Rev. bras. ortop. vol.51 número2

w w w . r b o . o r g . b r

Original

Article

Effects

of

ionizing

radiation

on

proteins

in

lyophilized

or

frozen

demineralized

human

bone

Uri

Antebi

,

Monica

Beatriz

Mathor

,

André

Ferreira

da

Silva

,

Rodrigo

Pereira

Guimarães

_,

_Emerson

_Kiyoshi

_Honda

a_{IrmandadedaSantaCasadeMisericórdiadeSãoPaulo,SãoPaulo,SP,Brazil} b_{InstitutodePesquisasEnergéticaseNucleares(IPEN),SãoPaulo,SP,Brazil} c_{InstitutoPaulistadeCirurgiadoQuadrileJoelho,SãoPaulo,SP,Brazil} d_{UniversidadeNovedeJulho,SãoPaulo,SP,Brazil}

e_{FaculdadedeCiênciasMédicasdaSantaCasadeSãoPaulo,SãoPaulo,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received28April2015 Accepted11May2015

Availableonline23February2016

Keywords:

Boneandbones Radiation,ionizing Tissuebanks

Bonemorphogeneticprotein2 Bonemorphogeneticprotein7

a

b

s

t

r

a

c

t

Objective:Theaimwastostudytheeffectsofapplicationofionizingradiation(gammaand electrons)assterilizingagentsatdosesof15kGy,25kGyand50kGy,onlyophilizedorfrozen demineralizedbonetissueforuseintransplants.

Methods:Fivehumanfemoraldiaphysesfromdifferentdonorsofmusculoskeletaltissue weredemineralizedandpreservedaslyophilizedorfrozenat−80◦_{C.Thesampleswere}

dividedintotwogroups:non-irradiated(control)andirradiatedbymeansofgammaraysor anelectronbeam.Theboneproteinswereextractedandusedtodeterminethe concentra-tionsoftotalproteinandBMP2and7.

Results:DecreasesintotalproteinandBMP2 and7concentrationswereobserved.The decreasesintotalproteinconcentrations,incomparisonwiththerespectivecontrolgroups, weresignificantinthelyophilizedandfrozensamplesthatwereirradiatedatadoseof 50kGyofgammaradiationandelectronbeam,withreductionsofmorethan30%. Signifi-cantdecreasesinthelevelsofBMP2and7werealsoobservedathigherdosesandespecially throughuseoftheelectronbeam.

Conclusion:Thereductionsintheconcentrationsoftotalproteinsandosteoinductive pro-teins(BMP2and7)wererelatedtotheradiationdose,i.e.theyincreasedwithhigherdoses ofionizingradiationtypeandthetypeofbonepreservation.Thelargestreductionsin con-centrationswereobservedinthebonesirradiatedbymeansofanelectronbeamandata doseof50kGy.However,thistypeofradiationandthishighdosearenotusualpracticesfor sterilizationofbonetissue.

©2016PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeOrtopedia eTraumatologia.

∗ _{Correspondingauthor.}

E-mail:uri@usp.br(U.Antebi).

http://dx.doi.org/10.1016/j.rboe.2016.02.006

Efeitos

da

radiac¸ão

ionizante

nas

proteínas

presentes

em

ossos

humanos

desmineralizados,

liofilizados

ou

congelados

Palavras-chave:

Ossoeossos Radiac¸ãoionizante Bancodetecidos

Proteínamorfogenéticaóssea2 Proteínamorfogenéticaóssea7

r

e

s

u

m

o

Objetivo: Estudarosefeitosdaaplicac¸ãodasradiac¸õesionizantes(gamaeelétrons)como agentesesterilizantes,nasdosesde15kGy,25kGye50kGy,nostecidosósseos desmineral-izadoscongeladoseliofilizadosparausoemtransplantes.

Métodos: Cinco diáfises femorais humanas de doadores distintos de tecidos muscu-loesqueléticosforamdesmineralizadasepreservadas comoliofilizadasoucongeladasa

−80◦C_{. Asamostras foram divididas em grupos nãoirradiados (controle) e irradiados}

por raiosgamaoufeixedeelétrons.Asproteínasósseasforamextraídasedosadasas concentrac¸õesdeproteínastotais,BMP2e7.

Resultados: Foiobservadadiminuic¸ãodasconcentrac¸õesdeproteínastotaiseBMP2e7. Adiminuic¸ãodasconcentrac¸õesdeproteínastotais,quandocomparadacomorespectivo controle,foisignificativanosgruposdeamostrasliofilizadasecongeladaseirradiadasna dosede50kGyporradiac¸ãogama efeixedeelétronscomreduc¸ãosuperioresa30%.A diminuic¸ãosignificativanasconcentrac¸õesdasBMP2e7tambémfoiobservadanasmaiores doseseprincipalmenteporfeixedeelétrons.

Conclusão: Asreduc¸õesnasconcentrac¸õesdasproteínastotaiseemproteínas osteoindu-toras(BMP2e7)foramrelacionadasàdosederadiac¸ão,ouseja,aumentamcommaiores doses,tipoderadiac¸ãoionizanteeaotipodepreservac¸ãodosossos.Asmaioresreduc¸ões dasconcentrac¸õesforamobservadasnosossosirradiadosporfeixedeelétronsenadose de50kGy.Porémessetipoderadiac¸ãoeessaaltadosenãosãopráticasusuaisparaa esterilizac¸ãodostecidosósseos.

©2016PublicadoporElsevierEditoraLtda.emnomedaSociedadeBrasileirade OrtopediaeTraumatologia.

Introduction

There isa growing demandforallogeneic musculoskeletal graftsfororthopedicanddentalreconstructioninBrazil.Bone tissuecanbehighlightedwithinthis,with21,681distributions in2014.

Musculoskeletaltissuebanksare organizationsthattake on the responsibility for selecting donors and obtaining, processing,storing,distributingandperformingquality con-trolontissues.

Bonetissuescanbeobtainedfrombothlivinganddeceased donors.Thesemusculoskeletaltissuesaremainlyusedin revi-sion arthroplasty procedures on hips or knees,in treating patientswithbonetumors,injointreconstructionfor treat-ingligamentinjuries,inraisingthemaxillarysinusincases ofatrophyandinmandibulargraftsforplacementofdental implants.1

Theidealallogeneicmaterialforuseasagraftingoption should have the followingproperties: biocompatibility, not causinginfection,lowimmunogenicity,osteoconductionand osteoinduction.

Osteoinductionisanosteogenesisprocessthatpromotes recruitment of mesenchymal pluripotent undifferentiated cells and stimulation toward differentiation into bone-forming cells. Bone morphogenic proteins (BMPs) are an importantgroupofglycoproteinsextractedfrom demineral-izedbonematrixthatareresponsibleforboneinduction.2

BMPs are classified as a subfamily withinthe superfa-milyoftransforminggrowthfactor-␤(TGF-␤).Accordingtothe

literature,BMPs2,4and7havethegreatestpotentialfor induc-ingosteoblasticdifferentiationfromprogenitormesenchymal cells.

Performingademineralizationprocedureoncorticalbone matrixincreasesthebiologicalavailabilityofBMPsandmakes thesegraftsosteoconductive.3_{Thereisapositivecorrelation}

between theBMP content thatispresent ingrafts and the degreeofosteoconductivity.4

Thereisgreatconcernaboutensuringtissuequalityand promotingsafetyamongpatientswhoreceive homogenous tissues,withregardtotransmissionofinfectiousand conta-giousdiseases.

Withtheaimofeliminatingpossiblecontamination,bone tissuedonorsundergoserologicalscreeningandassessment oftheirhistoriesandsocialbehavior,alongwithmolecular biologicalteststodetectviralRNAfromHIVandHCV,clinical examinationsandmicrobiologicalcontroltests.Aseptic tech-niquesareappliedduringtheprocedures.5_{However,thereis}

thepossibilityofcontaminationwithmicroorganismsduring tissueacquisition,processing,preservationandstorage.6

Manyprofessionalsresponsiblefortissuebanksconsider thatitisimportanttosterilizebiologicaltissuesusingan effec-tivemethodsuchasionizingradiation.

Sterilizationthroughionizingradiationisamethodthat presentsadvantagesoverothermethods.Itproducesonlya minimal increase intemperature and does notleave toxic residues, which makes it usable and also a form of final sterilization.7 _{However,some authors have concluded that}

Differenttypesofbonepreservationmayaffectboth pro-tein maintenanceand the interaction ofionizing radiation withthetissue.Differentdosesofionizingradiationmayalso influenceboneintegritywithregardtomaintainingthe osteo-conductivepotentialofthesegrafts.

Better knowledgeof theseparameters may help people inchargeoftissuebanksinchoosingthetissueirradiation andpreservationconditionsandensuringthequalityofgrafts usedintransplantations.

Theobjective ofthis study was toassess the effects of applyinggammaradiationandelectronbeamsproducedby

60_{Cosourcesandelectronaccelerators,respectively,atdoses}

of15kGy,25kGyand50kGytobonetissuethathadbeen dem-ineralized,preserved,frozenandlyophilized. Anadditional aimwastoevaluatethepossiblealterationstototalprotein andBMP-2andBMP-7concentrations,inordertodetermine thebestdoseforensuringsafetywithregardtosterilityand preservationoftheosteoinductiveproteinsofbonegrafts.

Materials

and

methods

After the project had been approved by the institution’s researchethicscommittee,underno.238/12,itwasstarted throughselectionofsamplesfromfivefemoraldiaphysesthat hadbeenacquiredasbonetissuedonationsfromfive differ-entdeceasedindividuals:twomalesandthreefemalesaged between39and65years(meanof52).Thematerialhadbeen obtainedinaccordancewiththemusculoskeletalbonetissue protocolofourdepartment.

The cortical bone tissue of the femoral diaphyses was groundup into particles andthose that were smallerthan 1mmwereselected.Fromeachdiaphysis,48gofthebone tis-sueparticlesthusselectedwasdividedinto16groups(3gper sample),thusproducingatotalof80samplescomingfromthe fivedonors.

Subsequently,partofthesample(42g)wasdemineralized in0.5NHClsolutioninaglassbeaker,intheproportionsof 50mlofsolutiontoonegramoftissue,for90minata temper-atureof18◦_C±2◦_{C.Anorbitalmechanicalstirrerwasused}

atlowrotationtokeepthesolutionconstantlymixedduring theentiredemineralizationprocess.3_{Someofthesamplewas}

thenlyophilizedorfrozen.

The demineralized bone tissue was divided into two groups:lyophilizedorfrozen.

Thesamplesinthesetwogroupswereirradiatedatdosesof 15,25and50kGyusingtworadiationsources:electron accel-erator(electronbeam)orcobalt-60(gammaray).Thesamples wereclassifiedas:LIO15;25;50AE,LIO15;25;50␥,CG15;25; 50AE;CG15;25;50␥.Twocontrolsamplesofnon-irradiated demineralizedtissuewerealsocreated.

The samples were irradiated at the Institute of Energy andNuclearResearch(IPEN).Thesamplesofthegroupswere irradiatedwithgammaraysfrom60_{Cosourcesfrom a}

mul-tipurposeirradiatoratadose rateof0.00138kGy/s;or with anelectronbeamfromanindustrialelectronacceleratorata doserateof3.92kGy/s.Toavoidtemperaturevariations,the lyophilizedand frozensampleswere irradiatedat4◦_{C and}

−70◦_{C,respectively.}

ToextractBMPsandtotalproteinsfromthebonematrix, abuffercontainingguanidine–HClwasusedasdescribedin the following: 0.5gof tissuefrom each samplewas added to 5mlof fresh solution of 4M guanidine–HCL and 0.05M Tris–HCL at pH 6.0, with a mixture of protease inhibitors: 5mMbenzamidine–HCL;0.1M6-aminohexanoicacid;0.5mM phenylmethylsulfonylfluoride;5mMN-ethylmaleimide.This waskeptunderagitationatatemperatureof4◦_Cfor24h.The

tubeswerethencentrifugedat697×gfor5min.The super-natantwasremovedandplacedinasecondtube.

Another5mloffreshsolutionof4Mguanidine–HCL,0.05M Tris–HCLand themixtureofprotease inhibitorswasadded inordertoresuspendthesediment.Thesampleswerekept underagitationatatemperatureof4◦_Cfor5h.3 _Afterthis,

thesampleswereagaincentrifugedandthesupernatantwas addedtothesecondtubedescribedabove.Thesupernatant solutionswerethendividedintoaliquotsandfrozenat−80◦_C.

ThetotalproteinsandBMPs2and7werequantifiedusing theBradfordandELISAmethods,respectively.Theassayswere performed intriplicate and theresult wastaken tobe the arithmeticmeanfromtheassays.

The resultsfrom each groupwere comparedwith their respectivecontrols,andthecontrolswerecomparedwitheach other.

Thestatisticalanalysisonthedataconsistedof compari-sonoftheresultsbymeansoftheone-wayANOVAmethod followedbytheTukeytestforstatisticaldifferencesofp<0.05.

Results

Table1andFig.1showthequantificationoftotalproteinsin mg/gofdemineralizedbonetissueandthegradedreductions inmeanconcentrationsintheirradiatedgroupsinrelation totheirrespectivenon-irradiatedcontrols.Statistically signif-icantalterationswereobservedinthefollowinggroups:LIO50

␥(31.9%);LIO50␥AE(35.6%);CG25␥(18.3%);CG50␥(39.8%); CG15AE(13.6%);CG25AE(18.3%);andCG50AE(36.1%).

14

12

10

8

6

4

2

0

Proteins (mg/g of demineralized

bone tissue)

*

_*

*

*

*

_*

*

LIO 15 Co-60LIO 25 Co-60LIO 50 Co-60LIO 15 AE

LIO 25 AE

LIO 50 AE

CG 15 CO-60CG 25 CO-60CG 50 CO-60 CG 15

AE

CG 25 AE

CG 50 AE

LIO 0 kGyCG 0 kGy

Table1–Decreaseinpercentagemeanconcentrationoftotalproteininthedifferentgroupsstudiedinrelationtothe control.

Lyophilizedgroups mg/gof dem-ineralized bonetissue

Reduction(%) Frozengroups mg/gof dem-ineralized bonetissue

Reduction (%)

LIOcontrol 10.7 – CGcontrol 11.54 –

LIO15␥ 9.35 12.5 CG15␥ 9.29 13.1

LIO25␥ 8.79 17.8 CG25␥ 8.74 18.3a

LIO50␥ 7.28 31.9a _{CG50␥ 6.44 39.8}a

LIO15AE 9.02 15.7 CG15AE 9.24 13.6a

LIO25AE 8.52 20.3 CG25AE 8.74 18.3a

LIO50AE 6.89 35.6a _{CG50AE 6.83 36.1}a

a _{Indicatesastatisticallysignificantdifferenceinrelationtothecontrol(p<0.05).}

*

*

*

4000

3500

3000

2500

2000

1500

1000

500 000

BMP-2 (µg/g of demineralized

bone tissue) control

LIO 15 yLIO 25 yLIO 50 y_{LIO 15} AE

LIO 25 AE

LIO 50 AE

CG 15 yCG 25 yCG 50 y_{CG 15} AE

CG 25 AE

CG 50 AE

LIO controlCG control

Fig.2–Effectsofgammaandelectron-beamradiationon theconcentrationsofconcentrationsofBMP-2pergramof demineralizedbonetissueinthegroupsstudied.(*) indicatesastatisticallysignificantdifferenceinrelationto thecontrol(p<0.05).

BMPs2and7werequantifiedwiththeaimofanalyzingthe effectsoftheionizingradiationandthepossiblealterations totheconcentrationsoftheseproteins.Theresultsobtained regardingconcentrationswerecorrelatedintermsofgramsof demineralizedbonetissueandgramsoftotalprotein.

Theresultsobtainedfrom the groupsanalyzedare pre-sentedinTables2and3.Theseshow theresultsregarding meanconcentrationsofBMP-2andBMP-7,in␮g/gandng/g, respectively.Inthelyophilizedandfrozengroups,wenoted percentagereductionsinthemeanconcentrationsofBMP-2 andBMP-7inthedifferentirradiatedgroupsinrelationtothe non-irradiatedcontrols.

Table2andFig.2showtheeffectsoftheionizing radia-tionon theconcentrationsofBMP-2inthegroupsstudied. Statisticallysignificantchanges,withreductionsinmean con-centrationsinrelationtotherespectivecontrol,wereobserved inthefollowinggroups:LIO50AE(32.6%);CG50␥(27.2%);and CG50AE(39.4%).

Table3andFig.3showtheeffectsofionizingradiationon theconcentrationsofBMP-7,inthedifferentgroupsstudied. Statisticallysignificantchanges,withreductionsinthemean concentrationsofBMP-7inrelationtotherespectivecontrol, wereobservedinthefollowinggroups:LIO15AE(26.9%);LIO 25AE(32.4%);LIO50AE(41.7%);CG50␥(44.1%);CG15 AE (36.4%);CG25AE(39.3%);andCG50AE(52.6%).

Figs.4and5showthemeanconcentrationsofBMP-2in relationtothe totalproteinsassayed inthe irradiatedand

180

160

140

120

100

80

60

40

20

0

BMP-7 (ng/g of demineralized bone

tissue) control

*

*

*

*

*

*

*

LIO 15 yLIO 25 yLIO 50 y_{LIO 15} AE

LIO 25 AE

LIO 50 AE

CG 15 yCG 25 yCG 50 y_{CG 15} AE

CG 25 AE

CG 50 AE

LIO controlCG control

Fig.3–Effectsofgammaandelectron-beamradiationon theconcentrationsofconcentrationsofBMP-7pergramof demineralizedbonetissueinthegroupsstudied.(*) indicatesastatisticallysignificantdifferenceinrelationto thecontrol(p<0.05).

600

500

400

300

200

100

0

BMP-2 (Pg/µg of protein) control

LIO 15 y LIO 25 y LIO 50 y _{LIO 15} AE

LIO 25 AE

LIO 50 AE

LIO control

*

Fig.4–RelationshipbetweentheconcentrationsofBMP-2 andspecifictotalproteinsineachlyophilizedtissuegroup: non-irradiatedandirradiatedwithgammaand

electron-beamradiation.(*)indicatesastatistically significantdifferenceinrelationtothecontrol(p<0.05).

non-irradiated groups. There were statistically significant changesinthegroupsLIO50␥andCG50␥inrelationtothe control.

Table2–RecordoftheBMP-2assaysinthegroupsstudied.ReductioninconcentrationofBMP-2intheirradiatedgroups asapercentageinrelationtothenon-irradiatedcontrol.

Lyophilizedgroups BMP-2(␮g/gof demineralized bonetissue)

Reduction (%)

Frozen groups

BMP-2(␮g/gof demineralized bonetissue)

Reduction (%)

LIOcontrol 2.82 – CGcontrol 3.27 –

LIO15␥ 2.84 0 CG15␥ 2.72 16.8

LIO25␥ 2.83 0 CG25␥ 2.7 17.4

LIO50␥ 2.81 0.3 CG50␥ 2.38 27.2a

LIO15AE 2.82 0 CG15AE 2.67 18.3

LIO25AE 2.82 0 CG25AE 2.66 18.6

LIO50AE 1.9 32.6a _{CG50AE 1.98 39.4}a

a _{Indicatesastatisticallysignificantdifferenceinrelationtothecontrol(p<0.05).}

Table3–ReductioninconcentrationofBMP-7intheirradiatedgroupsasapercentageinrelationtothenon-irradiated control.

Lyophilizedgroups BMP-7(ng/gof demineralizedbone

tissue)

Reduction (%)

Frozen groups

BMP-7(ng/gof demineralizedbone

tissue)

Reduction (%)

LIOcontrol 138.66 – CGcontrol 148.2 –

LIO15␥ 128.42 7.4 CG15␥ 133.29 10.1

LIO25␥ 125.46 9.5 CG25␥ 122.03 17.6

LIO50␥ 117.33 15.4 CG50␥ 82.85 44.1a

LIO15AE 101.25 26.9a _{CG15AE 94.2 36.4}a

LIO25AE 93.78 32.4a _{CG25AE 89.97 39.3}a

LIO50AE 80.77 41.7a _{CG50AE 70.18 52.6}a

a _{Indicatesastatisticallysignificantdifferenceinrelationtothecontrol(p<0.05).}

*

600

500

400

300

200

100

BMP-2 (Pg/µg of protein) control 0

CG 15 y CG 25 y CG 50 y _{CG 15} AE

CG 25 AE

CG 50 AE

CG control

Fig.5–RelationshipbetweentheconcentrationsofBMP-2 andspecifictotalproteinsineachfrozentissuegroup: non-irradiatedandirradiatedwithgammaand electron-beamradiation.(*)indicatesastatistically significantdifferenceinrelationtothecontrol(p<0.05).

inthegroupsLIO50␥,CG15AE,CG25AEandCG50AEin relationtotherespectivecontrols.

Discussion

Increaseduseofmusculoskeletaltissuegraftsin reconstruc-tivesurgery,alongwiththeaimofpromotinggreatersafetyin transplantationsandintissuequality,hasledtissuebanksto seekbettertechniquesforprocessing,preservationand ster-ilization.

LIO 15 y LIO 25 y LIO 50 y _{LIO 15} AE

LIO 25 AE

LIO 50 AE

LIO control

BMP-7 (Pg/µg of protein) control

*

18 16 14 12 10 8 6 4 2 0

Fig.6–RelationshipbetweentheconcentrationsofBMP-7 andspecifictotalproteinsineachlyophilizedtissuegroup: non-irradiatedandirradiatedwithgammaand

electron-beamradiation.(*)indicatesastatistically significantdifferenceinrelationtothecontrol(p<0.05).

Gamma radiationisthe typemostusedintissuebanks andhasbeenshowntobeaneffectivemethodforensuring final sterilization ofbiologicaltissues. However,therehave beenreportsofdeleteriouseffectsonthemechanicaland bio-logicalpropertiesoftissues,dependingontheradiationdose applied.5

*

*

*

CG 15 y CG 25 y CG 50 y _{CG 15} AE

CG 25 AE

CG 50 AE

CG control

BMP-7 (Pg/µg of protein) control

18 16 14

12

10 8

6 4

2 0

Fig.7–RelationshipbetweentheconcentrationsofBMP-7 andspecifictotalproteinsineachfrozentissuegroup: non-irradiatedandirradiatedwithgammaand electron-beamradiation.(*)indicatesastatistically significantdifferenceinrelationtothecontrol(p<0.05).

impossibletoperformirradiation,dependingonthestructure anddensityofthetissues.

TheactivityandconcentrationofBMPsandgrowthfactors thatformpart ofTGF-␤ are essential foreffective osteoin-ductionofbonegrafts.However,thesemaybecomedegraded duringtheprocessofsterilizationthroughionizingradiation andmaythereforecompromisethebonetransplantation.9

The protein quantification technique is an important parameter,becauseosteoinductionandosteogenesisare func-tionalprocessesofbonegraftsanddependquantitativelyon theexistenceofboneproteinsandgrowthfactors.Thereisa positivecorrelationbetweentheBMPcontentpresentingrafts andosteoinductivity.4,10

Thereare somestudieswithdivergent resultsregarding theeffectsofgammaradiationontheosteoinductive poten-tialof demineralizedbone grafts.In 1988,Munting et al.11

concludedthatosteoinductionwastotallyinhibitedthrough gammaradiationdosesgreaterthan20kGy.Thisstudywas conductedthroughimplantationofdemineralizedbone tis-sueintothemusclesofratsandrabbits.However,allthegrafts wereirradiatedatroomtemperature,whichmightexplainthe compromisedqualityofthetissue.

In contrast, other authors12,13 _{irradiated demineralized}

bonetissueusinggammaradiationundertemperature condi-tionsthatwerecontrolledthroughusingdryiceandobtained promisingresults.Thisreinforcesthehypothesisthatbone tis-suethatisirradiatedatlowtemperaturesislesssusceptibleto radiation.WientroubandReddi13_{concludedthatastandard}

dose(25kGy)ofgammaradiationdidnotalterthe osteoinduc-tivepotentialofdemineralizedbonetissuethatwasimplanted inmuscletissueinrats,andthathigherdoses(30–50kGy)even increasedthisproperty.

Likewise,Glowacki14_{reportedin2005thatdemineralized}

bonetissueirradiatedwithdosesbetween20kGyand40kGy maintained 80% of their osteoinductive activity. In 2002, Dziedzic-Goclawskareportedresultsfoundusingfrozenbone tissueata temperature of−72◦_{C that wasirradiated with}

dosesof35kGyto50kGyandconcludedthattherewereno differencesinosteoinductive potential,incomparisonwith non-irradiated bone tissue. However, lyophilized preserved tissuethatwasirradiatedatroomtemperatureatthesame

doseswascompletelyreabsorbedandlostthecapacityto pro-duceosteoinduction.

Throughtheresultsobtainedfromtheeffectsofthe ioniz-ingradiationontheconcentrationsofosteoinductiveproteins in the present study, as shown in Table 2 and Fig. 2, we observedthattherewere significantreductionsinBMP-2in relationtothenon-irradiatedcontrol,onlyinthegroups irra-diatedatdosesof50kGy(LIO50AE;CG50␥;andCG50AE). Thelargestreductions(greaterthan32%)werefoundinthe groupsirradiatedusinganelectronaccelerator.Atthedosesof 15and25kGy,underanyoftheconditions,wedidnotobserve anydose-dependentvariation.

TheresultsfromassayingBMP-7,asshowninTable3and

Fig.3,showthatthereweresignificantreductioninrelation totherespectivecontrolsinallthegroupsirradiatedusingan electronaccelerator(LIO15AE;LIO25AE;LIO50AE;CG15AE; CG25AE;andCG50AE)andinthefrozengroupwithgamma rayirradiationatadoseof50kGy(CG50␥).Inthegroupsin whichthereductionwasnotsignificant,wewereabletosee tendencytowardadose-dependentdecrease(from7to18%).

In comparing the effects from the radiation in the lyophilizedandfrozengroupsstudied,althoughtheresults fromthefrozengroupswerenotstatisticallysignificant,they showedalargerreductionintheconcentrationsoftotal pro-teins(13.1–36.1%),BMP-2(16.8–39.4%)andBMP-7(10.1–52.6%), inrelationtothe lyophilizedgroups(12.5–35.6%;0.0–32.6%; and7.4–41.7,respectively).Thisevidencecanbeexplainedby thegreaterindirectbiologicaleffectsofionizingradiationon tissuesthatpresentgreaterpresenceofwater,through radio-lysisonthewater,whichgenerateslargerquantitiesoffree radicalsandconsequentlyproducespossibledamagetothe tissues.15

InFigs.4and5,asignificantincreaseinthemean concen-trationBMP-2pergramofproteincanbeseenatthegamma radiationdoseof50kGyinboththefrozenandthelyophilized groups.InFig.6, itcanbeseenthattherewasasignificant increaseinthemeanconcentrationofBMP-7pergramof pro-teininthelyophilizedand irradiatedgroupsatthedoseof 50kGy.Fig.7showsthatthereweresignificantreductionsin themeanconcentrationsofBMP-7pergramofproteininthe samplesfromthefrozenandirradiatedgroupsthatreceived electronbeamsatthe dosesof15,25 and50kGy.Thiswas duetothedifferenteffectsofionizingradiationonthe con-centrationsoftotalproteinsandspecificproteins(BMP2and 7).

Thus,theeffectsofionizingradiationwithregardto caus-ing damageto bone grafts depend mainly on two factors: (1)theirradiationconditions(radiationdose,doserate,type ofionizingradiationandradiationtemperature);and(2)the physicalstateofthesamples,especiallythequantityofwater presentinthebonetissues.15

Conclusion

ofgamma radiation,with regard tothe quantitiesof bone proteinsstudies.However,theseconditionsare notusually appliedtosterilizationofbonetissues.

Underthesterilizationconditionsusuallyappliedtobone tissues(15–25kGy,usinggammarays),thereductionsinthe concentrations of the osteoinductive proteins (BMP-2 and BMP-7)werelessthan20%.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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