著者
ARAKI Michiko, ATSUMI Yui, UCHIDA Aki, OHSAKO
Masafumi
著者別名
荒木 美智子, 渥美 結衣, 内田 明希, 大迫 正文
journal or
publication title
Journal of Human Life Design
number
12
page range
183-199
year
2017-03
Effects of mechanical loading on bone formation in tibial
primary cancellous bone of growing rats
ARAKIMichiko,ATSUMIYui,UCHIDAAki,OHSAKOMasafumi
Summary Apurposeofthisstudywastoinvestigatehistologicallyeffectsofjumpingexercisesperformed withdifferentintensitiesanddurationsonstructuresofaprimarycancellousboneataproximal metaphysisoftibiaingrowingrats. Thirty-sixwistarmaleratsaged7weekswereusedasmaterials.Theyweredividedinto anexercisegroupandacontrolgroup（CO）randomly,andeachgroupwassubdividedintoa 7-daygroup（7D）,a14-daygroup（14D）anda21-daygroup（21D）foreachexperimentalperiod. Furthermore,theexercisegroupsweresubdividedintoE30,E45andE90randomly.Ratsin eachgroupperformedjumpingexercisesat30, 45,or90%oftheirmaximumjumpingheights measured,respectively, 100timesperday, 5daysperweek,foreachexperimentalperiod. Adensityandathicknessoftrabecularbonesattheprimarycancellousboneincreasedasthe exerciseintensitywasenhanced.Aboneadditiontothesurfaceofacalcifiedcartilagetrabecula alreadystartedfromthesitenexttothegrowthplateinE30andE45.However,theboneaddition ofE90wasdelayedatthatsite,comparedtotheothers. Inconclusion,itwasunderstoodthataboneformationwaspromotedattheprimarycancellous bonebyexercisebutcouldbesuppressedattheareanexttothegrowthplatebyhighintensity exercise. Keywords:primarycancellousbone,exerciseintensity,histologicalstructure FacultyofHumanLifeDesign,ToyoUniversity1. Introduction
Whenexperiencingvascularinvasion,acartilagematrixinthehypertrophiczoneofthegrowth plateisleftasascaffoldofaboneformation,andthenplaysaroleasacoreofthetrabecularbone afterbonematrixdepositionbyosteoblasts.Thetrabeculaejustafterthenewboneadditionare calledaprimarycancellousbone.Theprimarycancellousbonebecomesasecondarycancellous bonethroughaboneremodeling.Athicknessoftrabecularboneinprimaryandsecondary cancellousboneisincreasedwithgrowth1）.Thetrabecularboneoftheprimarycancellousboneis coveredwithnoorlittlebonematrix,soithaslowstrengthstructure.Then, interface between growth plate and primary cancellous bone is a common site for epiphysiolysisinchildren.Epiphysiolysisiscausedbyoveruseduringsportsactivity.Itaccounts for30%ofpubertalfracture2）andleadstogrowthdisorderatthesitebecauseofinappropriate treatment,soitisconsideredasacriticaldisorderatclinicalsite.Thintrabecularboneinthe growingperiodoralossofbonemasscausedbyagingandinactivityisabletoberiskfactors offracture.Then,appropriatemechanicalloadingisconsideredasawayofenhancementofthe bonemassandastrength.Inacorticalboneandthesecondarytrabecularbone,ithasbeen provedthatexercisessuchasrunningorjumpingareeffectiveforanincreaseinthebonemass bytheexperimentsusinganimals3, 4）andhumans5, 6）.Additionally,boneformationispromotedin anintensity-dependentmanneratthesesites7, 8）.Ontheotherhand,inprimarycancellousbone,it isknownthattheboneformationwasincreasedbyexercise9）,butitisunclearthatwhetherthe boneformationwasenhancedinanintensity-dependentmanner.Therefore,thepurposeofthis studyistoinvestigatehistologicallytheeffectsofjumpingexercisesthroughvariousconditions ofintensitiesanddurationsonstructuresofprimarycancellousboneataproximalmetaphysisof tibiaingrowingrats.
2. Materials and Methods
2.1Materials Malewistarstrainratsaged7weekswereusedasmaterialsinthisstudy.Theyweredivided intoanexercisegroupandacontrolgrouprandomly,andeachgroupwassubdividedinto7-day group（7D）, 14-daygroup（14D）and21-daygroup（21D）accordingtoeachexperimentalperiod. 2.2Methods 2.2.1Experiments Theexercisegroupswereaccustomedtojumpingexercisepriortotheexperimentalperiod. Byseveralelectronstimulationsonthefloorofjumpingapparatus,ratsacquiredjumpingreflex withinanacclimationperiodbeforetheexperiments.Therefore,ratswereabletojumpwithout
electronstimulationintheexperiments.Maximumjumpingheightwasmeasuredineachrat oftheexercisegroups.TheexercisegroupsweresubdividedintoE30,E45andE90randomly, andfortheexperimentalperiods,eachgroupperformedjumpingexerciseat30, 45,or90%of themaximumjumpingheightonthebasisofthemeasurements,respectively.Thejumping exercisewasperformed100timesperday, 5daysperweekforeachperiodbyeverygroup.The experimentalprotocolwasperformedasshowninFig.1.Themaximumjumpingheightwasalso measuredafterthefirstandsecondweekoftheexperimentsandthejumpingheightofeachrat wascorrected. 2.2.2Sampling Aftertheexperimentalperiods,ratswereeuthanizedbycarbonedioxideinhalation,softtissues wereremovedfromhindlimbsandtibiaewereexcisedafterconfirmingtheirdeath.Proximal portionsoftibiaeweredividedinsagittaldirectionandwerefixedin4%paraformaldehyde.All samplesweredecalcifiedin8%ethylenediaminetetraaceticacid（EDTA）at4℃ . 2.2.3Analyses Specimensfixedin4%paraformaldehydeweredehydrated,cleared,andembeddedinparaffin wax.Sectionsof4μmthicknesswerecutbymicrotome,stainedwithHematoxylin-Eosinstaining
Fig.1: Experimental protocol
At the end of the each experimental period, rats were euthanized
and their tibiae were sampled.
Exercise
condition
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Fig.1: Experimental protocol
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Fig.2: Images of growth plate chondrocytes.
Proliferative （PZ） and hypertrophic （HZ） chondrocytes in the exercise groups were smaller compared with those of control group and cartilage matrixes between columns were thicker in the exercise group than those in control group.
Yellow squares: proliferative zone Blue squares: hypertrophic zone
A-C: CO-7D, D, E: CO-21D, F-H: E90-7D, I, J: E90-21D Bar: A, F: 20μm, B-E, G-J: 10μm
methodsandthenobservedthroughalightmicroscopy.
3. Results
3.1Growthplate Inthegrowthplateofallgroups,smallchondrocytesofproliferativeandhypertrophiczone wereincreasedwithgrowth.（Fig.2.A-E）However,chondrocytesweresmallerinexercisegroups thanincontrolgroup.Thicknessofthecartilagematrixbetweenthechondrocyteswasincreased, accompaniedwithminiaturizationofchondrocytes.（Fig.2.F-J） 3.2Erosionzoneongrowthplateandprimarycancellousbone 3.2.1Thicknessofcalcifiedcartilagetrabeculaejustunderthegrowthplate InCO-7D,purple-stainedcalcifiedcartilageexisteddenselyintheerosionzone. （Fig.3.A）Most ofthecalcifiedcartilagetrabeculaewereextendedtothebonemarrow,andthereweresome cartilagetrabeculaeacrossthetrabeculaerunningalongthelongitudinalaxisoftibia.These imageswerenotobservedattheareafarfromthegrowthplate.（Fig4.A）InCO-14D,adensity ofthecalcifiedcartilagetrabeculaewasdecreasedcomparedtothoseofCO-7D.（Fig.3.B）Those trabeculaelinkedtoothertrabeculaeweredecreasedneartheerosionzone.Thetrabeculaein CO-14DwerethickerthaninCO-7D.InCO-21D,thetrabeculaelinkedacrosstheothertrabeculae wereonlyafew,especiallyaroundtheerosionzone.（Fig.3.C）CalcifiedcartilageinCO-21Dwas thickerthaninCO-14D,butthedensityofthetrabeculaewasdecreased.（Fig.4.B）Thus,the thicknessofthetrabeculaewasincreased,andthenumberofthemdecreasedwithgrowthinCO. InE30-7D,thedensityofthetrabeculaeincreasedobviously,comparedtoCO-7D.（Fig.3.D）The thicknessofthetrabeculaeofE30-7DwasalmostthesameasCO-7D.（Fig.4.C）Manycalcified cartilagetrabeculaerunningacrossthelongitudinaltrabeculaewereobserved.Differencesinthe trabeculaeobservedwerenotdifferentbetweenE30-14DandE30-7D.（Fig.3.E）Comparedto E30-14D,thenumberoflongitudinalandtransversecalcifiedcartilagetrabeculaewasincreasedin E30-21D.（Fig4.F） InE45-7D,thedensityofthetrabeculaewaslowerandhorizontallinkstotheotherswerefewer comparedtoE30-7D.（Fig.3.G）However,thicknessofthetrabeculaewereincreasedinE45-7D. （Fig.3.H）InE45-14D,thicknessanddensityofthetrabeculaewereslightlyincreasedinE45-14D comparedtoE30-14D.InE45-21D,thedensityofthetrabeculaewasdecreasedclearly.（Fig.3.I） Thicknesswasincreasedslightly,butthischangewasnotobviouscomparedtoday14groups. In E90-7D, the density of the trabeculae was decreased compared with E45-7D.（Fig.3.J andFig.4.E）ThenumberofthemwasalsoreducedinE90-14D.（Fig.3.K）Therewasnoclear differencebetweenE90-21DandE45-21D.（Fig.3.L）Howeverthetrabeculaewasthinnernearthe erosionzonethanintheothersite.（Fig.4.F）3.2.2Onsetofboneformationandthecellsaroundthesites InacancellousboneofCO-7D,theboneformationwasstartedatadistancefromthegrowth plate,andmanycalcifiedcartilageswithoutadditionofanewbonewereobservedneartheerosion zone.Thesecalcifiedcartilageswerecoveredwithmanymono-ormulti-nuclearcellsstaineddark pink.Atadistancefromthegrowthplate,purple-stainedsphericalcellswhosecytoplasmwas partlylightappearedonthesurfaceofthecalcifiedcartilagetrabeculae.Farfromthegrowth plate,bonematrixstainedpinkwasformedatthemarrowspacebetweenthecellsandthe calcifiedcartilagematrix.（Fig.5.A）InCO-14D,thestartingportionoftheboneformationslightly
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Fig.3: Low magnified images of each group.
Thickness of bone trabeculae was increased with growth in both control and exercise group. A: CO-7D, B: CO-14D, C: CO-21D, D: E30-7D, E: E30-14D, F: E30-21D, G: E45-7D, H: E45-14D, I: E45-21D, J: E90-7D, K: E90-14D, L: E90-21D
approachedtotheerosionzone.（Fig.5.B）InCO-21D,thatportionwasclosertothegrowthplate thaninCO-14D,andtheboneadditionwasobservedatthesurfaceofthecalcifiedcartilagearound theerosionzone.（Fig.5.CandFig.6.A）However,inbothCO-14DandCO-21D,pink-coloredmono- ormulti-nuclearcellswereobservedintheinterfacebetweenthegrowthplateandtheprimary cancellousbone.（Fig.6.B）Additionally,purple-stainedmononuclearcellscontainingalargerlight areaintheircytoplasmwereseenaroundthesecellscomparedtoCO,asbonematrixbetweenthe cellsandthecalcifiedcartilagebecamethicker.Tosumup,inCO,thestartingportionofthebone formationapproachedthegrowthplate,andbonematrixwasaddedatthispointandthelight areaincytoplasmofpurple-stainedcellswasincreased. InE30-7D,thecalcifiedcartilagetrabeculaethatwerenotcoveredwiththenewbonewere Fig.4: Images of primary cancellous bone.
There was no obvious difference in thickness of bone trabeculae between 7-day groups. However, at day 14 and 21, primary cancellous bone in both exercise groups was thicker than that in CO.
A: CO-7D, B: CO-21D, C: E30-7D, D: E30-21D, E: E90-7D, F: E90-21D Bar: 50μm
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observedthesameasinCO-7D,however,theboneformationwasstartedclosertothegrowth platethaninCO-7D,andthepurple-stainedsphericalcellswereseenonthesurface.Thebone matrixobservedatthissitewasthickerthaninCO-7D.（Fig.5.D）InE30-14D,thenewbone matrixwasrecognizednearthegrowthplateandwasthickerthaninE30-7D.Pink-colored mononuclearcellswerereducedandthepurple-stainedcellsappearedintheerosionzone.（Fig.5.E） InE30-21D,thereisnoobviousdifferenceinthestartingportionofboneformation,yetbone matrixaddedatthesitewasclearlythickened.Thereweremanypink-coloredmononuclearcells
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Fig.5: Magnified images of starting portion of the bone formation.
Bone formation was started closer to the growth plate and bone addition was increased as the experimental periods were proceeded.
A: CO-7D, B: CO-14D, C: CO-21D, D: E30-7D, E: E30-14D, F: E30-21D, G: E45-7D, H: E45-14D, I: E45-21D, J: E90-7D, K: E90-14D, L: E90-21D
intheerosionzonecomparedtoE30-14D,andthepurple-stainedcellswereobservedaroundthese cells.（Fig.5.FandFig.6.C,D） InE45-7D,thereisalsothecalcifiedcartilagetrabeculaecoveredwithnobonematrix,and thenumberofthesetrabeculaewaslimited.Thestartingportionofboneformationslightly approachedthegrowthplateandanincreaseofthicknessinbonematrixatthesitewasobvious. Pink-stainedcellsintheerosionzonewerebiggerthaninE30-7Dandwerealreadyappeared withthepurple-stainedcellsthathadthelightareaintheircytoplasm. （Fig.5.G）InE45-14D,an obviousdifferenceinthestartingportionofboneformationwasnotobserved.（Fig.5.H）InE45-21D,theboneadditiontothecalcifiedcartilagetrabeculaeintheerosionzonewasincreasedand thebonematrixwasthickerthaninE30-21D.（Fig.5.I）Withthesechanges,thepurple-stained cellsandthelightareaintheircytoplasmwerebiggernearthegrowthplatecomparedtoE45-14D. InE90-7D,thestartingportionofboneformationwasslightlyfarfromthegrowthplate,and theboneadditiontothesurfaceofcalcifiedcartilagehadnotoccurredjustunderthegrowth plate.However,bonematrixatthestartingportionofboneformationwasthickerthaninE45-7D.Thepink-coloredcellswerelocalizedintheerosionzoneandthepurple-stainedcellswere
observedonlyfarfromthegrowthplate,sotheywerenotco-localizedinE90-7D.Thepink-Fig.6: Delay of the bone formation in E90.
At day 21, in the primary spongiosa just under the growth plate in CO, little new bones were added around the calcified cartilage trabeculae. Bone formation was started near the cartilage lacunae in E45. However, bone formation was delayed in E90 and thin calcified cartilage trabeculae was not covered with the new bones.
Yellow arrow heads: starting portions of bone formation
Blue arrow heads: calcified cartilage trabeculare without addition of new bones A, B: CO-21D, C, D: E45-21D, E, F : E90-21D
Bar: A, C, E: 20μm, B, D, F: 10μm
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coloredcellswereoftenseenaroundthecalcifiedcartilagewithoutboneaddition.（Fig.5.J）In E90-14D,thestartingportionofboneformationwasalsofarfromthegrowthplatecompared toE45,andthecartilagelacunaeinwhichtransversecartilagetrabeculaewereremovedwere runninglongitudinally.（Fig.5.K）InE90-21D,thestartingportionofboneformationwasreceded fromthegrowthplatecomparedtoE45-21D,andatthissite,bonematrixbecamethickerthan inE45.（Fig.5.L）IntheerosionzoneofE90-7D,thepink-coloredcellswerereduced,however, thenewboneadditionwasnotstartednearthegrowthplate.Manycalcifiedcartilagetrabeculae werenotcoveredbyanybonematrixandcellnearthegrowthplate.Pink-stainedcellsappeared inthenotinferiorborderofthegrowthplatebutslightlyfarfromthisarea.Cartilagelacunae lyinglongitudinallyweresurroundedwithcalcifiedcartilagetrabeculaeinwhichboneadditionhad notoccurred.（Fig.6.E,F）Intheselacunae,thereweremanyerythrocytesandthecellsthathad extendedtheirflattenedprocesstothegrowthplate.Atsitesslightlyfarfromthegrowthplate, thesecellswerenotobserved,butthepink-coloredcellsandthepurple-stainedcellshademerged.
4. Discussion
The aim of this study was to investigateeffectsof differentheightjumpingexerciseson structuralchangesofprimarycancellousbone.
Ithasbeenknownthatdifferencesofwaysofmechanicalloadingleadtodifferentchangesof bonestructure.Intheexperimenttoinvestigatetheeffectsofrunningandjumpingontrabecular structure,ithasbeenreportedthatincreaseoftrabecularthicknessdistributestoincreased bonevolumeinjumpingexercise,ontheotherhand,intherunningexercise,thenumberof trabecularbonewasincreasedwithbonegain10）.Notomietal.11）havestatedthatinananaerobic exercisesuchasjumping,boneformationwasstartedmorequicklythaninanaerobicexercise. Also,notcontinuousbutintermittentloadingleadstoquickresponseandformationofwoven bone12）.Fromtheabove,bonemetabolicandstructuralchangesareaffectedbyloadingmanner. Intheseexperiments,thethicknessofprimarycancellousboneinbothcontrolgroupandexercise groupsisthesameasthereportbyTakizawaetal.1）.Especially,theprimarycancellousbonewas thickerinexercisegroupsthanincontrolgroups,andthedifferencesbetweenthe2groupswere remarkableasexperimentalperiodsproceeded.Additionally,increaseinthicknesswasobvious withelevationofexerciseintensity.Theresultsshowedincreaseofbonevolumeinanexercise intensity-dependentmannerinprimarycancellousbone,andthiswasconcurredwithprevious studyonsecondarycancellousboneandcorticalbone7, 8）.Moreover,Obuchietal.13）havereported thattheboneformationintibialprimarycancellousboneofratswaspromotedasthenumberof jumpsincreased.Fromtheabove,itisthoughtthattheboneformationintheprimarycancellous boneisinproportiontointensityorworkoutputofexercise.
reportsthatinvestigatedeffectsofdifferentloadintensity,indicatethatthelevelofeffectbythe loadingwhichreachesthethresholdofintensityisthesameasbytheloadingwhichexceedsthe threshold14-16）.Moreover,excessivemechanicalloadingcouldcausebreakdownoftissue.Actually, ithasreportedthatmechanicalloadingoverthephysiologicalconditioninhibitsbonegrowth17）. Besides, when the bone received a repetitive low-intense loading, stress fracture caused by accumulationofthesmallstressoccurs18）.Tosumup,thereisathresholdofeffectsonpromotion ofboneformationandinhibitionofboneresorptionbymechanicalloading. Inourobservation, theboneformationonprimarycancellousbonewaspromotedinallexercisegroups.Ontheother hand,theboneadditiontocalcifiedcartilagetrabeculaewasstartedjustunderthegrowthplate inE30andE45butnotinE90.Accordingly,osteoblastwithmaturegoldiareaclearlyobserved wasincreased.Elevationofthestartingportionofboneformationandmaturityofosteoblasts associatedwiththeelevationwereobservedingrowth19）andmechanicalloading20）.InE90,thehigh intensityexercisegroup,therewasdelayofboneadditiontocalcifiedcartilagetrabeculaejust underthegrowthplate,thereforejumpingexerciseat90%ofthemaximumjumpingheighthas anegativeeffectatthissite.Takahashietal.21）haveinvestigatedtheboneformationofsecondary cancellousboneusingthesameexerciseconditionsasourexperiment.Theyhavereportedthat theboneformationwasincreasedasintensitywenthigherandwasnotinhibited.Theirresultsat thesecondarycancellousbonecorrespondtoourobservationattheprimarycancellousbonefrom thisstudy.Itwasalsoobservedthatthethicknessoftrabeculaeboneincreasedinanintensity dependentmanner.However,thesechangeswerenotobservedjustunderthegrowthplate,and itisshownthatthereisaverydifferentresponsetomechanicalloadingbetweenthesitesof cancellousbone. Intheboneremodelingofcancellousandcorticalbonethatrespondedtoincreaseanddecrease ofmechanicalstress,itisknownthatosteocyte,mechanosensor,embeddedinbonematrixplays akeyroleasaregulatorofosteoblastandosteoclast22）.Factorsinvolvinginboneremodeling, receptoractivatorofnuclearfactorkappa-Bligand（RANKL）andosteoprotegerin（OPG）which aresynthesizednotbyosteoblastsbutratherbyosteocytesareimportantforregulationofbone remodeling23）.Variouspathwaysthatosteocytecontrolsboneremodelinghavebeenreported24-28）. Itisknownthatbyincreaseofmechanicalloading,boneremodelingispromotedbyactivation ofcanonicalWntsignalingpathwayinosteocyte24）,inhibitionoftranscriptionofsclerostinvia expressionoftransforminggrowthfactorbeta（TGF-β）inosteocyteacceleratesboneformation25）, andsecretionofinsulin-likegrowthfactor-1 （IGF-1）enhancesboneformation26）.Ontheother hand,ithasreportedthatosteoclastogenesisinthebonesurfaceispromotedbyamicrofracture inducedbyrepetitiveloadinganddeathofosteocytecausedbyunloading27, 28）.However,these responsestomechanicalstimulationoccurredinmaturebonematrixthatcontainsosteocytes.The sitesobservedinthisstudywereprimarycancellousbonecomposedofimmaturebonematrix andthecalcifiedcartilagetrabeculaewithoutnewbonematrixjustunderthegrowthplate.The
microenvironmentisgreatlydifferentbetweenthesecondarycancellousboneandtheprimary cancellousbone.Therefore,itcouldbethoughtthattheeffectofexerciseisdifferentbetween primaryandsecondarycancellousbone. Chondrocyte,amechanosensorincartilagematrix,isembeddedinextracellularmatrixjust likeosteocyte.IthasbeenalsoconfirmedbothproteinandmRNAexpressionsofRANKLand OPGinchondrocyte29, 30）.Infact,ithasbeenreportedthattherecouldbeaninteractionbetween chondrocyteandosteoblastorosteoclast31-33）.Thepreviousstudywhichcarriedoutco-culture has suggested that chondrocyte and osteoblast regulate matrix synthesis of each other by paracrine31）.Inlongbonespecificallydeletedbetacatenininhypertrophicchondrocytes,trabecular boneformationjustunderthegrowthplatewassignificantlyinhibited31）.Thisphenotypewas accompanied with elevation of RANKL expression, so it has been indicated that changes of Wntsignalingingrowthplatehypertrophicchondrocyteenhanceactivitiesofosteoclastsor chondroclastsneartheerosionzone.Moreover,sternalhypertrophicchondrocytefrommouseand chickembryoco-culturedwithosteoclastprecursorcontrolsosteoclastogenesisthroughRANKL expressionviabonemorphogeneticprotein2 （BMP2）33）.Fromtheabove,thereisapossibility thatchondrocytesintibialgrowthplateregulatetheearlytrabecularboneformation.However, onlyafewstudiesinvestigatetheeffectsofchangesingrowthplateontrabecularboneformation. Therefore,interactionbetweenthegrowthplatechondrocyteandosteoblastorosteoclastaround thetrabeculaeisstillunclear. Histomorphometricinvestigationoneffectsofmechanicalstressongrowthplateandtrabecular bonehasbeenperformedbyNyskaetal.34）.Theyhavestatedthatgrowthplateproliferative chondrocytesincreasedbyswimmingexercise,sometabolicaccelerationinthegrowthplate leadstoincrementoftrabecularbone.Nevertheless,whetherthereisarelationshipbetween eventsofgrowthplateandtrabecularboneornotisunknown.Besides,Takahashietal.20）have describedtheincreaseofosteocalcin-positivematureosteoblastsnearthegrowthplatebyshort-termjumpingexerciseaftershort-termimmobilization.Thisresultsupportsourobservationin thisstudy,Butitisunclearwhetherthesechangeswerecausedbyloadedchondrocyteinthe growthplate.Fromtheabove,therearemanyunclearpointsineffectofmechanicalloadingtothe growthplateonboneformation.Likethereportthatexaminedeffectofmechanicalstressonly inthegrowthplate,Tsutsumietal35）havereportedthatcalcificationofhypertrophiczonewas acceleratedwithelevationofrunningspeed.Katsuta36）hasnotedincreaseofsizeinchondrocyteof ratswithelevationofspeedintreadmillrunning.However,miniaturizationofchondrocytesinstead ofincreasinginsizewasobservedinthisstudy.Itisunclearwhetherthedifferencebetweenthese reportsisdependentonexercisepatternornot. Moreover,Reichetal.37）haveshowedthatexpressionsofmatrixmetalloproteinase（MMP）-9, MMP-13,andosteopontininthegrowthplatewereenhancedbycontinuousloading,andindicated thatincreaseofthesefactorscouldinduceappearanceofosteoblast,osteoclastandtheirprecursor
frombonemarrow.InE90showingthedelayofboneformation,thereweremanyerythrocytes andendothelial-likecellsbetweencalcifiedcartilagetrabeculaewithoutbonematrixoranycellon thesurface,yetitisanunclearassociationbetweensuppressionofboneformationandappearance thesecells.However,ithasbeenpreviouslyreportedthatincreaseofvascularinvasiontothegrowth platewithenhancementofmechanicalloading37）.theosteoblastanditsprecursordeliveredwith angiogenesisarecontainedinapartofboneformationcellsattrabecularbone38）.Increaseinsuch boneformationcellscouldpromoteboneformation.Fromtheexperimentthattreadmillrunning wasperformedonratstreatedwithanti-vascularendothelialgrowthfactor（VEGF）antibody, ithasbeenprovedthatangiogenesisinbonemarrowisessentialtogainofboneformationon trabeculaebyexercise39）.Thesereportssuggestthatangiogenesisisacriticaleventnotonlyin boneformationofdevelopmentalstage,butalsointhatofgrowingstage.However,inhibitionof boneformationjustunderthegrowthplatewithhighintensityexerciseisnotabletobeexplained byexercise-inducedactivationofangiogenesis.Infact,therehasbeenastudyperformingthe invivomechanicalloadingongrowthplatethatreportednorelationbetweenVEGFexpression andvascularinvasion17）.Furthermore,therearesomeevidencesforchangesinthesefactorsin thegrowthplateontrabecularboneformationandassociationbetweenthechangesandexercise intensity. Thesitejustunderthegrowthplateistheinterfaceofcartilageandbone,andisknownasa commonsiteofepiphysiolysisinchildren2, 40）.Moreover,ithasbeendescribedthatextensively strainedtibiahadshowedfractureinosteochondro-junction41）,andindicatedthatitisalow strengthsite.Inthisstudy,justunderthegrowthplateinhighintensityexercisegroups,bone formationdidn’tstartandremainedbareofthincalcifiedcartilagetrabeculae.Intheosteochondro-junction,boneformationinE90wasmoredelayedthaninCO.Highintensityexercisedoesnot enhancethebonestructureapplicabletomechanicalloading.Tothecontrary,itcouldreducethe strengthatthesite. Inthisstudy,jumpingexerciseswithvariousexerciseintensityconditionswereperformedand increaseofbonevolumeinprimarycancellousboneinanexerciseintensity-dependentmanner wasobserved.However,justunderthegrowthplate,delayofboneadditionwasrecognizedonly inthehighintensityexercisegroup.So,itisindicatedthatresponsetomechanicalloadingdiffered betweenthesites.Itisconsideredthatboneformationandresorptiononprimarycancellousbone arecontrolledbythegrowthplatechondrocyte,orinfluencedbyvascularinvasion.However, effects of these factors corresponding to changes of mechanical loading on trabecular bone formationwerenotinvestigated.Furtherstudyisneeded.
5. Conclusion
promotedyetboneappositionwassuppressednexttothegrowthplateinthecaseofhighexercise intensity. Acknowledgement WethankMr.TakahashiMasatoforguidanceandsupportinperformingtheexperiment. CommitteeofAnimalExperimentandEthics ThisstudywasapprovedbytheEthicalCommitteefortheresearchoftheFacultyofHuman LifeDesignandbytheAnimalCareandUseCommittee,ToyoUniversity. References １）TakizawaA.,etal.:Observationonboneformationprocessesoftibialcancellousboneingrowingrat. BulletinoftheGraduateSchool,ToyoUniversity47: 257-268, 2010. ２）DoverC.,etal.:Growthplateinjuriesandmanagement.OrthopaedicsandTrauma29: 261-267, 2015. ３）ShimanoR.C.,etal.:Biomechanicalandmicrostructuralbenefitsofphysicalactivityassociatedwith risedronateinbonesofovariectomizedrats.MicroscopyResearchandTechnique77: 431-438, 2014. ４）UmemuraY.,etal.:AtimecourseofboneresponsetojumpexerciseinC57BL/6Jmice.JournalofBone andMineralMetabolism20: 209-215, 2002. ５）MckayH.A.,etal.: “BounceattheBell”:anovelprogramofshortboutsofexxerciseimprovesproximal femurbonemassinearlypubertalchildren.BritishJournalofSportsMedicine39: 521-526, 2005. ６）KatoT.,etal.:Effectoflow-repetitionjumptrainingonbonemineraldensityinyoungwomen.Journalof AppliedPhysiology100: 839-843, 2006. ７）NagasawaA.,etal.:Effectsoflow-repetitionjumpexerciseonosteogenicresponseinrats.JournalofBone andMineralmetabolism26: 226-230, 2008. ８）KodamaY.,etal.:Exerciseandmechanicalloadingincreaseperiostealboneformationandwholebone strengthinC57BL/6JmicebutnotC3H/Hejmice.CalcifiedTissueInternational66: 298-306, 2000. ９）BourrinS.,etal.:Effectsofphysicaltrainingonboneadaptationinthreezonesoftherattibia.Journalof boneandmineralresearch10: 1745-1752, 1995. 10）YuY-I.,etal.:Differentialeffectsofjumpversusrunningexerciseontrabeculararchitectureduring remobilizationaftersuspension-inducedosteopeniaingrowingrats.JournalofAppliedPhysiology112: 766-772, 2012. 11）NotomiT.,etal.:Acomparisonofresistanceandaerobictrainingformass,strengthandturnoverofbone ingrowingrats.EuropeanJournalofAppliedPhysiology109: 1600-1607, 2010. 12）SugiyamaT.,etal.:Functionaladaptationtomechanicalloadinginbothcorticalandcancellousboneis controlledlocallyandisconfirmedtotheloadedbones.Bone46: 314-321, 2010. 13）ObuchiN.,etal.:Effectsofvariousexerciseloadonstructuresoftibialcancellousboneinrat.Journalof HumanLifeDesign6: 73-84, 2010. 14）UmemuraY.,etal.:Fivejumpsperdayincreasebonemassandbreakingforceinrats.JournalofBone andMineralResearch12: 1480-1485, 1997. 15）HagiwaraY.,etal.:HowmanydaysperweekshouldratsundergorunningexercisetoincreaseBMD? JournalofBoneandMineralMetabolism23: 289-294, 2005.
16）SwiftJ.M.,etal.:Increasedtrainingloadsdonotmagnifycancellousbonegainswithrodentjump resistanceexercise.JournalofAppliedPhysiology109: 1600-1607, 2010. 17）OhashiN.,etal.:Theeffectsofdynamicaxialloadingontheratgrowthplate.JournalofBoneand MineralResearch17（2）: 284-292, 2002. 18）NobleB.,etal.:Bonemicrodamageandcellapoptosis.EuropeanCellsandMaterials6: 46-56, 2003. 19）TakizawaA.,etal.Histologicalstudyofdifferentiationprocessaboutbonetissueingrowingrat.Bulletin oftheGraduateSchool,ToyoUniversity48: 145-161, 2011. 20）TakahashiM.,etal.:Effectsofexerciseaftershort-termimmobilizationonbonestructureingrowingrat. JournalofHumanLifeDesign8: 161-175, 2012. 21）TakahashiM.,etal.:Effectsofvariousmechanicalloadingonosteogenicprocessesoftibialcancellous boneinrats.BulletinoftheGraduatedSchool,ToyoUniversity52: 241-253, 2015. 22）TatsumiS.,etal.:Targetedablationofosteocytesinducesosteoporosiswithdefectivemechanotransduction. CellMetabolism5: 464-475, 2007. 23）XiongJ.,etal.:Matrix-embeddedcellscontrolosteoclastformation.NatureMedicine17: 1235–1241, 2011. 24）HensJ.R.,etal.:TOPGALmiceshowthatthecanonicalWntsignalingpathwayisactiveduringbone developmentandgrowthandisactivatedbymechanicalloadinginvitro.JournalofBoneandMineral Research20: 1103-1113, 2005. 25）NguyenJ.,etal.:LoadregulatesboneformationandsclerostinexpressionthroughaTGFβ-dependent mechanism.PlosOne8:e53813, 2013. 26）RejindersC.M.A.,etal.:Effectofmechanicalloadingoninsulin-likegrowthfactor-Igeneexpressionin rattibia.JournalofEndocrinology192: 131-140, 2007. 27）KennedyO.D.,etal.:Activationofresorptioninfatigue-loadedboneinvolvesbothapoptosisandactive pro-osteoclastogenicsignalingbydistinctosteocytepopulations.Bone50: 1115-1122, 2012. 28）AguireJ.I.,etal.:Osteocyteapoptosisisinducedbyweightlessnessinmiceandprecedesosteoclast recruitmentandboneloss.Journalofboneandmineralresearch21: 605-615, 2006. 29）SolbergL.B.,etal.:Tartrate-resistantacidphosphatase（TRAP）co-localizeswithreceptoractivator ofNF-KBligand（RANKL）andosteoprotegerin（OPG）inlysosomal-associatedmembraneprotein1 （LAMP1）-positivevesiclesinratosteoblastsandosteocytes.HistochemicalCellBiology143: 195-207, 2015. 30）SilvestriniG.,etal.:Detectionofosteoprotegerin（OPG）anditsligand（RANKL）mRNAandproteinin femurandtibiaoftherat.JournalofMolecularHistology36: 59-67, 2005. 31）JiangJ.,etal.:Co-cultureofosteoblastsandchondrocytesmodulatescellulardifferentiationinvitro. BiochemicalandBiophysicalResearchCommunications338: 762-770, 2005. 32）GolovchencoS.,etal.:Deletionofbetacatenininhypertrophicgrowthplatechondrocytesimpairs trabecularboneformation.Bone55: 102-112, 2013. 33）UsuiM.,etal.:MurineandchickenchondrocytesregulateosteoclastogenesisbyproducingRANKLin responsetoBMP2.JournalofBoneandMineralResearch23（3）: 314-325, 2008. 34）NyskaM.,etal.:Histomorphometryoflongbonegrowthplateinswimmingrats.InternationalJournalof ExperimentalPathology76: 241-245, 1995. 35）TsutsumiT.:Theeffectofphysicalexerciseonthebonecellsingrowingrat-SEMfindings-TheJournal oftheKyushuDentalSociety51: 821-841, 1997. 36）KatsutaS.,etal.:Theinfluencesofexerciseintensityonbonedevelopmentingrowingrats.JapanJournal ofPhysicalEducation36: 29-51, 1991.
ossificationandvascularization.JournalofAppliedPhysiology98: 2381-2389, 2005. 38）MaesC.,etal.:Osteoblastprecursors,butnotmatureosteoblasts,moveintodevelopingandfractured bonesalongwithinvadingbloodvessels. 19: 329-344, 2010. 39）YaoZ.,etal.:IncreaseofboneangiogenesisandbonemassinresponsetoexercisedependsonVEGF. JournalofBoneandMineralResearch19: 1471-1780, 2004. 40）SalterR.B.,etal.:Injuriesinvolvingepiphysealplate.TheJournalofBoneandJointSurgery45: 587-622, 1963. 41）ApteS.S.,etal.:Physealdistractionandcellproliferationinthegrowthplate.JournalofBoneandJoint SurgeryTheBone&JointJournal76-B: 837-843, 1994.
