The retention and ultrastructural appearances of various extracellular matrix molecules incorporated into three-dimensional hydrated collagen lattices☆

摘要

Artificialextracellularmatricescomposedofcollagen,glycosaminoglycans(GAG),proteoglycans(PG),plasmafibronectin(FN),andahyaluronate-bindingprotein(HABP)havebeenpreparedthatmorphologicallyresembleembryonicextracellularmatricesinvivoatthelightandelectronmicroscopelevel.Theeffectofeachoftheabovematrixmoleculesonthestructureanda€?self-assemblya€?oftheseartificialmatriceswasdelineated.(1)Matrixcomponentsassembledinvitromorphologicallyresembletheircounterpartsinvivo,forthemostpart.Scanningandtransmissionelectronmicroscopyindicatethatunderourassemblyandfixationconditions,collagenformsstriatedfibrilsthatare125nmindiameter,FNforms30-to60-nmgranules,chondroitinsulfateproteoglycan(CSPG)forms27-to37-nmgranules,chondroitinsulfate(CS)assemblesinto100-to250-nmspheres,andhyaluronate(HA)appearseitherasgranularmatswhenfixedwithcetylpyridiniumchloride(CPC)oras1.5-to3-nmmicrofibrilswhenpreservedwithrutheniumredplustannicacid.ThesemoleculesareknowntoassumethesameconfigurationsinembryonicmatriceswhenthesamepreservationtechniquesareusedwiththeexceptionofFN,whichgenerallyformsfibrillararrays.(2)Additionofvariousmatrixmoleculescanradicallychangetheappearanceofthecollagengels.HAgreatlyexpandsthevolumeofthegelandincreasesthespacebetweencollagenfibrils.CSPGatlowconcentrations(&1mg/ml)andCSathighconcentrations(&20mg/ml)bundlethecollagenfibrilsintotwistedropes.(3)Avarietyofassayswereusedtoexaminebindingbetweenvariousmatrixcomponentsandretentionofthesecomponentsinthehydratedcollagenlattices.Theseassaysincludedsolid-phasebindingassays,negativestainingofspreadmixturesofmatrixcomponents,cryostatsectionsofunfixedmixturesofmatrixcomponents,andretentionofradiolabeledmatrixmoleculesinfixedandwashedgels.Anumberofthesebindinginteractionsmayplayaroleintheassemblyandstabilizationofthematrix.(a)HA,CSPG,andFNbindtocollagen.CSappearstoonlyweaklybindtocollagen,ifatall.(b)FNpromotestheincreasedretentionofHA,CSPG,andtoaverysmalldegree,CS,incollagengels.Conversely,theGAGincreasetheretentionof3H-FNinthegels.Furthermore,FNbindstoHA,CS,andCSPGasdemonstratedbysolidsurfacebindingassaysandmorphologicalcriteria.TheincreasedretentionofGAGandCSPGbytheadditionofFNmaybeduetobothstabilizationofbindingtothecollagenandtrappingofmatrixcomplexeswithinthegel.(c)HAbindstobothCSandCSPG.ItsassociationwithCSPGmonomersproducesthetypicala€?bead-on-a-stringa€?appearancethathasbeenwellcharacterizedinvivo.HAassociationwithCSsuggestsyetanotherwaythatmatrixcanbestabilized.(d)A70-kDaproteinthathasbeenshowntobindtobothHAandcollagenincreasestheretentionofHAinthecollagengelsandpromotesHAfibrilformation.Fibronectin,GAG,andCSPGspontaneouslyorganizeinthecollagenmatricesinamannerthatisconsistentbothwithbindingcharacteristicsthathavebeenpreviouslypublishedandthosethatarereportedhere.