骨跟软骨的生物化学 bone and Cartilage biochemistry

BiochemistryofBoneandCartilagePholasitCHAMODTheMusculoskeleton,Jan.2005TopicsHomeostasisofcalciumRegulationofcalciumhomeostasisBiochemistryofcartilageClassificationArticularCartilagecompositionThefunctionsofboneClassificationBonecomposition(biochemicalaspects)BoneformationandOsteoblastBoneresorptionandOsteoclastBoneremodelingBone:Theminerizedconnectivetissue•BoneconstantlybeingcycleofBrokendown&Rebuild•Boneisnotastatictissuebutanactivetissue/dynamicstructure(alsoneedbloodsupply/nutrientarteries)Functionofbone•Mechanicalfunction:Support&siteofattachmentforlocomotion•Protectivefunction:Protectiveforimportantorgan&bonenarrow•Metabolismfunction:Reserveofions(Calcium&Phosphateions)formaintenanceserumhomeostasis•PrimarysiteofBloodproduction(hematopoiesis)FlatboneviewCorticalvs.Cancellousbone%Mineralizedintotalvolume80-9020%CorticalCancellous80BoneMarrow(bathedinfattyand/orhematopoietictissue)Corticalbone:mechanicalfunctionCancellousbone:metabolicfunctionCorticalbone:mechanicalfunctionCancellousbone:metabolicfunctionSuggestedfunctionThecompositionofbone•Matrix(ECM,extracellularmatrix)•Organicmatrix•Collagenousproteins•Non-collagenousproteins•Inorganicmatrix•Bonecells•Osteoblast•Osteocyte•OsteoclastInorganicBoneMatrixMajorinorganicconstituentsofboneConstituentTotalbodycontentPresentinbone(%)Calcium99Phosphorus85Carbonate80Magnesium50Sodium35Water9•Andotherminerals:Sodium,Magnesium,Carbonate,FluorideInorganicBoneMatrix•Themajormineralphaseofbone:>90%totalmineralweight•Spindle-orplate-shapedcrystalsHydroxyapatite(apatite)•Chemicalformula:Ca10[PO4]6[OH]2•Embeddedwithinthecollagenextracellularmatrix•TendtobeorientedinthesamedirectionascollagenfiberOrganicBoneMatrix•CollagenousProteinsTypeICollagen(majorprotein)90–95%•Non-collagenousProteins¾Osteocalcin¾Osteopontin¾Osteonectin¾MatrixGlaProteinImportantofmineralandcollageninboneCollagen•Themajorstructureofbonematrix•Themostabundantproteininthebody(≈25%inadult)TissueCollagencontent(%)DemineralizedBone90Tendons80–90Skin50–70Cartilage50–70Arteries10–25Lung10Liver4•Distribution:Strong,toughconnectivetissueCollagen•Collagenclassification:baseonstructuralfeatures¾FibrillarcollagenonlytypeI,II,III,V,VIIandXI¾Non-fibrillarcollagen•About19distincttypesofcollagenmadeupofabout30distinctpolypeptidechainsinhumantissues•AlargeproteinfamilyThefibrilarcollagen[α1(I)]2α2(I)Bone,skin,tendon,dentin,etc.α1(I),α2(I)IosteblastcellgeneproductCollagenfibrilmolecule•IntracellularBiosynthesis(Osteoblast,OB)ExtracellularPolymerization&mature(Posttranslationalmodification)•Consistof3polypeptidechainsTropocollagenFormatriplehelixRod-likestructure300nminlength1.5nmindiameter•Tropocollagen:2α1(I)polaypeptidechains1α2(I)polaypeptidechainsThepolypeptidechainofTropocollagenmolecule•EachchaincontainsatleastonestretchofRepeatingpatternofaminoacidsequence(Gly–X–Y)n----Gly-X-Y-Gly-X-Y-Gly-X-Y-Gly-X-Y-Gly-X-Y---Gly:GlycineXOtheraminoacid,oftenProlineYorHydroxyprolineorHydroxylysineHydroxyproline&Hydroxylysine•Proline&Hydroxyproline:25%totalaminoacidinthepolypeptidechainTriplehelixstabilityHydroxyproline&Hydroxylysine•Hydroxylysine:Essentialfor•Lys-OH----Glu-Galdisaccharide•Glycosylationofpolypeptidechains•Only0.5-1%carbohydratecontentintypeIcollagen•α1(I),α2(I)chainLeft-handedhelix“PolyprolinetypeIIhelix”(Moreextendthanαhelix)Conformationofchain(s)•TropocollagenRight-handedtriplehelixH–bondbetweenpeptidebondofdifferentpolypeptidesStericrepulsion:prolineandhydroxyprolinesidechainStabilizedbyHydroxylationGlycosylationS-SbondingNCterminaldirectionStaggereddepositionofTropocollagenmoleculesThefibrilformationThecross-linkingThetelopeptidecovalentcross-linkingAllysineHydroxylallysineHydroxylysinePyridinolinecross-linkVitaminCandCollagenbiosynthesis•Hydroxylationreactionofpropylandlysylsidechainsinprocollagenmolecule(Dioxygenase)ProlineresiduePeroxideformHydroxyprolineresidueNohydroxyprolineConnectivetissueweakness:hemorrhagictendency(scurvy),looseteeth,poorwoundhealingThetriplehelixDenatures&DegradedNon-collagenousProteins(NCPs)•10-15%ofbonematrixprotein•Netnegativecharge:HighCabindingpotential•InvolveinCalcification•UnknownspecificfunctionEndogenousmolecules(bonecellssynthesis)1.Proteoglycans2.Glycosylatedproteins3.Glycosylatedproteinswithpotentialcell-attachmentactivity(RGD-containingglycoproteins)4.Carboxylated(Gla)proteinsBoneproteinFunction2.Glycoproteins-Alkalinephosphatase-phosphotransferase-potentialCa2+carrier-Osteonectin-bindtocollagen-maymadiateddepositionofhydroxyapatite-mayassociatedwithosteoblastgrowth/proliferation1.Proteoglycans-controltransportandtensilestrength-maybindtocollagenNon-collagenousProteins(NCPs)BoneproteinFunctionNon-collagenousProteins(NCPs)-Osteopontin-bindtocells-inhibitmineralization-mayregulateproliferation-Bonesialoprotein-bindtocells,bindCa2+withhighaffinity(BSP)-mayinitiatemineralization3.RGD-containingglycoproteinsRGDdomain:Arg-Gly-AsnThecell-attachmentconsensusaminoacidsequencethatbindstointegrinclassofcell-surfacemoleculesBoneproteinFunctionNon-collagenousProteins(NCPs)4.Carboxylated(Gla)proteins-Matrixglaprotein-mayfunctionincartilagemetabolism(MGP)-inhibitmineralization-Osteocalcin-mayregulateactivityofosteoclasts(Bonegla-protein)-maymarkturningpointbet.Boneformationandboneresorption-regulatemineralmaturation-ProteinS-ProteinSdeficiencymayresultinosteopeniaGla(Carboxyglutamate)residue:highaffinitytoCalciumBynegativechargeofGlaresidueThebonecellsOsteoblastOsteocyteOsteoclastOsteoblast-TypeICollagen-Osteocalcin-Osteonectin.-Bonespecificalkalinephosphatase(membraneboundprotein)-etc.•Appearalongbonesurface(Groupofcubodialcellinmonolayer)•AlotofrERandgolgiapparatuswithincell(highproteinsynthesisactivities)•Producematrixconstituents“Osteoid”BoneformationorOsteoblasrticmarkersOsteoblastOsteocyteBoneliningcellProosteoblastOsteoblastUndifferentiatedMesenchymalStemcellOthermesenchymalderivedlineages(chondrocyte,adipocyte,stromalcell,etc.)differentiationOsteoblast:BoneformationThreestepsforboneformationprocess1.ProductionOsteoblastcellproducts(CollagenandNCPs)ExtracellularmatrixorOsteoids2.MaturationOsteoidreadyforcalcification3.MineralizationMineralizedosteoid(firstcome,firstgoprinciple)•MechanismofboneMineralizationRegulatepassageofCalcium&PhosphateionsacrossOBsurfacemembraneVia-AlkalinephosphataseonOBmembrane(Boneformationindex)-Matrixvesicleonsiteofmineralization(calcium&phosphate)-Roleofmitochondria:concentrateCa2+/PO4ion•Receptorforbonemetabolismmediators:PTHOsteoblast:BoneformationOsteocyte•Satellitecellshape•DifferentiatedOsteoblast(OB)•EntrappedOBinthecalcifiedbonematrix•LessactivemetabolicallythanOB/OC•ControlledbyOsteoblastliningcellOsteocyteinlacunaeCytoplasmicprocess&canaliculiCanaliculi:-ControlCalciumionexchangebetweenbonemineralpoolandextracellularfluidOsteocyteosteolysis:osteocytedemineralizationofthecavityOsteocyticosteolysisOsteocyte:boneresorptionOsteclast•Highspecializedcells:notalwaysseenonbonesurface•Multinucleatedgiantcell(4-20nuclei)•Foundincontactwithcalcifiedbonesurface•SpecialMb.Domain:“RuffledBorder”•KeyroleinboneresorptionBoneresorption&DemineralizationSpecialmembranedomain:“RuffledBorder”Foldingofplasmamembrane(toincreasesurfacearea)-Preparation:CollagenasesecretionbyOC-AttachmentIntegrin&BonematrixproteininteractionRGD-containingglycoproteins(Osteopontin,Bonesialoprotein)-MicroscopicsealedEnvironment:ClearzoneorSealingzoneOsteclast:BoneresorptionTheAttachmentBetweenIntegrin&RGD-containingglycoproteins(Osteopontin)•Releaseproteolyticenzymes:Cysteineprotease(CathepsinB)MatrixmetaloprotenaseCollagenase•Demineralizationby1.Acidification:CarbonicanhydraseIIH+/K+ATPasepumpPumpH+acrossborder2.Dissolution:IncreasesolubilityofHydroxyapatiteOsteclast:BoneresorptionBoneresorptionmarkers•Hydroxyproline•Hydroxylysine•Pyridinolinescross-linkCirculationUrineexcretionRegulationofOsteoclast’sfunctions1.Indirecteffect:viareceptoronOsteoblastcellsurfaceEndocrineregulators-ParathyroidHormone-VitaminD2.Directeffect:viareceptoronOsteoclastcellsurfaceEndocrineregulators-CalcitoninOtherregulatedcytolines-IL-1-IL-6-M-CSFcytolineThecouplingregulationbetweenOB&OCBoneremodeling•Boneremodelingoccursconstantlythroughoutlife.•Osteoclastsandosteoblastsarethemajorcelltypesinvolvedinboneremodeling.•Osteoclastserodebonematrixwhereasosteoblastssecreteit.•Convertwovenboneintolamellarbone•Involvedin-Bonegrowth-Changeinboneshape-Theadjustmentofbonetostress-Bonerepair-CalciumionregulationinthebodyBoneRemodelingRestingPhaseResorptionActivationReversalPhaseFormationResorptionphase:~10daysFormationphase:~3monthsBMU(BasicMulticellularUnit)orBRU(BoneRemodelingUnit)Insideharversiansystem:thecuttingconeHappenonthesurfaceofcancellousboneCalciumHomeostasisPlasmaCalcium2.38mmol/LIntestineKidneyBoneREABSORPTIONABSORPTIONRESORPTIONCalciumTurnoverinhumansPhoaphateTurnoverinhumansThemainhormonesinvolvedinregulatingplasmacalciumconcentration•Parathyroidhormone(PTH)•1,25DihydroxyVitaminD3•Calcitonin•Peptidehormone(84aa.):ChiefcellofparathyroidglandParathyroidhormone(PTH)Parathyroidhormone(PTH)Parathyroidhormone(PTH)•Toincreaseplasma[Ca2+]&decrease[PO4]•StimulaterenalCalcium“Reabsorption”•Promotesbone“Resorption”(PTHReceptoronOBcell)•Stimulateformationof1,25dideoxyVitaminD3inkidney(Viainduction1α-Hydroxylaseactivity)Enhancecalcium“Absorption”fromintestine¾DecreaserenalPhosphate“Reabsorption”TheactionofPTHonBone:Bone(Ca2+)resorptionTheactionofPTHontheKidney:Ca2+reabsorptionTheactionofPTHontheIntestine:Ca2+absorption(moreactiveformofvitaminD)ThePTHregulation•Dominantregulator:PlasmaCalciumlevel,[Ca2+][Ca2+][Ca2+]IncreasePTHsecretionSuppressPTHsecretion•Sensechangeinextracellularfluidconc.ofCalciumionbyUniqueCa2+receptorwithinparathyroidcellplasmamembrane•RegulationbyVitaminD:-InhibittranscriptionofPTHcodinggene-DecreasePTHsecretion-InhibitproliferationofparathyroidcellsTheInverserelationshipbetweenPTHandplasma[Ca2+]inhumanTheMechanismofregulationofPTHsecretion1,25dideoxyVitaminD3•VitaminD3:Cholecalciferol(Diet&Skin)•VitaminD2:Ergocalriferol(Plant)•Kidney1α-Hydroxylase:Pointofcontrols!•EnhancingdietaryCalcium“Absorption”fromintestineby-Calciumchannelabsorption-IncreaseCalbidinsynthesis(Ca-bindingprotein)•IncreaseCaBPsynthesis•IncreaseCaabsorption•IncreasePO4absorption•Increaseboneresorption(indirect)ThetargetgenesofvitaminDFactorsthatregulatedconversionof25-OH-Dtoeither1,25-(OH)2-Dor24,25-(OH)2-DParafollicularorCcellsofthyroidgland:peptidehormone(32aa.)CalcitoninCalcitonin•RapiddeclinelevelsofserumCalcium&Phosphate•ActiondirectlyonBoneandKidney•Bone:Directlyinhibitboneresorption-InhibitOsteoclast(OC)activity-CalcitoninreceptoronOCcell-RapidOCmorphologychange,Ruffledborder-DetachOCfrombonesurface•Kidney:¾IncreaseurinaryPhosphateexcretion¾IncreaserenalfractionalexcretionofCalciumTargetcellofCalcitonin:Osteoclast(membranereceptor)Cartilage•Templateoflongboneduringdevelopment(highervertebrates)•Remaininselectedregionsofadultbones•Resilienttissueandprovidesemi-rigidsupportforcertainpartsofskeleton(e.g.articularcartilage,respiratoryairways)•Majorcomponent:Extracellularmatrix(ECM)¾Networkofcollagenfibrils(mainlytypeIIcollagen,alsoVI,IXandXI)¾Highconcentrationofproteoglycans(mainlyaggrecan)•RetentionofaggrecanwithincollagennetworkcauseswellingpressureresistingcompressiveloadwithminimaldeformationClassificationofcartilage•Growthplatecartilagegrowth(physeal)plate•Fibrocatilagetendon&ligamentinsertionintobone•Elasticcartilagenose,epiglottis,auricle•Fibroelasticcartilagemenisci•Articular(hyaline)cartilagejointfunctionArticularcartilage•Metabolicallyactivetissue(limitedcapacityofrepair)•Theresilientload-bearingmaterialofdiarthrodialjoint•Excellentfriction,lubricationandwearcharacteristics•Aneural:nonerves•Avascular:noarteries•Alymphatic:nolymphaticchannels•ChondrocytesreceivenutrientsandO2viadiffusionthroughcartilagematrixOrganizationofnormalarticularcartilage•Consistsprimarilyofalargeextracellularmatrix(ECM)withhighlyspecializedcells,chondrocytes•Variationinstructure&compositionthroughoutitsdepthfromarticularcartilagesurfacetosubchondralbone(cellshape,cellvolume,collagenfibrildiameterandorientation,proteoglycanconcentration)•Cartilagecanbedividedintofourzones1.Superficialzone(STZ)2.Middleortransitionalzone3.Deepzone4.CalcifiedzoneSuperficialzone(tangentialzone)•Flattedchondrocytes•Lowproteoglycancontent•Thehighestconcentrationofwater•Thethinnestzoneofarticularcartilage•Denselypackedlayersofuniformcollagenfibers•BundleofcollagenarrangedparalleltoeachotherandtoarticularsurfaceTransitionalzone(middlezone)•RoundedchondrocytessurroundedbyECM•Collagenfiberarrangerandomly•Largerdiameterofcollagenfiber•IncreasinginproteoglycancontentCollagenfiberincrosssectionarticularcartilageLayerOrientationofcollagenfibersFunctionSuperficialTangential/ParallelVersusshear(highconcofcollagenfiber)MiddleOblique/RandomlyVersuscompressionDeepVerticalVersuscompressionTidemarkTangential/ParallelVersusshearCalcifiedzone---AnchorLayerCharacteristicWidth(um)SuperficialLowmetabolicactivity40MiddleHighmetabolicactivity500DeepLargercollagensize1000TidemarkUndulationbarrier5CalcifiedzoneHydroxyapatitecrystal300ArticularcartilagecompositionQuantitativelymajorcomponents(%wetweight)Water65–80Collagen(typeII)10–20Aggrecan4–7Quantitativelyminorcomponents<5Proteoglycans-biglycan-decorin-fibremodulinCollagens(typeV,VI,IX,X,XI)LinkproteinHyaluronateFibronectinLipidsChondroblast/Chondrocyte•Synthesisandmaintenanceofarticularcartilage•Stemcell:mesenchymalcells•Surroundedbyathin,pericellularmatrixthatprovidehydrodynamicprotectionfromphysiologicalloadingandplayametabolicrole•Metabolicallyactive•Abletoresponsetovarietyofstimuli;¾solublemediators:GF,ILs¾matrixcomposition¾mechanicalloads¾hydrostaticpressurechangeWaterinarticularcartilage•themostabundantcomponentofnormalarticularcartilage•Inearlyphaseofosteoarthritis,watercontentmayincreaseto>90%•About30%(fromtotal65-80%)intrafibrillarspacewithinthecollagen•Inorganicsalts(Na+,Ca2+,Cl-,K+)dissolvedinthetissuewater•Mostofwatermaybemovedthroughthetissueorsqueezedoutbyapplyingapressuregradientacrossthetissueorbycompressingthesolidmatrix•Frictionresistance:Biomechanics•Highaffinitytowatercausedmostlybyproteoglycan(hydrophilicnature)andlessfromcollagenExtracellularmatrixCollagen•Majorform:typeIIcollagen(80-90%oftotalcollagen)•TypeIIcollagenmolecule:Tropocollagen:3α-1(II)polaypeptidechainsinhelicalform(300-nmtriplehelix)•Minorforms:•typeXI----copolymerizedwithtypeII•typeIX----covalentlylinkedtothesurfaceoftypeIIcollagenfibrilmaymediatedfibril-fibrilandfibril-proteoglycaninteraction•alsotypeX,III,VI•TypeII,IXandXIarecartilage-specificandarecross-linkedtogetherinanetworkProteoglycans•ByDefinition:Proteoglycanmoleculesconsistsofglycosaminoglycanchainsboundstoaproteincore•AGGRECAN:80–90%ofallproteoglycansinarticularcartilages•Coreprotein~250,000daltons,encodedbysinglegeneDomains3globulardomains(G1,G2&G3)2extendeddomains(IGD,KSrish-CS1-CS2)AGGRECAN•Coreproteinishighlyglycosylatedwith~90%carbohydrate•Twomajortypeofglycosaminoglycanchains•Chondrotinsulfate(CS)~100CS/aggrecanmolecule•Keratinsulfate(KS)<60KS/aggrecanmolecule•Aggrecansbindtohyaluronanandlinkprotein•Collagenfibrilsinteractwithkeratansulfate-richregionsofseveralaggrecanmonomersExtracellularmatrix•Hyaluronicacdis(hyaluronan)issynthesizedinandsecretedfromchondrocytesAggrecandegradationCartilage(internal)remodelingInhibitionSynthesisSynthesisStimulationInhibitionDegradationDegradationStimulationGFsGrowthHormoneIL-1InterferonTNFaGlucocorticoidsProteolyticenzymesEnzymeinhibitors_+ROSProstagladinTNFaBalance1Balance2