synovial-fluid

5Balazs, LA.(1974). Thephysical properties ofsynovial fluidandthe special roleofhyaluronic acid.In Disorders oftheKnee. (Ed.Helfet, A.) T.B.Lippincott Company, Philadelphia. ,p".63-75. ThePhysicalProperties ofSynovial FluidandtheSpecialRole ofHyaluronic Acid EndreA.Balazs,M.D. Thischapter waswrittenwithtwoaimsin mind.First.togiveabriefreviewofthe chemical composition andrheological prop­ ertiesofthesynovial fluidinnormal and pathological joints;andsecond. topresent, withcritical comments, various current hypotheses ontheimportance ofthisfluid inthefunction ofthejointwithspecial emphasis onthebiological roleofhyalu­ ronicacid. Thereisconsiderable dataavailable inthe literature onthechemical composition and rheological properties ofsynovial fluidof variousspecies. Thisreviewdoesnotintend tobeall-inclusive. ratheritpresents primary dataonhuman jointfluidsonly.Alarge number ofspeculations andhypotheses are recorded intheliterature onthelubricating andnutritive functions ofthesynovial Iluid inthejoint.Thisreviewaimstobeselective inthisareaaswellandpresentonlythemost important recentfindings andspeculations onthissubject. SYNOVIAL FLUID ASAN INTERCELLULAR ~L\TRIX Fromarheological pointofview,the naturalenvironment ofceUsinatissuecan beliquidorsolid.Theliquidorsolidextra­ cellular matteraroundandbetween cellsis calledmatrix. Inthesolidmatrix. certain macromolecular components (mostly colla- 63genorelastin,orboth)andtheiraggregates formacontinuous solidphase.Therheo­ logicalqualities ofthesolidmatrix, thatis, therigidity, elasticity, andviscosity, vary considerably asexemplified bythediffer­ encesbetween cartilage andthevitreous of theeye. Thesolidmatrixconsists offixedandfluid components. Thefixedcomponents are madeupofsuchmicroscopic elements as collagen andelastinfibrils,basallaminae, andthestructural network ofproteoglycans. Thefluidcomponent comprises waterand thosesolutes(salts,smallorganic molecules, peptides, proteins, etc.)that,dissolved in water,aredistributed between thefixed components ofthesolidmatrix. Theliquidmatrix. ontheotherhand,is waterinwhichmolecules ofvarious sizes aredissolved ordispersed. Therheological qualities ofliquidmatrices alsovarycon­ siderably, asexemplified bythedifferences between theaqueous humoroftheeyeand synovial fluid. Thejointcontains bothsolidandliquid matrices. Fromthe[ourtissueelements [orming ajoint;articular cartilage, synovial tissue,intraarticular: ligaments, andsynovial Iluid,thefirstthreehavesolidmatrices and thelastisaliqUIdmatrix. Itisofprimary importance torecognize thatallthreesolidmatrixcompartments of '.w 64Physical Properties 01Synovial Fluid'SpecialHole01Hyaluronic Acid. j• TABLE5-1.Proteins andHyaluronic A~id·intheSynovial Auid ofKneeJointsofHuman Subjects ofVarious Ages· AgeKnees Protein J!ya/tiTonic AchILimiting Viseos;/yGroup (yrs.)l1Ivesrigatec! (mg.lml.) mg.!ml. Number (ml./g.) 18-20 36 20.5±0.8 3.8±0.1 5200±100 21-23 24 21.0±0.9 3.8±0.2 5300±100 24-27 20 19.4±1.3 3.4±0.1 5000±150 28-35 18 18.5±2.6 2.5±0.04 5800±300 52-78 34 18.5±1.6 2.5±0.2 5400±200 (Datatakenfromunpublished workofE.A.Balazs,N.W.Rydell, P.O.Seppala, J.F.Duff,E.W. MerrillandD.A.Gibbs) thejointaredirectly adjacent totheliquid matrixcompartment, thesynovial fluid. Another important morphological factis thatthesolidmatrixcompartmcnts ofthe articular cartilage, ligaments, andsynovial tissuearenotscparalcd fromthefluidmatrix compartmcnt byacontinuous celllaycror basallamina.Thcrefore, novisible,morpho­ logicalbarrierseparates thesetwotypesof matrixcompartments. Otberadajacent liquidandsolidmatrices suchasarepresent intheperitoneal, peri­ cardial, andpleural spaces andinthe anterior chamber oftbeeye,areseparated bymicroscopically recognizable cellular (epitbelium) andmatrix(basement mem­ brane)barriers. Thejoint,however, isnot tbeonlytissueinwhicbtheliquidandsolid matrices arcnotseparated bythesebarriers. Othertissuesinthemusculoskeletal systcms aresimilarly structured, suchasthetendons andthcirsheaths andthespacebetween ,fasci"eandthebursalspace. CHE~lICAL COMPOSITION OFTilESYl'iOVIAL FLUID Proteins TheprOleincontentofnormalsynovial fluidinallspecies studied ismuchlower thaninserum.3~ Thegeneralstatement can bemadethattheveryI",geproteinmole­ culesoftheserum.suchasy-lmacro­ globulin, ,a-lipoprotein, fibrinogen, and",-2 macroglobulin, areabsent,andothers,such asa-Iantitrypsin andplasminogen, arc presentintracesinthenormalsynovial fluid.Inaddition, somesmallerproteins, suchas haptoglobulins andprothrombin present in theserumarealsoabsentfromnormalsyno­ vial fluid.J,I~·HI,3J.J~ Because oftheabsence offibrinogen andprothrombin, thenormal fluiddocsnotclot. Thetotalproteincontentofsynovial fluid aspirated fromnormal humankneejoints doesnotchange withageofthedonor' (Table5-1).Inallinflammatory joint diseases, however, theconcentration of~ro­ teininsynovial fluidincreases, andthemiss­ ingplasma proteins appear. Fibrinogen appears, and,afteraspiration, thefibrin precipitates, oroften,thefluide1ots.From thepointofviewofproteincontent, inflam­ matorysynovial fluidismore"plasmalike" thannormalsynovial fluid. IIImostdegenerative typejointdiseases, theproteinconcentration alsoincreases in thefluid,butitdocsnotnecessarily become "pl"smalike." Whilethereisnoclearproof, thereareindications thatinthesecases, proteins appe",inthesynovial fluidthat originale fromthecellsandmatrixofneigb­ boringtissues.38 Proteins withenzym"tic activityalsoap­ pearintheinflammatory synovial fluid. Enzymes presentintheIysosomes ofleueo­ cytes,suchasacidphosphatases, ,a-glucu­ ronidase, and{J-N-acetylglucosaminidase, arcreleased fromthedestroyed cells.Other enzymes, suchaslacticdebydrogenases, collagenase. andmuramidase (lysozyme) arcalsofoundinpathological synovial nuids,1.1G.::1 Mostimportantly, innammatory synovial" Chemical Composillon oftheSynovial Fluid 65 fiuidcontains immune complexes andanti­ bodiesthatarenotpresent inthenormal fluid.Inrheumatoid arthritis, theappear­ anceoftheso-called rheumatoid factors (primarily antibodies toyG-globulin) and theircomplexes withimmunologically active proteins, aswellasactivation ofcomponents ofthecomplement system arctypicalex­ amples ofthedrasticchanges occurring in theprotein composition andimmunological characteristics ofsynovial fluidduringin­ flammation. :::(1.3;.4:l-r, Theextremely complex alteration intheimmunologically activepro­ teinsofthesynovial fluidindicate thatthe neighboring tissues-first ofall,thesynovial tissue-are thesitesofveryintensive protein synthesis duringinflammation. H)'aluronic Acid Theviscoelastic natureofsynovial fluid isduetoitshyaluronic acidcontent. This polyanionispresent inthesynovial fluidof allspecies investigated. Inequine, bovine, andhuman jointsaconsiderable variation wasfoundinthehyaluronic acidconcentra­ tionofsynovial fluidcollected fromvarious jointsofthesamesubject.4u Hyaluronic acidconcentration alsovaries withage."';Inthe;ynovial fluidofhuman kneejointsthehya~'Jronic: acidconcentration ishighest between 18and25years,after whichitdecreases. BCiwecn theagesof 30and80,nochange couldbeobserved innorma!juints.lj ThesiLeofthehyaluronic acidmolecules inthesynovial fluidofhuman kneejoints wasdetermined byvarious physicochemical methods. The[tsultsvarysomewhat. de­ pending Oilthemethod usedforthepuri­ fication ofthehyaluronic acidandforthe determination ofthemolecular weight. Thislargemolecule occupies anextremely largevolume whendissolved inwaterthat contains aphysiological concentration of saltsandhvdrogen ions.Asinglemolecule ofNa-hyaiuro~ate, withav7eight of5 million. occupies aspheroidal domain with adiJmct~r of0.5jJ..Thismeansthatone gramdissolved inphysiological salineflils3litersofsolvcnt. Inotherwords, the individual molecules, inclosecontact with oneanother butwithout overlapping, occupy thewholevolumeofsolution ataconcentra­ tionofonly0.33mg./ml. Theconcentration ofhyaluronic acidinsynovial fluidofthe humankneejointis2to3mg./ml. This meansthatthemolecules arc"crowded"; theyoverlap, and,therefore, suchasolution mustbeconsidered asacontinuous net\vork ofinteracting, entangled molecular chains. Anyothermolecules, largeorsmall,dis­ solvedinthesolution, arewithinthe_domain ofthishyaluronic acidnetwork, and any molecule orparticle thatmovesinitmust passthrough thisfeltlikemolecular network. Thechemical activity ofthemolecules that canpenetrate thisnetwork maybechanged, andotherlargemolecules cannotfindspace topenetrate thenetwork. Inthissense,one speaksabouttheexclusion ellectandthe dynamic fIltration ellectofhyaluronic acid solutions. :.'1) Experiments invitrocarriedoutbymany investigators, havedemonstrated thatlarge proteinmolecules canbefilteredfromsolu­ tionbypassage through afilterlayerof synovial fluidorhyaluronic acid.Further­ more,duetotheexclusion effect,molecules dissolved inhyaluronic acidsolutions are alteredintheirchemical activity( solubility, aggregation, osmotic effect,charge effect, etc.)." Limiting viscosity number (intrinsic vis­ cosity) isawidcly-used indexforcharacter­ izingpolymers suchashyaluronic acid.The limiting viscosity number ofhyaluronic acid insynoviai fluidcanbemeasured without separating thepolysaccharide fromproteins. Thismeasurement givesameaningful pa­ rameter ofonehyaluronic acidmolecule \....hichexpresses thesize,volume, andshape ofthemolecule as\\I'ellasitsinteraction with thesolvent (waterwithionsdissolved in it;';·H.:IG). Thelimiting viscosity number ofthe synovial fluidofnormal humankneejoint doesnotchange withage(Table 5-1).' Thisindicates thatthemolecular sizeofthe hyaluronic acidinthejointremains thesame 66Ph}'sical Properties ofSynovial Fluid'SpecialRoleofHyaluronic Acid duringalifetime, buttheconcentration of thispolymer dropssuddenly around28to 35yearsofage. THEELASTOVISCOUS NATURE OFSYNOVIAL FLUID Normal Fluid Synovial fluidexhibits viscousandelastic properties. Bothdepcudonitssize,con­ formation, interactions, andnumber ofhy­ aluronic acidmolecules presentinthefluid. Inrecent.years,aconsiderable amountof workhasbeenreported ontheelastoviscous natureofhumankneesynovial fluidobtained fromnormalandpathological joints.'·G,",I4 Thesestudies clearlyshowthat,froma rheological pointofview,thesynovial fluid andtheprotein-free «1.0percent)hy-aluronic acidprepared fromothertissues (umbilical cordandroostercomb)areiden­ tical.Uptonow,noevidence hasbeen foundthatwouldindicate thatthepresence ofproteins inthesynovial fluidsignificantly alterstheelastoviscous properties ofthe puresodium saltofhyaluronic acid.Of course,thisdocsnoteliminate thepossibility thatsomeinteraction mayoccurbetween proteins andNa-hyaluronate molecules, whichcouldcauseminormodifications in theviscoelastic properties ofsynovial fluid. Theimportant factis,however, thatNa­ hyaluronate without proteins exhibits the samemolecular relaxation mechanism as synovial fluidwhenitisexposed tostrain ofvariousfrcquencies.::!o Todemonstrate theelastoviscous nature ofsynovial fluid,onehastomeasure the '""~ ~ YOUNG \G" ""-l101l ;: (HA238mQ/ml) ~ [7]6400ml/Qm G' I OLDJG'I(HA223m91m1 :[ry15200mll,mL' 100~ G I CSTEOARTHRQS1S (HAI27mq/ml) ~'I~4000ml/9m• o, o6Cycle/Su (Wal ....), __----1I I I I I 25Cf'!~/S~c IRun} I 100 (JOT'RADIANS/SEC.10' FIG.5-1.Dynamic clasticmodulus. G'(opensymbols) anddynamic viscousmodulus.G"(filled symhols) ofthreehumansynovial fluidsamples <lspirated fromthenormalkneejointofoneyoung (20yrs.)andOlll'old(67yrs.)subjectanufromtheosteoarthritic kneejointofanolusubject (63yrs.),plottedagainststrainfn,'quency. Thebrokenvertical linesindicate thefrequencies that corrcspolll..1 approximately 10the1ll00\'ml.'111 ofthekneejointinwalking nndrunning. Theconcen­ tration(HA)anulimiting viscosity numher (111\)ofhyaluronic acidintheaspirated fluidarcgiven inparl.'llthescs. (FromBal;lzs.E.A.:Uni\'.ofr-.1iehigan Mcu.etr.July,[Speci;d Arthritis issue): TheEiasioviscOU5 NalureofSynovIal Fluid 67 dynamic shearmoduliatvarious strainfre· quencics andatvarioustemperatures.20rOle dynamic rigidity ofsynovial fluid(G'), whichistheraliooftbepeakslress10the peakslraininthefluid,canbedivided vectorially intotwocomponents (G*:::: v'G"-+-G"').Oneofthesecomponents iscalledthedynamic lossmodule(G")or viscous module because itrepresents the energythatisdissipated asheatwhentbe molecule issubmitted tostrain.Theother component iscalledtbedynamic storage module(G'),orelasticmodule, because it represents theenergystoredwhenthemole­ culeissubmitted tostrain. Thisenergy storedinthemolecules forashortperiod oftimeimpartsanelasticnaturetothefluid. Tbiselasticity issimilartotbatdescribed in rubber solutions, anditisbasedonthe interaction between varioussegments ofthe molecular chainofhyaluronic acid.Itis alsocalledentropy elasticity because it depends onthestalesoforderanddisorder inthearrangement ofthemolecular chains ofhyaluronic acid. Atypicalsetofvaluesofthestorage and lossmoduliasafunction ofstrainfrequency isshowninFigure 5-1.Thethreesets ofcurvesofdynamic shearmoduliarc selected asrepresentative samples ofsyno­ vialfluidobtailiedfromthenormal jointof ayoung(20yrs.)andanold(67yrs.) donorandfromano:-.teoarthritic jointofan old(63yrs.)donor. Inallthreefluids,asthestrainfrequency increases, boththelossandthestorage modules increase. Theabsolute valuesof thesemoduli atlowfrequencies varycon­ siderably. Thefluid obtain~d fromthe normal youngjointh<.J.s th~highest value J.Ddthatobtained fromtheosteoarthritic jointthe10\l,'cst. r.lostimportantly, asthe frequency increases, thecurvesrepresenting thelossandstorage modulicrosseachother inthetwonormal fluids,butnotinthe p:hological fluid.Thismeans thatthe ~,-~~malfluidsarepredominantly viscous at [0\\'strainfrequency andpredominantly c:JsticathighstrainfrcqucrlCY.'.Sincethis cru::;sover isnotobservable inthepatho-logicalfluid,onehastoconclude thatthis fluidbebaves intheentirefrequency range, asapredominantly viscous ratherthanan clasticfluid.Itisimportant tonotethat thestrainfrequencies atwhichthesemea­ surements weremadearcwithintherange to\vhichthefluidisexposed inthecourse ofthenormalmovement ofthekneejoint (flexing undernoload,walking, running). Theseobservations canheexplained on themolecular levelbyconfigurational adjust­ mentsoftbehyaluronic acidchains.Atlow strainfrequencies, theconfigurational adjust­ mentsofthechains, owingtothermal (Brownian) motions arcrapidenough to a(Jowthemolecule tomaintain itsoriginal conformation. Thatis,underimposed strain, thechainsslipbyeachother,whichresults inaviscollsflow.Therefore, therheological properties ofthefluidarepredominantly viscous. Athighstrainfrequency, thecon­ figurational readjustmcnt ofthechainscan­ notoccurbetween theshortpcriods ofthe oscillating strain,andthemolecules cannot maintain theiroriginal conformation. That is,undcrtheimposed strain,themolecules dc[ormsinusoidally andalternately storethe mechanical energyandthenreleaseitelastic­ ally.Under theseconditions, thefluid's rheological behavior ispredominantly that ofanelasticbody. Themostremarkable behavior ofsynovial fluidunderincreasing strainfrequency isits rapidtransition fromviscous Iluidtoclastic body.Thistransformation isreversible and hasnodeteriorating elfectonthemolecule. Natural rubberandindustrial polymers with elastoviscous properties showconsiderably different behavior inthatthistransition occursduringaconsiderably extended fre­ quency range. \Vhatarcthebiological implications of thisfrequency-dependent viscoelastic trans­ formation ofsynovial Iluid?Thes.ynovial Iluidoccupies narrO\I,/channels between the softtissuesofthejoint,anditi~sandwiched between the(\1,/0cartilage surfaces. The measurClllents ofdynamic shearmoduli tel! usthattheIluid\,,'illmoveasavi.-;cousliquid inthesechannels whenthejointmovesat 68Ph}'sical Properties ofS}'l1ovial Fluid'SpecialRole01H}'afurol1ic Acid TABLE5-2.Rheological Properties ofSynovial FluidsAspirated from theKneesofHumanSubjects ofVarious Ages.* Elasfic Viscous Crossover Point TwoModuli AgeAlodllles A10dllles ofTwoA1odu/i (G',G'I)atGroup SubjectsC' Coo (G',GIf )Crossover Poiflt (yrs.)(llyn.!sec.-2Jt(dYIJ.Isec.-I)t (dyn./sec.-1)(dyll.lsec.-') 18·27 16 1170:':130 450:':82 0.13:':0.02 332:':41 27-35 18 226±7 72± 8 0.21:':0.004 59:':6 52-78 26 189:':33 101:':12 0.41:':0.12 61:':7 *'Datatakenfromtheunpublished workofE.A.Balazs,N.W.Rydell,P.O.Seppala,I.F.Duff,D.A. GibbsandE.W.Merrill. tMeasured at2.5cycle/scc.- 1 lowshearfrequency. Underhighshearfre­ quency movements, thefluiddocsnotmove inthechannels; ratheritbehaves asan elasticsolid,storingthemechanical energy. Butthefluidalsoimpregnates thesurface layerofarticular cartilage andsynovial tissue.Thus,itseparates cells(synovial cells)andproteinfibrils(collagen), prevent­ ingthemfromdirectcontact witheach otber.Thismeansthatunderslowmechani­ calloadingofthejoint,whenarticular carti­ lageandsynovial tissueareexposed tolow frequency strain,hyaluronic acidbehaves asaviscousoil.Thefluidbetween thecells andcollagen fibrilsisdisplaced (flows) underthemechanical forcesandthetissue itselfdeforms. Ontheotherhand,athigh 'mechanical loadingratesofthejoint,when thesetissuelayersareexposed tohighfre­ quencystrain,hyaluronic acidtransforms intoahighlydeformable elasticsystem. Therefore, thefluid,whichcontainshyal­ uronicacid,isnotdisplaced, andthetissue itselfisnotdeformed. Thismeansthatthe hyaluronic acidinthesurface layersof articular cartilage andsynovial tissueab­ sorbsmechanical stressthereby protecting thecellsandcollagen network (romme­ chanical shockanddeformation. Inother words,thispolysaccharide servesinthese tissuelayersasashockabsorber. Thecells,whicharcextremely sensltlvc tomechanical stress,andtherigid,load­ supporting collagen fibrilsarcsurrounded by theelastoviscolls hyaluronic acidsolution. Thereisamechanical coupling bdwcen therigidsystemofcellsandfibrilsandthe energy-storing andenergy-dissipating system ofhyalurollic acid.Therefore, alargepart ofthestressimposed ontheentiresystem isconverted toelasticdeformation ofthe hyaluronic acid.Bythismechanism, the stress-sensitive elements ofthesystem(cells) arcprotected andthestructural integrity of thetissue(special organizational patternof thefibrils)ismaintained. EffectsofAging' Therheological properties ofsynovial fluidchangeconsiderably duringaging.The elastic(storage) modulus (G')andviscous (loss)modulus (GOO)decrease sharplyafter theageof27years.Theelasticmodulus dropsfurther aftertheageof52years (Table5-2).Aswepointed outabove, synovial fluids,likesolutions ofpurehyal­ uronicacid,showarapidtransition from viscollstoclasticbehavior whenthestrain frequency increases. Thefrequency atwhich thistransition occursor,moreprecisely, wherethecurvesbfthestorage andloss modulicrossoneanotherandbothmoduli havethesumenumerical value,isspecific foragivenfluid(Fig.5-1).Thisfrequency valueatthecross-oYer pointincreases with aging(Table5-2).Thevalueofthetwo moduliatthecross-over pointdropssharply afterthe27thyearbutdoesnotchange *Thedalareported herearefromtheworkof Balazs.Ryucll. Sepp;;I~i, Duff,MerrillanuGibbs. While th~detailsofthisworkarcasyetunpublished, abriefrc\"il..'\\ CUllbefounuinBalazs,1969.n TheEIJ510viscous NalureofSynovial Fluid 69 TABLE 5~3.Concentration andLimiting Viscosity Number ofHyaluronic Acid andtheRheological Properties ofSynovial FluidsAspirated fromHuman Pathological Knees. <t Elastic ViSCOllS Croysover Pathological FluidsVoll/me Hyaluronic Acid fdodtlles Modllies Poirltof Fluid [,j G' GnTwoA10dules Corldilion AnalysedCal/eered (dy!!.! IdYll.! (G',Gn) 1",/.) IIlg./mi.ml.!g.sec.-t)sec.-f)(cycleslsec.-J) Osteoarthritis II 1.55"0.143800"35085"5448"284.7"1.9 Traumatic Arthritis 37·20 0.69-1.76 2iOU-42UO 2-41 2-29 1.3-2.9 Gout 4 3-51.28"0.143500" 69U3U"IU15"70.9"0.3 ChonJrocalcinosis 22.5 0.75 37UU 5 5 1.7 1.22 3900 22 13 0.9 *Dalalakenfromunpublished workofE.A.Balazs,P.O.Seppala, N.W.Rydell,I.F.Duff,E.W. ~lerrillandD.A.Gibbs. later.Thus,therheological properties of synovial fluidsinallthreeagegroupsstudied arcsignificantly different. Thefluidfrom youngsubjects isveryhighlyelasticand rigidatrelatively lowfrequencies. Thefluid frommiddle-aged subjects islessrigidbut stillhighlyelasticathigherfrequencies. The synovial fluidfromoldersubjectsisless rigid,lessviscous, andlesselasticatall frequencies. Thefrequencies atwhich th~semeasure­ mentswerecarried outwereinthesame rangeasthefrequencies atwhichthejoints areloadedandflexedduringnatural I11ove­ mentsofthebody.Therefore, somecon­ clusions canbedrawnabouttherhrological behavior ofthefluidinthejointsubmitted tovarious ratesofstrain. B~tween theages of18and39,thesynovial fluidundergoes asubstantial decrease inrigidity, butretains itsgenerally elasticcharacter. Withfuriher agingtheelasticity decreases insuchaway thatunderthefrequency conditions of norillal kneemolion thesynovial fluid cbanges fromthehighlyclasticfluidinthe youngtoanonelastic viscous fluidintheold. Sincetheconcentration, size,shape,and limiting viscosity number ofindividual hyal­ uronicacidmolecules docsnotchangein thesynovial IJuidofnormal human knee Jointbetween theagesof27and78butthe elastoviscous properties ofthefiuiJriJdicalty d~crcase, onehastoassume thattheinter­ actionbetween thechainsoftheneighbor-ingmolcules isaltered. Recent studies on hyaluronic acid,carried outusingX-ray diffraction andoptical rotation measure· ments,indicate thatacertain amountof theindividual polysaccharide chainsform double helicaljunction pointsorcrosslinks thatincrease theelastoviscous properties of thepolymer. Itispossible thatduringaging theamounto[thesedoublehelicalcrosslinks between thechainsofneighboring molecules decreases. Thisinturnwouldmakethe molecular chainsegments lessstillandthe solution lesselastic. Pathological Floids' Inthesynoviailluids aspirated fromjoints withosteoarthritis, traumatic arthritis, gOllt, chondrocalcinosis, andrheumatoid arthritis, theconcentration, limiting viscosity number, andmolecular weightofhyaluronic acidis lowerthaninnormal joints. \1,35.40Conse­ quently, allrheological properties ofthe fluid,suchasthedynamic viscousandclastic moduli andthecrossover pointofthetwo moduli arealsomuchbelownormal values Cfable5-3;Fig.5-1).Thus,inthepatho­ logicaljoint,thesynovial fluiddocsnot havethoserheological properties thatpro­ tectthesynovial lissu~andcartilage against mechanical stress. '"Datareported herearefromtheworkofBalazs, Sepp;iL.l, Ryuell,Gibbs,DuffandMerrill. Whilethe delailsofthisworkarenotyetpublished, abrief reviewofIIc"nbefoulloinBalazs1968.'-' 70Physical Properlies ofSynovial Fluid'SpecialRole01Hyaluronic Acid THESURFACE OF 'HIEARTICULAR CARTILAGE Tbereissomeindication thathyaluronic acidisnotevenlydistributed intheentire jointspace.Onthesurfaceo[thearticular cartilage andonthesur[aeeo[thesynovial tissue,ahyaluronic acidlayerwasobserved whiebcannotbeeasilywashed away[rom thesetissuesudaees. Theconcentration of thehyaluronic acidintheselayersishigher thaninthefluidaspirated fromthejoint. Itisnotclearwhatkindo[molecular inter­ actionisresponsible [orthe"accumulation" ofhyaluronic acidonthesetissuesurfaces. Electron microscopic studiesshowed that a1-to2-p.-thick layerofhyaluronic acid­ proteincomplex coversthesudace o[tbe articular cartilage. Thislayerisanchored to thefibrillarcollagen matrixo[thecartilage andcanberemoved fromitbytreatment withproteolytic enzymes orhyaluronidase, Witbaging,andinosteoarthritic cases,this layerbecomes thicker.Itisnowknown, however, bowthehyaluronic acid-and pro­ teincontentofthelayerchangewithaging andwitbvarious pathological conditions' Histochemical andchemical analyses car­ riedoutonthecartilage closetothearticular surfaceindicate thepresence ofhyaluronic acid50to100P.deepintothecartilage whereitsharestbespacebetween thecolla­ genfibrilswithsul[ated proteoglycans .•The deeplayerso[thecartilage matrixcontain onlysulfated proteoglyeans (proleoglyeans o[cbondroitin 4-sul[ate andkenllansul[ate) butnohyaluronic acid. Scanning electron microscopy alsoshowed anaccumulation ofsynovial fluid(hyal­ uronicacidandproteins) unthesurfaceof articular cartilage'"'""" (1970), Undercon­ ditionsofextreme load,experiments invitro show,thisfluidlayerprotects thecartilage surface.":: Onecanonlyspeculate abouttheimpor­ tanceofthislayerinthenormal [unction andpathology o[thejoint.Sinceitrcpre­ sentstbeonlymorphologically visiblebar­ rierbetween thecartilage matrixandthejointspacc,itistempting toassumethat itisresponsible [ortheprotection o[the canilage surface. Sincethesynovial fluid o[thenormaljointdocsnotcontainappre­ ciableamounts o[sul[ated proteoglyeans, it ispossible thatthesurface layo<ofthe canilage impregnated withhyaluronic acid formsaneffective barrieragainstdiffusion orflow(underpressure causedbycom­ pressing thecanilage whenthejointis loaded) o[thesul[ated proteoglycans into thesynovial fluid.Invarious pathological conditions sul[ated glycosaminoglyeans were foundinthesynovial fluid,"suggesting that theintegrity o[thesurface layero[the articular canilage impregnated withhyal­ uronicacidisresponsible [ortbenormal maintenance o[thecartilage bypreventing theleakageofproteoglycans intothesyno­ vialspaceandtbesubsequent losso[this important component ofthecartilagc matrix. Themissinglinkinthisargument, o[course, isthecomplete lacko[knowledge o[the chemical composition o[thecaniluge surface layerinpathological conditions. THEEFFECT OFHYALURONIC ACID ONCELLACTIVITIES Recently, several eflectso[hyaluronic acidoncellsinvitroandinvivohavebeen reponed. Noneo[theseeflectshavebeen directly connecled tothepathological pro­ cessesinthejoint.Nevenheless, theeflect ofhyaluronic acidoncellactivities isso generalthaItheassumption thatitisopera­ tiveinwoundhealingandinOammution o( thejointisjustified." Na-hyaluronate wasround10beacell­ immobilizing agent.Themovement ofcells o[thelymphomyeloid system(lymphocytes, granulocytes, macrophagcs) isinhibited by thisbiopolymer andbythetissueIluidsthat containthismolecule (synovial fluid,IiquiC vitreous). Thelast-moving cellso[the Iymphomyeloid systemexhibitditkrent sen· sitivities tohyaluronic acid.Thiscell­ immobilizing eflectisspecific[orthesetype, o[cellsbecause cellsthatmoveslowlyir Ifie:LI!t:"Ll VIII,aIUrV'''L "....'......,.n,'-......""....'''..,....-1 vitro(fibroblasts) arcnotaflected. Tbe eflectofhyaluronic acidonthemotility of cellsisnotdirectly related tothebulk viscosity ofthesolution inwhichthecells aremoving. Theeffectisdependent onthe limiting viscosity number ofthehyaluronic acidused.Hyaluronic acidpreparations withlowlimiting viscosity numbers arcless effective cellmovement inhibilors than preparations withhighlimiting viscosity numbers. Pathological synovial Ouids,with lowlimiting viscosity numbers, arelesseffec­ tivethannorma!fluids.!3 Hyaluronic acidalsoinhihits themodula­ tionoflymphocytes tolymphoblasts. When bloodlymphocytes, stimulated bymitogens (phytohemagglutinin, pokeweed mitogen, streptolysin 0orpurified proteinderivative oftuberculin) arcplacedinviscous hyal­ uronicacidsolution, thctransformation of lymphocytes tolymphoblasts andthesub­ sequent mitosis isprcv~nted aslongastbe cellsarcsurrounded andseparated fromeach otherbyviscous Na-hyaluronic solutions,!; Na-hyaluronate canalsoprevent thetar­ getscellsfromkillingsensitized lymphocytes. Thecytotoxic effectoflymphocytes isinhib­ ited\vhentheNa-hyaluronate concentration inthemedium separating thetargetceUs fromthelymphocytes reaches acertain concentration.11 Thegraft-versus··host reaction couldbe inhibited withNa-hyaillronak whenthe donorcellsarcinjected intotheperitoneal cavity:Apparently, thespleencellsinjected \vithNa-hyaluronate donotfindtheirtarget organ(splecn, liver);or,iftheydo,their proliferation isinbibitcd.!3 i\a-hyaluronate seemsalsotoinfluence the\vound healing ofarticular cartilage, kndons, and fasci~\e. Relatively fewexperi­ mentsbavebeenreported inthisareaand th\?refore. onehastoregardtheseresultsas preliminary. Whenthedorsalfasciae of rabbitsandguineapigsandthelongex­ t~nsortendons ofthelegsofrabbits\\'crc traumatized bymechanical damage and, afterthetrauma, viscous Na-hyaluronate solution (sterile andpyrogen-free) wasapplied tothedamaged area,thesubse­ quentformation ofgranulation tissueand fibrous adhesions wasconsiderably sup­ pressed. Na-hyaluronate alsosuppressed formation ofgranulation tissuearound foreign bodies (polyethylene) .1'30.:" All theseinvestigations suggest thathyaluronic acidhasaceBregulatory function which specilically affectstheIymphomyeloid system duringtheinflammatory process. EffectonFrictional Resistance Ithasbeengenerally accepted foralong timethatthefrictional resistance ofthose partsofthejointthatmoveadjacent toeach other(articular cartilagc, synovial tissue, ligaments, tendons withintheirsheaths, walls ofbursae)isdecreased bytheviscous s~mo­ vialftuid.Theviscosity oftheftuidhas beenregarded asthekeytothislubricating effect,andthehigh-molecular-weight hyal­ uronicacidastheessential component of thelubricating Ouid."·"" Recently, thisjointlubrication wasfurther defined byseparating itintotwoproblem areas:thelubrication ofthe"softtissues" (ligaments andsynovial tissue) andthe lubrication ofthecartilage surfaces." The roleofhyaluronic acidindiminishing the frictional resistance of"softtissues" sliding acrosseachotherwasconfirmed. Onthe otherhand,thesameactionofhyaluronic acidoncartilage slidingovercartilage was questioned. Aglycoprotein fraction was foundinthesynovial fluidthatdecreased thecocflicicl1t offriction between moving cartilage surfaces. nTheimportance of h,Y'uluronic acidasanagentthatreduces thefrictional resistance between themoving surfaces insidethejoint,andbetween ten­ donsandtheirsheaths, is stiH notfully understood. RoleoftheCartiluge Surface Asdescribed above,thesurfac.e layerof articular cartilage isimpregnated withhyal­ uronicacid.Onecanvisualize thetwo opposing cartilage surfaces andtbethin layerofsynovial fluidbetween asacon- 72I'hl'sicall'roperlies ofSl'novial Fluid'SpecialRole01J-fl'a/uronic Acid tinuoushyaluronic acidnctwork. Thchyal­ uronicacidnetwork isanchored ontothe collagcn fibrillarmatrixofthesurfacelayer ofcartilagc ofbothsides.Thcspacebewecn thetwocollagen matriccs isfilledwiththe samchyaluronic acidmolecular network that impregnates tbecollagen matrix. Therefore, dislocations between thetwomovingcarti­ lagesurfaces occurnotbetween tworheo­ logically different systems (solidcartilage andsynovial fluid),butwithinthebyal­ uronieacidnetwork. Thisconcept hastwo important biological implications. One,there arenoasperities orripplesonthesliding surfaces. Thatis,thebeautiful scanning elec­ tronmicrographs showing theunbydrated cartilage-synovial fluidsurfacewithitsmany crevices donotpicturetherealslidingsur­ face.Therealslidingsurface isnotthat whichoneexposes bybreaking theconti­ nuityofthehyaluronic acidnetwork andits debydrated pictureintheelectron micro­ scopedoesnotgivetheproperimpression ofabighlybydrated hyaluronic acidmolecu­ larnetwork. Two,thehyaluronic acid impregnated cartilage surfacetbatservesas abarrieragainstthemovements ofmacro­ molecules inandoutofthecartilage isnot distributed bythemovements inthejoint. Therefore, theintegrity oftbislayerandthe composition ofthecartilage matrixismain­ tained. According tothishypothesis, the majorroleofhyaluronic acidonthecarti­ lagesurfacc istoprovide therealsliding surfaces andtomaintain theintegrity ofthe cartilage matrix. ControlofCellInvasion Thereisnoepithelial barrieronthesur­ faceofthesynovial tissuesthatwouldpre­ ventcellmigr:.ilion fromthesetissuesinto thesynovial spaceandonthesurfaceofthe softtissuesandthecartilage ofthejoint.It wasproposed thathyaluronic acidprevcnts theinvasion ofcellsintojointspace.i.!! Thisconcept isespecially important invicw ofthefactthatinallacuteorchronic in­ flammatory processes ofthejoint.boththe concentration andsizeofthehyaluronic acidmolecules decrease and,atthesametime, thecellpopulation inthejointspacein­ creases.Itisimportant tonotethatthe pathological fluidislesseffective inpre"ent­ ingthemigration oftbelymphomycloid cells invitrothanthenormalfluid.Thesefind­ ings,whilesuggestive) donotpresentdirect proofoftheroleofhyaluronic acidasacell movement controlling factorinthejoint. HYALURONIC ACID ASATHERAPEUTIC AGENT Inacuteandchronicinflammation andin mostdegenerative proccsses ofthejoint,the concentration andmolecular sizeofhyal­ uronicaciddecreases inthesynovial fluid. Consequently, theviscosity andelasticity of thefluidalsodecreases. Wesuggested that intraarticular application ofbighlypurified (protcin content<0.3%)concentrated (10 to20mg./ml.) Na-hyaluronate thatcontains fairlylargemolecules (molccular weight1 to3million) ofthisbiopolymer caninllu­ encethehealing andrcgeneration ofthe cartilage andsofttissuesofthejoint.The rationale forthissuggestion isthatthein­ jectedhyaluronic acidwillaccumulate on thearticular andsynovial tissuesurfaces, thereby "reinforcing" tbenatural barriers whicharemostprobably deteriorated inthe courseofthcpathological process. Thus, theinjection ofhyaluronic acidintoa diseased connective tissuecompartment in whichitisnormally present canproperly becalledamacromolecular implantation. Themainobjective oftheimplantation is toincreasc'thehyaluronic acidconcentration inthejointwellabovethcpathological and eventhenormallevel.Sincethisbiopolymer isanaturalcomponcnt ofthejoint,it metabolizes bydiffusion andprobably by phagocytic activity ofmacrophages. Con­ sequently, theclevatcd concentration intbe joilltcausedbytheinjection dccreases to normalIcvelwithind<JYs.Nevertheless, one cxpects thattheinvasion oftheIympho­ myeloid cellsintothejointspaccishalted bythclemporary increase ofhyaluronic acid concentration bythesamemechanism as themovement ofthesecellsisinhibited by thisbiopolymer invitro.Furthermore, the hyaluronic acidaccumulated onthesurface ofthecartilage andsofttissuesmayblock inflowofproteins (immune complexes) and proteoglycans intothejointspace,thereby triggering ahealing process inthecartilage anddecreasing inflammation inthesynovial tissue, Experiments carriedoutindogandrabbit jointsindicate thatintraarticular cartilage healsbetterwhentheNa-hyaluronate con­ centration ofthesynovial spaceisincreased afterwounding byimplantation ofthisbio­ polymer.:" Itwasalsofoundthatthegranu­ lationreaction insubcutaneous tissueafter surgical wounds:wandinadhesion formation between tendonandtendonsheaths after mechanical damage isdecreased whenNa­ hyaluronate isapplied tothewounded surfaces.:~:! Treated withinlraarticular administration ofNa-hyaluronate traumatic arthritis in horses,rapidlyimproved and,inmostcases, thenormalfunction ofthejointwasrestored afteroneortwotreatments,:Jl* Na-hyaluronate wasadministered intfJ­ articularly forhumanosteoarthritis bysev­ eralinvestigators, (Ryc1ell, Helfet,Peyron), Results oftheseinvestigations arcreported elsewhere inthishook(secp,1.42)!7:J Na-hyaluronatc wasalsoimplanted into humanvitreous duringsurgical procedures forretinaldetachment. Sincehyaluronic acidispresentinthevitreous initshighest concentration adjacent totheretina,itwas thought thatinchronicinflammation caused byretinalwounds, thehealing wouldbe promoted byimplantation ofthisbio­ polymer. Furthermore, itwasstipulated thatviscoelastic Na-hyaluronatc solution wouldfacilitate thereattachment ofthe retinaincaseswhereothersurgical tech­ niquesfailed. ~Severa! investigators found "J.L.BUller:..LnuA.Asheim. personal communi­ cations. t:\vailable fromBiotrics. Inc..Arlington. r-.!Jss., USA.References 73 thatNa-hyaluronate implanted intohuman vitreous incomplicated casesofretinal detachment facilitated thereattachment o[ retinatochoroid, thusimproving theheal­ ingofthevitreoretinal wound.: U::i.:!:.!.:!8 OnehastopointoutthattheNa-hyal­ uronate usedforimplantation intothejoint, vitreous, orotherconnective tissuecompart­ mentsmustbe.freefromimpurities that cancauseimmunological reaction ortissue irritation, Furthermore, thepreparation mustexhibitspecific biological activity on Iymphomyeloid cells,SuchNa-hyaluronate fractions havebeenprepared fromboth human(umbilical cord)andavian(comb) tissues;t itwasusedinmostoftheexperi­ mentalandclinicalworkcitedabove, REFERENCES I.Alexandersson, R.,Nettelbladl, E.,and Sundblad, L.:Lacticdehydrogenase isoen­ zymesinarthritic synovial fluid:Effectof cortisol. ActaRheumatol. Scand., 14:243. 1968. 2.Algvcre, P.:Intravitrcal injection ofhigh­ molecular-weight hyaluronic acil!inretinal dctachment surgery. ActaOphthalmol.. 49:975,1971, 3.Anderser:, R.B.,andGormsen, J.'Fibrin dissolution insynovial fluid.ActaRheuma­ tal.Scand.,16:)19,1970, 4.Balazs,E.A.:Physical chemistry ofhyalu­ ronicacid.Fed.Proc.,17:1086.1958. 5,--~: Viscoelastic Properties ofHyalu­ ronicAcidandBiological Lubrication. Univ.t-.Iichigan Med.Or.J.,[Spccial Is· sue]: 255~159, December, 1968. 6.---. Someaspectsoftheagingand radiation sensitivity oftheintercellular matrix withspecial regardtohyaluronic acidinsynovial fluidandvitreous. InEngel. A.,andLarsson, T.(cds.):ThuleInterna­ tionalSymposium: AgingofConnective andSkeletal Tissue. Stockholm, Nordiska Bakhandelns Forlag,1969. 7,---: Structure andmetaboiism ofcon­ nective tissueunderphysiological and pathological conditions. IIIRiittner. J.,ct al.(eds.): Arthritis andOsteoarthrosis. 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