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Methods in Molecular Biology 2598 Martin J. Stoddart Elena Della Bella Angela R. Armiento Editors C artilage Tissue Engineering M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Cartilage Tissue Engineering Edited by Martin J. Stoddart and Elena Della Bella AO Research Institute Davos, Davos Platz, Switzerland Angela R. Armiento UCB Pharma, Slough, UK Editors MartinJ.Stoddart ElenaDellaBella AOResearchInstituteDavos AOResearchInstituteDavos DavosPlatz,Switzerland DavosPlatz,Switzerland AngelaR.Armiento UCBPharma Slough,UK ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2838-6 ISBN978-1-0716-2839-3 (eBook) https://doi.org/10.1007/978-1-0716-2839-3 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. CoverIllustrationCaption:Scaffold-filledosteochondraldefect. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface Therepairoftraumaticcartilageinjuriesisoneofmedicine’sremainingdilemmas,withfew clinicaltreatmentoptionsavailable.Currenttreatmentsincludedebridement,microfracture, and various implementations of autologous chondrocyte implantation, yet the results are largelyunsatisfactoryoverthemediumterm(5yearsonwards).Forthisreason,thereisstill significant research being performed on novel therapies for cartilage regeneration. Within this methods book, we attempt to bring together a collection of methodologies, from the mostbasictomorecomplex,thatprovideresearcherswithaplatformtheycanusetoembark on a cartilage research career. One of the factors delaying research on cartilage is likely the fact that 3D cartilage models are more challenging to work with compared to cells in monolayer. Such challenges are associated with RNA and protein extraction, imaging, gene transfer, and for some laboratories even access to such tissues. Stable differentiation and variations in cell phenotype from different tissue origins adds to the complexity. As technologyevolves(omics,bioinformatics,datamining,organ-on-a-chip,tocitebutafew) and scientists have a better appreciation of the relevant in vitro and in vivo models, the researchoncartilageismoreexcitingthanever. DavosPlatz,Switzerland MartinJ.Stoddart DavosPlatz,Switzerland ElenaDellaBella Slough,UK AngelaR.Armiento v Contents Preface ..................................................................... v Contributors................................................................. xi 1 CartilageTissueEngineering:AnIntroduction...... ........ ....... ........ 1 MartinJ.Stoddart,ElenaDellaBella, andAngelaR.Armiento 2 ChondrocyteIsolationandExpansion....... ....... ........ ....... ........ 9 PaulHarrison,TimothyHopkins,CharlotteHulme, HelenMcCarthy,andKarinaWright 3 IsolationofChondronsfromHyalineCartilage...... ........ ....... ........ 21 JasmijnV.Korpershoek,MargotRikkers, andLucienneA.Vonk 4 ArticularCartilageChondroprogenitors:Isolation andDirectedDifferentiation ........ ....... ..... .. ...... .. ...... ... ...... 29 IlyasM.Khan,JoshuaMcKenna,andYadanZhang 5 PhysioxicCultureofChondrogenicCells .... ....... ........ ....... ........ 45 GirishPattappa,BrandonD.Markway, DenitsaDocheva,andBrianJohnstone 6 IsolationandInVitroChondrogenicDifferentiationofHumanBone Marrow-DerivedMesenchymalStromalCells........ ........ ....... ........ 65 AngelaR.Armiento,YannD.Ladner,ElenaDellaBella, andMartinJ.Stoddart 7 Adipose-DerivedStromalCells:Isolation,Expansion,andDifferentiation ..... 75 JustynaM.Buchert,BenedictLotz,SolvigDiederichs, andWiltrudRichter 8 ChondrogenicDifferentiationofHuman-InducedPluripotent StemCells ......... ....... ....... ... ..... ....... ........ ....... ........ 87 AmandaR.Dicks,NancySteward,FarshidGuilak, andChia-LungWu 9 TheDimethylmethyleneBlueAssay(DMMB)for theQuantification ofSulfatedGlycosaminoglycans...... ....... ....... ........ ....... ........ 115 YannD.Ladner,MauroAlini,andAngelaR.Armiento 10 ExtracellularVesicleIsolationandCharacterizationforApplications inCartilageTissueEngineeringandOsteoarthritisTherapy.......... ........ 123 AlexanderOtahal,AndreaDeLuna,AliMobasheri, andStefanNehrer 11 MetabolomicProfilingtoUnderstandChondrocyteMetabolism ..... ....... . 141 PriyankaP.Brahmachary,HopeD.Welhaven, andRonaldK.June vii viii Contents 12 TechniquesforVisualizationandQuantificationofPrimaryCilia inChondrocytes.... ....... ........ ....... ....... ........ ....... ........ 157 HuanMeng,ClareL.Thompson,ClarissaR.Coveney, AngusK.Wann,andMartinM.Knight 13 Co-cultureofHumanArticularChondrocytesSeededinPolyurethane ScaffoldsandHumanMesenchymalStromalCellsEncapsulated inAlginateBeads .......... ........ ....... ....... ........ ....... ........ 177 TeresaZ.Brose,YannD.Ladner,E.JohannaKubosch, MartinJ.Stoddart,andAngelaR.Armiento 14 IsolationofMurineArticularChondrocytesforSingle-CellRNA orBulkRNASequencingAnalysis ...... .... ...... ....... .. ..... .. ........ 187 JillianL.McCool,NicholasR.Hum,AimySebastian, andGabrielaG.Loots 15 ModulationofMicroRNAExpressionDuringInVitroChondrogenesis....... 197 AustinBell-Hensley,HongjunZheng,andAudreyMcAlinden 16 PurificationandIsolationofProteinsfromHyalineCartilage......... ........ 217 MakennaJ.Hardy,XinzhuPu,andJuliaThomOxford 17 HistologicalPreparationandEvaluationofCartilageSpecimens...... ........ 227 SalimE.Darwiche,MilenaTegelkamp,KatjaNuss, andBrigittevonRechenberg 18 ArticularCartilageImagingintheContextoftheSuperficialChondrocyte SpatialOrganization(SCSO)asaSurrogateMarker forFunctionalPathology ........... ....... ....... ........ ....... ...... .. 265 BodoKurz,ThomasLange,MaritaVoelker, andBerndRolauffs 19 UnconfinedCompressionExperimentalProtocolforCartilageExplants andHydrogelConstructs:FromSamplePreparation toMechanicalCharacterization...... ....... ....... ........ ....... ........ 271 SeyedAliElahi,RocıóCastro-Vin˜uelas,AnkeGovaerts, RikLories,NeleFamaey,andIlseJonkers 20 ViralGeneDeliveryinChondrocytes........ ....... ........ ....... ........ 289 ChristopherV.Nagelli,ChristopherH.Evans, andRodolfoE.DeLaVega 21 DecellularizationofArticularCartilage:AHydrochloricAcid– BasedStrategy...... ....... ........ ...... ....... ......... ....... ........ 301 CorneliaSchneiderandSylviaNu¨rnberger 22 Self-AssemblyCultureModelforEngineeringMusculoskeletalTissues........ 313 NicholasN.DePhillipo,JerahmeMartinez, andGeorgeR.Dodge 23 OsteochondralExplantIsolationandCultureUnderaCompression andShearBioreactor....... ........ ....... ....... ........ ....... ........ 325 M.LetiziaVainieriandSibylleGrad 24 AMouseModelofAcuteCartilageInjuryandRepair........ ....... ........ 337 Anne-SophieThorup,FrancescoDell’Accio, andSuzanneE.Eldridge Contents ix 25 Small-AnimalCompressionModelsofOsteoarthritis......... ....... ........ 345 BlaineA.Christiansen,DevaD.Chan, MarjoleinC.H.vanderMeulen,andTristanMaerz 26 ModelingandAssessingOsteoarthritisinMicebyDestabilization oftheMedialMeniscus(DMM)..... ....... ....... ........ ....... ........ 357 JadwigaMiotla-Zarebska,IdaParisi,VickyBatchelor, ToniaL.Vincent,andPaulK.Potter 27 Collagenase-InducedOsteoarthritisinMice......... ........ ....... ........ 375 PeterM.vanderKraan 28 Platelet-RichPlasmaforIntra-articularInjections:Preclinical andClinicalEvidence....... ........ ... .... ....... ........ ....... ........ 381 AngeloBoffaandGiuseppeFilardo Index ...................................................................... 391 Contributors MAUROALINI • AOResearchInstituteDavos,DavosPlatz,Switzerland ANGELAR.ARMIENTO • AOResearchInstituteDavos,DavosPlatz,Switzerland;UCB Pharma,Slough,UK VICKYBATCHELOR • CentreforOsteoarthritisPathogenesis,KennedyInstituteof Rheumatology,UniversityofOxford,Oxford,UK AUSTINBELL-HENSLEY • DepartmentofBiomedicalEngineering,WashingtonUniversity,St Louis,MO,USA ANGELOBOFFA • AppliedandTranslationalResearchCenter,IRCCSIstitutoOrtopedico Rizzoli,Bologna,Italy PRIYANKAP.BRAHMACHARY • DepartmentofMechanical&IndustrialEngineering, MontanaStateUniversity,Bozeman,MT,USA TERESAZ.BROSE • MedicalCenter-Albert-Ludwigs-University,DepartmentofOrthopedics andTraumaSurgery,FacultyofMedicine,Albert-Ludwigs-UniversityofFreiburg, Freiburg,Germany JUSTYNAM.BUCHERT • ResearchCentreforExperimentalOrthopaedics,Heidelberg UniversityHospital,Heidelberg,Germany ROCIÓCASTRO-VIN˜UELAS • HumanMovementBiomechanicsResearchGroup,Department ofMovementSciences,KULeuven,Leuven,Belgium;TissuehomeostasisandDiseaseGroup, SkeletalBiologyandEngineeringResearchCenter,DepartmentofDevelopmentand Regeneration,KULeuven,Leuven,Belgium DEVAD.CHAN • PurdueUniversity,WeldonSchoolofBiomedicalEngineering,West Lafayette,IN,USA BLAINEA.CHRISTIANSEN • UniversityofCaliforniaDavisHealth,Departmentof OrthopaedicSurgery,Sacramento,CA,USA CLARISSAR.COVENEY • KennedyInstituteofRheumatology,MedicalSciencesDivision, UniversityofOxford,Oxford,UK;DepartmentofHumanEvolutionaryBiology,Harvard University,Cambridge,MA,USA SALIME.DARWICHE • MusculoskeletalResearchUnit(MSRU),VetsuisseFaculty,University ofZu¨rich,Zu¨rich,Switzerland;Center forAppliedBiotechnologyandMolecularMedicine (CABMM),VetsuisseFaculty,UniversityofZu¨rich,Zu¨rich,Switzerland RODOLFOE.DELAVEGA • MusculoskeletalGeneTherapyLaboratory,MayoClinic, Rochester,MN,USA FRANCESCODELL’ACCIO • CentreforExperimentalMedicineandRheumatology,William HarveyResearchInstitute,BartsandtheLondonSchoolofMedicineandDentistry,Queen MaryUniversityofLondon,London,UK ELENADELLABELLA • AOResearchInstituteDavos,DavosPlatz,Switzerland ANDREA DELUNA • Center forRegenerativeMedicine,UniversityForContinuing Education,Krems,Austria NICHOLASN.DEPHILLIPO • UniversityofPennsylvania,DepartmentofOrthopaedic Surgery,McKayOrthopaedicResearchLaboratory,Philadelphia,PA,USA;Mechano- TherapeuticsLLC,Philadelphia,PA,USA AMANDAR.DICKS • DepartmentofOrthopaedicSurgery,WashingtonUniversity,St.Louis, MO,USA;ShrinersHospitalsforChildren–St.Louis,St.Louis,MO,USA;Departmentof xi