Author's Accepted Manuscript
Nucleotomy Reduces the Effects of Cyclic Compressive Loading with Unloaded Recovery on Human Intervertebral Discs Brent L. Showalter, Neil R. Malhotra, Edward J. Vresilovic, Dawn M. Elliott
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S0021-9290(14)00329-7 http://dx.doi.org/10.1016/j.jbiomech.2014.05.018 BM6673
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Journal of Biomechanics
Accepted date: 24 May 2014 Cite this article as: Brent L. Showalter, Neil R. Malhotra, Edward J. Vresilovic, Dawn M. Elliott, Nucleotomy Reduces the Effects of Cyclic Compressive Loading with Unloaded Recovery on Human Intervertebral Discs, Journal of Biomechanics, http://dx.doi.org/10.1016/j.jbiomech.2014.05.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
NucleotomyReducestheEffectsofCyclicCompressiveLoading withUnloadedRecoveryonHumanIntervertebralDiscs BrentL.Showalter1,NeilR.Malhotra2,EdwardJ.Vresilovic3,andDawnM.Elliott4* (1)DepartmentofBioengineering UniversityofPennsylvania (2)DepartmentofNeurosurgery UniversityofPennsylvania (3)DepartmentofOrthopaedicsandRehabilitation PennStateUniversity (4)DepartmentofBiomedicalEngineering UniversityofDelaware OriginalArticle WordCount:3965 Keywords:IntervertebralDisc;Spine;CyclicLoading;NucleusPulposus;Nucleotomy *Correspondingauthor: DawnM.Elliott BiomedicalEngineering UniversityofDelaware 125EastDelawareAve Newark,DE19716 USA Email:[email protected]
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Abstract Thefirstobjectiveofthisstudywastodeterminetheeffectsofphysiologicalcyclicloadingfollowedby unloadedrecoveryonthemechanicalresponseofhumanintervertebraldiscs.Thesecondobjectivewas toexaminehownucleotomyaltersthedisc’smechanicalresponsetocyclicloading.Tocompletethese objectives,15humanL5S1discsweretestedwhileintactandsubsequenttonucleotomy.Thetesting consistedof10,000cyclesofphysiologicalcompressiveloadsfollowedbyunloadedhydratedrecovery. Cyclicloadingincreasedcompressionmodulus(3%)andstrain(33%),decreasedneutralzonemodulus (52%),andincreasedneutralzonestrain(31%).Degenerationwasnotcorrelatedwiththeeffectofcyclic loadinginintactdiscs,butwascorrelatedwithcyclicloadingeffectsafternucleotomy,withmore degeneratesamplesexperiencinggreaterincreasesinbothcompressiveandneutralzonestrain followingcyclicloading.Partialremovalofthenucleuspulposusdecreasedthecompressionandneutral zonemoduluswhileincreasingstrain.Thesechangescorrespondtohypermobility,whichwillalter overallspinalmechanicsandmayimpactlowbackpainviaalteredmotionthroughoutthespinal column.Nucleotomyalsoreducedtheeffectsofcyclicloadingonmechanicalproperties,likelydueto alteredfluidflow,whichmayimpactcellularmechanotransductionandtransportofdiscnutrientsand waste.Degenerationwasnotcorrelatedwiththeacutechangesofnucleotomy.Resultsofthisstudy provideanidealprotocolandcontroldataforevaluatingtheeffectivenessofamechanicallybaseddisc degenerationtreatment,suchasanucleusreplacement.
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Introduction Theintervertebraldiscperformsthemechanicalrolesofsupportingloads,permittingmotion, anddissipatingenergy.Discdegenerationisastronglyimplicatedcauseoflowbackpainandwithinthe U.S.resultsinannualcostsover$100billion(Katz,2006).Degenerationisamultifacetedprocessthat manifestsearlyinthenucleuspulposus(NP)andsubsequentlyextendstootherdisccomponents (VernonRobertsetal.,2007).Despitediscdegenerationprevalence,manycurrenttreatmentsare palliativeonlyandultimatelyfail(Andersson,1999;Luoetal.,2004;Rajaeeetal.,2012).Minimally invasivetreatmentsofearlydiscdegeneration,suchasinjectableNPreplacements,areunder development(Lewis,2012;Malhotraetal.,2012;Reitmaieretal.,2012).Forsuchmechanicallybased treatmentstobesuccessfultheymustreplicatenaturalNPmechanicalfunction.Inhealthydiscs, proteoglycanscreateanosmoticpressurewhichdrawsfluidintotheNP(UrbanandMcMullin,1988). Thisfluidbothdirectlysupportscompressiveloadsandplacesthecollagenfibersoftheannulusfibrosus intension,permittingthedisctosupportloadswhileundergoingawiderangeofmotion.
Physiologicalcycliccompressiveloadingandunloadedrecoveryisanidealtestingmodalityfor examiningtheloadingcontributionofthenucleuspulposusbecauseitsimulatesthedisc’stime dependentmechanicalresponse.Theinterplaybetweenosmoticpressureandexternalloadscauses20 25%ofthedisc’swatercontenttobeexpressedandreimbibeddaily(Sivanetal.,2006).Reduced hydrationdecreasesdischeight,increasesstiffness,andincreasesrangeofmotion,withproperties returningtobaselineafterrehydration(Adamsetal.,1990;Wilkeetal.,1999).Thesediurnalchanges havebeenreplicatedinseverallargeanimalmodelsbycompressivecyclicloadingandunloaded recovery(Johannessenetal.,2004;Koreckietal.,2008;Thoresonetal.,2010).Whileanimalandhuman discsaresimilar,thesefindingsmaynotberepresentativeofcyclicloadinginhumandiscsduetolower waterandproteoglycancontentinhumans,variedappliedloadsandloadingdurationsbetweenstudies, anddifferencesincartilageendplate(Becksteinetal.,2008;Demersetal.,2004;Showalteretal.,2012; 3
Wilkeetal.,1997;Zhangetal.,2014).Further,themajorityofpreviouscyclicloadingstudieswere designedtotestdamagemechanismsbyemployinghighloadsandnumberofcyclesandbynot includingunloadedrecovery(AdamsandHutton,1985;Hasegawaetal.,1995).Dischydrationeffects havealsobeenstudiedusingcreeptests(Adamsetal.,1987;Koelleretal.,1984).However,creep induceslowerfluidflowratesthancyclicloadingandthusdoesn’tsimulatethedynamicnatureofdisc loading(WhiteandMalone,1990).ThisstudyexaminesthecontributionoftheNPtodiscloadingby testinghumandiscsundergoingaphysiologicallyrelevanttestingmodality,whichiscompressivecyclic loadingwithhydratedunloadedrecovery.
Themechanicalroleofthenucleuspulposuscanbeevaluatedbycomparingtheeffectofcyclic loadingonintactandpartiallynucleotomizedsamples.Nucleotomy,alsoknownasdiscectomy,isa clinicaltreatmentfordischerniation(Friedman,1983;KambinandBrager,1987).Theprocedurealters discmechanicsbydecreasingdischeight,NPpressure,andstiffnesswhileincreasingrangeofmotion andcreep(BrinckmannandGrootenboer,1991;Cannellaetal.,2008;Freietal.,2001;Goeletal.,1986; Ishiharaetal.,1993;MeakinandHukins,2000;O’Connelletal.,2011).Whiletheacuteeffectsof nucleotomyhavebeendetermined,itsimpactonthedisc’sfluidflowrelatedmechanicalresponsehas not.
Thefirststudyobjectivewastodeterminetheeffectsofphysiologicalcyclicloadingfollowedby unloadedrecoveryonthemechanicalresponseofhumanintervertebraldiscs.Thesecondobjectivewas toexaminehownucleotomyaltersthedisc’smechanicalresponsetocyclicloading.
Methods SamplePreparation Fifteenhumanlumbarspinesegmentswithameandonorageof50.1years(2275years)were obtainedfrominstitutionallyapprovedsources.ThesespinesunderwentMRimagingtodetermine 4
degenerationgradeandT2relaxationtimes(Kerttulaetal.,2001;Marinellietal.,2010;Pfirrmannetal., 2001).TheL5S1discwasselectedfortestingbecauseherniationsanddiscectomiesaremostcommon atthislevel(Weinsteinetal.,2006).Sampleshaddegenerationgradesrangingfrom1to5andT2 relaxationtimesrangingfrom66to204,withhigherrelaxationtimesindicatinghealthierdiscs.T2 relaxationtimewasnotacquiredforonesample.Fortheremaining14samples,T2relaxationtimes correlatedwithPfirrmanngrades(r=0.76)andprovideaquantitative,continuousmeasurementof degeneration(Figure1).DisccrosssectionalareawasmeasuredfromMRimages.Afterimaging,L5S1 bonediscbonesegmentswerepreparedbyfirstremovingtheposteriorelementsandextraneoussoft tissue.Althoughinvivoloadsaresharedbetweenthediscandfacets,posteriorelementswereremoved inordertoisolatethecontributionsofthediscincompression(Becksteinetal.,2008;Meakinand Hukins,2000).Followingdissection,1.25mmKirschnerwireswereplacedintheL5vertebraeand sacrum.TheL5S1discswerewedgeshaped,witha13±4°anglebetweentheL5andS1endplates, whichlikelyresultsinsomeappliedshearloadsevenduringsimplecompressionexperiments.For consistencyinmechanicaltesting,loadswereappliedperpendiculartotheL5vertebrae.Thiswas ensuredbypottingthemotionsegmentsinPMMAwiththeL5endplateparalleltothepottingfixtures usingfluoroscopicguidance.Sampleswerestoredinafreezerat20°Cwhennotinuse. Mechanicaltesting Themechanicaltestconsistedofsixsteps(Figure2).1.Overnighthydration,alsocalledinitial hydration.2.Measuremechanicalparameters(Initial).3.10,000cyclesofcompressiveloading.4. Measuremechanicalparameters(Cyclic).5.Overnighthydration,alsocalledunloadedrecovery.6. Measuremechanicalparameters(Recovery).Duringthefirststep,overnighthydration,sampleswere submergedina0.15MPBSbathcontainingproteaseinhibitors(PhenylmethylsulfonylFluroide,N EthylmalemideBioxtra,andBenzamidineHydrochloridefromSigmaAldrich,St.Louis,MO)at4°Cfor16 24hours.Second,mechanicalparametersweremeasuredbyapplying60cyclesat0.2Hzbetween0.48 5
MPa(734±122N)incompressionand375Nintensioncycles,whichissimilartopreviousstudies (Becksteinetal.,2008).Thestressof0.48MPacorrespondstoonebodyweightortolowintensity activitiessuchassittinguprightwithoutsupportorrelaxedstanding(ElliottandSarver,2004; Nachemson,1981;Wilkeetal.,1999).Thetensileloadof375Nwasappliedtoallsamplesinsteadofa uniformstresstopreventsamplepulloutfromthePMMAfordiscswithlargecrosssectionalareas whileensuringsufficientloadtodescribethemechanicalparametersofinterest.Thethirdstepwasto apply10,000cyclesofcompressiveloadsat2Hzbetween0.12and0.96MPa(1467±244N)whichare loadssimilartomoderatephysicalactivitiessuchasjoggingorliftinga10kgweight(Nachemson,1981; Wilkeetal.,1999).Thenumberofcyclesandcyclefrequencywereselectedforcomparisonwithearlier studiesandbecauseattheseloadsarerepresentativeofmoderatephysicallabor(Brinckmannetal., 1987;Johannessenetal.,2004).Fourth,mechanicsweremeasuredimmediatelyaftercyclicloadingin thesamemannerasthesecondstep.Forthefifthandsixsteps,discswererehydratedovernight followedbymeasurementofmechanicalparameters.
SamplesweretestedonanElectroPulsE3000testsystem(Instron,Norwood,MA).Alltestswere runusingtrimodalcontrol,inwhichthesystemreachesthetargetpeakloadwhilerunningsinewavesin positioncontrol.Ifcyclepeakloadsdonotmatchthetargetloads,thepeakpositionsofthesinewave areadjustedonthenextcycle.Duringtesting,targetloadswerereachedbetweenthe30thand40th cycles.Whilethisismorepreconditioningthanappliedinmanystudies,previousworkshowed20cycles wereneededtosufficientlypreconditionthediscformeasuringmechanicalparametersandpreliminary studiesforthisworkshowednodifferenceinmechanicalpropertiesmeasuredbetweenthe20thand 50thcycles(Boxbergeretal.,2006).
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Nucleotomy Toevaluatetheeffectofnucleotomy,samplesweretestedusingthe6stepmethoddescribed abovewhileintactandagainfollowingnucleotomy.Nucleotomywasperformedbyfirsthydratingthe discsinPBSforeighthours.Next,acruciateincisionwascreatedintheposterolateralannulususinga #11scalpelblade.Afterwards,either2or4mmpituitaryrongeurswereusedtoremove1.71±0.38gof nuclearmaterial,whichisapproximately50%ofNPvolume(Castroetal.,1992;Dullerudetal.,1993). AmountofNPremovedwasnotcorrelatedwithsampledegeneration(r=0.19,p=0.5).Following nucleotomy,sampleswerefrozenuntilfurthertesting. DataAnalysis DiscareawasmeasuredfromMRimagesbyoutliningthediscinamidaxialsliceusingthe DICOMviewerOsiriX(Pixmeo,Switzerland).Discheightwascalculatedfromalateralfluoroscopicimage ofthediscbydividingdiscareabytheanteroposteriorwidthusingMatlab(MathWorks,Natick,MA) (O'Connelletal.,2007).
Themaximumpositionofasingleloadingcycledecreasesoverthe10,000cyclesofcompressive loading(Figure3).Creepwasdefinedasthedecreaseinmaximumcyclepositionfromcycle50to10,000 ofthecyclicloadingandcreepstrainwascalculatedbydividingcreepbyinitialdischeight. Mechanicalparametersweremeasuredonthe50thcycleofthemechanicalmeasurementsteps ofthetestingprocedure(steps2,4,and6)toensuresufficientpreconditioningandeliminatesuper hydration(Adamsetal.,2000;Johannessenetal.,2006).Parametersweremeasuredusingapreviously describedcustomMatlabprogramthatperformsatrilinearfittothedata(Figure4A)(Becksteinetal., 2008).Thisprogramfirstdeterminestheminimumslopelocationofa5thorderpolynomialfittothe data(Figure4B).Neutralzonestiffnesswasdefinedastheslopeoftheloadingdataatthislocation
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(Figure4C).Next,thedatawasseparatedintotensionandcompressionloadingcurvesandcompressive rangeofmotionwasdefinedasthedisplacementbetween0MPaand0.48MPaofappliedstress(Figure 4D).Compressivestiffnesswasdefinedastheslopeofthelinefitthroughthedatapointsabove80%of themaximumcompressiveload(Figure4E).Neutralzonelengthwasdefinedasthedisplacement betweentheintersectionoftheneutralzonelinefitwiththecompressionandtensioncurves(Figure 4F).Compressionandneutralzoneapparentmoduluswerecalculatedfromstiffnessbymultiplyingby discheightandthendividingbydiscarea.Similarly,compressionandneutralzonestrainwere calculatedbydividingthecompressionrangeofmotionorneutralzonelengthbydischeight.Intactdisc heightandareawereusedforthisnormalization. Statistics Theeffectsofloadinghistory(Initial,Cyclic,andRecovery)andtreatment(Intactand Nucleotomy)onthemechanicalparameterswereanalyzedusinga2WayRepeatedMeasuresANOVA. SignificantloadinghistoryandtreatmentwereexaminedwithaTukey’sorSidak’sMultipleComparison Test,respectively.Degenerationeffectsoncyclicloadingwereexaminedusinglinearcorrelations betweenT2relaxationtimeandthepercentchangeofparametersbetweenloadinghistorystates. Similarly,degenerationeffectsonthechangescausedbynucleotomywereanalyzedbyperforming linearcorrelationsbetweenT2relaxationtimeandthepercentchangebetweenintactandnucleotomy parametervaluesatrespectiveloadinghistorystate.ToincludethesamplewithnomeasuredT2 relaxationtimeinthedegenerationanalysis,itsT2relaxationtimewasestimatedbasedonitsPfirrmann gradeandtheregressionlinecalculatedfromtheother14samples.Theeffectsofnucleotomyondisc heightandcreepstrainwereanalyzedusingpairedStudent’sttest.Significancewassetatp0.05).However, followingnucleotomy,degenerationinfluencedcyclicloadingeffectsincompressivestrain,neutralzone modulus,andneutralzonestiffness(Figure7).Specifically,sampleswithlowerT2relaxationtimes(more degeneratesamples)hadgreaterincreasesincompressivestrainbetweentheinitialandcyclictime points(r=0.53,p=0.04)andsimilarlyhadlargerdecreasesincompressivestrainbetweenthecyclic andrecoverytimepoints(r=0.54,p=0.04)(Figure7B).Moredegeneratesamplesalsoexperienced greaterchangesinneutralzonemechanics.LowT2relaxationtimescorrespondedtogreaterdecreases inpercentchangeofneutralzonemodulus(r=0.72,p=0.002)andincreasesinpercentchangeof neutralzonestrain(r=0.70,p=0.004)betweentheinitialandcyclictimepoints(Figure7C&D).These greaterchangesbetweentheinitialandcyclictimepointswerematchedbysimilargreatermagnitudes ofrestorationtowardsinitialvaluesbetweenthecyclicandrecoverytimepoints.
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EffectsofNucleotomy Nucleotomyreduceddischeightby9%andcauseda29%increaseincreepstrain(Figure8). Compressionapparentmodulusdecreased19,15,and9%attheInitial,Cyclic,andRecoverytimepoints, respectively(Figure9A)andwasaccompaniedbycorrespondingincreasesincompressivestrainof38, 19,and19%(Figure9B).Nucleotomydecreasedneutralzonemodulusby16and13%(Figure9C)atthe initialandrecoverytimepoints,butincreaseditby31%atthecyclicloadingcondition.Theprocedure alsocausedincreasesinneutralzonestrainof18,11,and15%atthethreeloadingconditions(Figure 9D).Thesefindingsindicatethatnucleotomyinduceshypermobilityregardlessofthedisc’shydration state.Sampledegenerationdidnotaffectthechangescausedbynucleotomy(p>0.28).
Followingnucleotomy,cyclicloadingaffectedthecompressiveandneutralzonemechanical propertiesinthesamedirectionastheintactsamples(e.g.compressivemodulusincreasedforboth intactandnucleotomysamples),butchangeinmagnitudesweredifferent.Specifically,after nucleotomy,cyclicloadingincreasedcompressivemodulusby7%andcompressivestrainby15%. Neutralzonemodulusdecreasedby25%andneutralzonestrainincreasedby23%.Fornucleotomy samples,allmechanicalparametersreturnedtoinitialvaluesfollowingaperiodofunloadedhydrated recovery(Figure6AD). InteractionofNucleotomyandCyclicLoading Therewasasignificantinteractionbetweennucleotomyandloadinghistoryforcompression modulus,compressionstrain,andneutralzonemodulus.Nucleotomyincreasedthemagnitudeofthe percentincreaseincompressionmoduluscausedbycyclicloading(Figure2,step3)from3%forintact samplesto7%followingnucleotomy(Figure6A).However,theprocedurealsoreducedthepercent changecausedbycyclicloadingincompressionstrainfrom33%forintactsamplesto15%(Figure6B) andpercentchangeinneutralzonemodulusdecreasedfrom52%forintactsamplesto25%following
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nucleotomy(Figure6C).Nucleotomyalsoaffectedthetrendscausedbyunloadedrecovery(Figure2, step5).Compressivemodulusdecreasedby12%followingunloadedrecoveryforintactsamples,but wasonly5%lowerfornucleotomysamples.Forintactsamples,compressivestraindecreasedby12% followingunloadedrecovery,whichincreasedto13%fornucleotomysamples.Unloadedrecovery increasedneutralzonemodulusby94%forintactsamples,butonly29%fornucleotomysamples.
Discussion Thefirststudyobjectivewastodeterminetheeffectsofphysiologicalcyclicloadingfollowedby unloadedrecoveryonthemechanicalresponseofhumanintervertebraldiscs.Wedeterminedthat humandiscswerelessaffectedbycyclicloadingthananimalmodelsinpreviousstudiesandthatcyclic loadingaffectedneutralzonepropertiesmorethancompressiveproperties.Thesecondobjectivewas toexaminehownucleotomyaltersthedisc’smechanicalresponsetocyclicloading.Wefoundthat partialremovalofthenucleuspulposusdecreasesintervertebraldiscmoduluswhileincreasingstrain (rangeofmotion),whichcorrespondstohypermobility.Nucleotomyalsomitigatedcyclicloadingeffects onmechanicalproperties,indicatingalteredfluidflow.Thisalteredfluidflowmayinturnaffectcellular mechanotransductionandtransportofdiscnutrientsandwaste.
Inhumandiscs,cyclicloadingandhydratedrecoveryaffectedbothcompressiveandneutral zoneproperties.Forexample,cyclicloadingincreasedcompressivemodulusby3%whilerecovery decreaseditby12%(Figure6A).Thisislowerthanthe1533%changesseeninbovineandovinetests (Johannessenetal.,2004;Koreckietal.,2008).However,thelowermagnitudeswereexpectedbecause themechanicalchangesareduetoredistributionoffluidwithinthediscandhumandiscshavealower fluidcontentthantheanimaldiscs(Adamsetal.,1990;Johannessenetal.,2004).
Cyclicloadingcausedgreaterchangesinneutralzonepropertiesthancompressiveproperties (Figure6).Cyclicloadingeffectsonneutralzonepropertieshavenotpreviouslybeenreported;however, 11
theroleofthenucleusismorepronouncedintheneutralzonethanathigherloads(Cannellaetal., 2008).Giventhatcyclicloadingcausesfluidtobeeitherredistributedwithinorextrudedfromthedisc andthattheNPhashigherwatercontentthantheannulus(Antoniouetal.,1996;Johannessenetal., 2004),higherfluidflowfromthenucleusmayexplainwhyneutralzonepropertiesweremoreaffected bycyclicloadingthancompressiveproperties.
Anintriguingresultofthestudyisthelimitedroledegenerationplaysinaffectingthedisc’s mechanicalresponsetocyclicloadingandhydratedrecovery.Degenerationwascorrelatedwithcyclic loadingeffectsafternucleotomy(Figure7),withmoredegeneratesamplesexperiencinggreater increasesinbothcompressiveandneutralzonestrainfollowingcyclicloading.However,degeneration wasnotcorrelatedwiththeacutechangesofnucleotomyoreffectofcyclicloadinginintactdiscs.The moredisorganizedannularstructureofthedegeneratesampleslikelypermitsgreaterredistributionof fluidthanispossibleforintactsamples,whichwasmagnifiedbythenucleotomyincision(Haefelietal., 2006;MichalekandIatridis,2011).Thisfindinghasclinicalrelevance,indicatingthatnucleotomycauses morehypermobilityinextendedphysiologicalloadingfordegeneratediscsthannondegeneratediscs.
Theacuteeffectsofnucleotomywerealossofdischeight,decreaseinbothcompressiveand neutralzonemodulus,andincreaseincompressiveandneutralzonerangeofmotion(Figures8&9). Decreaseddischeightandincreasedrangeofmotioncausedbynucleotomyindicatethatinorderto compensateforthedisc’salteredmechanicalloadingfollowingnucleusdepressurization(viaagerelated proteoglycanloss,herniation,ordiscectomy)theremainingannulusandsurroundingspinalelements mustresistmoremotion.Thiscouldinducebackpainbyplacingadditionalstressesontheseinnervated structuresandadvancingthedegenerativecascade.Inaddition,spinalmotionofthetreatedand adjacentlevelsmaybealterediffacetjointsinduceflexionmomentswithinthedisc.Also,the mechanicalchangesofnucleotomyaresimilartothemechanicalchangesofnaturaldegeneration.Thus,
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nucleotomyisapotentialplatformforevaluatingmechanicallybasedspinaltreatments,suchnucleus pulposusreplacement(Omloretal.,2012;Ruanetal.,2010;Woiciechowskyetal.,2012).
Ourmeasuredacuteeffectsofnucleotomyarecomparabletoliteraturevalues.Specifically,our measureddischeightlossof0.8mmfor1.71gofNPremovediscomparabletoareportedheightlossof 0.5mmfor1.75gremoved(Wilkeetal.,2001).Ourobservationthatnucleotomyinitiallycausesa0.35 mmincreaseincompressiverangeofmotionislowerthanliteraturevaluesof0.60.8mm(Freietal., 2001;O’Connelletal.,2011).However,thesestudiesalsoappliedhighercompressiveloads,whichmay accountforsomeofthisdifference.Inaddition,theuniquegeometryoftheL5S1discrelativetothe otherlumbarlevelsusedintheexistingliteraturemayfurtherexplainthesedifferences.Ourobserved 20%decreaseincompressivestiffnessfitswithintheliteraturerangeof350%decreasefortrans annularnucleotomystudies.(Cannellaetal.,2008;MarkolfandMorris,1974).However,transendplate nucleotomydidnotaffectcompressivestiffness(Johannessenetal.,2006),whichsuggestsdecreased compressivestiffnesscausedbynucleotomymaybemoretheresultoftheannularinjurythanthe removalofthenucleus.Thisiscorroboratedbyexistingliteraturethatshowstheannulusplaysa significantroleinsupportingcompressiveloads(Iatridisetal.,1998;Vresilovicetal.,2006).
It’sinterestingthattheNZmodulusatthecyclictimepointishigherforthenucleotomysample thanfortheintactsamples(Figure9C).Thehighermodulusafternucleotomymaybeattributedtofluid flowthroughtheannularnucleotomyincision.Inintactsamples,theannulusservesasabarrierforfluid flowtoandfromthenucleus.Theincisionmayincreasepermeabilityorcreateafluidflowchannelin theinjuryregion(MichalekandIatridis,2011).Asaresult,followingnucleotomy,PBSfromthebathmay morefreelyenterandexitthedisc.Thus,theneutralzonemodulusofnucleotomysamplesatthecyclic timepointmaybehigherthanintactsamplesbecausethereismorePBSwithinthenuclearregion.
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Inadditiontotheacuteeffectsofnucleotomy,wealsofoundthatnucleotomyreducedcyclic loadingeffects.Thisisclearlyseeninthereductionofpercentchangebetweencyclicandrecovery valuesforcompressivemodulus(12to5%)andneutralzonemodulus(94to29%)(Figure6A&C). Reducedeffectsofcyclicloadingindicatealteredfluidflow,whichhastwosignificantramifications. First,italtersthestressesandstrainsexperiencedbythedisc’scells,potentiallyalteringcellular mechanotransductionandsubsequentproteinsynthesis(SettonandChen,2004).Thisinturn,mayalter thediscsbiochemicalcompositionandmechanicalfunction(Baeretal.,2003).Thesecondpotential effectofalteredfluidflowismodifiednutrientdeliveryandwasteremoval.Theintervertebraldiscisa largelyavasculartissueand,asaresult,itscellsaredependentonnutrientdiffusion(Moore,2000; UrbanandRoberts,2003).Thus,anyalterationstothedeliverymechanismhavethepotentialtocreate widereachingeffects.
Thefailureofcompressivemodulusandcompressivestraintoreturntoinitialvaluesforthe intactsamples(Figure6A&B)indicatesastudylimitationinthatcyclicloadingmayhavecausedsome nonrecoverabledamagetotheannulusfibrosus.However,followingannularnucleotomy,the compressivepropertiesattheinitialandrecoveryhydrationstatesareidentical.Thissuggeststhe damagecausedtotheintactsamplesisduetoresistingfluidflowthroughtheannulus,because followingnucleotomyfluidhadalessobstructedpathway.Thispotentialannulardamagewas unexpectedbecauseitwasnotseeninpreviousovinestudieswithsimilarloadsorinthepreliminary studiesforthiswork(Johannessenetal.,2004;Malhotraetal.,2012).However,mostofthehuman tissueusedwasmoderatelydegenerated,incontrastwiththenondegeneratedsamplesusedinanimal studies.Thisinitialdegenerationmayhavecausedthesamplestobemoresusceptibletoinjury.In contrasttocompressiveproperties,cyclicloadingeffectsinneutralzonepropertieswerecompletely recoverablefollowinghydration.
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Thisstudyhasdemonstratedthatcyclicloadingincreasescompressivemodulus,compressive strain,andneutralzonestrainwhiledecreasingneutralzonemodulus.Thesechangesaretheresultof alteredhydrationlevelscausedbythecyclicloadingprotocolandaresimilartonaturallyoccurring diurnalchanges.Nucleotomyreduceddischeightandmoduluswhileincreasingrangeofmotion.In additiontotheseacutechanges,nucleotomyalsoreducedthedifferencebetweenmechanical propertiesbeforeandaftercyclicloading.Theobservedchangesinmechanicalbehaviorinducedby nucleotomyaresimilartonaturallyoccurringdegenerativechanges.Resultsofthisstudyprovidean idealprotocolandcontroldataforevaluatingtheeffectivenessofamechanicallybaseddisc degenerationtreatment,suchasanucleusreplacement.
Acknowledgements WewouldliketothankDharaB.AminandIanS.MacLeanforperformingsomeofthe mechanicaltesting.SpineswereacquiredfromNationalDiseaseResearchInterchange,Philadelphia,PA andPlatinumTraining,Henderson,NV.ThisstudywasfundedbyNIHR01AR050052,NSFGraduate ResearchFellowship#DGE0822,andaNeurosurgeryResearchandEducationFoundationgrant.Noneof thefundingagenciesplayedadirectroleinstudydesign,datacollection,ordataanalysis.
Conflictofintereststatement Noneoftheauthorshaveconflictsofinteresttodisclose.
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Goel, V.K., Nishiyama, K., Weinstein, J.N., Liu, Y.K., 1986. Mechanical properties of lumbar spinal motion segments as affected by partial disc removal. Spine 11, 1008-1012. Haefeli, M., Kalberer, F., Saegesser, D., Nerlich, A.G., Boos, N., Paesold, G., 2006. The course of macroscopic degeneration in the human lumbar intervertebral disc. Spine 31, 15221531. Hasegawa, K., Turner, C., Chen, J., Burr, D.B., 1995. Effect of disc lesion on microdamage accumulation in lumbar vertebrae under cyclic compression loading. Clinical Orthopaedics & Related Research, 190-198. Iatridis, J.C., Setton, L.A., Foster, R.J., Rawlins, B.A., Weidenbaum, M., Mow, V.C., 1998. Degeneration affects the anisotropic and nonlinear behaviors of human anulus fibrosus in compression. Journal of Biomechanics 31, 535-544. Ishihara, H., Tsuji, H., Hirano, N., Ohshima, H., Terahata, N., 1993. Biorheological responses of the intact and nucleotomized intervertebral disks to compressive, tensile, and vibratory stresses. Clinical Biomechanics 8, 250-254. Johannessen, W., Cloyd, J., O'Connell, G., Vresilovic, E., Elliott, D., 2006. Trans-endplate nucleotomy increases deformation and creep response in axial loading. Annals of Biomedical Engineering 34, 687-696. Johannessen, W., Vresilovic, E.J., Wright, A.C., Elliott, D.M., 2004. Intervertebral disc mechanics are restored following cyclic loading and unloaded recovery. Annals of Biomedical Engineering 32, 70-76. Kambin, P., Brager, M.D., 1987. Percutaneous posterolateral discectomy - anatomy and mechanism. Clinical Orthopaedics and Related Research, 145-154. Katz, J.N., 2006. Lumbar disc disorders and low-back pain: Socioeconomic factors and consequences. Journal of Bone and Joint Surgery 88A, 21-24. Kerttula, L., Kurunlahti, M., Jauhiainen, J., Koivula, A., Oikarinen, J., Tervonen, O., 2001. Apparent diffusion coefficients and t2 relaxation time measurements to evaluate disc degeneration. A quantitative mr study of young patients with previous vertebral fracture. Acta Radiol 42, 585-591. Koeller, W., Funke, F., Hartmann, F., 1984. Biomechanical behavior of human intervertebral discs subjected to long lasting axial loading. Biorheology 21, 675-686. Korecki, C.L., MacLean, J.J., Iatridis, J.C., 2008. Dynamic compression effects on intervertebral disc mechanics and biology. Spine 33, 1403-1409. Lewis, G., 2012. Nucleus pulposus replacement and regeneration/repair technologies: Present status and future prospects. Journal of Biomedical Materials Research Part B: Applied Biomaterials 100B, 1702-1720. Luo, X., Pietrobon, R., X Sun, S., Liu, G.G., Hey, L., 2004. Estimates and patterns of direct health care expenditures among individuals with back pain in the united states. Spine 29, 79-86. Malhotra, N.R., Han, W.M., Beckstein, J., Cloyd, J., Chen, W., Elliott, D.M., 2012. An injectable nucleus pulposus implant restores compressive range of motion in the ovine disc. Spine 37, E1099-E1105. Marinelli, N.L., Haughton, V.M., Anderson, P.A., 2010. T2 relaxation times correlated with stage of lumbar intervertebral disk degeneration and patient age. American Journal of Neuroradiology 31, 1278-1282. Markolf, K.L., Morris, J.M., 1974. The structural components of the intervertebral disc: A study of their contributions to the ability of the disc to withstand compressive forces. Journal of Bone and Joint Surgery 56A, 675-687. Meakin, J.R., Hukins, D.W.L., 2000. Effect of removing the nucleus pulposus on the deformation of the annulus fibrosus during compression of the intervertebral disc. Journal of Biomechanics 33, 575-580.
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Michalek, A.J., Iatridis, J.C., 2011. Penetrating annulus fibrosus injuries affect dynamic compressive behaviors of the intervertebral disc via altered fluid flow: An analytical interpretation. Journal of Biomechanical Engineering 133, 084502. Moore, R.J., 2000. The vertebral end-plate: What do we know? European Spine Journal 9, 9296. Nachemson, A.L., 1981. Disc pressure measurements. Spine 6, 93-97. O'Connell, G.D., Vresilovic, E.J., Elliott, D.M., 2007. Comparison of animals used in disc research to human lumbar disc geometry. Spine 32, 328-333. O’Connell, G., Malhotra, N.R., Vresilovic, E.J., Elliott, D.M., 2011. The effect of nucleotomy and the dependence on degeneration of human intervertebral disc strain in axial compression. Spine 36, 1765-1771. Omlor, G., Nerlich, A., Lorenz, H., Bruckner, T., Richter, W., Pfeiffer, M., Gühring, T., 2012. Injection of a polymerized hyaluronic acid/collagen hydrogel matrix in an in vivo porcine disc degeneration model. European Spine Journal 21, 1700-1708. Pfirrmann, C.W.A., Metzdorf, A., Zanetti, M., Hodler, J., Boos, N., 2001. Magnetic resonance classification of lumbar intervertebral disc degeneration. Spine 26, 1873-1878. Rajaee, S.S., Bae, H.W., Kanim, L.E.A., Delamarter, R.B., 2012. Spinal fusion in the united states. Spine 37, 67-76. Reitmaier, S., Wolfram, U., Ignatius, A., Wilke, H.-J., Gloria, A., Martín-Martínez, J.M., SilvaCorreia, J., Miguel Oliveira, J., Luís Reis, R., Schmidt, H., 2012. Hydrogels for nucleus replacement—facing the biomechanical challenge. Journal of the Mechanical Behavior of Biomedical Materials 14, 67-77. Ruan, D.K., Xin, H., Zhang, C., Wang, C., Xu, C., Li, C., He, Q., 2010. Experimental intervertebral disc regeneration with tissue-engineered composite in a canine model. Tissue Engineering: Part A 16, 2381-2389. Setton, L.A.P., Chen, J.P., 2004. Cell mechanics and mechanobiology in the intervertebral disc. Spine 29, 2710-2723. Showalter, B.L., Beckstein, J.C., Martin, J.T., Beattie, E.E., Espinoza Orías, A.A., Schaer, T.P., Vresilovic, E.J., Elliott, D.M., 2012. Comparison of animal discs used in disc research to human lumbar disc: Torsion mechanics and collagen content. Spine 37, E900-E907. Sivan, S., Merkher, Y., Wachtel, E., Ehrlich, S., Maroudas, A., 2006. Correlation of swelling pressure and intrafibrillar water in young and aged human intervertebral discs. Journal of Orthopaedic Research 24, 1292-1298. Thoreson, O., Baranto, A., Ekström, L., Holm, S., Hellström, M., Swärd, L., 2010. The immediate effect of repeated loading on the compressive strength of young porcine lumbar spine. Knee Surgery, Sports Traumatology, Arthroscopy 18, 694-701. Urban, J.P.G., McMullin, J.F., 1988. Swelling pressure of the lumbar intervertebral disks influence of age, spinal level, composition, and degeneration. Spine 13, 179-187. Urban, J.P.G., Roberts, S., 2003. Degeneration of the intervertebral disc. Arthritis Research & Therapy 5, 120-130. Vernon-Roberts, B., Moore, R.J., Fraser, R.D., 2007. The natural history of age-related disc degeneration: The pathology and sequelae of tears. Spine 32, 2797-2804. Vresilovic, E.J., Johannessen, W., Elliott, D.M., 2006. Disc mechanics with trans-endplate partial nucleotomy are not fully restored following cyclic compressive loading and unloaded recovery. Journal of Biomechanical Engineering 128, 823-829. Weinstein, J.N., Lurie, J.D., Tosteson, T.D., Skinner, J.S., Hanscom, B., Tosteson, A.N.A., Herkowitz, H., Fischgrund, J., Cammisa, F.P., Albert, T., Deyo, R.A., 2006. Surgical vs nonoperative treatment for lumbar disk herniation. JAMA: The Journal of the American Medical Association 296, 2451-2459. White, T.L., Malone, T.R., 1990. Effects of running on intervertebral disc height. Journal of Orthopaedic and Sports Physical Therapy 12, 139-146. 18
Wilke, H.J., Kavanagh, S., Neller, S., Haid, C., Claes, L.E., 2001. Effect of a prosthetic disc nucleus on the mobility and disc height of the l4-5 intervertebral disc postnucleotomy. Journal of Neurosurgery 95, 208-214. Wilke, H.J., Kettler, A., Claes, L.E., 1997. Are sheep spines a valid biomechanical model for human spines? Spine 22, 2365-2374. Wilke, H.J., Neef, P., Caimi, M., Hoogland, T., Claes, L.E., 1999. New in vivo measurements of pressures in the intervertebral disc in daily life. Spine 24, 755-762. Woiciechowsky, C., Abbushi, A., Zenclussen, M.L., Casalis, P., Kruger, J.P., Freymann, U., Endres, M., Kaps, C., 2012. Regeneration of nucleus pulposus tissue in an ovine intervertebral disc degeneration model by cell-free resorbable polymer scaffolds. Journal of tissue engineering and regenerative medicine, DOI: 10.1002/term.1582. Zhang, Y., Lenart, B.A., Lee, J.K., Chen, D., Shi, P., Ren, J., Muehleman, C., Chen, D., An, H.S., 2014. Histological features of endplates of the mammalian spine: From mice to men. Spine (Phila Pa 1976) 39, E312-317. Figure1:T2relaxationtimesarestronglycorrelatedtoPfirrmannscores(r=0.76,p=0.001)forthe humanL5S1discsusedinthisstudy. Figure2:Themechanicaltestingprocedurealtereddischydration.DiscsarefullyhydratedinaPBSbath containingproteaseinhibitors(Steps1and5),andhavereducedhydrationaftercyclicloading(Step3). Mechanicalparametersaremeasuredaftereachchangeinhydrationlevel(Steps2,4,and6). Figure3:Themaximumpositionofsinglecycledecreasesthroughoutthe10,000cyclesofcompressive loading.Creepisdefinedasthedropinmaximumcycledisplacementover10,000cycles,afterwhich creepstrainiscalculatedbydividingcreepbyinitialdischeight. Figure4:Mechanicalparametersarefoundusingatrilinearfitofforcedisplacementcurvefrom50th cycleofthemechanicalmeasurementsteps(steps2,4,and6ofthetestingprocedure).A.Originaldata B.Theminimumslopeofa5thorderpolynomialfitislocated.C.Neutralzoneisfitthroughdataatthe pointofminimumslope.Neutralzonestiffnessistheslopeofthisline.D.Tension(redcircles)and compression(bluediamonds)loadingcurvesareseparatedfromthedata.Compressionrangeofmotion isthedisplacementbetween0and0.48MPaonthecompressionloadingcurve.E.Tensionand compressionlinefitsthroughthemaximum80%oftherespectiveloadingcurves.Compressionstiffness istheslopeofthecompressionline.F.Linesarefitintheneutralzoneregionsofthetensionand compressionloadingcurves.Neutralzonelengthisthedisplacementbetweentheintersectionofthese lineswitheitherthetensionandcompressionlines.Allmechanicalparametersarethennormalizedby initialdiscareaandheight. Figure5:Forcedisplacementcurvesforasinglesampleoverthecourseoftheexperiment.Cyclicloading increasedrangeofmotionanddecreasedneutralzonemodulus,whichrecoveredfollowinghydration. Nucleotomyincreasedrangeofmotionwhiledecreasingstiffnessandtheamountofchangein mechanicalparameterscausedbycyclicloading.
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Figure6:Discmechanicalpropertiesarealteredbycyclicloading(cyclic)andgenerallyrecoverafter unloadedhydration(recovery).Thesechangesmimicnaturallyoccurringdiurnalchanges.Nucleotomy reducedthehydrationeffectsonmechanicalproperties.Inparticular,nucleotomyreducedthepercent changebetweentheInitialandCyclichydrationstatesforcompressivestrain(B),neutralzonemodulus (C),andneutralzonestrain(D).*p
Nucleotomy Reduces the Effects of Cyclic Compressive Loading with Unloaded Recovery on Human Intervertebral Discs Brent L. Showalter, Neil R. Malhotra, Edward J. Vresilovic, Dawn M. Elliott
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S0021-9290(14)00329-7 http://dx.doi.org/10.1016/j.jbiomech.2014.05.018 BM6673
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Journal of Biomechanics
Accepted date: 24 May 2014 Cite this article as: Brent L. Showalter, Neil R. Malhotra, Edward J. Vresilovic, Dawn M. Elliott, Nucleotomy Reduces the Effects of Cyclic Compressive Loading with Unloaded Recovery on Human Intervertebral Discs, Journal of Biomechanics, http://dx.doi.org/10.1016/j.jbiomech.2014.05.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
NucleotomyReducestheEffectsofCyclicCompressiveLoading withUnloadedRecoveryonHumanIntervertebralDiscs BrentL.Showalter1,NeilR.Malhotra2,EdwardJ.Vresilovic3,andDawnM.Elliott4* (1)DepartmentofBioengineering UniversityofPennsylvania (2)DepartmentofNeurosurgery UniversityofPennsylvania (3)DepartmentofOrthopaedicsandRehabilitation PennStateUniversity (4)DepartmentofBiomedicalEngineering UniversityofDelaware OriginalArticle WordCount:3965 Keywords:IntervertebralDisc;Spine;CyclicLoading;NucleusPulposus;Nucleotomy *Correspondingauthor: DawnM.Elliott BiomedicalEngineering UniversityofDelaware 125EastDelawareAve Newark,DE19716 USA Email:[email protected]
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Abstract Thefirstobjectiveofthisstudywastodeterminetheeffectsofphysiologicalcyclicloadingfollowedby unloadedrecoveryonthemechanicalresponseofhumanintervertebraldiscs.Thesecondobjectivewas toexaminehownucleotomyaltersthedisc’smechanicalresponsetocyclicloading.Tocompletethese objectives,15humanL5S1discsweretestedwhileintactandsubsequenttonucleotomy.Thetesting consistedof10,000cyclesofphysiologicalcompressiveloadsfollowedbyunloadedhydratedrecovery. Cyclicloadingincreasedcompressionmodulus(3%)andstrain(33%),decreasedneutralzonemodulus (52%),andincreasedneutralzonestrain(31%).Degenerationwasnotcorrelatedwiththeeffectofcyclic loadinginintactdiscs,butwascorrelatedwithcyclicloadingeffectsafternucleotomy,withmore degeneratesamplesexperiencinggreaterincreasesinbothcompressiveandneutralzonestrain followingcyclicloading.Partialremovalofthenucleuspulposusdecreasedthecompressionandneutral zonemoduluswhileincreasingstrain.Thesechangescorrespondtohypermobility,whichwillalter overallspinalmechanicsandmayimpactlowbackpainviaalteredmotionthroughoutthespinal column.Nucleotomyalsoreducedtheeffectsofcyclicloadingonmechanicalproperties,likelydueto alteredfluidflow,whichmayimpactcellularmechanotransductionandtransportofdiscnutrientsand waste.Degenerationwasnotcorrelatedwiththeacutechangesofnucleotomy.Resultsofthisstudy provideanidealprotocolandcontroldataforevaluatingtheeffectivenessofamechanicallybaseddisc degenerationtreatment,suchasanucleusreplacement.
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Introduction Theintervertebraldiscperformsthemechanicalrolesofsupportingloads,permittingmotion, anddissipatingenergy.Discdegenerationisastronglyimplicatedcauseoflowbackpainandwithinthe U.S.resultsinannualcostsover$100billion(Katz,2006).Degenerationisamultifacetedprocessthat manifestsearlyinthenucleuspulposus(NP)andsubsequentlyextendstootherdisccomponents (VernonRobertsetal.,2007).Despitediscdegenerationprevalence,manycurrenttreatmentsare palliativeonlyandultimatelyfail(Andersson,1999;Luoetal.,2004;Rajaeeetal.,2012).Minimally invasivetreatmentsofearlydiscdegeneration,suchasinjectableNPreplacements,areunder development(Lewis,2012;Malhotraetal.,2012;Reitmaieretal.,2012).Forsuchmechanicallybased treatmentstobesuccessfultheymustreplicatenaturalNPmechanicalfunction.Inhealthydiscs, proteoglycanscreateanosmoticpressurewhichdrawsfluidintotheNP(UrbanandMcMullin,1988). Thisfluidbothdirectlysupportscompressiveloadsandplacesthecollagenfibersoftheannulusfibrosus intension,permittingthedisctosupportloadswhileundergoingawiderangeofmotion.
Physiologicalcycliccompressiveloadingandunloadedrecoveryisanidealtestingmodalityfor examiningtheloadingcontributionofthenucleuspulposusbecauseitsimulatesthedisc’stime dependentmechanicalresponse.Theinterplaybetweenosmoticpressureandexternalloadscauses20 25%ofthedisc’swatercontenttobeexpressedandreimbibeddaily(Sivanetal.,2006).Reduced hydrationdecreasesdischeight,increasesstiffness,andincreasesrangeofmotion,withproperties returningtobaselineafterrehydration(Adamsetal.,1990;Wilkeetal.,1999).Thesediurnalchanges havebeenreplicatedinseverallargeanimalmodelsbycompressivecyclicloadingandunloaded recovery(Johannessenetal.,2004;Koreckietal.,2008;Thoresonetal.,2010).Whileanimalandhuman discsaresimilar,thesefindingsmaynotberepresentativeofcyclicloadinginhumandiscsduetolower waterandproteoglycancontentinhumans,variedappliedloadsandloadingdurationsbetweenstudies, anddifferencesincartilageendplate(Becksteinetal.,2008;Demersetal.,2004;Showalteretal.,2012; 3
Wilkeetal.,1997;Zhangetal.,2014).Further,themajorityofpreviouscyclicloadingstudieswere designedtotestdamagemechanismsbyemployinghighloadsandnumberofcyclesandbynot includingunloadedrecovery(AdamsandHutton,1985;Hasegawaetal.,1995).Dischydrationeffects havealsobeenstudiedusingcreeptests(Adamsetal.,1987;Koelleretal.,1984).However,creep induceslowerfluidflowratesthancyclicloadingandthusdoesn’tsimulatethedynamicnatureofdisc loading(WhiteandMalone,1990).ThisstudyexaminesthecontributionoftheNPtodiscloadingby testinghumandiscsundergoingaphysiologicallyrelevanttestingmodality,whichiscompressivecyclic loadingwithhydratedunloadedrecovery.
Themechanicalroleofthenucleuspulposuscanbeevaluatedbycomparingtheeffectofcyclic loadingonintactandpartiallynucleotomizedsamples.Nucleotomy,alsoknownasdiscectomy,isa clinicaltreatmentfordischerniation(Friedman,1983;KambinandBrager,1987).Theprocedurealters discmechanicsbydecreasingdischeight,NPpressure,andstiffnesswhileincreasingrangeofmotion andcreep(BrinckmannandGrootenboer,1991;Cannellaetal.,2008;Freietal.,2001;Goeletal.,1986; Ishiharaetal.,1993;MeakinandHukins,2000;O’Connelletal.,2011).Whiletheacuteeffectsof nucleotomyhavebeendetermined,itsimpactonthedisc’sfluidflowrelatedmechanicalresponsehas not.
Thefirststudyobjectivewastodeterminetheeffectsofphysiologicalcyclicloadingfollowedby unloadedrecoveryonthemechanicalresponseofhumanintervertebraldiscs.Thesecondobjectivewas toexaminehownucleotomyaltersthedisc’smechanicalresponsetocyclicloading.
Methods SamplePreparation Fifteenhumanlumbarspinesegmentswithameandonorageof50.1years(2275years)were obtainedfrominstitutionallyapprovedsources.ThesespinesunderwentMRimagingtodetermine 4
degenerationgradeandT2relaxationtimes(Kerttulaetal.,2001;Marinellietal.,2010;Pfirrmannetal., 2001).TheL5S1discwasselectedfortestingbecauseherniationsanddiscectomiesaremostcommon atthislevel(Weinsteinetal.,2006).Sampleshaddegenerationgradesrangingfrom1to5andT2 relaxationtimesrangingfrom66to204,withhigherrelaxationtimesindicatinghealthierdiscs.T2 relaxationtimewasnotacquiredforonesample.Fortheremaining14samples,T2relaxationtimes correlatedwithPfirrmanngrades(r=0.76)andprovideaquantitative,continuousmeasurementof degeneration(Figure1).DisccrosssectionalareawasmeasuredfromMRimages.Afterimaging,L5S1 bonediscbonesegmentswerepreparedbyfirstremovingtheposteriorelementsandextraneoussoft tissue.Althoughinvivoloadsaresharedbetweenthediscandfacets,posteriorelementswereremoved inordertoisolatethecontributionsofthediscincompression(Becksteinetal.,2008;Meakinand Hukins,2000).Followingdissection,1.25mmKirschnerwireswereplacedintheL5vertebraeand sacrum.TheL5S1discswerewedgeshaped,witha13±4°anglebetweentheL5andS1endplates, whichlikelyresultsinsomeappliedshearloadsevenduringsimplecompressionexperiments.For consistencyinmechanicaltesting,loadswereappliedperpendiculartotheL5vertebrae.Thiswas ensuredbypottingthemotionsegmentsinPMMAwiththeL5endplateparalleltothepottingfixtures usingfluoroscopicguidance.Sampleswerestoredinafreezerat20°Cwhennotinuse. Mechanicaltesting Themechanicaltestconsistedofsixsteps(Figure2).1.Overnighthydration,alsocalledinitial hydration.2.Measuremechanicalparameters(Initial).3.10,000cyclesofcompressiveloading.4. Measuremechanicalparameters(Cyclic).5.Overnighthydration,alsocalledunloadedrecovery.6. Measuremechanicalparameters(Recovery).Duringthefirststep,overnighthydration,sampleswere submergedina0.15MPBSbathcontainingproteaseinhibitors(PhenylmethylsulfonylFluroide,N EthylmalemideBioxtra,andBenzamidineHydrochloridefromSigmaAldrich,St.Louis,MO)at4°Cfor16 24hours.Second,mechanicalparametersweremeasuredbyapplying60cyclesat0.2Hzbetween0.48 5
MPa(734±122N)incompressionand375Nintensioncycles,whichissimilartopreviousstudies (Becksteinetal.,2008).Thestressof0.48MPacorrespondstoonebodyweightortolowintensity activitiessuchassittinguprightwithoutsupportorrelaxedstanding(ElliottandSarver,2004; Nachemson,1981;Wilkeetal.,1999).Thetensileloadof375Nwasappliedtoallsamplesinsteadofa uniformstresstopreventsamplepulloutfromthePMMAfordiscswithlargecrosssectionalareas whileensuringsufficientloadtodescribethemechanicalparametersofinterest.Thethirdstepwasto apply10,000cyclesofcompressiveloadsat2Hzbetween0.12and0.96MPa(1467±244N)whichare loadssimilartomoderatephysicalactivitiessuchasjoggingorliftinga10kgweight(Nachemson,1981; Wilkeetal.,1999).Thenumberofcyclesandcyclefrequencywereselectedforcomparisonwithearlier studiesandbecauseattheseloadsarerepresentativeofmoderatephysicallabor(Brinckmannetal., 1987;Johannessenetal.,2004).Fourth,mechanicsweremeasuredimmediatelyaftercyclicloadingin thesamemannerasthesecondstep.Forthefifthandsixsteps,discswererehydratedovernight followedbymeasurementofmechanicalparameters.
SamplesweretestedonanElectroPulsE3000testsystem(Instron,Norwood,MA).Alltestswere runusingtrimodalcontrol,inwhichthesystemreachesthetargetpeakloadwhilerunningsinewavesin positioncontrol.Ifcyclepeakloadsdonotmatchthetargetloads,thepeakpositionsofthesinewave areadjustedonthenextcycle.Duringtesting,targetloadswerereachedbetweenthe30thand40th cycles.Whilethisismorepreconditioningthanappliedinmanystudies,previousworkshowed20cycles wereneededtosufficientlypreconditionthediscformeasuringmechanicalparametersandpreliminary studiesforthisworkshowednodifferenceinmechanicalpropertiesmeasuredbetweenthe20thand 50thcycles(Boxbergeretal.,2006).
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Nucleotomy Toevaluatetheeffectofnucleotomy,samplesweretestedusingthe6stepmethoddescribed abovewhileintactandagainfollowingnucleotomy.Nucleotomywasperformedbyfirsthydratingthe discsinPBSforeighthours.Next,acruciateincisionwascreatedintheposterolateralannulususinga #11scalpelblade.Afterwards,either2or4mmpituitaryrongeurswereusedtoremove1.71±0.38gof nuclearmaterial,whichisapproximately50%ofNPvolume(Castroetal.,1992;Dullerudetal.,1993). AmountofNPremovedwasnotcorrelatedwithsampledegeneration(r=0.19,p=0.5).Following nucleotomy,sampleswerefrozenuntilfurthertesting. DataAnalysis DiscareawasmeasuredfromMRimagesbyoutliningthediscinamidaxialsliceusingthe DICOMviewerOsiriX(Pixmeo,Switzerland).Discheightwascalculatedfromalateralfluoroscopicimage ofthediscbydividingdiscareabytheanteroposteriorwidthusingMatlab(MathWorks,Natick,MA) (O'Connelletal.,2007).
Themaximumpositionofasingleloadingcycledecreasesoverthe10,000cyclesofcompressive loading(Figure3).Creepwasdefinedasthedecreaseinmaximumcyclepositionfromcycle50to10,000 ofthecyclicloadingandcreepstrainwascalculatedbydividingcreepbyinitialdischeight. Mechanicalparametersweremeasuredonthe50thcycleofthemechanicalmeasurementsteps ofthetestingprocedure(steps2,4,and6)toensuresufficientpreconditioningandeliminatesuper hydration(Adamsetal.,2000;Johannessenetal.,2006).Parametersweremeasuredusingapreviously describedcustomMatlabprogramthatperformsatrilinearfittothedata(Figure4A)(Becksteinetal., 2008).Thisprogramfirstdeterminestheminimumslopelocationofa5thorderpolynomialfittothe data(Figure4B).Neutralzonestiffnesswasdefinedastheslopeoftheloadingdataatthislocation
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(Figure4C).Next,thedatawasseparatedintotensionandcompressionloadingcurvesandcompressive rangeofmotionwasdefinedasthedisplacementbetween0MPaand0.48MPaofappliedstress(Figure 4D).Compressivestiffnesswasdefinedastheslopeofthelinefitthroughthedatapointsabove80%of themaximumcompressiveload(Figure4E).Neutralzonelengthwasdefinedasthedisplacement betweentheintersectionoftheneutralzonelinefitwiththecompressionandtensioncurves(Figure 4F).Compressionandneutralzoneapparentmoduluswerecalculatedfromstiffnessbymultiplyingby discheightandthendividingbydiscarea.Similarly,compressionandneutralzonestrainwere calculatedbydividingthecompressionrangeofmotionorneutralzonelengthbydischeight.Intactdisc heightandareawereusedforthisnormalization. Statistics Theeffectsofloadinghistory(Initial,Cyclic,andRecovery)andtreatment(Intactand Nucleotomy)onthemechanicalparameterswereanalyzedusinga2WayRepeatedMeasuresANOVA. SignificantloadinghistoryandtreatmentwereexaminedwithaTukey’sorSidak’sMultipleComparison Test,respectively.Degenerationeffectsoncyclicloadingwereexaminedusinglinearcorrelations betweenT2relaxationtimeandthepercentchangeofparametersbetweenloadinghistorystates. Similarly,degenerationeffectsonthechangescausedbynucleotomywereanalyzedbyperforming linearcorrelationsbetweenT2relaxationtimeandthepercentchangebetweenintactandnucleotomy parametervaluesatrespectiveloadinghistorystate.ToincludethesamplewithnomeasuredT2 relaxationtimeinthedegenerationanalysis,itsT2relaxationtimewasestimatedbasedonitsPfirrmann gradeandtheregressionlinecalculatedfromtheother14samples.Theeffectsofnucleotomyondisc heightandcreepstrainwereanalyzedusingpairedStudent’sttest.Significancewassetatp0.05).However, followingnucleotomy,degenerationinfluencedcyclicloadingeffectsincompressivestrain,neutralzone modulus,andneutralzonestiffness(Figure7).Specifically,sampleswithlowerT2relaxationtimes(more degeneratesamples)hadgreaterincreasesincompressivestrainbetweentheinitialandcyclictime points(r=0.53,p=0.04)andsimilarlyhadlargerdecreasesincompressivestrainbetweenthecyclic andrecoverytimepoints(r=0.54,p=0.04)(Figure7B).Moredegeneratesamplesalsoexperienced greaterchangesinneutralzonemechanics.LowT2relaxationtimescorrespondedtogreaterdecreases inpercentchangeofneutralzonemodulus(r=0.72,p=0.002)andincreasesinpercentchangeof neutralzonestrain(r=0.70,p=0.004)betweentheinitialandcyclictimepoints(Figure7C&D).These greaterchangesbetweentheinitialandcyclictimepointswerematchedbysimilargreatermagnitudes ofrestorationtowardsinitialvaluesbetweenthecyclicandrecoverytimepoints.
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EffectsofNucleotomy Nucleotomyreduceddischeightby9%andcauseda29%increaseincreepstrain(Figure8). Compressionapparentmodulusdecreased19,15,and9%attheInitial,Cyclic,andRecoverytimepoints, respectively(Figure9A)andwasaccompaniedbycorrespondingincreasesincompressivestrainof38, 19,and19%(Figure9B).Nucleotomydecreasedneutralzonemodulusby16and13%(Figure9C)atthe initialandrecoverytimepoints,butincreaseditby31%atthecyclicloadingcondition.Theprocedure alsocausedincreasesinneutralzonestrainof18,11,and15%atthethreeloadingconditions(Figure 9D).Thesefindingsindicatethatnucleotomyinduceshypermobilityregardlessofthedisc’shydration state.Sampledegenerationdidnotaffectthechangescausedbynucleotomy(p>0.28).
Followingnucleotomy,cyclicloadingaffectedthecompressiveandneutralzonemechanical propertiesinthesamedirectionastheintactsamples(e.g.compressivemodulusincreasedforboth intactandnucleotomysamples),butchangeinmagnitudesweredifferent.Specifically,after nucleotomy,cyclicloadingincreasedcompressivemodulusby7%andcompressivestrainby15%. Neutralzonemodulusdecreasedby25%andneutralzonestrainincreasedby23%.Fornucleotomy samples,allmechanicalparametersreturnedtoinitialvaluesfollowingaperiodofunloadedhydrated recovery(Figure6AD). InteractionofNucleotomyandCyclicLoading Therewasasignificantinteractionbetweennucleotomyandloadinghistoryforcompression modulus,compressionstrain,andneutralzonemodulus.Nucleotomyincreasedthemagnitudeofthe percentincreaseincompressionmoduluscausedbycyclicloading(Figure2,step3)from3%forintact samplesto7%followingnucleotomy(Figure6A).However,theprocedurealsoreducedthepercent changecausedbycyclicloadingincompressionstrainfrom33%forintactsamplesto15%(Figure6B) andpercentchangeinneutralzonemodulusdecreasedfrom52%forintactsamplesto25%following
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nucleotomy(Figure6C).Nucleotomyalsoaffectedthetrendscausedbyunloadedrecovery(Figure2, step5).Compressivemodulusdecreasedby12%followingunloadedrecoveryforintactsamples,but wasonly5%lowerfornucleotomysamples.Forintactsamples,compressivestraindecreasedby12% followingunloadedrecovery,whichincreasedto13%fornucleotomysamples.Unloadedrecovery increasedneutralzonemodulusby94%forintactsamples,butonly29%fornucleotomysamples.
Discussion Thefirststudyobjectivewastodeterminetheeffectsofphysiologicalcyclicloadingfollowedby unloadedrecoveryonthemechanicalresponseofhumanintervertebraldiscs.Wedeterminedthat humandiscswerelessaffectedbycyclicloadingthananimalmodelsinpreviousstudiesandthatcyclic loadingaffectedneutralzonepropertiesmorethancompressiveproperties.Thesecondobjectivewas toexaminehownucleotomyaltersthedisc’smechanicalresponsetocyclicloading.Wefoundthat partialremovalofthenucleuspulposusdecreasesintervertebraldiscmoduluswhileincreasingstrain (rangeofmotion),whichcorrespondstohypermobility.Nucleotomyalsomitigatedcyclicloadingeffects onmechanicalproperties,indicatingalteredfluidflow.Thisalteredfluidflowmayinturnaffectcellular mechanotransductionandtransportofdiscnutrientsandwaste.
Inhumandiscs,cyclicloadingandhydratedrecoveryaffectedbothcompressiveandneutral zoneproperties.Forexample,cyclicloadingincreasedcompressivemodulusby3%whilerecovery decreaseditby12%(Figure6A).Thisislowerthanthe1533%changesseeninbovineandovinetests (Johannessenetal.,2004;Koreckietal.,2008).However,thelowermagnitudeswereexpectedbecause themechanicalchangesareduetoredistributionoffluidwithinthediscandhumandiscshavealower fluidcontentthantheanimaldiscs(Adamsetal.,1990;Johannessenetal.,2004).
Cyclicloadingcausedgreaterchangesinneutralzonepropertiesthancompressiveproperties (Figure6).Cyclicloadingeffectsonneutralzonepropertieshavenotpreviouslybeenreported;however, 11
theroleofthenucleusismorepronouncedintheneutralzonethanathigherloads(Cannellaetal., 2008).Giventhatcyclicloadingcausesfluidtobeeitherredistributedwithinorextrudedfromthedisc andthattheNPhashigherwatercontentthantheannulus(Antoniouetal.,1996;Johannessenetal., 2004),higherfluidflowfromthenucleusmayexplainwhyneutralzonepropertiesweremoreaffected bycyclicloadingthancompressiveproperties.
Anintriguingresultofthestudyisthelimitedroledegenerationplaysinaffectingthedisc’s mechanicalresponsetocyclicloadingandhydratedrecovery.Degenerationwascorrelatedwithcyclic loadingeffectsafternucleotomy(Figure7),withmoredegeneratesamplesexperiencinggreater increasesinbothcompressiveandneutralzonestrainfollowingcyclicloading.However,degeneration wasnotcorrelatedwiththeacutechangesofnucleotomyoreffectofcyclicloadinginintactdiscs.The moredisorganizedannularstructureofthedegeneratesampleslikelypermitsgreaterredistributionof fluidthanispossibleforintactsamples,whichwasmagnifiedbythenucleotomyincision(Haefelietal., 2006;MichalekandIatridis,2011).Thisfindinghasclinicalrelevance,indicatingthatnucleotomycauses morehypermobilityinextendedphysiologicalloadingfordegeneratediscsthannondegeneratediscs.
Theacuteeffectsofnucleotomywerealossofdischeight,decreaseinbothcompressiveand neutralzonemodulus,andincreaseincompressiveandneutralzonerangeofmotion(Figures8&9). Decreaseddischeightandincreasedrangeofmotioncausedbynucleotomyindicatethatinorderto compensateforthedisc’salteredmechanicalloadingfollowingnucleusdepressurization(viaagerelated proteoglycanloss,herniation,ordiscectomy)theremainingannulusandsurroundingspinalelements mustresistmoremotion.Thiscouldinducebackpainbyplacingadditionalstressesontheseinnervated structuresandadvancingthedegenerativecascade.Inaddition,spinalmotionofthetreatedand adjacentlevelsmaybealterediffacetjointsinduceflexionmomentswithinthedisc.Also,the mechanicalchangesofnucleotomyaresimilartothemechanicalchangesofnaturaldegeneration.Thus,
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nucleotomyisapotentialplatformforevaluatingmechanicallybasedspinaltreatments,suchnucleus pulposusreplacement(Omloretal.,2012;Ruanetal.,2010;Woiciechowskyetal.,2012).
Ourmeasuredacuteeffectsofnucleotomyarecomparabletoliteraturevalues.Specifically,our measureddischeightlossof0.8mmfor1.71gofNPremovediscomparabletoareportedheightlossof 0.5mmfor1.75gremoved(Wilkeetal.,2001).Ourobservationthatnucleotomyinitiallycausesa0.35 mmincreaseincompressiverangeofmotionislowerthanliteraturevaluesof0.60.8mm(Freietal., 2001;O’Connelletal.,2011).However,thesestudiesalsoappliedhighercompressiveloads,whichmay accountforsomeofthisdifference.Inaddition,theuniquegeometryoftheL5S1discrelativetothe otherlumbarlevelsusedintheexistingliteraturemayfurtherexplainthesedifferences.Ourobserved 20%decreaseincompressivestiffnessfitswithintheliteraturerangeof350%decreasefortrans annularnucleotomystudies.(Cannellaetal.,2008;MarkolfandMorris,1974).However,transendplate nucleotomydidnotaffectcompressivestiffness(Johannessenetal.,2006),whichsuggestsdecreased compressivestiffnesscausedbynucleotomymaybemoretheresultoftheannularinjurythanthe removalofthenucleus.Thisiscorroboratedbyexistingliteraturethatshowstheannulusplaysa significantroleinsupportingcompressiveloads(Iatridisetal.,1998;Vresilovicetal.,2006).
It’sinterestingthattheNZmodulusatthecyclictimepointishigherforthenucleotomysample thanfortheintactsamples(Figure9C).Thehighermodulusafternucleotomymaybeattributedtofluid flowthroughtheannularnucleotomyincision.Inintactsamples,theannulusservesasabarrierforfluid flowtoandfromthenucleus.Theincisionmayincreasepermeabilityorcreateafluidflowchannelin theinjuryregion(MichalekandIatridis,2011).Asaresult,followingnucleotomy,PBSfromthebathmay morefreelyenterandexitthedisc.Thus,theneutralzonemodulusofnucleotomysamplesatthecyclic timepointmaybehigherthanintactsamplesbecausethereismorePBSwithinthenuclearregion.
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Inadditiontotheacuteeffectsofnucleotomy,wealsofoundthatnucleotomyreducedcyclic loadingeffects.Thisisclearlyseeninthereductionofpercentchangebetweencyclicandrecovery valuesforcompressivemodulus(12to5%)andneutralzonemodulus(94to29%)(Figure6A&C). Reducedeffectsofcyclicloadingindicatealteredfluidflow,whichhastwosignificantramifications. First,italtersthestressesandstrainsexperiencedbythedisc’scells,potentiallyalteringcellular mechanotransductionandsubsequentproteinsynthesis(SettonandChen,2004).Thisinturn,mayalter thediscsbiochemicalcompositionandmechanicalfunction(Baeretal.,2003).Thesecondpotential effectofalteredfluidflowismodifiednutrientdeliveryandwasteremoval.Theintervertebraldiscisa largelyavasculartissueand,asaresult,itscellsaredependentonnutrientdiffusion(Moore,2000; UrbanandRoberts,2003).Thus,anyalterationstothedeliverymechanismhavethepotentialtocreate widereachingeffects.
Thefailureofcompressivemodulusandcompressivestraintoreturntoinitialvaluesforthe intactsamples(Figure6A&B)indicatesastudylimitationinthatcyclicloadingmayhavecausedsome nonrecoverabledamagetotheannulusfibrosus.However,followingannularnucleotomy,the compressivepropertiesattheinitialandrecoveryhydrationstatesareidentical.Thissuggeststhe damagecausedtotheintactsamplesisduetoresistingfluidflowthroughtheannulus,because followingnucleotomyfluidhadalessobstructedpathway.Thispotentialannulardamagewas unexpectedbecauseitwasnotseeninpreviousovinestudieswithsimilarloadsorinthepreliminary studiesforthiswork(Johannessenetal.,2004;Malhotraetal.,2012).However,mostofthehuman tissueusedwasmoderatelydegenerated,incontrastwiththenondegeneratedsamplesusedinanimal studies.Thisinitialdegenerationmayhavecausedthesamplestobemoresusceptibletoinjury.In contrasttocompressiveproperties,cyclicloadingeffectsinneutralzonepropertieswerecompletely recoverablefollowinghydration.
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Thisstudyhasdemonstratedthatcyclicloadingincreasescompressivemodulus,compressive strain,andneutralzonestrainwhiledecreasingneutralzonemodulus.Thesechangesaretheresultof alteredhydrationlevelscausedbythecyclicloadingprotocolandaresimilartonaturallyoccurring diurnalchanges.Nucleotomyreduceddischeightandmoduluswhileincreasingrangeofmotion.In additiontotheseacutechanges,nucleotomyalsoreducedthedifferencebetweenmechanical propertiesbeforeandaftercyclicloading.Theobservedchangesinmechanicalbehaviorinducedby nucleotomyaresimilartonaturallyoccurringdegenerativechanges.Resultsofthisstudyprovidean idealprotocolandcontroldataforevaluatingtheeffectivenessofamechanicallybaseddisc degenerationtreatment,suchasanucleusreplacement.
Acknowledgements WewouldliketothankDharaB.AminandIanS.MacLeanforperformingsomeofthe mechanicaltesting.SpineswereacquiredfromNationalDiseaseResearchInterchange,Philadelphia,PA andPlatinumTraining,Henderson,NV.ThisstudywasfundedbyNIHR01AR050052,NSFGraduate ResearchFellowship#DGE0822,andaNeurosurgeryResearchandEducationFoundationgrant.Noneof thefundingagenciesplayedadirectroleinstudydesign,datacollection,ordataanalysis.
Conflictofintereststatement Noneoftheauthorshaveconflictsofinteresttodisclose.
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Wilke, H.J., Kavanagh, S., Neller, S., Haid, C., Claes, L.E., 2001. Effect of a prosthetic disc nucleus on the mobility and disc height of the l4-5 intervertebral disc postnucleotomy. Journal of Neurosurgery 95, 208-214. Wilke, H.J., Kettler, A., Claes, L.E., 1997. Are sheep spines a valid biomechanical model for human spines? Spine 22, 2365-2374. Wilke, H.J., Neef, P., Caimi, M., Hoogland, T., Claes, L.E., 1999. New in vivo measurements of pressures in the intervertebral disc in daily life. Spine 24, 755-762. Woiciechowsky, C., Abbushi, A., Zenclussen, M.L., Casalis, P., Kruger, J.P., Freymann, U., Endres, M., Kaps, C., 2012. Regeneration of nucleus pulposus tissue in an ovine intervertebral disc degeneration model by cell-free resorbable polymer scaffolds. Journal of tissue engineering and regenerative medicine, DOI: 10.1002/term.1582. Zhang, Y., Lenart, B.A., Lee, J.K., Chen, D., Shi, P., Ren, J., Muehleman, C., Chen, D., An, H.S., 2014. Histological features of endplates of the mammalian spine: From mice to men. Spine (Phila Pa 1976) 39, E312-317. Figure1:T2relaxationtimesarestronglycorrelatedtoPfirrmannscores(r=0.76,p=0.001)forthe humanL5S1discsusedinthisstudy. Figure2:Themechanicaltestingprocedurealtereddischydration.DiscsarefullyhydratedinaPBSbath containingproteaseinhibitors(Steps1and5),andhavereducedhydrationaftercyclicloading(Step3). Mechanicalparametersaremeasuredaftereachchangeinhydrationlevel(Steps2,4,and6). Figure3:Themaximumpositionofsinglecycledecreasesthroughoutthe10,000cyclesofcompressive loading.Creepisdefinedasthedropinmaximumcycledisplacementover10,000cycles,afterwhich creepstrainiscalculatedbydividingcreepbyinitialdischeight. Figure4:Mechanicalparametersarefoundusingatrilinearfitofforcedisplacementcurvefrom50th cycleofthemechanicalmeasurementsteps(steps2,4,and6ofthetestingprocedure).A.Originaldata B.Theminimumslopeofa5thorderpolynomialfitislocated.C.Neutralzoneisfitthroughdataatthe pointofminimumslope.Neutralzonestiffnessistheslopeofthisline.D.Tension(redcircles)and compression(bluediamonds)loadingcurvesareseparatedfromthedata.Compressionrangeofmotion isthedisplacementbetween0and0.48MPaonthecompressionloadingcurve.E.Tensionand compressionlinefitsthroughthemaximum80%oftherespectiveloadingcurves.Compressionstiffness istheslopeofthecompressionline.F.Linesarefitintheneutralzoneregionsofthetensionand compressionloadingcurves.Neutralzonelengthisthedisplacementbetweentheintersectionofthese lineswitheitherthetensionandcompressionlines.Allmechanicalparametersarethennormalizedby initialdiscareaandheight. Figure5:Forcedisplacementcurvesforasinglesampleoverthecourseoftheexperiment.Cyclicloading increasedrangeofmotionanddecreasedneutralzonemodulus,whichrecoveredfollowinghydration. Nucleotomyincreasedrangeofmotionwhiledecreasingstiffnessandtheamountofchangein mechanicalparameterscausedbycyclicloading.
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Figure6:Discmechanicalpropertiesarealteredbycyclicloading(cyclic)andgenerallyrecoverafter unloadedhydration(recovery).Thesechangesmimicnaturallyoccurringdiurnalchanges.Nucleotomy reducedthehydrationeffectsonmechanicalproperties.Inparticular,nucleotomyreducedthepercent changebetweentheInitialandCyclichydrationstatesforcompressivestrain(B),neutralzonemodulus (C),andneutralzonestrain(D).*p
