组织工程骨血管化策略ppt课件.ppt

VascularizationinBoneTissueEngineering Physiology CurrentStrategies MajorHurdlesandFutureChallenges dw20110809 1 期刊 MacromolecularBioscience 影响聚合物和生物材料领域的优秀期刊MacromolecularBioscience涉及再生医学 组织工程 给药和生物医药领域 是聚合物 材料科学和生物医药学交叉科学领域的先进杂志 在2010年 庆祝创刊10年之际 MBS杂志的引用率比2009年增加了20 Macromol Biosci 影响因子 3 298Macromol Biosci 2010 10 12 27 2 作者 3 abstract Thelackofafunctionalvascularsupplyhas toalargeextent hamperedthewholerangeofclinicalapplicationsof successful laboratory basedbonetissueengineeringstrategies Tothepresent graftshavebeendependentonpost implantvascularization whichjeopardizesgraftintegrationandoftenleadstoitsfailure Forthisreason thedevelopmentofstrategiesthatcouldeffectivelyinducetheestablishmentofamicrocirculationintheengineeredconstructshasbecomeamajorgoalforthetissueengineeringresearchcommunity Thisreviewaddressestheroleandimportanceofthedevelopmentofavascularnetworkinbonetissueengineeringandprovidesanoverviewofthemostuptodateresearcheffortstodevelopsuchanetwork 4 Introduction Oneofthemostwidelytestedtissueengineeringapproachfortherepairandregenerationofbonedefectshasbeentheinvitrocultureofathreedimensional 3D scaffoldingmaterial seededwithautologouscells followedbyimplantationinthepatient Despiteitsenormouspotential thisregenerativestrategyisgenerallyassociatedwithamajorpitfall Whentheengineeredtissueconstructisimplanted theseededcellswillhavealimitedcapacitytobothuptakesubstratemolecules oxygen glucose andaminoacids andtoclearbyproductsofmetabolism CO2 lactate andurea 5 Introduction Theselimitationsimpaircellviabilitytosuchanextentthatithindersthesuccessoftheengineeredtissue Thelackofafunctionalmicrovasculatureconnectedtothehostbloodsupplyhasbeenidentifiedastheculpritforimplantfailureandiscurrentlyacknowledgedasthemajorchallengeintissueengineering 6 Introduction Boneisametabolicallyactivetissuesuppliedbyanintraosseousvasculaturewithosteocytesdistancingnomorethan100mmfromanintactcapillary Intheabsenceofavascularsupply thetransportofnutrientsoccursmainlybydiffusion atransportmechanismthatisonlyefficientovershortdistancesorfortissueswithlowmetabolicactivity e g cartilage Theoreticalmodelingpredictsthatacentimeterthickscaffoldwithoutavascularsupplycansupportthemetabolismof280000cellscm3withoutcentralnecrosis innativecancelousbone thisvalueis1000 foldhigher 7 Introduction Diffusionconstrainsofengineeredconstructsarealreadyanissueduringinvitroculture inwhichnutrienttransportisonlyassuredtocellsonthesuperficialareasofthescaffold thosecellsgrowingatagreaterdepthintheconstructfacenutrientdeprivationandultimatelycelldeath Fromaclinicalpointofviewthisisanimportantaspectbecauseresectionofbonetumorsortraumaoriginatelargeskeletaldefects whichrequirelargesegmentsofimplantmaterialtobegrafted Masstransferinagraft definedastheinandoutmovementofmolecules isaphenomenonhighlyimpairedbythethicknessoftheimplant 8 Introduction Invitrodeliveryofnutrientstoengineeredconstructscanbefurtherimprovedwithdynamiccultureusingbioreactorsystems 12 13 however thiswillinmostcasesonlydelayfurthertheproblemofmasstransfertotheinvivosituation Upongraftimplantation theformationofnewbloodvesselsfrompre existingones angiogenesis willoccurspontaneously Thisvascularresponseisinducedbyinflammationanditispartofthewound healinggeneratedbythehostasaresponsetotheischemia reperfusioninjuryformedduringsurgery However capillarynetworksinducedbyinflammatoryprocessesarerelativelytransientinnatureandwillregresswithinafewweeks 1 15 16 9 Introduction Inadditiontothecapillarynetworksformedduringwoundhealing neovascularizationofthescaffoldoccurs However theslowrateofinfiltrationofbloodvesselsintothescaffold 1mm d makesitaninsufficientprocesstovascularizetissuesofclinicallyrelevantsize Anotherproblemassociatedwiththelackofvascularizationistheremovalofdegradationproductsfrombiodegradablescaffolds Intheabsenceofafunctionalvasculaturethecapacityofthesurroundingtissuetoeliminatethedegradationproductsderivedfromtheimplantmaterialisverylow Consequently thereisamassivereleaseofby productsthataccumulatelocallyandtriggerinflammatoryresponses 19 10 Introduction Thechallengefollowingthedevelopmentofmicrocirculationintheengineeredconstructistoconnectittothehosts systemiccirculation aphenomenondesignatedasinosculationoranastomose 20 Inosculationbetweenthehost sandconstruct svasculatureisnotanimmediateprocessandmaytakeuptoeightdays thismayleadtoischemiaandahostileenvironment 21 Consequently thespontaneouspost implantationneovascularizationfromthehostisnotsufficienttoassureimplantintegration Thiscreatesaneedfornewstrategiestoacceleratetheonsetofneovascularization 11 Introduction Thefocusofthecurrentreviewisonvascularization inthecontextofbonetissueengineering Itaimstocovertheintricateconnectionbetweenvascularizationandbone startingwiththedevelopment movingthroughremodeling andendingwithrepair Furthermore wewillreviewthemostrecentstrategiesproposedtoacceleratetheestablishmentofafullyfunctionalvascularnetworkwithinbone engineeredconstructs 12 IntraosseousVasculatureinBoneFormation Remodeling andFracture IntramembranousandEndochondralOssificationAsidefromassuringthenutrienttransportandremovalofwasteproducts intraosseousvasculatureaccomplishesotherimportantfunctionsthatrangefrombonedevelopment toremodelingandfracturerepair Dependingontheirorigin boneisformedbytwodistinctmodesofossification byintramembranousossification characteristicofflatbonessuchasthosefromskullandclavicle andbyendochondralossification whichisinvolvedinthedevelopmentofbonesfromloadbearingjoints 22 23 Despitethedifferences thetwotypesofossificationhaveascommonfeature thepre requisiteofvascularization 24 26 13 IntraosseousVasculatureinBoneFormation Remodeling andFracture Vasculaturealsoplaysanimportantroleonboneformationthroughtheproductionofgrowthfactorsthatcontroltherecruitment proliferation differentiation function and orsurvivalofvariouscellincludingbone formingosteoblastsandbone resorbingosteoclasts 26 14 IntraosseousVasculatureinBoneFormation Remodeling andFracture Thesebioactivefactorsaresecretedbyendothelialcells ECs thecelltypethatformstheinnerliningofbloodvessels 28 30 Therefore angiogenesisnotonlyprecedesosteogenesisbutitisalsorequiredforitsoccurrence 31 Thisisaccomplishedbyacombinationoffactors whichincludeadequateoxygentension compressionforces nutrients andgrowthfactors 32 15 IntraosseousVasculatureinBoneFormation Remodeling andFracture VascularOrganizationofBoneAdultlongboneissuppliedbyfourarterialinputs namedaccordingtotheirlocation nutrientarteryordiaphyseal periostealarteries metaphysealarteries andepiphysealarteries Thenutrientarteryisthelargestvesselandresponsibleformorethan50 oftotalbloodsupplytothelongbones 33 34 Assuggestedbythename periostealarteriessupplyperiosteum themembranethatcoverstheexteriorofbonesandisarichsourceofstemcells whichhasbeentestedinseveralregenerativestrategies 16 IntraosseousVasculatureinBoneFormation Remodeling andFracture BoneRemodelingTheinvadingvasculature therefore servesasbothareservoirandconduitfortherecruitmentofessentialcelltypesinvolvedinboneresorptionanddeposition regulatesthefunctionalactivitiesofsuchcells andprovideskeysignalsnecessaryforbonemorphogenesis 32 Insummary alterationsofthemicrovascularsupplynetworkwillultimatelyaffectthetightlyregulatedresorptionsequencetoresultinadecreaseinboneformation regeneration andrepair 17 IntraosseousVasculatureinBoneFormation Remodeling andFracture FractureRepairFracturerepairisacomplexregenerativemechanismasevidencedbythedistinguishableprocessesthatitinvolves suchastheimmediateresponsetoinjury intramembranousboneformation endochondralboneformation andboneremodeling Whenboneisinjurednotonlyskeletalintegrityiscompromisedatthefracturesite butintraosseousvasculatureisclearlydisrupted 22 Firstlyahematomaisformed secondly thefracturemilieubecomeshypoxicbecauseofthedisruptionofoxygensupply 18 IntraosseousVasculatureinBoneFormation Remodeling andFracture HypoxiaisanimportantphysiologicalsignalinbonerepairbecauseitregulatestheproductionofkeymodulatorsbyosteoblaststhatinfluenceECproliferation 32 directcellulardifferentiation andinduceECstosecretosteogenicgrowthfactors 46 47 Inthehematomaphase thefirstoutoffouroverlappingphasesthatcharacterizebonerepair anormalhealingresponseistriggered TheinflammatoryresponseandthehypoxicenvironmentareassociatedwiththereleaseofseveralgrowthfactorsandcytokinesthattriggerECmigrationandtheformationofnewcapillaries 26 48 19 IntraosseousVasculatureinBoneFormation Remodeling andFracture Infact thereconstructionofintraosseouscirculationisoneoftheearliesteventsduringbonerepair 49 50 Meanwhile thehypoxicmicroenvironmentsupportsthedifferentiationofMSCsintochondrocytesthatstabilizethefracturebycartilageformation knownasaninternalcallus 14 Theperiosteumthendirectlyundergoesintramembranousboneformation whichleadstotheformationofanexternalcallus Inthenextphase hardcallusformation theinternalcallusbecomesmineralizedandformsahardcallusofwovenbone Finally intheremodelingphase thecallusisreplacedbylamellarbone 48 20 IntraosseousVasculatureinBoneFormation Remodeling andFracture Externalfactorssuchasthebiomechanicalenvironmentalsoinfluencetheboneregenerativeprocessbyaffectingangiogenesisandconsequentlycelldifferentiation 51 52 Inafixatedfracturethevascularnetworkisrapidlyreestablishedandhealingoccursmainlybyintramembranousossification 51 52 Conversely inanunstablemechanicalenvironmentthespreadingcapillariesaredisruptedandthehypoxicenvironmentpromotesthedifferentiationofchondrocytesthatstabilizethefracturebycartilageformation 14 Lackofangiogenesishasbeenpointedout 51 asoneofthemainreasonsfornon healingbone Forinstance usingaratdistractionosteogenesismodel Fangetal 53 haveshownthattheadministrationofananti angiogenicdrugpreventednormalosteogenesis whichresultedinafibrousnon union 21 HeterotypicCommunicationbetweenOsteoblastsandEndothelialCells Consideringtheintricateconnectionbetweenangiogenesisandosteogenesis itisnotsurprisingthatcommunicationbetweenosteoblastsandECsisoneofthemostimportantcellularinteractionsthatorchestrateboneformation 32 54 Thecross talkbetweenosteoblastsandECsoccursattwolevels 1 byindirectcellcontact 55 56 throughthereleaseofsolublefactorswithparacrineandautocrineaction and2 bydirectcell cellcontact 57 mediatedbyproteinsatgapjunctions Oneofthemoststudiedgrowthfactorsisvascularendothelialgrowthfactor VEGF 49 58 59 apotentandspecificangiogeniccytokineproducedatthefracturesitebynumerouscelltypes includingosteoblasts 22 HeterotypicCommunicationbetweenOsteoblastsandEndothelialCells BesidesbeinganEC specificmitogen VEGFalsoinducesincreasedvascularpermeabilityandmonocytemigrationthroughendotheliallayers 60 TheactionofVEGFisnotlimitedtoECs andtherearesomestudiesreportingthatosteoblastsalsorespondtomembersoftheVEGFfamily 61 63 Arecentstudy 24 hasshedsomelightonthecellularandmolecularmechanismsresponsibleforcontrollingVEGF dependentosteoblast ECscrosstalkbyconfirmingthatVEGFisreleasedpredominantlybyhumanosteoblastsanditsprimaryactionisbyECs Anotherangiogenicgrowthfactor fibroblastgrowthfactor FGF isalsoproducedbyosteoblastsamongothercelltypes 64 65 FGFfunctionsasaparacrinefactortostimulateECproliferationandmigration 65 andasanautocrinefactortoinducetheproliferationanddifferentiationofosteoblasts 22 23 HeterotypicCommunicationbetweenOsteoblastsandEndothelialCells Furthermore FGF 2exertsitsangiogeniceffectindirectlybymodulatingVEGFexpressionthroughitsactionoverosteoblasts 66 OncethecrosstalkbetweenECsandosteoblastsisbidirectional ECsalsosecretnumerousregulatorymoleculesthatexertmajoreffectsincontrollingthedifferentiationandactivityofbone formingcells 32 Bonemorphogenicprotein 2 BMP 2 andendothelin 1 ET 1 aretwoofthegrowthfactorsproducedbyECsthatpromoteosteoblasticproliferationanddifferentiation 67 69 24 HeterotypicCommunicationbetweenOsteoblastsandEndothelialCells Inaddition platelet derivedgrowthfactorBB PDGF BB releasedatthefracturesitebyseveralcelltypesthatincludeECs hasamitogenicandchemotacticeffectoverosteoblasts 70 71 OsteogenicgrowthfactorsproducedbyECs BMP 2 PDGF BB andbyosteoblasts insulin likegrowthfactor IGF andtransforminggrowthfactor TGF alsohaveanangiogeniceffectbyinducingVEGFmRNAexpressioninosteoblasts 70 72 74 VeilletteandSchroeder 75 proposedaninhibitoryroleofET 1onVEGFsynthesisinosteoblasticcellsasafeedbackmechanisminthetemporalandspatialcouplingofangiogenesistoboneformationandresorption 25 HeterotypicCommunicationbetweenOsteoblastsandEndothelialCells Externalfactorssuchasthehypoxicmicroenvironmentoffracturehealingstimulatetheexpressionofavarietyofcytokinesfrominflammatorycells ECs osteoblasts andfibroblasts 76 ECsandosteoblastsrespondtohypoxiabyupregulatingtheexpressionofnumerousosteogenicfactors ET 1 PDGF BB BMP 2 IGF II andTGF b1 andoftheangiogenicgrowthfactorVEGF 68 76 80 Themechanismunderlyingtheresponseofcellstothehypoxicmicroenvironmentisprimarilymediatedthroughgeneswhoseexpressioncontainahypoxia induciblefactor 1 HIF 1 bindingsite 81 82 Nevertheless ithasbeenreported 76 79 83 thatthisenvironmentoflowoxygentensionhasnoeffectontheexpressionofgrowthfactorssuchasFGF 2andmembersofIGFandTGFfamilies 26 HeterotypicCommunicationbetweenOsteoblastsandEndothelialCells Thefactthathypoxiaisadrivingfactorforthecreationofagrowthfactorrichmilieuinspiredresearcherstotestitspotentialbenefitsinengineeredtissues Severalstudieshaveaddressedoxygentensionasanimportantvariableinoptimizinginvitroconditionsforstemdifferentiation 84 87 whileothers 45 havehypothesizedthatadaptingthegrafttohypoxiapriortoengraftmentmightinduceangiogenesisafterimplantation Itisobviousthatinordertoavoidthenoxiouseffectsoflowoxygentension cultureconditionsmustbewelldefined Ontheonehand 48hofculturedonothaveasignificanteffectoncelldeath 88 ontheotherhand long termcultureswillhaveanegativeeffectonboneformationpartlybecauseofdecreasedosteoblastproliferation 89 27 StrategiestoIncrementVascularization Inordertoacceleratetheestablishmentofafunctionalvascularnetworkinboneengineeredtissuesseveralstrategieshavebeenproposed Figure1 Thefollowingsectionswillreviewthemainapproaches theirprinciples outcomes andlimitations 28 StrategiestoIncrementVascularization ScaffoldArchitectureEngineeredbonemustassurethemechanicalstabilityoftheosseousdefect whilesimultaneouslystimulatingthehealingcapacityofthetissue Thevastmajorityofboneregenerationstrategieshavebeencenteredonthescaffoldmaterial however withinthelastfewyearsaconceptualshifthasbeentakingplaceinthedevelopmentofscaffoldsforboneengineering fromasupportroleforbone formingcells toascaffoldthathomesavascularnetwork 29 StrategiestoIncrementVascularization Regardingthebulkpropertiesofascaffoldingmaterial porosityhasbeenoneofthemostdiscussedissues 93 95 butwithafocusmainlyonosteoblastproliferation matrixdeposition andcalcificationratherthantheinpenetrationofvasculature RecentworkfromNarayanandVenkatraman 96 evaluatedtheeffectofporesizeandinterporedistanceoverECsgrowthon3Dpolymericscaffoldsandfoundthatcellgrowthwasenhancedbyasmallerporesize 5 20mm andlowerinterporedistance Asimilarbehaviorwasalsoreportedforosteoblasts wherelowerporositystimulatesosteogenesisbysuppressingcellproliferationandforcingcellaggregationinvitro 95 However theinvivoscenarioiscompletelydifferentandahigherporosityandporesizeresultingreaterbonein growthandvascularization 95 97 30 StrategiestoIncrementVascularization Thisisexplainedbythefactthatwhenimplanted scaffoldswithsmallerporestendtobehypoxic favoringchondrogenesis whereasinconstructswithlargerporesthehigheroxygentensionpromotesthedifferentiationofMSCsintoosteoblastlineage favoringosteogenesis 31 StrategiestoIncrementVascularization HierarchicalStructuresThedesignandarchitectureofthescaffoldaretwootherfeaturescriticalfortheformationofavascularnetwork Oneexampleofaninnovativearchitectureisthenano microfiber combinedscaffold Figure2a 98 Thisscaffoldmadefromablendofstarchwithpolycaprolactone SPCL 13 94 99 101 combinesinthesamestructuremicroandnanofibers Microfibersobtainedbyfiberbondingwerecombinedwithnanofibersproducedbyelectrospinninginasinglestructure Inthiswaythescaffoldmaterialiscapableofsimultaneouslyprovidingthemechanicalsupportforbonerepairandmimickingthephysicalstructureoftheextracellularmatrix ECM 32 StrategiestoIncrementVascularization Aswillbedescribedlateringreaterdetail strategiesthatincludeseedingECsonbiomaterialsandpromotingtheiradhesion migration andfunctionalitymightbeasolutionfortheformationofvascularizedbone Therefore thenanonetworkthatresemblestheECMphysicalstructureonSPCLnano microfiber combinedscaffoldswasdesignedwiththeaimtopromoteECmigration andtoestablishavascularnetwork Underpro angiogenicconditionsinvitro thisnanonetworkprovidedthestructuralandorganizationalstabilityforthemigrationandorganizationofECsintocapillary likestructures 102 33 StrategiestoIncrementVascularization Alternatively Yangetal 105 usedextrusionfreeforming arapidprototypingtechniquetofabricateceramicscaffoldswiththreedistinctstructurelevels submicrometerpores aimedtoenhancecell surfaceinteractions poresoftensofmicrometerstoencourageboneingrowth andcorridors hundredsofmicrometers forvascularization AnotherratherinterestingconcepthasbeenrecentlyintroducedbyBellanetal 106 whopromotedtheconstructionofavascularnetworkusingsacrificialsugarstructures Briefly macro andmicrofibersmadeofsugarwereproducedbymelt spinningandincludedinapolymericmatrix suchaspoly dimethylsiloxane PDMS Thestructureisthensoakedinwaterandthesugar fiberssolubilized leavinginthepolymericmatrixmacrochannelsthatcanbeattachedtoabloodsupplyandmicrochannelswithdiametersclosetothatofcapillaries 34 MicrofabricationofNetworkswithVascularGeometry Anotherinterestingconceptistheinclusionofanetworkwithavasculargeometryinabiocompatiblepolymer Thisisachievedbymicrofabricationtechniquesthatcompriseafullrangeofprocessesandtoolsoriginallydevelopedforapplicationsinthemicroelectronics automotive aerospace anddefensefields 107 Photolithography aprocessbasedonsemiconductorwafertechnologies hasbeenattractingattention Thismethodisatwo stepprocesswhereamoldisfirstproduced generallymadeofsilic