacteristicsrelevanttobonehealing,modeling,andremodeling[12].Painanddonorsite
morbidityaswellasotherriskssuchasmajorvesselorvisceralinjuriesduringharvestingaresomeofthe
disadvantagesofautografts[13].Forthesereasons,severalalternativeoptionshavebeenintroducedandtested
[14,15].Allograftsareanalternativeoptionwithmajorlimitationsassociatedwithrejection,transmissionof
diseases,andcost.Allograftshavelowerincorporatingpropertieswiththehosthealingtissuesascomparedwith
autografts[13,16,17].Xenografts,inadditiontothedisadvantagesofallografts,carrytherisksoftransmissionof
zoonoticdiseases,andrejectionofthegraftismorelikelyandaggressive[17,18].Giventheseproblems,tissue
engineeringhasbeenintroducedinthelastdecade.Tissueengineeringinvolvesusingrelevantscaffolds,
introducingappropriategrowthfactorsandcells,and,morerecently,theuseofstemcells[17].Usingtissue
engineeringtechniques,itispossibletodesignnewscaffoldsandtissuegraftsaimingtodecreasethedisadvantages
oftraditionalgraftsandimprovegraftincorporation,osteogenicity,osteoconductivity,andosteoinductivity[10,17].
Tissueengineeringhaslimitations,includinguseofawidevarietyofmaterialsinproducingtissueengineeredgrafts
orscaffolds.Consequently,translationalinvestigationstestingeachmaterialarelimited,reducingtheirclinical
applicability.Therefore,someimportantaspectsofhostgraftinteractionandimmuneresponsetotheseimplants,
scaffolds,andviablegraftsarestillnotclear[17].Withadvancesoftissueengineering,theabilitytorepairor
regeneratebonetissueisdeveloping,anditsapplicationsareexpanding.Inthisreview,wediscusssomeofthe
availablescientificevidenceondifferenttypesofbonegraft,theircharacteristics,andtheiradvantagesand
disadvantages.Moreover,wehighlighttheapplicationoftissueengineeringtechniquestoovercomethelimitations
oftheavailablegraftsandtoimproveboneregeneration.
Structureandpropertiesofgraftsandbonesubstitutes
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Structureofbonegrafts
Thecorticalbonehashighermineralcontentsthanthetrabecularorcancellousbone[9].Inaddition,giventhe
presenceofspaceswithinthestructureofcancellousbone,thelatterismoreosteogenicthancorticalbone[2].The
compressivestiffnessandstrengthofthecorticalbonearemuchhigherthanthoseofthecancellousbone.In
selectingagraftorcombinationofgrafts,thesurgeonmustbeawareofthesefundamentaldifferencesinbony
structures[2,7].Bonegraftsmaybecortical,cancellous,orcorticocancellous(scanningultramicrographsof
differentbonegraftsarepresentedinFigure1)[2].
Figure1
SEMultramicrographsofmicrostructureofnaturalbonegrafts.(A)
Trabecularorcancellousbonegraft.Notetheporoushoneycomblike
microstructureofcancellousbonegraft.(B)Corticocancellousbonegraft.
(C)Corticalorcompactbonegraft(scalebars...
Corticalbonegraftsareusedmostlyforstructuralsupportandstrength,andcancellousbonegraftsforosteogenesis.
Structuralsupportandosteogenesismaybecombined,oneofthemostimportantadvantagesofusingcortico
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cancellousbonegrafts[9].Cancellousbonegraftsarecommonlyusedinfracturenonunion,dentaldefects,
maxillofacialdefects,spinalfusion,andothersmallbonedefects[19,20].Thesegraftslackmechanicalstrength,but
areeasytouse.Theporousstructureofcancellousbonegraftscanenhancebonyingrowthandimprovehealing,
allowingfasterrevascularization[19].
Corticalbonegraftsareappliedlessfrequently,andtheymaybeusedasonlaygrafts[21].Onlaybonegraftisused
toaugmentatrophicboneoutsidetheanatomicalboundariesoftheskeleton.Anexampleofanonlaygraftisthe
graftneededtoincreasetheatrophicalveolarbonewidthofafutureimplantsite.Thisthreedimensionalpositioning
ofthegrafthasamajorroleonthecourseofhealingandthusonthegraftsuccessandoutcomeofincorporation
[7].Whenagraftisusedtofillabonedefectwithintheconfinesoftheanatomicalskeleton,theterminlaygraftis
moreappropriate[16].Onlaygraftsundergoamorecomplicatedhealingcoursethaninlaygrafts[7].
Theresorptionrateoftheonlaygraftsishigherthantheinlaygraftsfortworeasons:(1)theonlaygraftsareless
exposedtotherecipientbonevasculature,whichresultsindecreasedboneremodeling(2)theonlaybonegraftsare
exposedtoforcesfromthesurroundingsofttissuesleadingtomoreosteoclasticresorptionintheareasexposedto
theseforces[7,16].
Propertiesofbonegrafts
Todecidewhichgraftismoreappropriateforagivencondition,understandingofthebiologicalpropertiesofeach
graftisnecessary.Anidealbonegraftmaterialshouldhaveosteogenesis,osteoinductivity,osteoconductivity,and
osseointegrationcharacteristics[2,8,22].
Osteogenesisisthecapacitytoproducenewbonebytheosteoblastsbydifferentiationofosteoprogenitorcells
eitherpresentintherecipientboneorcomingfromthegraftmaterial.Thispropertyismainlypresentinautogenous
graftsascomparedwithallograftsandxenografts,becausethecellularstructuresoftheallograftsandxenografts
havelowviabilityafterimplantation[23,24].
Osteoinductionisthecapabilityofthegraftmaterialstoinduceformationoftheboneformingcellsvia
differentiationofmultipotentmesenchymalstemcells(MSCs)ofthesurroundinghosttissuestoproduce
osteoprogenitorcellsfollowedbydevelopmentofosteoblasts.Suchabilityhasbeendiscoveredingrowthfactors
includingbonemorphogeneticproteins(BMPs)suchasBMP2andBMP7,transforminggrowthfactor(TGF
),fibroblastgrowthfactor(FGF),insulinlikegrowthfactor(IGF),andplateletderivedgrowthfactor(PDGF)
[2,8,22,24,25].
Osteoconductionisacharacteristicwherebythegraftactsasapermanentandresorbablescaffold,mechanically
supportingingrowthofvesselsandnewbonefromthebordersofthedefectintoandontoitssurfaces.This
characteristicinitiatesorinducesnewboneformation[8,22,24,25].Finally,osseointegrationistheabilitytobindto
thesurroundingbonewithoutaninterveninglayeroffibroustissue,allowingincorporationofthegraftatthehost
site[8].Allbonegraftsandbonegraftsubstitutematerialscanbedescribedbytheseprocesses[23].
Amongalltypesofbonegrafts,onlyautograftspossessalltheabovefeatures.Allograftsandxenograftsexhibit
onlytwoorthreeofthefourfeaturesofanidealbonegraftmaterial(osseointegration,osteoconduction,and
perhapsosteoinduction)andlackosteogenicproperties[6,8].
Incorporationofbonegrafts
Incorporationofbonegraftswithintherecipientbonebeddependsonfactorssuchasgraftrevascularization.Fast
incorporationandsuitablehealingofthegraftcanbeobtainedwithoptimalqualityandspeedofrevascularizationif
thereisadequateindependentvascularsupplytothedefectsite[7,13].Innonvascularizedgrafts,thebloodvessels
slowlypenetratefromtherecipientboneintothegraft,andthehealingtimeisthusprolonged[16,26].Incorporation
ofeachgraftdependsontheseproperties,whichcanvarybasedonthesource(auto,allo,orxenograft),and
structureofthegraft(cancellousorcorticalbone)[13,16].
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Regardlessofthesourceorstructure,alltransplantedbonegraftsproceedthroughfivestages:inflammation,
revascularization(capillarybudsinvadethegraft),osteoinduction(differentiationofmultipotentcellsinto
osteoblasts),osteoconduction(ingrowthintothegraftbymeansofthehost),andfinallyremodeling[13,21].The
durationofeachphasecanvarydependingonthecharacteristicsofthegraft.Interferencewithvascularization,
includinginfectionsandexcessivemicromotionatthereconstructedarea,willdelayincorporation[7,16].For
corticalgrafts,vascularizationisslowerandoccursalongHaversiancanals,whileitisperformedbycreeping
substitutionincancellousgrafts[13,16].Inthelatterprocess,thenewlyformedosteoblastslinethetrabeculaeto
formnewbonesimultaneoustoresorptionofbonebyosteoclastswhileincorticalbonegrafts,osteoclastic
resorptionisaprerequisitebeforeosteoblastscanproducenewbone.Inotherwords,fromtheperspectiveof
incorporation,themajordifferencebetweenthecancellousandcorticalautograftsisthatboneresorptionprecedes
boneformationinthelatter[13,16].
Duringthesecondandthirdstages,theimmunesystemoftherecipientbecomessensitivetothedonorantigenicity
[21].Incorporationofcancellousautograftsisfastestandmostcomplete,followedbycorticalautografts,cancellous
andcorticalallografts,andxenografts,respectively.Becauseallograftsandxenograftsarenotgeneticallymatched,
theycaninitiateanimmuneresponseintherecipient.Whenallograftsandxenograftsareused,therefore,itismore
likelythatthegraftwillfailandthedonortissueisrejected[13,16,21].Freshallograftsandxenograftsproduce
strongerimmunologicresponsesthanfreshfrozenorfreezedriedallograftsandxenografts[27].
Immuneresponseagainstbonegraftsandsubstitutes
Th1lymphocytesproduceproinflammatorycytokinessuchasinterleukin2(IL2),interferon(IFN),and
tumornecrosisfactor(TNF)leadingtomacrophageactivation,andcanbeassociatedwithpoortissue
remodelingandrejectionofbothalloandxenografttransplants[28,29].Ontheotherhand,Th2lymphocytes
produceIL4,IL5,IL6,andIL10cytokinesthatdonotactivatemacrophagesandareprobablyassociatedwith
graftincorporation[28,29].
MacrophagesarecharacterizedasM1orM2basedonreceptorexpression,function,andproductionofcytokines
[30].M1macrophagesproducelargeamountsofproinflammatorycytokinessuchasIL12andTNF,which
promoteinflammationandexpressCD68andCD80surfacemarkersinrats.Ontheotherhand,M2macrophages
producelargeamountsofIL10andTGF,inhibitthereleaseofproinflammatorycytokines,promoteconstructive
tissueremodeling,andexpressCD163surfacemarkersinrats[30,31].M2macrophagesinducetheTh2
lymphocyteresponsewhichisbeneficialfortissueremodeling[30].Thepresenceofcellularmaterialwithin
extracellularmatrix(ECM)ofscaffoldmodulatesthephenotypeofthemacrophagesandlymphocytesinvolvedin
therecipientimmunityresponseafterimplantationthiscanberelatedtotissueremodelingoutcomeintermsof
acceptanceorrejection[30,31].Indeed,acellulargraftelicitsM1macrophageandTh1lymphocyteresponseand
canresultinthedepositionofconnectivetissueandrejectionofthegraft.AnacellulargraftelicitsM2macrophage
andTh2lymphocyteresponse,leadingtomoreconstructivetissueremodelingoutcomeandacceptanceofthegraft
[30].
Typesofbonegrafts
Auto,allo,andxenograftsaswellasbonegraftsubstitutesareallusedtoimproveandenhancehealingofbone
defects(Figure2).Autograftshavelimitationsinpathologicfracturesandmassivebonedefectstherefore,other
typesofgraftshavebeenintroducedtoovercomethelimitationsofautograftsinsuchsituations[2,10,13].Allthe
availableandalternativeoptionshavelimitationsandmerits(Table1),andselectionofapropergraftor
combinationofthemdependsonthesurgeon'spreferenceandexperience.
Figure2
Typesofbonegrafts.(A)Autograft:Thesurgeonharvestsbonefromanothersiteofthe
patient'sskeleton,oftenfromtheiliaccrest,andimplantsitintothebonedefectsite.Thistype
ofbonegraftsleadstotwosurgeries,thus,twoscars,morepain,...
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Table1
Someadvantagesanddisadvantagesofthemostcommonlyusedthreetypes
ofbonegrafts
Autografts
Bonegraftsthatareharvestedfromonesiteandimplantedintoanothersitewithinthesameindividualaretermed
autografts,autologous,orautogenousbonegrafts[32].Theymaybecancellousorcortical(nonvascularizedor
vascularized)bone,andinsomeinstancesacombinationofboth,corticocancellousgrafts[2,33].Freshautografts
containsurvivingcellsandosteoinductiveproteinssuchasBMP2,BMP7,FGF,IGF,andPDGF[9,22].Froma
biologicalpointofview,theyarethebestmaterialavailable,sincetheytotallylackimmunogenicity.Theyretain
theirviabilityimmediatelyaftertransplantation,andthelackofimmunogenicityenhancesthechancesofgraft
incorporationintothehostsite[34].Furthermore,theosteogenic,osteoinductive,andosteoconductivepropertiesof
freshautograftsareoptimal,giventhepresenceofMSCs,osteoprogenitorcells,osteogeniccells,andgrowth
factors[24,35].Autograftshavenoassociatedriskofviraltransmissionmoreover,theyofferstructuralsupportto
implanteddevicesand,ultimately,becomemechanicallyefficientstructuresastheyareincorporatedintothe
surroundingbonethroughcreepingsubstitution[23].Themaindrawbackisthatautograftsmustbeharvestedfrom
anotherbodysite,whichimpliesadditionalsurgerywithahigherchanceofdonorsitepain,morbidity,and
complications[34].Ifmassivegraftingisneeded,adequateamountsofautograftmaynotbeavailable,andother
bonegraftmaterialshavetobeconsidered[18,21].
Varioussiteshavebeenusedforharvestingthegrafts'tissues.Graftscanbeobtainedfromtheiliacwingorcrest,
theproximalordistaltibiaandradius,theproximalhumerus,thedistalulna,ribs,calcaneus,andtheproximal
olecranon[3641].Thesesourceseachhaveadvantagesanddisadvantages(Table2).Amongthem,theiliaccrest
hasnotableadvantages,suchaseasyaccessandavailabilityofsufficientamountsofbothcorticalandcancellous
bones[37,38,42].Nevertheless,graftharvestingfromthissitecanproducenerve,arterial,andurethralinjury
pelvicfractureandpainatthedonorsite[37,41].Therefore,othersitessuchasthedistalradiushavebeenused
[43,44].Kitzingeretal.[44]comparedtheiliaccrestanddistalradiusasthesourcesofbonegraftin18patientsthe
distalradiuscanbeamoresuitablealternative,as,intheirsetting,itobviatedtheneedforgeneralanesthesia,
reducedthedurationoftheoperation,andinvolvedasmallersurgicalexposure.Thegreatestchanceforsuccessful
transplantationofliveboneisassociatedwithacancellousautograftorpedicled,vascularizedcorticalautograft
[16,26].Ingeneral,thesuccessofgraftingtheautogenicbonedependsonthesurvivalandproliferationofthe
osteogeniccells,conditionsattherecipientbed,typeofgraftchosen,handlingofthegraft,andshapingofthegraft
duringtheoperativeproceduretoadaptitintothehost'sbone[39].Whilefreshautologousgrafthasthecapability
ofsupportingnewbonegrowthbyallfourmeans(induction,genesis,conduction,andintegration),itmaynotbe
necessaryforabonegraftreplacementtoinherentlyhaveallfourpropertiesinordertobeclinicallyeffective.When
inductivemoleculesarelocallydeliveredonascaffold,themesenchymalstemcellsareultimatelyattractedtothe
siteandarecapableofreproduciblyinducingnewboneformation,providedthatminimalconcentrationanddose
thresholdsaremet[23].
Table2
Advantagesanddisadvantagesofsomesourcesofharvestingbonegrafts
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Allografts
Allograftsareharvestedfromoneindividualandimplantedintoanotherindividualofthesamespecies[13].Given
thelimitationsassociatedwithharvestingautografts,allograftshavebeenappliedclinicallyandexperimentallyasa
commonalternativetoautografts[45].Boneallograftsaredistributedthroughregionaltissuebanksandbymost
majororthopedicandspinalcompanies[23].
Allograftsareusedinbothmorselizedandstructuralforms[24]andareprovidedascortical,cancellous,orcortico
cancellousgrafts[22]andinvariousshapessuchaspowder,corticalchips,andcancellouscubes.Theyalsocanbe
processedasmineralizedordemineralized,fresh,freshfrozen,orfreezedriedforms[27,32].Allograftscanbe
obtainedfromcadaversorlivingdonors.Thecadavericformisavailableasacommercialproduct[32].Allografts
obtainedfromfreshcadaverswithpreservationoftheircellularandorganiccontentareminimallyprocessed[27].
Themajoradvantagesofallograftsaretheirreadyavailabilityinvariousshapesandsizes,avoidanceoftheneedto
sacrificehosttissues,andnochallengesofdonorsitemorbidity[21,23].Allograftshavevariableosteoinductiveand
osteoconductivepropertiesbutlackviablecellsand,therefore,havelowerosteogenicpotentialthanautografts[32].
Boneallograftscarrytheriskoftransmittingbacterialcontaminationandviraldiseases,suchasHIVandhepatitisB
andC,andalsotheymayinduceimmunologicalreactionsthatinterferewiththebonehealingprocessandcanlead
torejectionofthegraft[17,18,21,22].Inaddition,therateofhealing,usingallografts,isgenerallylowerthanthe
autografts.
Giventhehigherchanceofimmuneresponseinitiationandtheriskofdiseasetransmission,freshboneallografts
areseldomused.Withfrozenandfreezedriedallografts,theseconcernsarepartlyobviated[24],asthepotentialfor
immunereactionsrelatedtoallograftsisminimized,andthebiologicalandbiomechanicalpropertiesareonly
partiallyaffected[21,46].Still,thegraftsarenotfreeofcontroversy,particularlyregardingtheirassociationwiththe
transmissionofinfectiousagents.Sometissueprocessorsincorporatemethodsthatmaymarkedlyreducethisrisk.It
iscriticaltoknowyourtissuebankprovidertoensuretheirprocessingandpreservationmethodsinactivateviruses
butdonotnegativelyalterthebiomechanicalandbiochemicalpropertiesofthetissuesintendedforaparticular
clinicaluse[23].
Freshfrozenandfreezedriedboneallograftsinducemorepromptgraftvascularization,incorporation,andbone
regenerationthanfreshallograft[46].Freezedryingproducesasafergraftintermsofreducingtheriskof
immunologicresponsesinthedonorandtransmissionofviraldiseases.However,despitemodernsterilizationand
storagemethods,processingofallograftsusingfreezedryingtechniquesandtreatingthegraftbyhypotonic
solutions,acetone,ethyleneoxide,orgammairradiationwhichcaneliminatecellularandviralparticlesand
thereforereducetheriskofinfectiousandtransmissiblediseases[45],theuseofallograftsisnotcompletelysafe
[32,46].Theseprocessesmaydestroythebonecellsanddenatureproteinspresentinthegraftandalter
osteoconductiveandosteoinductivecharacteristics,essentiallyeliminatingtheosteogenicproperties[24].Therefore,
freezedriedallograftsincomparisontoautograftstakelongertobecomerevascularizedandincorporatedthan
autografts[21].Freezedryingprocedurealsoreducesthemechanicalstrengthofthegraft,andthecostofprocessed
andreadytouseallograftsishigh[46,47].Themineralcomponentoftheallogeneicbonecanberemovedby
demineralizationtoobtaindemineralizedbonematrix(DBM)whichhasosteoinductiveandpartlyosteoconductive
properties[32].DBMrevascularizesquickly,anditsbiologicalactivityisattributedtoproteinsandvariousgrowth
factorspresentintheextracellularmatrix[22].Giventhesemajordisadvantages,allograftsarenottheperfect
substituteforautograft.
Xenografts
Anotheralternativetoautogenousbonegraftsarexenografts,alsoknownasheterologousorxenogenicgrafts[48].
Xenograftsareharvestedfromoneindividualandtransplantedintoanotherindividualofadifferentspecies.The
commonavailablexenograftsarederivedfromcoral(Biocoral,naturalcoralBiocoralInc,Wilmington,New
Castle,DE,USA),porcine,andbovinesources[48,49].Xenogenousbonegraftsareatheoreticallyunlimited
supplyofavailablematerialiftheycouldbeprocessedtobesafefortransplantationinhumans[48].Amajor
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concernwithbovinederivedproductsisthepotentialtransmissionofzoonoticdiseasesandprioninfectionssuchas
bovinespongiformencephalitis(BSE)[17,49].Xenografts,similartoallograft,losetheirosteogenicandpartly
osteoinductivepropertiesduringtheprocessingtocounteracttheirantigenicpropertiesandpreventtransmissionof
infection.Xenograftsproducepoorclinicaloutcome[17]however,newinsightshavebeenpresented.
Severalstudieshavebeenconductedtotreatbonydefects,nonunions,andpathologicbonefracturesby
applicationofdifferenttypesofbonegrafts(Table3)[5053].Inmostofthem,autologousbonegraftshavebeen
suggestedasthegoldstandard,andothermethodsarecomparedwiththem[4,54],yieldingvariablelevelsof
effectivenesswhencomparedtoautogenousbonegrafts.
Table3
Comparisonofvariousbonegraftsinseveralexperimentalandclinical
studies
Keskinetal.[11]evaluatedtheeffectivenessofautologousbonemarrowonthehealingofulnarbonedefectsfilled
withbovinederivedxenograftsinrabbits.Thebonydefectintheulnaewasproducedbyexcisinga1cmlong
bonesegmentfromthe3cmproximalsegmentoftherightdistalradioulnarjoint.Thebonedefectsweretreated
simultaneouslywithbovinederivedxenograft,acombinationofxenograftandbonemarrowor,onthefifthday
followingthefillingofthesegment,withthexenograftandautogenousbonegraft.Theyconcludedthatwhen
xenograftswerecombinedwithautogenousredbonemarrow,thedrawbacksofxenograftswerecompensated
therefore,theirincorporationintotherecipientbedwassignificantlyenhanced.Inaddition,theyconcludedthatthe
spongyxenograftmayprovideasuitablemediumforosteogenesisbybonemarrowcells.Furthermore,someother
studieshavesuggestedthatbonemarrowinjectioncouldhavemorepromisingeffectswithnosignificant
complicationonbonehealingincomparisonwithbonegrafting[50,55].
Lubboc(LubbocOsteocellSA,Athens,Greece)isaxenogenousbasedpurifiedtrabecularbonematrixwhich
mainlycontainscollagentypeIandhydroxyapatite[12].Athanasiouetal.[12]comparedthehistologicalproperties
ofseveralwidelyusedbonegraftsubstitutes.Forthispurpose,theyproduceda4.5mmdiameterholeinthelateral
femoralcondyleofbothkneesof90rabbits,allocatedtosixexperimentalgroups.Thebonedefectswerefilledwith
variousgraftsincludingautograft,demineralizedbonematrixcrunchallograft(GraftonOsteotech,Inc.,
Eatontown,NJ,USA),bovinecancellousbonexenograft(Lubboc),calciumphosphatehydroxyapatitesubstitute
(CeraformTeknimed,VicenBigorre,France),calciumsulfatesubstitute(OsteosetSynthes,WestChester,PA,
USA),andnofilling(control).Theanimalswereeuthanizedat1,3,and6monthsafterimplantation,andthetissue
samplesfromtheimplantedsiteswerehistologicallyevaluated.Thehighesthistologicalgradeswereobtainedwith
theuseofcancellousboneautograft.Bovinexenograftwasthesecondbestinthehistologicalscalegrading.The
othersubstitutesweresimilarbutinferiortobothallograftandxenograft.
Recently,theeffectsofxenogenicbovinefetalDBM,commercialDBM,omentum,omentumcalffetalDBM,
corticalautograft,andxenogeniccartilagepowderonthehealingoftibialdefectsinadogmodelhasbeen
investigated[56].Overall,theomentumandomentumDBMgroupsweresuperiortothecontrolgroupbutinferior
totheautograft,commercialDBM,calffetalDBM,andcalffetalcartilagegroups.
Acellularizationofsoftandhardconnectivetissuessuchastendons,ligaments,andbonesreducesoreven
eliminatestheimmunogenicityassociatedwithallograftsandxenograftsand,therefore,maybeeffectivein
enhancingincorporationofthesegrafts[5760].Multiplephysical,chemical,andenzymaticmethodshavebeen
usedtoremovecytoplasmicandnuclearantigenswithpreservationoftheextracellularmatrixstructureand
maintenanceofmechanicalandfunctionalcharacteristics[58,60].Ionicdetergentssuchassodiumdodecylsulfate
(SDS)solubilizecellmembranesand,giventheirtendencytodenatureproteins,alsoimpairtissuestructure.Using
nonionicdetergents(suchasTritonX)resultsinpartialpreservationofthestructureoftheacellularizedtissue
[57,60].Zhangetal.[59]compareddifferentmethodssuchasNaCl+SDS,trypsin/EDTA,trypsin/EDTA+Triton
X100,TritonX100,TritonX100+SDS,andfreezingat70CfollowedbyusingTrypsin/EDTA+TritonX100
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toacellularizeintrasynovialflexortendonsofrabbit.Amongtheseagents,thebestresultsregardingacellularization
wereobtainedbyfreezingat70Cfollowedbytrypsin/EDTA+TritonX100.Moreover,Elderetal.[57]
evaluatedtheeffectsofvariousacellularizationtreatmentsontissueengineeredarticularcartilageconstructs,
includingcellularityandbiochemicalandbiomechanicalpropertiesaswellascollagencontent,usingatwophased
approachafter4weeksofculture.Inthefirstphase,fivetreatmentregimenswereassessed,including1%SDS,2%
SDS,2%TritonX100,2%tributylphosphate(TnBP),andhypotonicfollowedbyhypertonicsolutionwhichwere
usedforeither1or8h,andthena2hwashinPBS.InphaseII,thebesttreatmentfromphaseI(2%SDS)was
appliedfor1,2,4,6,or8h.Treatmentwith2%SDSfor1or2hsignificantlydecreasedtheDNAcontentofthe
tissuewhilemaintainingthebiochemicalandbiomechanicalproperties.Ontheotherhand,2%SDSfor6or8hled
tocompletehistologicalacellularization,withcompleteeliminationofcellnuclei,butsubstantialreductionofthe
compressiveproperties.Followingthisstudy,thetreatmentwith2%SDSfor1or2hwasthemosteffectiveand
promisingmethodforcartilageacellularization.Indeed,itresultedincompleteacellularizationwhilemaintaining
thephysiologicalfunctionalproperties.Finally,Vavkenetal.[58]comparedtheeffectivenessofTritonX,trypsin,
andSDSinacellularizationoftheporcineanteriorcruciateligament(ACL)andshowedthatTritonXisthemost
effectivereagentforacellularizationoftheporcineACL.
Bonetissueengineering
Tissueengineeringisthefinaloptioninmanagingboneloss.Tissueengineeringcaninvolvetheuseofscaffolds,
healingpromotivefactors(e.g.,growthfactors),andstemcells[17].Tissueengineeringisdefinedasaprocessthat
affectsthestructureandarchitectureofanyviableandnonviabletissuewiththeaimtoincreasetheeffectivenessof
theconstructinbiologicenvironments[61].Therefore,allthenonfreshgraftswhichareprocessedfor
acellularizationbelongtothetissueengineeringcategory.Infact,acellularizationisthebasictissueengineering
technologydescribedforallograftandxenografts[58].Thismethodoftissueengineeringhasbeenusedformany
yearstodecreasetheantigenicityoftheviablegrafts[58,59].Newerapproacheshavebeendeveloped,andnewer
tissueengineeredproductshaverecentlybeenintroduced.
Tissuescaffolds
Scaffoldsarethemostimportantissueintissueengineeringandcouldbedividedintotwomaincategoriesincluding
biological(naturalororganic)andsynthetic(artificial)materials[5,22].Theformerarenaturalpolymerssuchas
collagentypeIorDBM[24,62,63].Porousmetals,bioactiveglasses,syntheticpolymerssuchaspolylacticacid
(PLA)andpolyglycolicacid(PGA),andcalciumphosphateceramicssuchashydroxyapatite(HA)andtricalcium
phosphates(TCP)areexamplesofsyntheticmaterials[2,24,27,32,64].
Thetraditionaltechnologiesusedtoacellularizethebiologicgraftsaimingtomimiccharacteristicsoftheautografts
inthedefectareastartedthebasisofbonetissueengineering[58,59].Withthisapproach,thearchitectureofthe
graftwasnotaltered,andthehealingcharacteristicsofsuchnonviableacellularizedgraftcouldnotbeincreased
[47].Newertechnologiesuseddifferentmaterialstoproducegrafts,thearchitectureofwhichcouldbedesigned
accordingtoclinicalneeds.Syntheticmaterialsareoftenselectedastissueengineeringmaterialinproducing
scaffoldsbecausetheirpolymericmoleculesarecommerciallyavailableandthereisnoneedforspecialprocessing
priortouse[65,66].Usingvarioustissueengineeringtechnologies,differentscaffolds,eachwithadvantagesand
disadvantages,havebeenproducedforbonetissueengineeringapplications(Table4).Inaddition,scanning
electronmicroscopy(SEM)imagesofseveralpolymershavebeenprovidedinFigure3.
Table4
Advantagesanddisadvantagesofsomeofthebiologicandsynthetictissue
engineeredpolymers
Figure3
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SEMimages.Alginate(A),alginatechitosan(B),chitosan(C),chitosan
collagen(D),mesenchymalstemcellsculturedonthescaffoldcollagen(C)
DL lacticacidglycolicacid(PLGA)(P)medium(E),andsynthesized
porousHAscaffold(F).Thescaffolds...
Toproduceascaffold,thebiologictissuesshouldbedegradedtotheircomponentsandthenredesignedaccording
tothetissueengineeringgoals[67,68].Suchconstructsarespecificforoneortwomaterials(e.g.,collagen+
hydroxyapatite,collagen+chitosan)andthereforehavelowantigenicitycomparedwithcadavericgrafts[66,67].
Severalcharacteristicshavebeensuggestedforthetissueengineeredconstructs,includingporosity,suitablepore
sizeandshape,fiberalignmentandorientation,fiberdensity,internalandexternalarchitecture,hydrophilicity,and
hydrophobicity,includingwateruptake,binding,anddelivery[65,69].Regardlessofthecompositionofthe
scaffolds,theabovementionedcharacteristicsarecrucialfortheirosteoconductiveproperties[34].Sincethe
materialsshouldbesubstitutedbythenewbone,anotherimportantcriterionistheirresorbability.Degradationof
thepolymersisbasedonenzymaticorhydrolyticpathways[34,70].Polymericscaffoldshaveuniqueproperties,
suchasbiodegradation.Naturalpolymersareconsideredasthefirstbiodegradablebiomaterials,whilesynthetic
biodegradablepolymerscanbeproducedundercontrolledconditions[34,71].Bioactiveceramics,suchasHA,
TCP(TCPismorequicklybiodegradablethanHAP),andbioactiveglasses,reactwithphysiologicalfluids[71].
However,theirbiodegradabilityisofteninsufficient,limitingtheirpotentialclinicaluse.Thisissuecanbeovercome
byblendingsyntheticandnaturalpolymersorusingcompositematerialsthatimprovethescaffoldpropertiessuch
asbiodegradability.Theseproductsareoftennamedhybrid[17,7173].
Ingeneral,tissueengineeredconstructshavelowmoleculardensityandhighporesize,andtheirfiberalignment
andsizecouldvarybasedonthenatureoftheinjuryandtherecipienttissue[74].Scaffoldsmustbehighlyporous
toallowcellingrowthandfacilitateneovascularizationoftheconstruct[71].Averageporesize,poresize
distribution,porevolume,poreinterconnectivity,andporeshapeareimportantparameterstoconsiderwhen
designingascaffold[74].Poresizeisveryimportant:iftheporesaretoosmall,theywillbeoccludedbythecells
thiswillpreventcellularpenetration,extracellularmatrixproduction,andneovascularizationintheinner
architectureofthescaffold.Aporesizeof200350misoptimalforboneingrowthandfacilitates
osteoconduction[71].
Naturalbasedmaterialsusedfortissuescaffolding
Naturalorbiologicbasedmaterialsaretakenfrombiologicbasedtissues,andxenograftsmaybethebestsource
fortheselaterproducts.Theadvantagesofnaturalbasedscaffoldsarethattheyhavesignificantlysuperior
biocompatibility,biodegradability,regenerativecharacteristics(e.g.,osteoinduction,osteoconduction,osteogenesis,
andosteointegration)thanthoseofsyntheticmaterials,buttheirimmunologicalbehaviorisvariableindifferent
speciesandisalsorelatedtothetypeofapplication[7579].
Collagen
Collagensareamongthemostwidelypresentinthehumanbody,providingstrengthandstructuralstabilityto
varioustissues,fromtheskintobone[78].Collagen(collagentypeI),themajororganiccomponentofECMofthe
bone,isthemostpopularbiologicmaterialsusedtoproducebiologicallybasedtissueengineeredgrafts(Table4)
[75,78,80].Recentinvestigationstriedtoincreasethebiocompatibility,biodegradability,andregenerativecapability
oftissueengineeredbasedscaffoldsbyincorporationofcollagenintothestructureofdifferentcomposites.Surface
modificationofdifferentbonescaffoldswithcollagenhasbeeninthefocusofmanyrecentstudies.Usingaligned
collagenfibersinthescaffoldimprovedcellularproliferation,anddifferentiationtoosteoblastsareobtained[78].
Collagenscaffoldshavealsobeenusedasbiomaterialcarriersforbonemorphogeneticproteins.Usingaratectopic
boneformationmodel,rhBMP2wasinjectedintoacollagenmatrixandtheresultsshowedthatusingcollagen,it
ispossibletofunctionallydeliverbonebasedgrowthfactorstoproducenewboneformationinvivo[81].Despite
theirexcellentbioactivity,collagenbasedscaffoldshavelowmechanicalpropertiesandaresusceptibleto
substantialshrinkageduringcellculture,whichlimitstheirpotentialapplicationsintissueengineeringofbone.The
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effectsoffishcollagenpeptides(FCP)oncollagensynthesis,quality,andmineralization,usinganosteoblastic
MC3T3E1cellculturesystem,havebeenrecentlyinvestigated:FCPexertsapositiveeffectonosteoblasticcellsin
termsofcollagensynthesis,quality,andmineralization,therebysuggestingthepotentialutilityofFCPforbone
tissueengineering[82].Moreover,acombinationofcollagenchitosancalciumphosphatemicroparticleshasbeen
producedwhichwasthenfusedwithglycolicacid.Incorporationofcollagenintothisbonegraftsubstitute
increasesthebiocompatibilityanddegradationprofileofthecomposite[83].
Chitosan
Chitosan,alinearpolysaccharidewithmanycommercialandbiomedicalusesduetoitspropertieswhichallowitto
rapidlyclotblood,hasrecentlygainedapprovalintheUSAandEuropeforuseinbandagesandotherhemostatic
agents,quicklystoppingbleedingandreducingbloodloss,witha100%survivalofotherwiselethalarterial
woundsinswine[84].Thechitosansaltscanbemixedwithothermaterials(suchasalginate)tomakethemmore
absorbentortovarytherateofsolubilityandbioabsorbabilityofthechitosansalt.Chitosansaltsarebiocompatible
andbiodegradablemakingthemusefulasabsorbablehemostats[85].Thechitosansaltmaybeplacedonan
absorbablebacking.Theabsorbablebackingmaybesynthetic(forinstance,madefromexistingabsorbablesuture
materialse.g.,TephaflexpolymerTephaMedicalDevices,Lexington,MA,USA)ornatural(e.g.,celluloseor
gelled/solidifiedhoney)[84].Thepropertiesofchitosanalsoallowittobeusedintransdermaldrugdeliveryitis
mucoadhesive,reactive,canbeproducedinmanydifferentforms,andhasapositivechargeunderacidic
conditions.Thismoleculewillmaintainitsstructureinaneutralenvironmentbutwillsolubilizeanddegradeinan
acidicenvironment.Hence,chitosancanbeusedtotransportadrugtoanacidicenvironment,wherethechitosan
packagingwillthendegrade,releasingthedrugtothedesiredenvironment[85].Givenitsuniqueproperties,
chitosanhasbeenusedincombinationofvariousmaterialsforbonetissueengineeringpurposes.Tridimensional
compositescaffoldscomposedofchitosanandcalciumphosphatehavebeendevelopedandcharacterized.Air
dryingofthisscaffoldenhancesitsbioactivity.Givenitsoptimumstrength,degradationresistance,andcell
supportivecharacteristics,chitosancanbeusedforbonetissueengineering[86].
Alginate
Alginicacid,alsocalledalginoralginate,isananionicpolysaccharidedistributedwidelyinthecellwallsofbrown
algaewhere,bindingwithwater,itformsaviscousgum.Inextractedform,itquicklyabsorbswater,withawater
absorptioncapacityof200300timesitsownweight.Itissoldinfilamentous,granular,orpowderedforms.A
novelstemcelldeliverysystemcomposedofcollagenandalginateasthecoreandshell,respectively,hasbeen
developed.Thisfibrouscarrierhasbeenshownpromisingtoenabletheencapsulationoftissuecellsandtheir
deliveryintodamagedtargettissues,includingbonewithdefecttunabilityforbonetissueengineering[87].
Elastin
Similartocollagen,elastinisakeystructuralproteinfoundintheECMofmosttissuesyet,verylittleisknown
abouttheresponseofbonecellstoelastinoritsderivatives.Recently,anovelclassofECMbasedcomposite
scaffoldswithcollagenandageneticallyengineeredpolymer,elastinlikepolypeptide(ELP)hasbeendesignedand
produced.Byembeddingtheelastinwithincollagenscaffolds,itispossibletoexpectsuperiormechanical
propertiesanddrugreleasecharacteristicscomparedtocollagenscaffoldsalone.Elastinalsoenhancesosteogenic
differentiationofstemcellsandregulatescellsbehaviorinvitro[88].
Cellulose
Celluloseisanorganiccompoundandanimportantstructuralcomponentoftheprimarycellwallofgreenplants,
manyformsofalgae,andtheoomycetes,andissecretedbysomebacteriatoformbiofilms.Celluloseisthemost
abundantorganicpolymeronEarth[89].Celluloseisusedtomakehydrophilicandhighlyabsorbentsponges,
beneficialincombinationwithothermaterialsforbonetissueengineeringapplications[90].Acelluloseand
collagenbasedmicro/nanostructuredscaffoldhasbeenrecentlyproduced.Afterculturinghumanosteoblastson
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thescaffold,thescaffoldsupportedanoptimumadhesionandphenotypemaintenanceofculturedcellsasreflected
byhigherlevelsofosteogenicenzymealkalinephosphataseandmineraldepositioncomparedtocontrolpolyester
micro/nanostructuredscaffoldsofidenticalporeproperties[90].
Syntheticpolymericmaterials
Severalinvitroandinvivoresearchestriedtooptimizesyntheticbased,tissueengineeredscaffoldsinordertobe
usefulinboneregenerativemedicine.Asinglewalledcarbonnanotube(SWCNT)andpolylacticcoglycolicacid
(PLGA)compositehasbeendevelopedrecently.AfterseedingwithhumanMSCsandosteoblasts,thecomposite
impartedbeneficialcellulargrowthcapabilityandgeneexpression,andmineralizationabilitieswerewell
establishedsuggestingitspotentialapplicationinboneregeneration[91].Asanotherstrategy,acombinationof
differentpolymershasbeentriedtoincreasethecellcytocompatibilityofthesyntheticbasedscaffolds.Poly(l
lactide)andpoly(caprolactonetriol)aresomeexamples.Usingsuchcombination,newmembranespromotedtherat
osteoblasticcellbehaviorinvitro(e.g.,migration,attachment,proliferation,andmatrixproduction)[92].Surface
modificationandcoatingisanotherstrategytoenhancebioactivityofthesyntheticscaffolds.Silicananoparticles
havebeenappliedontothefibersurfaceofaninterbondedthreedimensionalpolycaprolactone(PCL)fibroustissue
scaffold.Thenanoparticlelayerwasfoundtoimprovethefiberwettabilityandsurfaceroughness.Thus,it
enhancedosteoblastattachment,proliferation,andalkalinephosphataseactivities[93].Despitemanybeneficial
characteristicsofsyntheticmaterialsinbonehealingandregeneration,theirbiocompatibility,biodegradability,and
regenerativepropertiesarestillsuboptimalcomparedtonaturalbasedscaffolds.Therefore,manyattemptshave
beenmadetocombinesyntheticwithnaturalmaterials.Recently,poly(D,L lactidecoglycolide)hasbeencombined
withanaturallybioceramichybridmaterial,nanonizedpearlpowder,asanosteoinductivematerial:thescaffold
wasabletoinfluenceosteoblastbehaviorinvitro[94].Thebenefitsassociatedwithpolyhydroxyalkanoates(PHA)
intissueengineeringincludehighimmunotolerance,lowtoxicity,andbiodegradability.Poly(3hydroxybutyrateco
3hydroxyhexanoate)(PHBHHx),amoleculefromthePHAfamilyofbiopolymers,sharesthesefeatures.Collagen
hasbeenusedwithPHAtoincreasethebiocompatibilityofthescaffoldandtosupportcellproliferationand
osteogenicdifferentiationinvitro[95].
Thereisanincreasingdemandforaninjectablecellcoupledthreedimensional(3D)scaffoldtobeusedasbone
fractureaugmentationmaterial.Toaddressthisdemand,anovelinjectableosteogenicscaffoldcalledPNCOLwas
developed,usingcells,anaturalpolymer(collagentypeI),andasyntheticpolymer(PCL).Theinjectable
nanofibrousPNCOLisproducedbyinterspersingPCLnanofiberswithinpreosteoblastcellembeddedcollagen
typeI.Thissimpleyetnovelandpowerfulapproachprovidesagreatbenefitasaninjectablebonescaffoldover
othernonlivingbonefracturestabilizationpolymers,suchaspolymethylmethacrylateandcalciumcontentresin
basedmaterials.Theadvantagesofinjectabilityandthebiomimicryofcollagenwerecoupledwiththestructural
supportofPCLnanofiberstocreatecellencapsulatedInjectable3Dbonescaffoldswithintricateporousinternal
architectureandhighosteoconductivity.TheeffectsofPCLnanofiberinclusionwithinthecellencapsulated
collagenmatrixhavebeenevaluatedforscaffoldsizeretentionandosteocompatibility,aswellasforMC3T3E1
cellsosteogenicactivity.Atstructuralanalysis,thisnovelbioactivematerialwaschemicallystableenoughinan
aqueoussolutionforextendedperiodswithoutusingcrosslinkingreagents,butitisalsoviscousenoughtobe
injectedthroughasyringeneedle.DatafromlongterminvitroproliferationanddifferentiationsuggeststhatPN
COLscaffoldspromoteosteoblastproliferation,phenotypeexpression,andformationofmineralizedmatrix[96].A
noveldicalciumphosphateanhydrate/poly(lacticacid)(DCPA/PLA)compositenanofiber,whichmimicsthe
mineralizedcollagenfibrilsviabiomimeticinsitusynthesisandelectrospinningforhardtissueregenerative
medicine,hasbeenproduced.Additionofpoly(ethyleneglycol),asacopolymersource,producedmorestableand
efficientelectrospunjetsandaidedintheelectrospunabilityofthePLAnanofibersincorporatingthe
nanocrystallites[97].
Calciumphosphateanditsderivatives
Differenttypesofmono,bi,andtricalciumphosphatebioceramicsandmoleculeshavebeenextensivelyusedin
bonetissueengineeringresearchesanddevelopments[68].Hydroxyapatiteisanaturallyoccurringmineralformof
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calciumapatite.Upto50%ofthebone'sweightisamodifiedformofhydroxyapatite(knownasbonemineral).
Carbonatedcalciumdeficienthydroxyapatiteisthemainmineralofwhichdentalenamelanddentinarecomposed
[98].Hydroxyapatitecrystalsarealsofoundinthesmallcalcifications(withinthepinealglandandotherstructures)
knownascorporaarenaceaorbrainsand.Boththecalciumphosphateandapatiteformshavewideapplicationsin
bonetissueengineering[99].Severalauthorshaveusedsuchmaterialsaloneorincombinationwithothermaterials
suchascollagen,alginate,andchitosaninordertodevelopnewscaffoldsandtissueengineeringstrategies[98
107].
Recently,abiomimeticandbioactivetissueengineeredboneconstruct(porousnanocrystallinehydroxyapatite
(nHA)/chitosanscaffolds)viaacoldatmosphericplasma(CAP)treatmentfordirectedosteogenicdifferentiationof
humanbonemarrowMSCshasbeenintroduced[98].Anewhybridmaterial(CMCHA)containingHAina
carboxymethylcellulose(CMC)basedhydrogelwasdeveloped.ThestrategyforinsertingHAnanocrystalswithin
thehydrogelmatrixconsistsofmakingthefreezedriedhydrogeltoswellinasolutioncontainingHA
microcrystals.WhenthecompositeCMCHAhydrogelwascharacterizedandseededwithosteoblastsMG63line,
thescaffoldwithHAenhancedcellproliferationandmetabolicactivityandpromotedproductionofmineralized
extracellularmatrixmorethanthatobservedforthescaffoldwithoutHA[102].Sagaretal.[108]evaluatedthe
completehealingofcriticalsizedefectmadeintheproximaltibiaofrabbits,usingnanohydroxyapatite/gelatinand
chemicallycarboxymethylatedchitin(nHA/gel/CMC)scaffoldconstruct.Thearchitectureindicesanalyzedby
microcomputedtomographyshowedasignificantincreaseinthepercentageofbonevolumefraction,with
reconciledcorticotrabecularboneformationatthesitestreatedwithnHA/gel/CMCconstructscomparedto
controls.Athistologyandfluorescencelabeling,theuniformlyinterconnectedporoussurfaceofthescaffold
constructenhancedosteoblasticactivityandmineralization.
CollagenhasbeenextensivelyusedwithHAandorTCPtoproducebonescaffolds.Aboneinspiredmaterialhas
beenrecentlyobtainedbyincorporatingcollagenintheliquidphaseoftricalciumphosphatecement,eitherin
solubilizedorinfibrilizedform.Thismaterialwasabletosetinsitu,givingrisetoacalciumdeficient
hydroxyapatite(CDHA)/collagencomposite.Thecompositecontrolsthecellbehaviortoaccelerateandtrigger
osteogenicdifferentiationinvitro[99].Acollagenhydroxyapatite(ColHA)compositethroughcontrolledinsitu
mineralizationontypeIcollagenfibrilswithnanometersizedapatitecrystalswasdesignedandproduced.After
culturingthescaffoldswithMSCs,theporousColHAcompositeshadgoodbiocompatibilityandbiomimetic
propertiesandsupportedboneregenerationandformation[101].
AcombinationofcollagenandHAhasbeenusedinvivo.Recently,abiomimeticcollagenapatitescaffold
composedofcollagenfibersandpoorlycrystallinebonelikecarbonatedapatitenanoparticleswasdevelopedto
improvebonerepairandregeneration.Invivo,thescaffoldenhancednewboneformationinmice[106].In
addition,theeffectofresorbablecollagenmembranesoncriticalsizedefectsinrabbittibiaefilledwithbiphasic
calciumphosphatehasbeeninvestigated:biphasiccalciumphosphatefunctionedwellasascaffoldandallowed
mineralizedtissueformation.Furthermore,theadditionofabsorbablecollagenmembranesenhancedbonegain
comparedwithnonmembranetreatedsites[109].
TheapplicationofporousHAcollagenasabonescaffoldrepresentsanewtrendofmimickingthespecificbone
extracellularmatrix.ApplicationofHAinreconstructivesurgeryhasshownthatitisslowlyinvadedbythehost
cells.Therefore,implantcompatibilitymaybeaugmentedbyseedingcellsbeforeimplantation.Humanprimary
osteoblastswereseededontoinnovativecollagengelatingenipin(GP)HApscaffolds.Invitroattachment,
proliferation,andcolonizationofhumanprimaryosteoblastsoncollagenGPHApscaffoldswithdifferent
percentagesofHA(10%,20%,and30%)allincreasedovertimeinculture,butcomparingdifferentpercentagesof
HA,theyseemtoincreasewithdecreasingofHAcomponent[105].Atricomponentosteogeniccompositescaffold
madeofcollagen(Coll),HA,andpoly(llactidecocaprolactone)(PLCL)hasbeenrecentlydeveloped.This
Coll/HA/PLCLcompositescaffoldwascombinedwithhumanosteoblastlikecells.Thecompositeishighly
porous,enablingosteoblastlikecelladhesionandgrowth[100].Jungetal.[110]elucidatedtheroleofcollagen
membranes(CMs)whenusedinconjunctionwithbovinehydroxyapatiteparticlesincorporatedwithcollagen
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matrix(BHC)forlateralonlaygraftsindogs.Thisstrategyleadstosuperiornewboneformationandbonequality
comparedwithbonegraftalone.
Calciumphosphateceramics,specificallytricalciumphosphate(TCP)andsyntheticHA,haverecentlybeen
usedincompositesandinfibrouscompositesformedusingtheelectrospinningtechniqueforbonetissue
engineeringapplications.Calciumphosphateceramicsaresoughtbecausetheycanbebonebioactive,sothat
apatiteformsontheirsurface,facilitatesbondingtobonetissue,andisosteoconductive[103].Inarecentstudy,the
bioactivityofelectrospuncompositescontainingcalciumphosphatesandtheircorrespondingosteogenicactivity
wasinvestigated.Electrospuncompositesconsistingof(20/80)HA/TCPnanoceramicsandpoly(caprolactone)
werefabricated,andtheresultsdemonstratedthatafterseedingthehybridscaffoldwithhumanMSCs,thecellsnot
onlyshowedgreaterosteogenicdifferentiationbutalsoproliferatedandproducedmorebonymatrixinvitro[103].
ThecombinationofTCPandHAisanotherstrategy.Bonehealingandbiodegradationpatternsofthreetypesof
CaPceramicparticleswithvariousHA/TCPweightratios(pureTCP,biphasicCaP(BCP)withaHA/TCP
weightratioof60/40(BCP60/40),andBCPwithanHA/TCPweightratioof20/80(BCP20/80))were
investigated[106].Four8mmdiameterdefectswereproducedintenrabbits.Threeofthedefectsineachrabbit
wereseparatelyandrandomlyfilledwithoneofthethreeexperimentalCaPceramicparticles,andthefourthwas
filledwithbloodclots(control).After2and8weeks,BCP60/40andBCP20/80exhibitedasimilarbonehealing
andbiodegradationpatternswithregardtobothindividualparticlesandthetotalaugmentedareainvivo.
Piccininietal.[111]implantedanewmineralogicalformulation,HA/tetracalciumphosphate(TTCP),asa
biomaterialforboneregenerationinthemaxillarysinusofrats.After17weeksfromimplantation,HA/TTCP
syntheticbonegraftperformedverywellasosteoconductivematerial:bonegraftcontactwasfoundveryhigh,and
bonevolumeandvitalboneshowedanidealbonedensityforimplantplacement.Farahpouretal.[112]
radiographicallyevaluatedtheeffectsofbiphasiccalciumphosphatescaffoldwith5%,10%,and20%ofporosity
oncorticalbonerepairinrabbits.TheyshowedthatTCP+HAscaffoldisanosteoconductiveandosteoinductive
biomaterial.TCP+HAscaffoldcanincreasetheamountofnewlyformedboneandmorerapidregenerationof
bonedefects.Velasquezetal.[104]reportedtheinvitroandinvivobehavioroftricalciumphosphate(TCP)
andalsoTCPwitheither1.5or3.0wt.%ofdicalciumsilicate(C2S).Theinvivobehavioroftheceramics
matchedtheinvitroresults,independentoftheC2ScontentinTCP.Afullymineralizednewbonegrowingin
directcontactwiththeimplantswasfoundundertheinvivoconditions.Thebioactivityandbiocompatibilityofthe
implantsincreasedwiththeC2ScontentinTCP.TheC2Sdopedceramicsalsofavoredaphasetransformation
ofTCPintoCHA,whichisimportantforfullimplantintegrationduringthenaturalbonehealingprocesses.
Thehybridrapidprototyping(RP)scaffoldofPLGA/TCPskeletonwithcollagenI/apatitespongecomposite
coatingisapromisingcandidateforbonetissueengineering.Theosteogenicpotentialofsynthetictricalcium
phosphateinahydroxylsulfatematrix(TCP/HS)andhumanDBMputtyhasbeeninvestigatedinrabbits[113].
Ineachanimal,twobonedefects(8mmlength3mmwidth3mmdepth)wereproducedintheleftandright
regionsofthemandible,respectively.ThedefectononesidewasfilledwithTCP/HS(groupA)orDBMputty
(groupB),whilethedefectontheoppositesidewasleftunfilledtoserveasacontrolsite.After1to6weeks,
TCP/HSandhumanDBMputtyshowedosteogenicactivityandsupportednewboneformation.
Bioactiveglass
SeveralvariationsofglassbeadscalledBioglass(USBiomaterials,Alachua,FL,USA)arecurrentlybeing
developed,andoneformulation(PerioGlas)hasbeenapprovedintheUSAforperiodontaluse.Thebeadsare
composedofsilica(45%),calciumoxide(24.5%),disodiumoxide(24.5%),andpyrophosphate(6%).When
implanted,theybindtocollagen,growthfactors,andfibrintoformaporousmatrixtoallowinfiltrationof
osteogeniccells[114].Recently,anovelnanocompositehydrogelmadeofcollagenandmesoporousbioactiveglass
nanoparticleswithsurfaceaminationhasbeendeveloped[115].Theadditionofbioglassintothecollagenhydrogel
significantlyincreasesthebioactivityofthescaffoldandimprovesitsmechanicalpropertiesthisnovelstrategy
wouldthereforebesuitableforbonetissueengineeringapplications[115].Moreover,thebioactiveglassfoam
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producedbysolgelisanosteoinductivematerialwithanetworkofinterconnectedmacroporesnecessaryforcell
colonization.Ithasbeenshownthatbioactiveglasscandifferentiatehumanadiposederivedstemcellsinto
osteoblasts,invitro[116].Moreover,Guetal.[117]usedscaffoldscomposedofamixtureoftwodifferentbioactive
glasses(silicate1393andborate1393B3)toevaluatetheirresponsetoosteogenicMLOA5cellsinvitroand
theircapacitytoregenerateboneinratcalvarialdefectsinvivo.Thescaffoldscanguideboneregenerationandhave
acontrollabledegradationrate.AcombinationofglassandHAhasalsobeenusedinboneregeneration.Fredericks
etal.[118]determinedtheperformancecharacteristicsofanovelsilicatesubstitutedHAbonegraftsubstitute
(BGS),SiCaPEP(BaxterHealthcare/ApaTech,Elstree,UK),inastandalonemode,astandalonewithbone
marrowaspirate(BMA)mode,andanextendermodewithiliaccrestautograft(ICBG)inarabbitposterolateral
spinefusionmodel.TheSiCaPEPutilizedasastandalone,asastandalonewithBMA,andasanautograft
(ICBG)extenderproducedresultsthatwereclinicallyandradiographicallysimilartoICBG.
Healingpromotivefactors
Healingpromotivefactorssuchasgrowthfactorshavebeenextensivelyusedtotreatbonydefectsandfor
osteoinduction.Somegrowthfactorssuchasvascularendothelialgrowthfactor(VEGF),TGF,PDGF,and
BMPssuchasBMP2,BMP7,andIGFarepresentinthehealthybonematrixandareexpressedduringbone
healing[32,34,119].Thesefactorscanregulatevascularizationandinduceproliferationanddifferentiationofthe
osteoblastsandtheirprecursors,sotheycanbeusefulinimprovingthehealingprocesses[32].
Bonemorphogeneticprotein2(BMP2)isapotentosteoinductivecytokinethatplaysacriticalroleduringbone
regenerationandrepair.Intheextracellularenvironment,sulfatedpolysaccharidesanchoredcovalentlyto
glycoproteinssuchassyndecanandalsononcovalentlytofibronectinfibershavebeenshowntobindtoBMP2
throughaheparinbindingdomainandregulateitsbioactivity[37].Thesupramolecularpeptideamphiphile
nanofibers,whichintegratethebiologicalroleofsyndecanandfibronectin,havebeencontrolledanddesignedto
formasanetworkwithintheporesofanabsorbablecollagenscaffold.Thehybridbiomaterialenhanced
significantlyboneregenerationinaratcriticalsizefemoraldefectmodelusingBMP2inamountsthatareone
orderofmagnitudelowerthanthatrequiredforhealinginthisanimalmodel.Presenceofmorematureboneinthe
newossifiedtissuewasnotedwhenalowdoseofBMP2wasdeliveredusingthebiomimeticsupramolecular
system[80].Chaetal.[120]determinedtheefficacyofBMP2inaBHCcarriertoaugmentboneformationina
caninenasalsinusmodel.Followingpreparationofbilateralsinusaccesswindows,BHCalone(control)orloaded
withEscherichiacoliderivedBMP2at0.1mg/mLwasimplantedinfouranimals,andBHCloadedwithE.coli
derivedBMP2at0.5and1.5mg/mLwasimplantedinfouranimals.Theanimalswereeuthanizedat20weeks.
HistometricanalysisshowedsignificantlyenhancedboneformationfortheBMP2groupscomparedwithcontrol.
BMP2inaBHCcarrier,evenatthelow0.1mg/mLconcentration,inducedosteogenicactivity,thusenhancedthe
localboneformationinacaninesinusmodel.Jangetal.[121]determinedwhetheraHA/TCPratioof20/80
impregnatedwithrhBMP2enhancesnewboneformationinratcalvarialdefectmodel.rhBMP2significantly
inducednewboneformation.Inaddition,Stancovenetal.[122]evaluatedthepotentialofrhBMP2soakloaded
ontoanabsorbablecollagensponge(ACS)toinducelocalboneformationcomparedwiththeclinicalreference
DBMandtoinvestigatepotentialadditive/synergisticeffectsofexogenousparathyroidhormone(PTH).Critical
size(8mm)throughthroughcalvariaosteotomydefectsin160adultmaleratswererandomizedtoreceiveoneof
theeightinterventions:rhBMP2/ACS,DBM,ACS,orserveascontrols(emptydefects)combinedornotwith
systemicPTH.Fourtoeightweekspostsurgery,rhBMP2/ACSsignificantlystimulatedlocalboneformation,
whereasboneformationwassignificantlylimitedintheDBMgroup.SystemicapplicationofPTHprovidedno
discernibleadditive/synergisticeffectsonlocalboneformation.Liuetal.[123]producedanovelbiomimeticbone
scaffoldcomposedofcalciumsulfatehemihydrate(CSH),collagen,andnanohydroxyapatite(nHAC).rhBMP2
wasintroducedintoCSH/nHAC.ThescaffoldswithorwithoutrhBMP2wereimplantedintoacriticalsizedefect
modelinthefemoralcondyleofrabbit.Theresultsofplainradiography,microCT,andhistologicalobservation
indicatedthatmorenewbonewasformedinrhBMP2group.
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AlthoughmanyreportsconfirmedthebeneficialeffectsofBMPonboneregenerationandquality,someothers
showedtheirineffectivenessonregenerationofnonweightbearingbonehealing.Inaninvestigation,the
biomechanicalpropertiesofcalvarialboneregeneratedwithderivationsofacommerciallyavailablerhBMP2
basedsystemwereevaluated.Standardizedcalvarialdefectswereproducedin23adultmalecanines.Thesedefects
weretreatedwithrhBMP2ononeoftheseveralcarriers.After24weeks,thebiomechanicalpropertiesofthe
rhBMP2generatedbonewerecomparedtothoseofthecontrolswithamodifiedpunchouttest.Theyconcluded
thatrhBMP2generatedcalvarialboneissignificantlylessprotectiveagainsttraumathannativeboneat6months.
Hsuetal.[124]evaluatedacombinationtherapy(TrioMatrixPioneerSurgical,Inc.,Marquette,MI,USA)
composedofDBM,hydroxyapatite,andananofiberbasedcollagenscaffoldinarodentspinefusionmodel.Thirty
sixathymicratsthatunderwentaposterolateralintertransversespinalfusionwererandomlyassignedtooneofthe
fivetreatmentgroups:ACSalone(negativecontrol),10grhBMP2onACS(positivecontrol),TrioMatrix
(Osteotech,Inc.,Eatontown,NJ,USA),Grafton,andDBX(SynthesInc.).BothTrioMatrixandrhBMP2
treatedanimalsdemonstrated100%fusionratesasgradedbymanualpalpationscores8weeksafterimplantation.
ThisratewassignificantlygreaterthanthoseoftheACS,Grafton,andDBXgroups.Notably,theuseof
TrioMatrixquantifiedbymicroCTledtoagreaterfusionmassvolumewhencomparedtoallothergroups,
includingtherhBMP2group.T2weightedaxialMRIimagesofthefusionbeddemonstratedasignificanthost
responseassociatedwithalargefluidcollectionwiththeuseofrhBMP2thisresponsewassignificantlyreduced
withtheuseofTrioMatrix.Inanotherstudy,BMP7orDBMwasimplantedinarabbittibialdistractionmodel,
andhealingwascomparedtoanontreatedcontrolgroup.Neitherofthetreatmentsshowedachangedhealing
pattern.DensitiesasmeasuredbyCTscanwerenotincreased,andtheonlysignificantfindingwasanincreased
areaofboneformationintheDBMtreatedgroup(65%increase).Theseexperimentalresultsdonotshowaneffect
ofthesesubstancesinthismodelofbonelengthening[125].
CombinationofzoledronateandBMP7hasalsobeentestedinvivotooptimizebonehealing.Zoledronicacid
(INN)orzoledronate(marketedbyNovartis(Basel,Switzerland)underthetradenamesZometa,Zomera,Aclasta,
andReclast)isabisphosphonategivenintravenously.Zometaisusedtopreventskeletalfracturesinpatientswith
cancerssuchasmultiplemyelomaandprostatecancer,aswellasfortreatingosteoporosis.Itcanalsobeusedto
treathypercalcemiaofmalignancyandcanbehelpfulfortreatingpainfrombonemetastasesandfractures[126].
Yamanetal.[127]examinedtheeffectsofsystemiczoledronicacidadministrationontheosseointegrationofHA
coatedandresorbableblastmaterialsurface(RBM)implantsinarabbittibialmodel.Histomorphometricanalyses
showedsignificantimprovementintheosseointegrationofimplantsintheRBMsurfacezoledronicacidgroup
comparedwiththeHAcoatedzoledronicacidgroup.Theresultssuggestthatsystemiczoledronicacid
administrationmayimproveosseointegrationoftitaniumimplantsinbone.Inrats,Mathavanetal.[128]
investigatedtheroleofcombinationofallograft+BMP7+systemiczoledronate(ZA)onbonehealingand
regeneration.Femoralosteotomieswereperformedon82maleSpragueDawleyratsandfixedwithintramedullary
Kirschnerwires.Theratswererandomizedintosevengroups:(i)saline,(ii)autograft,(iii)allograft,(iv)allograft+
BMP7,(v)autograft+ZA,(vi)allograft+ZA,and(vii)allograft+BMP7+ZA.Theratswereeuthanizedat
6weeks.CompleteradiologicalhealingwasseeninallratsintheBMP7groups.Thecallusvolumewaslarger,
andthecallusesweredenserwithallograft+BMP7+ZAthaninallothergroups.Mechanicaltestingyieldeda
substantiallyhigherpeakforcewiththeallograft+BMP7+ZAcombinationthanallothergroups,witha59%
increaseinthepeakforceobservedintheosteotomizedfemursoftheallograft+BMP7+ZAgroupcomparedto
thecontrolfemurs,whereassignificantdecreasesof22%27%wereobservedinthesalineorbonegraftalone
groups.AllograftcombinedwiththeanaboliceffectofBMP7andtheanticataboliceffectofzoledronateismore
efficientthanautograftalone.
TGF1iscrucialinthedevelopment,induction,andrepairofbone.TheeffectoflocalapplicationofagraftDBM
alongwithTGF1inamodelofopenosteotomyinducedexperimentallyindogshasbeeninvestigated[129].An
openosteotomyofthetibiawasproducedinyoungmaledogs.Onthefifthweek,therewasanimprovementand
restorationofbonearchitectureinanimalstreatedwithagraftcontainingTGF1(5ng/mL)comparedwiththe
controlandgraftgroups,asevidencedbyearlyformationofwidecallusandboneregeneration.Inaddition,local
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applicationofTGF1ledtoanincreaseincollagenandproteolyticactivity.Moreimmunopositiveosteoclastand
mesenchymalcellswerefoundinthebonetissuefromanimalstreatedwithTGF1comparedwiththecontrol
group.Ozturketal.[130]investigatedtheroleofanovelhydroxyapatitecontaininggelatinscaffoldwithand
withoutlocalvascularendothelialgrowthfactorasthesyntheticgraftmaterialinthetreatmentofcriticalsizedtibial
bonedefectsinrabbits.After6weeks,theadministrationofVEGFonthegraftexertedapositiveeffectintheearly
phasesoffracturehealingbuthadnoeffectafter12weeks.
Alendronicacidoralendronatesodium,soldasFosamaxbyMerck(WhitehouseStation,NJ,USA),isa
bisphosphonatedrugforosteoporosisandseveralotherbonediseases.Itismarketedaloneaswellasin
combinationwithvitaminD.OnFebruary6,2008,theUSFDAapprovedthefirstgenericversionsofalendronate,
whichweremarketedbyBarrPharmaceuticals(Montvale,NJ,USA)andTevaPharmaceuticals(HorshamRoad
NorthWales,PA,USA)[131].Mathijssenetal.[132]investigatedtheroleofseveralmaterialsanddrugsonbone
healing.Twentyfivegoatsreceivedeightboneconductionchambersinthecorticalboneoftheproximalmedial
tibia.Fiveconcentrationsofalendronate(0,0.5,1,2,and10mg/mL)weretestedincombinationwithallograft
boneandsupplementedwithcefazolin(200g/mL).Allograftnotsupplementedwithalendronateandcefazolin
servedascontrol.Inaddition,allograftmixedwithDBM,withandwithoutalendronate,wastested.After
12weeks,adoseresponserelationshipforlocalapplicationofalendronatewasdetected.Localapplicationof
cefazolinhadnoeffectonboneremodeling.
Simvastatinisconsideredastimulatorofboneformation.However,thehalflifeforsimvastatinisgenerally2h,
thereforenotlikelytobebiologicallyactiveinvivo.Toovercomethislimitation,Jiangetal.[133]createdasystem
toslowlyreleasesimvastatininvitroandinvivo.Theyconstructedapolylacticcoglycolicacid/hydroxyapatite
nanofibrousscaffoldtocarrysimvastatinandimplantedtheconstructintocalvariabonedefectmodels.After4to
8weekspostimplantation,theyindicatedthatpolylacticcoglycolicacid/hydroxyapatite/simvastatinscaffold
inducedboneformationmoreefficientlythancontrols.
Plateletrichplasma(PRP)isasimplewayofdeliveringgrowthfactors[134,135].ThecombinationofhumanPRP
withHAmaybeapromisingalternativeforreconstructionandregenerationofcriticalsizedefectsinanimalmodels
[134,136].Morerecently,acombinationofPRPwithsiliconstabilizedHA/TCPscaffoldhasbeeneffectivein
rabbitcalvarialdefect(SkeliteMilleniumBiologixCorporation,Kingston,Ontario,Canada).Usingsuch
strategy,significantosteoidlikematrixandnewbonedepositiontogetherwithhighercellularity,moreabundant
osteoiddeposition,andmoreregularcollagenfiberscouldbeseeninmicroCTandhistologicanalysiswhen
comparedtoscaffoldalone.Moreover,invitromigrationassaysconfirmedthechemotacticeffectofPRPto
endothelialandosteoprogenitorcells.AdditionofPRPinfluencedthelocaltissuemicroenvironmentbyproviding
keycrypticfactorsforregeneration,therebyenhancingtheprogenitorcellrecruitmentandcollagenandbonematrix
depositionandcreatingabridginginterfacebetweenthescaffoldandbone[137].Somecontroversiesexistinthe
effectivenessofPRP.TheeffectofautologousPRPontheearlyphasesofosteoinductionbyallogenicDBMin
rabbitintramuscularpositionshasshownthatadditionofPRPtoDBMhadanegativeeffectontheearlyphasesof
osteoinductionat3weeks[138].Faratzisetal.[139]investigatedtheeffectofautologousPRPontheosteogenic
potentialofabiphasicsyntheticgraftmaterialcomposedofHA/TCPincriticalsizecranialdefectsinrabbits.
AutologousPRPinadditiontoabiphasicHA/TCPsyntheticgraftmaterialhadnoeffectonbonehealingafter2,
4,and6weeksofimplantation.
Inanotherstudy,boneregenerationinthreegroupsofratcalvariatreatedwithDBMfromtheIranianTissueBank
ResearchandPreparationCenter(Tehran,Iran),DBMfromHansBiomedCorporation(Seongdong,Seoul,
Korea),andorleavingthecavityemptywerestudied[140],usingalbuminascarrier.Boneregenerationafter1,4,
and8weeksofimplantationwasevaluated.ThetwotypesofDBMhadasignificantdifferenceinbone
regeneration.Thisdifferencewasattributedtothetypeofcarriers.Albumincouldimprovemineralizationand
bioactivitycomparedwithcontrolcarriers.
Stemcells
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Thecombinationofstemcellswithscaffoldsasapolytherapyisanewoption.Collagenanddemineralizedbone
powderhavebeenusedtoproduceanovelscaffoldsforbonetissueengineering.Humanperiosteumderivedcells
(PDcells)wereculturedonthisscaffold:thehybridscaffoldsexhibitedgreaterosteoinductivepotentialthan
collagenscaffolds.ThePDcellswithhybridscaffoldspossessedhigherALPactivity,calciumdeposition,and
superiorbehavior(e.g.,attachment,differentiation,andproliferation)thanthosewithcollagenscaffolds[141].
Thefeasibilityofapplyingcalcinedbovinebone(CBB)coatedwithallograftbonemarrowmesenchymalstemcell
(BMSC)sheetasa3Dscaffoldmaterialinbonehealinghasbeeninvestigatedrecently.Thenewscaffoldmaterial
wasimplantedintoosteoporosisratcranialbonedefectsandcriticalsizebonedefects(8mmdiameter).TheCBB
BMSCsheetcombinationhadastrongerpotentialinosteogenicdifferentiationandmineralizedformationbothin
vitroandinvivothanCBBBMSCcombination.ThreedimensionalreconstructionofmicroCT,H&Estaining,
andbonestrengthresultsshowedthattheareaandvolumeofthenewlyformedboneinCBBBMSCsheetgroup
wassignificantlyhigherthanthatoftheCBBBMSCgroupafter4to12weeks[142].
Adiposederivedstemcells(ASCs)withmultilineagedifferentiationcapacitieshavebeendemonstratedasan
alternativecellcandidateforinvitroandinvivoboneregeneration.Thissuggeststhattheymaybeapotential
candidatetorepairthebonedefects.Yangetal.[143]attemptedtodemonstratetheuseofnewbiomimetic
constructionsofundifferentiatedrabbitASCswithfullyinterconnectedporousTCPscaffoldsencapsulatedby
collagenIhydrogelintheregenerationofacriticalsizeddefectofrabbitradii.Criticalsizeddefectsintheleftradii
ofrabbitswerepreparedandinsertedwithrASCs/collagenI/TCPscaffoldcompositesorcollagenI/TCP
scaffoldcomposites.Twelveweeksafterimplantation,thedefectswerealmostcompletelyrepairedasconfirmedby
thepresenceofthecorticalboneandmedullarycavity.Inaddition,agreaternumberofASCsinthescaffold
enhancedosteogenesisincriticalsizeddefects.Pourebrahimetal.[144]comparedboneregenerationoftissue
engineeredbonefromASCsandautogenousbonegraftinacaninemaxillaryalveolarcleftmodel.The
undifferentiatedcellswereincubatedwithaHA/TCPscaffoldinspecificosteogenicmediumfor21days.Four
mongreldogswerepreparedbyremovaloftwoofthethreeincisorsbilaterallyanda15mmdefectinbonewas
createdfromthecresttothenasalfloor.Afterhealing,repairwasfollowedbyatissueengineeredbonegraftfrom
ADCsononesideandcorticocancelloustibialautograftontheotherside.Boneregenerationwasevaluatedby
histomorphometryondays15and60afterimplantation.Theboneformationontheautograftsideswashigherthan
onthestemcellsidesat15and60days,and45%and96%versus5%and70%,respectively.
Pangetal.[145]observedthetherapeuticeffectsofhybridRPscaffoldscomprisingPLGA,TCP,collagenI,and
apatite(PLGA/TCPcollagenI/apatite)onsegmentalbonedefectsinconjunctionwithcombinedbonemarrow
MSCs.ThebonedefectremainedunconnectedintheoriginalRPscaffolds(PLGA/TCP)duringthestudy.In
hybridRPscaffoldgroup,wovenboneunitedtheradialdefectat12weeksandconsecutivelyremodeledinto
lamellarboneat24weekspostoperationandfinallymaturedintocorticalbonewithnormalmarrowcavityafter
another12weeks.NoboneformationbutconnectivetissuewasdetectedintheRPscaffoldatthesametime.Xuan
etal.[146]producedpolycaprolactone/HAtissuescaffoldswithindividualizedgroovestorepairthesternaldefect.
ThedefectsweresurgicallyproducedinasternocostaljointofBeagledogs.Theanimalswereallocatedtooneof
thethreegroups(n=6):notreatmentgroup,PCL/HAgroup,andPCL/HA/BMSCsgroup.Theapplicationofthe
PCL/HAscaffoldswithspecificgroovesresultedinsatisfactoryrepairofthesternaldefect.Furthermore,the
BMSCsseededscaffoldsenhancedtheamountofboneingrowthandseemedtobemorepromising.Calcium
phosphatenanoparticlessuchasHA/fluorapatite(FA),withchitosangelfilledwithunrestrictedsomaticstemcells
(USSCs),havebeenusedforhealingcalvarialboneinaratmodel[147].Thecombinationofscaffoldespecially
withUSSCcouldbeconsideredausefulmethodforboneregeneration.
Inanotherstudy,autogenicandallogenicbonemarrowderivedMSCswerecomparedforrepairofbonegapdefect
inrabbits.ThedefectswerefilledwithHAalone,HAwithautogeneicBMMSCs,andHAwithallogenicBM
MSCs.Histologically,increasedosteogenesis,earlyandbetterreorganizationofcancellousbone,andmorebone
marrowformationwerediscernibleintreatmentgroupsascomparedtothecontrolgroup.Invitrocultureexpanded
allogenicandautogenicBMMSCsinducedsimilarbutfasterandbetterhealingascomparedtocontrol[148].
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Thenewestapproachintissueengineering:threedimensionalprinting
Threedimensionalprinting(3DP)isarapidprototypingtechniquethatcancreatecomplex3Dstructuresbyinkjet
printingofaliquidbinderontopowderbiomaterialsfortissueengineeringscaffolds.Directfabricationofscaffolds
from3DP,however,imposesalimitationonmaterialchoicesbymanufacturingprocesses.Noveladditive
manufacturingprocessesareincreasinglyrecognizedasidealtechniquestoproduce3Dbiodegradablestructures
withoptimalporesizeandspatialdistribution,providingadequatemechanicalsupportfortissueregenerationwhile
shapingingrowingtissues[149].Withregardtothemechanicalandbiologicalperformancesof3Dscaffolds,pore
sizeandgeometryplayacrucialrole.Domingosetal.[150]usedanovelintegratedautomatedsystemfor
productionandinvitrocultureof3Dconstructs,knownasBioCellprinting,tomanufacturepoly(caprolactone)
scaffoldsfortissueengineering.TheresultsclearlydemonstratedthepotentialoftheBioCellprintingprocessto
produce3Dscaffoldswithreproduciblewellorganizedarchitecturesandtailoredmechanicalproperties.More
recently,Billietetal.[151]reportedonthecombinedeffortsofmaterialchemistry,engineering,andbiologyasa
systemicapproachforthefabricationofhighviability3Dprintedmacroporousgelatinmethacrylamideconstructs.
Leeetal.[152]reportedanindirect3DPapproachwhereinapositivereplicaofdesiredshapeswasprinted,using
gelatinparticles,andthefinalscaffoldwasdirectlyproducedfromtheprintedmold.Toproducepatientspecific
scaffoldsthatmatchpreciselyapatient'sexternalcontours,theauthorsintegratedtheirindirect3DPtechniquewith
imagingtechnologiesandsuccessfullyproducedcustomscaffoldsmimickinghumanmandibularcondyleusing
polycaprolactoneandchitosanforpotentialosteochondraltissueengineering.Totesttheabilityofthetechniqueto
preciselycontroltheinternalmorphologyofthescaffolds,theyproducedorthogonalinterconnectedchannelswithin
thescaffoldsusingcomputeraideddesignmodels.Becauseveryfewbiomaterialsaretrulyosteoinductive,they
modifiedinert3Dprintedmaterialswithbioactiveapatitecoating.Thefeasibilityofthesescaffoldstosupportcell
growthwasinvestigatedusingbonemarrowstromalcells(BMSC).TheBMSCsshowedgoodviabilityinthe
scaffolds,andtheapatitecoatingfurtherenhancedcellularspreadingandproliferation.Thistechniquemaybe
valuableforcomplexscaffoldfabrication.
Theabilitytothreedimensionallyinterweavebiologicaltissuewithfunctionalelectronicscouldenablethecreation
ofthebionicorganspossessingenhancedfunctionalitiesovertheirhumancounterparts.Mannooretal.[153]
presentedanovelstrategytoovercomethesedifficultiesviaadditivemanufacturingofbiologicalcellswith
structuralandnanoparticlederivedelectronicelements.Asaproofofconcept,theygeneratedabionicearvia3D
printingofacellseededhydrogelmatrixintheanatomicgeometryofahumanear,alongwithanintertwined
conductingpolymerconsistingofinfusedsilvernanoparticles.Thisallowedforinvitroculturingofcartilagetissue
aroundaninductivecoilantennaintheear,whichsubsequentlyenablesreadoutofinductivelycoupledsignalsfrom
cochleashapedelectrodes.Theprintedearexhibitedenhancedauditorysensingforradiofrequencyreception,and
complementaryleftandrightearscouldlistentostereoaudiomusic.Overall,theirapproachsuggestsameansto
intricatelymergebiologicandnanoelectronicfunctionalitiesvia3Dprinting.
Genetherapy
Genetherapyconsistsoftransferofgeneticinformationtothetargetcellsandmayintroduceasafeandeffective
strategytoinducebonehealing.Genetherapycanbeusedfordeliveryofgrowthfactorsintissueengineering[1,2].
Thevehicleforgenedeliverycanbeeitherviral(adenovirus,retrovirus,andadenoassociatedvirus)ornonviral
(liposomes)[22,154].However,thisapproachhasaseriesoflimitations,includingtransinfectionofthetargetcells
withtheforeigngenes[1,154].Furthermore,anunresolvedissueofgenetherapyistotargettherightgeneatthe
rightlocationintherightcellsandexpressitforsufficientlylongattherighttime,whileminimizingadverse
reactions[155].Ashortcontrolledexpressionisdesirableandoftensufficienttoacceleratebonehealing,while
achievingpermanentorlongtermexpressionofatherapeuticgeneismoredifficult.Therefore,providingcontrolled
andsufficientexpressionbyadaptationofgenetherapytotissueengineeringisakeyandcriticalaspectinthisfield
[61,156].
Commercialbonegraftsubstitutes
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Theidealbonegraftsubstituteisbiocompatible,biodegradable,osteoconductive,osteoinductive,structurally
similartobone,easytouse,andcosteffective.Withintheseparameters,agrowingnumberofbonegraft
alternativesarecommerciallyavailablefororthopedicapplications,includingreconstructionofcavitarybone
deficiencyandaugmentationinsituationsofsegmentalbonelossandspinefusion.Theyarevariableintheir
compositionandtheirclaimedmechanismsofaction[70].BasedontheFDAandAmericanAcademyof
OrthopedicSurgeons'reportsandthemanufacturers'information,manybonegraftsubstituteshavebeenapproved
forclinicaluse.Thislargevariabilityandoptionsmakeithardtoselectagraftwhenreconstructionofaninjured
boneisapurpose.AsummaryofcommerciallyavailablebonegraftsubstituteshasbeenprovidedinTables5,6,7
and8.
Table5
Commerciallyavailablebonegraftsubstitutes:part1
Table6
Table7
Table8
BasedonthedataprovidedinTables5,6,7and8,mostofthebonegraftsubstituteshavebeenlicensedunder
FDA510(k)program,butsomeofthemhavebeenregulatedunderCFR1270and1271programsinadditionto
510(k).ManufacturersofAlloSource,BiometOsteobiologics,DePuySpine,Exactech,Integra
Orthobiologics/(IsoTisOrthoBiologics),LifeNetHealth,MedtronicSpinal&Biologics,MTF/Synthes,
NovaBone/MTF,Orthovita,Osteotech,RegenerationTechnologies,Smith&Nephew,StrykerBiotech,Synthes,
WrightMedicalTechnology,andZimmeraresomeexamplesofcompaniesthathaveproducedseveral
commerciallyavailablebonegraftsubstitutesapplicableinclinicalpractice.
Despitemanyadvancesintissueengineeringtechnologies,mostofthecommerciallyavailablebonegraft
substitutesarenaturalbasedmaterialsthathavebeenusedandprocessedwiththecadaverallograftandxenograft
origins.
Regardingtheavailableproducts,DBM,themostpopularoption,hasbeenextensivelyproducedandisavailable
onmarket.DBMhasbothosteoconductionandosteoinductionandisbiodegradable,makingitanidealgraft
substitute.Itisavailableasinjectablepasteandputty,graft,gel,crunch,andflexandoftenisconjugatedor
embeddedwithcollagentypeI,alginate,gelatin,sodiumhyaluronate,glycerol,starch,andcalciumsulfate.
RegardlessoftheDBMcarrier,thematerialcanalsobemixedwithbonemarrowaspiratepriortosurgery.The
secondwellknownoptionisthecalciumphosphategroupincludingmono,biandtricalciumphosphate.Theyare
oftenavailableinconjugationwithcollagentypeIandcarboxymethylcellulose,andallofthemareosteoconductive
andbiodegradableproductsasinjectablepaste,moldableputty,andvarioussizedpellets.rhBMP2andrhBMP7
proteinsonanabsorbablecollagenspongeareotherbiodegradableoptionswithsomeosteoinductive
characteristics.Commercialbovinebonematrix,allograftbonematrix,calciumsulfate,andbioactivesilicateare
otheravailableoptionswithvariableosteoconductivityandosteoinductivity.
Discussion
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Discussion
Goto:
Todesignandproduceanefficientbonegraft,theresearchersandorthopedicsurgeonsshouldhavesufficient
knowledgeofthecharacteristicsofgraftssuchasosteogenesis,osteoinductivity,andosteoconductivity,andtheir
otheradvantagesanddisadvantages.Autograftsarethegoldstandardforboneregeneration.Amongtheavailable
strategiestoimprovefracturehealingandenhancetheoutcomeofincorporationofthegrafts,tissueengineeringisa
suitableoption[9,10,17].Anidealtissueengineeredproductshouldhavecharacteristicssimilartothoseof
autograftswithouttheirlimitations[17].
Thebonescaffoldsshouldadditionallybehighlyporousandhaveporesofsuitablesizesatalllocationsofthe
scaffoldtoprovideanoptimalenvironmentfornewbonematrixandboneregeneration[17].Furthermore,growth
factorssuchasbasicfibroblasticgrowthfactor[74,157,158]whichmayaffectcellfunctions,proliferation,or
differentiationhealingpromotiveagentssuchashPRP[15,35,136]andalsoTarantulacubensisextract[134,159]
canbeincludedinthescaffoldstoenhancethehealingperformanceoftheinjuredconnectivetissues.Agentssuch
asglycosaminoglycans,includinghyaluronicacid,chondroitin,anddermatansulfatehavemodulatoryrolesinbone
andfracturehealingandcanimprovethequalityofthetissueengineeredscaffolds[17,159].Morerecently,
zoledronate,simvastatin,andalendronatehavebeenshowntohavepromisingeffectsonbonehealingand
regeneration[128,132,133].
Thevascularityofthescaffoldiscriticalbecauseifnotpresent,thescaffoldwillundergoischemiaandthecellswill
die.Therefore,applicationofgrowthfactorssuchasVEGF,PDGF,andFGFcanbeusefultostimulate
angiogenesisinthescaffoldsandthegrafts[32,34].Acombinationofstemcellswithscaffoldsandhealing
promotivefactors,especiallythegrowthfactors,couldbeonepossiblestrategyprovidingallthenecessary
characteristicsforbonerepairandregeneration.
Noneoftheappliedgraftshasallthedesirablecharacteristicssuchasbiologicalsafety,lowdonormorbidity,no
sizerestriction,longshelflife,efficientcostandosteogenic,osteoinductive,osteoconductive,andangiogenic
properties.Tissueengineeringattemptstoprovidemostorallofthesefeatures[18,69].Tissueengineeringisalso
abletoinducerepairandreconstructionofbonedefects[17].Combiningthefundamentalsoforthopedicsurgery
withknowledgefromvarioussciencessuchasengineering,biology,chemistry,physics,andmaterialssciencecan
overcomethelimitationsofcurrenttherapies[9].Advancesintissueengineeringandbiomaterialswillprovide
moresuitabletoolstopromotethemigration,proliferation,anddifferentiationofbonecellsandenhancebone
fracturehealing.Severalproblemsremainthatlimitwideutilizationofsuchoptions,includingregulatory
requirements,highcosts,lackofrandomizedcontrolledhumanandanimalstudies,uncertainlongtermresults,and
methodspecificlimitations.Althoughtheliteraturecontainsalargenumberofstudiesontheeffectsofvarious
agentsonbonehealing,itstillisunclearwhichthebestoptionis.Nevertheless,autograftremainsthegoldstandard
ofgrafting[12,20].
Thenearfutureofbonehealingandregenerationiscloselyrelatedtoadvancesintissueengineering.Perhaps
polytherapybyusingscaffolds,healingpromotivefactors,andstemcellstogetherwiththenewadvancesinthree
andfourdimensionalprintingoftissueengineeredconstructswouldbeabletosolvethecurrentlimitationsin
managingboneinjuries.
Conclusion
Goto:
Bonegraftingisoneofthemostcommonlyusedoptionstotreatlargebonedefects.Autograftsremainthegold
standard.Allografts,xenografts,andtissueengineeredbasedgraftsallhaveshortcomings.Newstrategiessuchas
genetherapy,polytherapybyusingscaffolds,healingpromotivefactorsandstemcells,andfinallythree
dimensionalprintingareintheirpreliminarystagesbutmayoffernewexcitingalternativesinthenearfuture.
Competinginterests
Goto:
Theauthorsdeclarethattheyhavenocompetinginterests.
Authorscontributions
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Authorscontributions
Goto:
Allauthorshadequalcontributioninallpartsofthestudy.AO,SA,AM,andNMdesignedthestudyand
participatedindatacollection,manuscriptpreparation,andrevision.Allauthorsreadandapprovedthefinal
manuscript.
References
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