Expression of differentiated function by mineralizing cultures of chicken osteoblasts☆

摘要

Thisreportdocumentsosteoblastdifferentiationinvitro,asdemonstratedbythe50a€“100?—increaseofproteinswhichareknownmarkersoftheosteoblastphenotype.CollagentypeIandosteocalcinsynthesisandaccumulation,alkalinephosphataseactivity,andmatrixcalcificationshowsimilartemporalrelationshipsthatareanalogoustothoseseenduringinvivobonedevelopment.Chickenembryonicosteoblastprogenitorcellswereselectedbyinitialgrowthatlowdensitiesinminimalmedium.Uponsubcultivationintonutrient-enrichedmediumathighercelldensities,nearhomogeneouspopulationsofosteoblastswereobtainedasdemonstratedbythegreaterthan80%enrichmentofcellspositiveforalkalinephosphataseactivity.Acomparisonwasmadebetweencellsgrowninthepresenceorabsenceof10mM?2-glycerolphosphate(?2-GPO4),achemicalstimulantofmatrixcalcification,asafunctionoftime.Culturestreatedwith?2-GPO4showedvisiblecalcificationatDay12whenculturemonolayersbecameconfluent.ByDay30,numerouslargefociofcalcificationwerevisibleanda20-foldincreaseincalcium(Ca)contentwasobserved.Incontrast,untreatedcultureshadonlya3-foldincreaseinCacontentwithmanysmallerdiffuseareasofcalcification.DNA,RNA,andtotalproteinlevelswerenearlyidenticalbetweenthetwocultures,indicatingthat?2-GPO4hadnomarkedeffectoneithercellproliferationortranscriptionalactivity.ThemajorcollagentypeproducedbyeitherculturewastypeI,withnodetectabletypeIIIasdeterminedbyCNBrpeptidemappinganddelayedreductionanalysis.Alkalinephosphataseactivityshowedarapida??50-foldinductionbyDay18andremainedelevatedincontrolcultures.However,culturestreatedwith?2-GPO4demonstratedarapid80%declineofenzymeactivityafter18days.Incontrast,totalosteocalcinlevelsshoweda100-foldinductionbyDay18andremainedelevatedinbothcontroland?2-GPO4-treatedculturesthroughoutthetimeperiodexamined.Whiletheoveralllevelsofosteocalcinwerethesamein?2-GPO4-treatedanduntreatedcultures,2-to5-foldmoreosteocalcinwasassociatedwiththemoremineralizedmatricesofthe?2-GPO4-treatedcultures.Inordertoconfirmtheassociationofosteocalcinwithareasofmineralization,co-localizationofmineraltoosteocalcinandcollagenwascarriedoutbycombiningvitallabelingwithtetracyclineandimmunofluorescentstainingwithanti-osteocalcinandanti-collagenantibodies.Bothcollagenandosteocalcinshowedstronglocalizationwithareasofmineralization.Thisculturesystemdefinedtheexpressionofspecificmarkersofosteoblastfunctionduringdifferentiationandtheirrelationshipstomatrixcalcification.Thus,theseosteoblastculturesprovideauniquemodelinwhichtostudytheregulationofbone-specificproteinsandgenesduringosteoblastdifferentiationandmatrixcalcification.