胶原-透明质酸-硫酸软骨素复合三维支架体外构建组织工程软骨的实验研究

22(collagen,Col)2(hyaluronicacid,HA)2(chondroitinsulfate,CS)2(32)Col2HA2CSColHECol2HA2CS,,;2DNA(glycosaminoglycan,GAG);RT2PCRCol2HA2CSColÊCol2HA2CS,ColÊ,21d,DNAGAGGAG,Col,(P0.05)Col2HA2CS,:R318.08R322.7:AAPOTENTIALUSEOFCOLLAGEN-HYALURONAN-CHONDROITINSULFATETRI-COPOLYMERSCAFFOLDFORCARTILAGETISSUEENGINEERINGöYANJihong,LIULingrong,LIXuemin,etal.InstituteofBiomedicalEngineering,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Tianjin,300192,P.R.ChinaCorrespondingauthor:ZHANGQiqing,E2mail:[e-mail]luatecollagen(Col)2hyaluronan(HA)2chondroitinsulfate(CS)tri2copolymerasanewbiomimeticbiodegradablepolymerscaffoldforapplicationofthearticularcartilagetissueengineering.MethodsTheCol2HA2CStri2copolymerwaspreparedbyfreezingandlyophilizationandwascross2linkedby12ethyl232(32dimethyinaminoproyl)carbodiimide(EDC).ThemorphologicalcharacteristicsofthematriceswereevaluatedbytheSMEandHEstainings.Therabbitchondrocyteswereisolatedandseededinthetri2copolymerscaffold.Morphology,proliferationanddifferentiationofglycosaminoglycan(GAG),andphenotypicexpressionoftherabbitarticularchondrocytesculturedwithinthetri2copolymerscaffoldwereindicatedbythehistologicalexamination,SEM,biochemicalanalysis,andreversetranscriptasePCRforcollagentypeÊ(ColÊ).ResultsThechondrocytesproliferatedanddifferentiatedwell,andtheypreservedthephenotypicexpressionofColÊintheCol2HA2CSscaffold.Afterthe212daycellculturewithintheCol2HA2CSscaffolds,thecartilage2specificmorphologyandthestructuralcharacteristicssuchaslacunaeappeared,andDNAandGAGcontentsincreasedwiththetime.Inaddition,DNAandGAGcontentsweresignificantlyhigherintheCol2HA2CSmatrixthaninthecollagenmatrixalone(P0.05).ConclusionTheseresultsshowthattheCol2HA2CStri2copolymermatricescanprovideanappropriateenvironmentforthegenerationofcartilage2liketissuesandhaveapotentialapplicationinthecartilagetissueengineeringscaffoldfield.KeywordsTissueengineeredcartilageCollagenHyaluronanChondroitinsulfateFoundationitems:NationalHighTechnologyResearchandDevelopmentProgramofChina(2002AA326040);NationalKeyBasicScienceResearchandDevelopmentProgramofChina(G19905430924):(863)(2002AA326040);(973)(G19990543094):(,300192):,,,:,E2mail:[e-mail],[123],[4],,,,,(collagen,Col),031ChineseJournalofReparativeandReconstructiveSurgery,2006,Vol20(2)(extracellularmatrix,ECM),,,[5](hyaluronicacid,HA)(chondroitinsulf2ate,CS)ECM(glycosaminogl2ycan,GAG),[6]ECM,Col,HACS,11.1Col2HA2CSColÉ(),0.3%0.6%,HA,1%HA,Col1%Col2HA,63h,Col2HA50mmolöL22N2(22N2morpholinoethanesulfonicacid,MES),20ml40%30min,50mmolöLMES33mmolöL12232(32)[12ethyl232(32dimethylaminopropyl)carbodiimide,EDC]8mmolöLN2(N2hydroxysuccinimide,NHS)2%CS40%,4h0.1molöLNa2HPO4(pH9.1)1h,1molöLNaCl2h,2molöLNaCl1d;10,,,Col2HA2CSCol:016%,CS16mm,60Co1.21.2.1,1.2.2HE21d,,HE,1.331(),,,PBS(200Uöml)212mm3,PBS1;0125%45min,,PBS2;0.2%Ê374h,70215cm,1500römin10min,,PBS2;,1105ömlDMEM2F12(10%,L2584Lgöml,C50Lgöml,100Uöml,100Lgöml),25cm2,37,5%CO2,21,90%,0115%,21.422107öml24Col2HA2CSCol,100Ll,,,1.51.5.1721döCol2HA2CS4%,,,HE,;2%,,,,1.5.2DNA:öCol2HA2CS2Col,Hoechst33258[7],1371421dDNA,DNAGAG:2[8],GAG,CS1.5.3ColÉÊRT2PCR714d,TrizolRNA,RT2PCRÉÊmRNA,GAPDH(PCR411bp)ÉmRNA:52CCGAAGGTTCCCCTGGACGA23;:52CGCCCTGTTCGCCTGTCTCA23;PCR250bpÊmRNA:52GCACCCATGGACATTGGAGG23;:52AGCCCCGCACGGTCTTGCTT23;PCR323bpPCR,5LlPCR115%13120062021.6SPSS1010,t,,P0.0522.1Col2HA2CS,80150Lm,90%(1);21d,(2)2.222.2.17d,,;21d,(3)2.2.2,,14d,,ECM,(4)2.3DNA,,14d,Col,(P0.05),5,,GAG,Col,(P0.05),62.4ColÉÊRT2PCRRT2PCR,ÊmRNA,,ColÊColÉ,14(7)3,,,[9],,,,pH,[10],,,1Col-HA-CS(60)2Col-HA-CS21d,(HE100)3Col-HA-CSa7d(HE100)b21d(HE400)4Col-HA-CS14da(3000)b(4000)Fig.1TheCol-HA-CStri-copolymerscaffold(SEM60)Fig.2TheCol-HA-CSscaffoldincubationinDMEMmediumafter21daysandtheporousscaffoldstructuremaintainningintegrityduringculture(HE100)Fig.3ThechondrocytesseededintheCol-HA-CSmatrixaThecellsatthescaffoldshowedaroundmorphologyafterthe72dayseeding(HE100)bThecellspresentedcartilage2specificlacunaeafterthe212dayseeding(HE400)Fig.4ScanningelectronmicrographyshowingthechondrocytesseededintheCol-HA-CSscaffold14daysafterthecell-seedingaThecellsatthescaffoldshowedasphericitygrowthafterthe142dayseeding(SEM3000)bThecellsmatixdiffusedintothescaffoldpore(SEM4000)231ChineseJournalofReparativeandReconstructiveSurgery,2006,Vol20(2)5ColCol-HA-CSDNA6ColCol-HA-CSGAG7Col-HA-CS14dColÉÊ1:ColÊGAPDH2:ColÉGAPDHM:MarkerFig.5TheDNAcontentofthechondrocytesintheColandCol-HA-CSscaffoldsatthedifferenttimesofcultureFig.6TheGAGcontentofthechondrocytesintheColandCol-HA-CSscaffoldsatthedifferenttimesofcultureFig.7Thereversetranscriptase-polymerasechainreactionresultsagainstcollagentypesÉandÊofthechondrocytesafterthe14-dayculturewithintheCol-HA-CSscaffold1:CollagentypeÊandGAPDH2:CollagenÉandGAPDHM:Marker[11]ColHACS,ECMCol,ECM,,,ColHA,,[12]ColHAGAG,[13],HA,HA(CD44),CD44HA[14]ColHAEDCCS,,,,Col2HA2CS,,,21d,,,ECM,,ColÊ,21d:Col2HA2CS,41MinasT,NethrerS.Currentconceptsinthetreatmentofarticularcartilagedefects.Orthopedics,1997,20(6):525238.2NewmanAP.Articularcartilager