Study of the healing process after transplantation of pasteurized bone ...

Acta Medica Okayama Volume 56, Issue 3

2002

Article 1

J UNE 2002

Study of the healing process after transplantation of pasteurized bone grafts in rabbits. Sanja Zoricic∗

Dragica Bobinac†

Boris Lah‡

Ivana Maric∗∗

Olga Cvijanovic††

Snjezana Bajak‡‡

Vesna Golubovic§

Radovan Mihelic¶

∗

University of Rijeka, University of Rijeka, ‡ University of Rijeka, ∗∗ University of Rijeka, †† University of Rijeka, ‡‡ University of Rijeka, § University of Rijeka, ¶ University of Rijeka, †

c Copyright 1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.

Study of the healing process after transplantation of pasteurized bone grafts in rabbits.∗ Sanja Zoricic, Dragica Bobinac, Boris Lah, Ivana Maric, Olga Cvijanovic, Snjezana Bajak, Vesna Golubovic, and Radovan Mihelic

Abstract Different bone allografts (pasteurized, autoclaved, and frozen) were compared based on their osteoinductive properties. Our primary purpose was to examine the biologic qualities of pasteurized allografts, as pasteurization inactivates most viruses transmitted by transplantation. Frozen, pasteurized, and autoclaved allografts were packed into a standard defect of rabbit ulna. The animals were sacrificed at 2 and 4 weeks after surgery. The parts of bones with experimental defects were explored en bloc, and a roentgenogram was carried out. Ulna bone samples were then embedded in methyl-methacrylate. Roentgenograms showed that after 2 weeks, calluses were well-formed, but irregular in shape in all 3 types of allografts. After 4 weeks, the calluses were regular in shape in all but the autoclaved grafts. After 2 weeks, the healing processes had begun in the frozen and pasteurized grafts, with the reaching approximately the same stage, while in the autoclaved grafts these processes were not seen and the bone particles were surrounded by connective tissue without any changes. After 4 weeks, osteoinductive processes were very strong, with the first signs of complete bone remodeling at the bone edges of the defect in pasteurized and frozen allografts. The osteoinductive values of these 2 types were very high and similar. Autoclaved allografts, on the other hand, had very low osteoinductive values, as they were still at the very beginning of the healing process. Histomorphometric analysis revealed a significant difference in both newly formed osteoid thickness and osteoblast number per microm of bone surface in all experimental groups (P < 0.005). Values of osteoid thickness and osteoblast number were significantly higher in both frozen and pasteurized grafts when compared with the autoclaved ones (P < 0.005). Osteogenic properties of pasteurized bone allografts were preserved, and the allografts have been gradually replaced with newly formed bone. As such, pasteurized bone grafts from a bone bank have approximately the same biologic validity as frozen grafts, while autoclaved grafts impair bone healing. KEYWORDS: bone allograft, pasteurization, headling, rabbits

∗

PMID: 12108582 [PubMed - indexed for MEDLINE] Copyright (C) OKAYAMA UNIVERSITY MEDICAL SCHOOL

Zoricic et al.: Study of the healing process after transplantation of

Act aMed.Okay ama,2002 Vol .56,No.3,pp.121128 ｃ2002byOka Copyr i ght ya maUni ve r s i t yMe di c a lSc hool .

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Die r e ntboneal l ogr af t s( pas t e ur i z e d,aut oc l ave d,andf r oz e n)we r ec ompar e dbas e don t he i r os t e oi nduc t i vepr ope r t i e s .Ourpr i mar ypur pos ewast oe xami net hebi ol ogi cqual i t i e sofpas t e ur i z e d al l ogr af t s , aspas t e ur i z at i on i nac t i vat e smos tvi r us e st r ans mi t t e d by t r ans pl ant at i on. Fr oz e n, pas t e ur i z e d,andaut oc l ave dal l ogr af t swe r epac ke di nt oas t andar dde f e c tofr abbi tul na.The ani mal swe r es ac r i fic e dat2and4we e ksaf t e rs ur ge r y.Thepar t sofbone swi t he xpe r i me nt alde f e c t s we r ee xpl or e d ,andar oe nt ge nogr am wasc ar r i e dout .Ul nabones ampl e swe r et he n e mbe dde di n me t hyl me t hac r yl at e .Roe nt ge nogr amss howe dt hataf t e r2we e ks ,c al l us e swe r e we l l f or me d,buti r r e gul ari ns hapei nal l3t ype sofal l ogr af t s .Af t e r4we e ks ,t hec al l us e swe r e r e gul ari ns hapei nal lbutt heaut oc l ave dgr af t s .Af t e r2we e ks ,t hehe al i ngpr oc e s s e shadbe gun i nt hef r oz e nandpas t e ur i z e dgr af t s ,wi t ht her e ac hi ngappr oxi mat e l yt hes ames t age ,whi l ei nt he aut oc l ave dgr af t st he s epr oc e s s e swe r enots e e nandt hebonepar t i c l e swe r es ur r ounde dbyc onne c t i vet i s s uewi t houtanyc hange s .Af t e r4we e ks ,os t e oi nduc t i vepr oc e s s e swe r eve r ys t r ong,wi t ht he fir s ts i gnsofc ompl e t eboner e mode l i ngatt hebonee dge soft hede f e c ti npas t e ur i z e dandf r oz e n al l ogr af t s .Theos t e oi nduc t i veval ue soft he s e2t ype swe r eve r yhi gh ands i mi l ar .Aut oc l ave d al l ogr af t s ,ont heot he rhand,hadve r yl ow os t e oi nduc t i veval ue s ,ast he ywe r es t i l latt heve r y be gi nni ngoft hehe al i ngpr oc e s s .Hi s t omor phome t r i canal ys i sr e ve al e das i gni fic antdie r e nc ei n bot hne wl yf or me dos t e oi dt hi c kne s sandos t e obl as tnumbe rpe rμm ofbones ur f ac ei nal le xpe r i me nt algr oups( ＜ 0 . 0 0 5 ) .Val ue sofos t e oi dt hi c kne s sandos t e obl as tnumbe rwe r es i gni fic ant l yhi ghe r i n bot hf r oz e n and pas t e ur i z e d gr af t swhe nc ompar e d wi t ht heaut oc l ave d one s( ＜ 0 . 0 0 5 ) . Os t e oge ni cpr ope r t i e sofpas t e ur i z e dboneal l ogr af t swe r epr e s e r ve d,andt heal l ogr af t shavebe e n gr adual l yr e pl ac e dwi t hne wl yf or me dbone .Ass uc h,pas t e ur i z e dbonegr af t sf r om abonebank haveappr oxi mat e l yt hes amebi ol ogi cval i di t yasf r oz e ngr af t s ,whi l eaut oc l ave dgr af t si mpai rbone he al i ng. Ke ywor ds :b o n ea l l o g r a f t ,p a s t e u r i z a t i o n ,h e a l i n g ,r a b b i t s

o n et r a n s p l a n t a t i o ns u r g e r yh a sb e c o mei n t e g r a l t o o r t hope di c a nd t r a uma t ol ogy. Al t hough a u t o l o g o u sb o n eg r a f t sa r et h emo s tf a v o r a b l ef o rb o n e ［1 ］ t r a n s p l a n t a t i o np u r p o s e(g o l d e ns t a n d a r d) ,2 ,3 , t h e i rr e s o u r c ec a p a c i t yi sl i mi t e d .Co n s e q u e n t l y ,t r a n s -

B Re c e i v e dJ u l y3 ,2 0 0 1;a c c e p t e dDe c e mb e r1 7 ,2 0 0 1 . ＋3 ＋3 -5 -6 -1 Co r r e s p o n d i n ga u t h o r .Ph o n e: 8 5 5 1 6 5 1 1 4 3 ;Fa x: 8 5 1 5 1 4 3 ＠me Ema i l : Dr a g i c a . Bo b i n a c d r i . h r( D.Bo b i n a c )

Produced by The Berkeley Electronic Press, 2002

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Acta Medica Okayama, Vol. 56 [2002], Iss. 3, Art. 1

122 Zor i ci cetal .

p l a n t a t i o no fd e e p f r o z e na l l o g e n o u sb o n eh a sb e c o me ［］ p r e v a l e n ti nc l i n i c a lp r a c t i c e 1.Th i sp r o c e d u r eh a s s e v e r a lc o mp l i c a t i o nr i s k s ,i n c l u d i n gt r a n s p l a n tr e j e c t i o n h o s tv e r s u sg r a f tr e a c t i o n ,i n s t a b i l i t y ,a n di n f e c t i o n . Af t e rt h ea p p e a r a n c eo fHI Va n do t h e rr e t r o v i r a l i n f e c t i o n s ,t r a n s mi s s i o nr i s k sh a v eb e c o meo n eo ft h e mo s ti mp o r t a n tp r o b l e msi nd e e p f r o z e na l l o g e n o u sb o n e ［4 ］ t r a n s p l a n t a t i o ns u r g e r y ,5 .Si n c et h et i mewh e nt h e fi r s tc a s eo fHI Vt r a n s mi s s i o nb ya na l l o g e n i cb o n ewa s r e p o r t e d ,t h en u mb e ro fb o n ea l l o g r a f tt r a n s p l a n t a t i o n s ［6 ］. h a sd e c r e a s e d ［7 ］ I nt h eme a n t i me ,c e r t a i nme d i c a la s s o c i a t i o n s h a v ei n t r o d u c e dn e wme d i c a lg u i d e l i n e si no r d e rt or e d u c e ［8 ］.I t h er i s ko fHI Vt r a n s mi s s i o n na d d i t i o n ,n u me r o u s e x p e r i me n t sh a v eb e e nc a r r i e do u twi t ht h ei n t e n to f d e v e l o p i n ga na d e q u a t ep r o c e d u r ef o rv i r u si n a c t i v a t i o n ［9 ］.Ne wi t h o u tc o mp r o mi s i n gg r a f tb i o l o g i c a l v a l u e s v e r t h e l e s s ,t h e r ea r es t i l ls e r i o u sp r o b l e msi nb o n eb a n k i n g ［1 ］. ,1 0 Pr e v i o u ss t u d i e sh a v es h o wnt h a tHI Va n do t h e r r e t r o v i r u s e sa r en o ts e n s i t i v et ol o wt e mp e r a t u r e sa n d ］. t h e r e f o r ec a n n o tb ei n a c t i v a t e db yf r e e z i n g［5 ,1 1 Nu me r o u ss t u d i e sh a v ed e s c r i b e dt h eee c t sa n de c a c y o fv a r i o u sme t h o d sa n dp r o c e d u r e sd i r e c t e da tHI V d i s i n f e c t i o ns u c ha st h eu s eo fc h e mi c a la g e n t s ,g a mma ］. a n dd e l t ai r r a d i a t i o n ,a n dt h e r ma le n e r g y［1 2 ,1 3 Al t h o u g ht h e s eme t h o d sh a v eb e e ns u c c e s s f u li nv i r u s i n a c t i v a t i o n ,t h e yh a v eb e e ni n a p p l i c a b l et oa l l o g e n i cb o n e g r a f tt r a n s p l a n t a t i o n s ,a st h e ys e v e r e l ya l t e rt h ep h y s i c a l p r o p e r t i e sa n do s t e o g e n i cp o t e n t i a lo ft h eb o n ea l l o g r a f t ［1 ］. 4 Mo r e o v e r ,a l lg r o wt hf a c t o r s( TGFβ,BMPs ,I GFs e t c . )o ft h eb o n ema t r i xr e s p o n s i b l ef o ro s t e o g e n i cp r o p ［］ e r t i e sa r ei n a c t i v a t e da t8 0° C 1 5.Ass u c h ,a u t o c l a v e d b o n ea l l o g r a f t sh a v eb e e nc o n s i d e r e dt ob ei n a p p r o p r i a t e f o rb o n et r a n s p l a n ts u r g e r yd u et ot h ea s s o c i a t e dl o s so f ［1 ］.Ma p h y s i c a la n db i o l o g i c a lp r o p e r t i e s 6 n i f e s t y ,a l l o g r a f td i s i n f e c t i o nf r o mv i r u s e ss h o u l db ep e r f o r me db y h e a t i n ga tl o we rt e mp e r a t u r e s ,a si ti sd o n ewi t hb l o o d -6 ［1 ］. d e r i v a t e s( 5 0 0° C) 7 -6 I th a sr e c e n t l yb e e ns h o wnt h a th e a t i n ga t5 0 8° C ［1 ］h i n a c t i v a t e sHI V.Sp i r ee ta l . 1 a v es h o wnt h a ta f t e r 3 0mi no fi n c u b a t i o ni nawa t e r b a t h ,v i r a ls a mp l ei n a c t i v a t i o ni s0 ,4 0 ,6 3 ,a n d1 0 0 a t3 7° C,4 2° C,4 8 ° C,a n d5 6° C,r e s p e c t i v e l y .Th e yh a v ea l s oo b s e r v e d t h a tHI Vr e v e r s et r a n s c r i p t a s ea c t i v i t yr e a c h e sz e r oa f t e r ］.Th 2 0mi no fi n c u b a t i o na t5 6° C［1 8 i si mp o r t a n t fi n d i n gi n s p i r e du st oe l u c i d a t et h eo s t e o g e n i cp r o p e r t i e s

http://escholarship.lib.okayama-u.ac.jp/amo/vol56/iss3/1

Act aMed.Okay ama Vol .56,No.3

o fp r e v i o u s l yp a s t e u r i z e db o n eg r a f t( 5 6° Cf o r2 0mi n ) wi t hr e g a r dt oi t su s ei nb o n et r a n s p l a n t a t i o n .Th e r e f o r e , o u ra i mi nt h ep r e s e n ts t u d ywa st oe s t a b l i s ht h ee c a c y o fp a s t e u r i z e db o n ea l l o g r a f t si nt h eh e a l i n gp r o c e s sf o r b o n ed e f e c t si nr a b b i t s .Au t o c l a v e da n df r o z e na l l o g r a f t s we r eu s e da sc o n t r o la l l o g r a f t so fk n o wnb i o l o g i c a lv a l i d i t y .Du et oi t sg o o do s t e o g e n i cp o t e n t i a l ,t h er a b b i ti st h e b e s te x p e r i me n t a la n i ma l f o rs u c has t u d y .Mo r e o v e r ,t h e s u r g i c a lp r o c e d u r ep e r f o r me do nt h er a b b i t su l n ai s s i mp l ea n dr e a s o n a b l yr e p r o d u c i b l e ,a st h es k e l e t a l d i me n s i o n sa r ea p p r o p r i a t e .Th ep r e s e n tr e s u l t ss u g g e s tt h a t t h et e mp e r a t u r ea p p l i e d ,q u i t es uc i e n tt od i s i n f e c tt h e b o n ea l l o g r a f to fHI V, d o e sn o ti n t e r f e r ewi t ht h e o s t e o g e n i cp r o p e r t i e so fap a s t e u r i z e db o n eg r a f t .

Mat e r i al sandMe t hods Fu l l yg r o wnma l er a b b i t s ,we i g h t3 4 k g ,we r eh o u s e di ns t a n d a r dc a g e sa tc o n t r o l l e dt e mp e r a t u r e( 2 5° C)wi t hf r e ea c c e s st owa t e ra n ds t a n d a r dp e l l e t f o o d( MK Mo s l a v k a ,Ku t i n a ,Cr o a t i a ) .Si x t ya n i ma l s we r ed i v i d e di n t o3t e s tg r o u p sa c c o r d i n gt ot h ek i n d so f v a r i o u s l yp r e p a r e da l l o g r a f t s .Te na n i ma l si ne a c ht e s t g r o u pwe r es a c r i fi c e da f t e r2we e k sa n d4we e k s ,r e s p e c t i v e l y .Fi v emo r ea n i ma l swe r eu s e da sb o n ed o n o r sf o r p r e p a r i n gb o n et i s s u ei mp l a n t s .Th ea n i ma le x p e r i me n t wa sc a r r i e do u ti na c c o r d a n c ewi t h Me d i c a lSc h o o l Po l i c i e sa n dGu i d e l i n e sf o rt h eCa r ea n dUs eo fLa b o r a t o r yAn i ma l s .Al leo r t swe r ema d et omi n i mi z ea n i ma l s ue r i n g . Th ef e mo r a ,t i b i a ,a n dc o a x e s we r ei s o l a t e df r o mb o n ed o n o ra n i ma l su n d e ra s e p t i c c o n d i t i o n s .Th ep e r i o s t e u ma n da r t i c u l a rc a r t i l a g ewe r e c a r e f u l l yr e mo v e df r o mt h eb o n es u r f a c e .Th e r e a f t e r ,t h e b o n e swe r ec u tu pi n t os ma l lf r a g me n t swi t hLu e rs r o n g e u r( p a r t i c l e sc a .1mm ) .Su c hf r a g me n t e db o n e s we r ek e p ts t e r i l ea n df r o z e ni nt h eb o n eb a n ka t−7 0° C f o r6we e k s . Fr o z e na l l o g e n o u sb o n eg r a f t s :c o r t i c o c a n c e l l o u sb o n e g r a f t sf r o mt h eb o n eb a n kwe r et h a we di ns a l i n es o l u t i o n a tr o o mt e mp e r a t u r ei mme d i a t e yp r i o rt ot h e i ru s e . Pa s t e u r i z e da l l o g e n o u sb o n eg r a f t s :c o r t i c o c a n c e l l o u s b o n eg r a f t sf r o mt h eb o n eb a n ks t o r e di ns t e r i l et e s tt u b e s we r et h a we da t5 6° Cf o r2 0mi ni nawa t e r b a t h . Au t o c l a v e da l l o g e n o u sb o n eg r a f t s :c o r t i c o c a n c e l l o u s b o n eg r a f t sf r o mt h eb o n eb a n k ,s t o r e di ns t e r i l et e s t t u b e swe r es t e r i l i z e da t1 2 0° Cf o r2 0mi n .

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June2002

Ra b b i t swe r ea n e s t h e t i z e dwi t hi n t r a / v e n o u si n j e c t i o no fMi d a z o l a m( 0 . 2 5mgk g )a n dFa n t a n y l /k ( 0 . 0 0 5mg g )v i at h ema r g i n a le a rv e i n .Th emi d d l ep a r t o ft h eu n l ass h a f twa se x p o s e de x t r a p e r i o s t e a l l yu n d e r t o u r n i q u e ti s c h e mi aa n da s e p t i cc o n d i t i o n s .Th ep e r i o s t e u mwa si n c i s e dc i r c u mf e r e n t i a l l ya n dr e mo v e d .Th e r e a f t e r ,ab o n ed e f e c twa sc r e a t e dt h a twa sa sc l o s et ot h e s a mel e n g t ha sp o s s i b l ea st h ed i a me t e ro ft h eb o n es h a f t a tt h i sp o i n t( a p p r o x i ma t e l y4mm) .A d e n t a ld r i l lwa s u s e df o rt h i sp u r p o s et oa v o i dt h e r ma l d a ma g et ot h eb o n e e n d st h a tc a nb ec a u s e db yas a w.Th eb o n ed e f e c twa s c o mp l e t e l yfi l l e dwi t hp r e v i o u s l yp r e p a r e db o n ea l l o g r a f t t a k e nf r o mt h eb o n eb a n k .Th es o f tt i s s u e swe r er e s t o r e d , a n ds k i nwa sc a r e f u l l ys u t u r e d .On eo fu s( B. L. )p e r f o r me da l lt h es u r g i c a lp r o c e d u r e si nt h i ss t u d y ,a c h i e v i n g wh a twa sc o n s i d e r e dt ob eau n i f o r mt e c h n i q u e .Af t e r s u r g e r y ,a n i ma l sr e c e i v e dp e n i c i l l i n( 7 5 0 , 0 0 0I Ui n t r a mu s c u l a r l y ) .Noi n t e r n a lo re x t e r n a lfi x a t i o nwa su s e d ,s i n c e t h ewe i g h t b e a r i n gl o a di sn o tt r a n s mi t t e dt h r o u g ht h eu l n a i nr a b b i t s .Nos i g no fi n f e c t i o no rl o c o mo t i o nd i s t u r b a n c e wa so b s e r v e dd u r i n gt h ee x p e r i me n t a lp e r i o d . Att h ee n do ft h ee x p e r i me n t a lp e r i o d ,t h ea n i ma l s we r es a c r i fi c e db ya no v e r d o s eo ft h ep e n t o b a r b i t a l .A s e g me n to ft h eu l n ac o n t a i n i n gt h ee x p e r i me n t a lg r a f twa s e x c i s e de nb l o c ,i n c l u d i n gp o r t i o n so fn o r ma lu l n ap r o x i ma la n dd i s t a lt ot h eg r a f ta l o n gwi t hag e n e r o u si n v e s t me n to fs o f tt i s s u e . Ro e n t g e n o g r a mswe r ema d eo fa l l a n i ma l s2d a y sa f t e r s u r g e r yt oc o n fi r mg r a f tp l a c e me n t .Af t e rt h ea n i ma l s we r es a c r i fi c e d ,r o e n t g e n o g r a mswe r eo b t a i n e da g a i nt o c h e c kt h ea p p e a r a n c eo ft h eb o n ed e f e c th e a l i n g .Ho we v e r ,t h er o e n t g e n o g r a msp r o v e dt ob eo fl i t t l ev a l u ei n a s s e s s i n go ft h eee c t i v e n e s so ft h ev a r i o u st y p e so f g r a f t sa n dwe r et h e r e f o r en o tu s e di nt h efi n a le v a l u a t i o n . Ti s s u es p e c i me n swe r e fi x e di n4 p a r a f o r ma l d e h y d e ,d e h y d r a t e di ne t h a n o l , c l e a r e di nx y l e n e ,a n de mb e d d e du n d e c a l c i fi e di nme t h y l me t h a c r y l a t e .Se r i a lt i s s u es l i c e s ,7μmt h i c k ,we r ec u t p a r a l l e lt ot h eb o n ea x i s ,wi t haLe i c aRM 2 1 5 5mi c r o t o mee q u i p p e dwi t hat u n g s t e nc a r b i d ek n i f ea n dp l a c e d o n t os i l i n a t e dg l a s ss l i d e s( Si l a n e Pr e ps l i d e s ,Si g ma Ch e mi c a lCo . ,St .Lo u i s ,MO,USA) ,d r i e da t3 7° Ci n at h e r mo s t a ta n ds t a i n e dwi t hh e ma t o x y l i n e-e o s i n ea n d t o l u i d i n eb l u e . Ti s s u emo r p h o l o g y a n dc e l l u l a rme c h a n i s mso ft h eh e a l i n gp r o c e s si n d u c e db y i mp l a n t a t i o no fd ie r e n ta l l o g r a f t swe r ea n a l y z e d .Du r i n g t h ec o u r s eo ft h i ss t u d y ,i tb e c a mea p p a r e n tt h a tt h e r e


Pas t eur i z edBoneAl l ogr af t si nt heHeal i ngPr oces s 123

we r e5k e yh i s t o l o g i c a lf e a t u r e si nt h eh e a l i n gp r o c e s st h a t mu s tb ec o n s i d e r e da n di n c l u d e di na ne v a l u a t i o no ft h e e x p e r i me n t :t h ea p p e a r a n c eo ft h ea l l o g r a f tp a r t i c l e s ,t h e r e s t o r a t i o no fc o n t i n u i t yb e t we e nb o n ee d g e s ,t h ec a l l u s , t h eb o n ee d g e s ,a n dt h eq u a l i t yo fn e wl yf o r me db o n e . Ev e nt h o u g hh i s t o l o g yc l e a r l yr e v e a l e dd ie r e n c e si n t i s s u er e a c t i o na f t e rd ie r e n ta l l o g r a f tt r a n s p l a n t a t i o n ,i t wa sn o tp o s s i b l et oc o mp a r et h es t a t u so fd ie r e n t s p e c i me n so nt h eb a s i so fd e s c r i p t i v eh i s t o l o g y .Th e r e f o r e ,ak i n do fs e mi q u a n t i t a t i v es y s t e mwa sa p p l i e dwi t h n u me r i c a lr a t e sf o re a c ho ft h ek e ymo r p h o l o g i c a ls t r u c t u r e sl i s t e da b o v e .As i mi l a rme t h o do fn u me r i c a le v a l u a ［3 ］ t i o nwa sd e s c r i b e db yHe i p l e ,b u ta sh i se x p e r i me n t d ie r e df r o mo u r s ,wemo d i fi e dt h ep a r a me t e r si n c l u d e d i nt h ee v a l u a t i o n .I no r d e rt oo b t a i no b j e c t i v ea n a l y s i s , t h ee v a l u a t i o nwa sc a r r i e do u tb y2i n d e p e n d e n to b s e r v e r s ( S. Z. ,D. B. ) .Th ema x i mu mq u a l i t yo ft h eb o n eh e a l i n g p r o c e s swa se v a l u a t e db yas c o r eo f1 3 .Th ea r b i t r a rs c a l e u s e di no u rn u me r i c a le v a l u a t i o nwa s : 1 .t h ea p p e a r a n c eo ft h ea l l o g r a f t :p a r t i c l e so fa l l o g r a f t a r ei n t a c t( 0 ) ,n e wc e l l u l a ra c t i v i t yo nt h es u r f a c eo f b o n ep a r t i c l e s( 1 ) ,a l l o g r a f ti sf u l l yi n c o r p o r a t e d( 2 ) . 2 .r e s t o r a t i o no fc o n t i n u i t yb e t we e nb o n ee d g e s :n o( 0 ) , y e s( 1 ) . 3 .c a l l u s :n oc a l l u s( 0 ) ,fi b r o u sc a l l u s( 1 ) ,c h o n d r a l c a l l u s ( 2 ) ,b o n ec a l l u s( 3 ) . 4 .b o n ee d g e s :n e c r o t i c( 0 ) ,r e s o r b e d( 1 ) ,n e wb o n e a p p o s i t i o n( 2 ) . 5 .q u a l i t yo fn e wb o n e :i n i t i a la p p o s i t i o no fn e wb o n eo n t h et r a n s p l a n ts u r f a c e( 1 ) ,s i n g l en o d u l e so fwo v e n b o n e( 2 ) ,t r a b e c u l a so fwo v e nb o n e( 3 ) ,b r i d g i n g t r a b e c u l a s( 4 ) ,l a me l l a rb o n et r a b e c u l a s( 5 ) . Hi s t o mo r p h o me t r i ca n a l y s i swa so b t a i n e db ya ni ma g e a n a l y z e r( Sf o r m,VAMS,Za g r e b ,Cr o a t i a )u s i n gt h e Ol y mp u sBX5 0mi c r o s c o p e( Ol y mp u sOp t i c a lCo .Lt d . , To k y o ,J a p a n ) .Os t e o i dt h i c k n e s swa sme a s u r e d( O.Th . ( μm) ) ,o nt h es u r f a c eo ft h eb o n eg r a f tp a r t i c l e sa n do n n e wl yf o r me db o n e .Os t e o i dwa si d e n t i fi e di nt o l u i d i nb l u e s t a i n e dt i s s u es e c t i o n sa sap a l eb l u el a y e ro v e r l y i n gt h e n e wl yf o r me db o n eo rg r a f tp a r t i c l e s ,a n dc o v e r e dwi t ha l a y e ro fc u b o i d a lo s t e o b l a s t so ni t ss u r f a c e .Th et h i c k n e s s o ft h eo s t e o i dwa sme a s u r e da t4e q u a l l yd i s t a n tp o i n t s ［1 ］. a n de x p r e s s e da si t sme a nv a l u e 9 Os t e o b l a s tn u mb e rwa se x p r e s s e da st h er a t i oo f o s t e o b l a s tn u mb e r( NbOb )p e rb o n es u r f a c e( Tb .Pm. ［1 ］ ( μm) ) 9 . Os t e o b l a s t swe r ei d e n t i fi e da sc u b o i d a l b l u e s t a i n e dc e l l swi t hab a s a ln u c l e u s . Me a s u r e me n t swe r et a k e no n5r a n d o ml yc h o s e n

3




p o we rfi e l d sa l o n gt h ee n t i r et i s s u es e c t i o n ,i n c l u d i n g6 s e c t i o np e rs p e c i me n .Al lh i s t o mo r p h o me t r i cd a t aa r e g i v e na sme a nv a l u e s± s t a n d a r de r r o ro ft h eme a n . St a t i s t i ca n a l y s i swa sp e r f o r me du s i n gt h eSt u d e n tst t e s t f o ri mp a i r e dd a t a( St a t i s t i c af o rwi n d o ws )a n dPv a l u e s＜ 0 . 0 5we r ec o n s i d e r e ds t a t i s t i c a l l ys i g n i fi c a n t .

Re s ul t s Th ea p p e a r a n c eo f t h ei mp l a n t e dg r a f ta t2d a y sa f t e rs u r g e r yi ss h o wni n Fi g .1 A.Twowe e k sa f t e rs u r g e r y ,a l l3t y p e so f a l l o g r a f t ss h o we da ni r r e g u l a r l ys h a p e dc a l l u so nr a d i o g r a p h i ce x a mi n a t i o n( d a t an o ts h o wn ) .Fo u rwe e k sa f t e r s u r g e r y ,mo s tp a s t e u r i z e da n df r o z e nt y p e so fg r a f t sh a d ar e g u l a r l ys h a p e dc a l l u s ,wh i l et h ec a l l u so fa u t o c l a v e d g r a f t swa ss t i l li r r e g u l a r l ys h a p e d( Fi g .1 B,1 C,1 D) . Al l o g r a f tp a r t i c l e swe r ei d e n t i fi e db yt h e i ri r r e g u l a rs h a p e , s h a r pa n di r r e r g u l a re d g e s ,i r r e g u l a r l yo r i e n t e dl a me l l a s , a n de mp t yo s t e o c y t el a c u n a s( Fi g .2 A,2 B,2 C) . Fr o z e na n dp a s t e u r i z e da l l o g r a f tb o n ep a r t i c l e swe r e s u r r o u n d e db yd e n s ec o n n e c t i v et i s s u ewi t hc e l li n fi l t r a t e , b l o o dv e s s e l s ,a n db l o o dc e l l s( Fi g .2 B,2 A) .Ac c u mu l a t e do s t e o b l a s t swe r es e e no nt h es u r f a c eo ft h e g r a f tp a r t i c l e s .Sc a t t e r e dn e wl yf o r me db o n ewi t hp a l i -

s a d e so fo s t e o b l a s t swi t hu n d e r l y i n go s t e o i dwa sf o u n d wi t h i nab o n ed e f e c t .Th ec o r t i c a lb o n es h o we dt h ei n i t i a l e n d o s t e a la n dp e r i o s t e a lr e a c t i o nwi t hc e l l u l a ra c c u mu l a t i o n .Os t e o b l a s t i ca c t i v i t yo ft h eh o s tb o n ewa sa l s of o u n d o nt h ee d g e so ft h eb o n ed e f e c t( Fi g .2 D,2 E) . I nc o n t r a s t ,o s t e o b l a s t swe r er a r e l yf o u n do nt h e s u r f a c e so fb o n ep a r t i c l e si nt h ea u t o c l a v e da l l o g r a f t s . Th eb o n ep a r t i c l e ss e e me dt ob ep r e s e r v e d ,a n ds o meh a d e v e nb e e nr e s o r b e d( Fi g .2 C) .Nor e s t i n gl i n i n gc e l l s we r ef o u n do nt h es u r f a c eo fg r a f tp a r t i c l e s .Th eb o n e e d g e swe r ec l e a rwi t hn os i g no fc e l la c t i v i t y( Fi g .2 F) . On l yawe a ke n d o s t e a la n dp e r i o s t e a lc e l l u l a rr e a c t i o n c o u l db es e e na r o u n dt h eh o s tc o r t i c a lb o n e . I n p a s t e u r i z e da n df r o z e na l l o g r a f t s ,mo s to ft h ep a r t i c l e s we r er e s o r b e da n ds u b s t i t u t e dwi t hn e wl yf o r me db o n eb y t h ep r o c e s so fr e mo d e l i n g .Ne wl yf o r me dt r a b e c u l a so f wo v e nb o n eo re v e nl a me l l a rb o n eh a db r i d g e do v e r o p p o s i t ee d g e so ft h eb o n ed e f e c t( Fi g .3 D,3 E) .Th e r e ma i n i n ga l l o g r a f tp a r t i c l e swe r ei n c o r p o r a t e di n t ot h e n e wl yf o r me db o n et i s s u e .Av e r ys t r o n gp e r i o s t e a la n d e n d o s t e a lr e a c t i o nwi t hb o n er e mo d e l i n gc o u l db es e e ni n t h eh o s tc o r t i c a lb o n e .Th ec a l l u so fc a r t i l a g i n o u st i s s u e wi t hp e r i p h e r a lo s s i fi c a t i o nwa swe l l d e v e l o p e d .La r g e a mo u n t so fo s t e o i da n dn u me r o u so s t e o b l a s t swe r ep l a c e d o nt h es u r f a c e so fr e ma i n i n gp a r t i c l e sa n dn e wl yf o r me d

Fi g.1 Ro e n t g e n o g r a mss h o wt h ea p p e a r a n c eo ft h eb o n ea l l o g r a f t sa t2d a y sa f t e rs u r g e r y( A) ,t h e np a s t e u r i z e d( B) ,f r o z e n( C)a n d a u t o c l a v e d( D)g r a f t sa t4we e k sa f t e ri mp l a n t a t i o n .


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June2002


Fi g.2 Th er e s u l t so ft i s s u ea n dc e l l u l a re v e n t sd u r i n gt h eh e a l i n gp r o c e s si n d u c e db y3t y p e so fb o n ea l l o g r a f t sa t2we e k sa f t e r t r a n s p l a n t a t i o n( t o l u i d i nb l u e ,2 0 0×) .St r o n gc e l l u l a ri n fi l t r a t i o n( CI )a r o u n dp a s t e u r i z e db o n ea l l o g r a f tp a r t i c l e s( p b p ) ,n u me r o u so s t e o b l a s t s ( O)f o r mi n gn e wb o n e( n e wb )( A) ;c e l l u l a ri n fi l t r a t i o n( CI )a r o u n dt h ef r o z e nb o n ea l l o g r a f tp a r t i c l e s( f o p ) ,n u me r o u so s t e o b l a s t a( O)f o r mi n g n e wwo v e nb o n e( n e wb )( B) ;c e l l u l a ri n fi l t r a t i o n( CI )a r o u n dt h ea u t o c l a v e db o n ea l l o g r a f tp a r t i c l e s( a b p )( C) ;p a s t e u r i z e da l l o g r a f t t h eh o s t ↓)s b o n ee d g e( h o wss t r o n g e rc e l l u l a ra c t i v i t ywi t ht h eb e g i n n i n go fr e mo d e l i n g( D) ;f r o z e na l l o g r a f t c e l l u l a ra c t i v i t yo nt h eh o s tb o n ee d g e ↓)wi ↓)s ( t ht h eb e g i n n i n go fr e mo d e l i n g ,s t r o n gp e r i o s t e a lr e a c t i o n( p r )( E) ;a u t o c l a v e da l l o g r a f t t h ee d g eo ft h eh o s tb o n e( h o wsn ov i s i b l e c h a n g e s ,we a kp e r i o s t e a l( p r )a n de n d o s t e a lr e a c t i o n s( e r )( F) .

b o n et r a b e c u l a s( Fi g .3 A,3 B) . I na u t o c l a v e db o n eg r a f t s , mo s to ft h ep a r t i c l e s r e ma i n e du n c h a n g e d( Fi g .3 C) .He r ea n dt h e r e ,f o r ma t i o no fn e wb o n ewi t ha d j a c e n to s t e o b l a s t sc o u l db es e e n . Ons o mep a r t so ft h eb o n ee d g ean e wb o n ea p p o s i t i o n c o u l db es e e n ,b u tt h ec o n n e c t i o nwi t ht h en e wl yf o r me d r e g e n e r a t i v eb o n ei nt h ed e f e c th a dn o ty e tb e e ne s t a b l i s h e d( Fi g .3 F) . Nu me r i c a le v a l u a t i o n o ft h emo r p h o l o g i c a ls t r u c t u r e si n v o l v e di nt h eb o n e h e a l i n gp r o c e s ss h o we da l mo s tt h es a men u me r i c a lv a l u e s f o rp a s t e u r i z e da n df r o z e nb o n ea l l o g r a f t sa t2we e k sa n d 4we e k sa f t e rt r a n s p l a n t a t i o n .I nc o n t r a s t ,t h en u me r i c a l


v a l u e sf o ra u t o c l a v e db o n ea l l o g r a f t swe r ema r k e d l yl o we r i nb o t he x p e r i me n t a lp e r i o d s( Ta b l e1 ) . Th eme a n ± o s t e o i dt h i c k n e s s( O. Th )i nf r o z e n( 4 . 4 5 0 . 0 6μm)a n d p a s t e u r i z e d( 3 . 5 1± 0 . 0 6μm)g r a f t sd ie r e ds i g n i fi c a n t l y f r o mt h a ti na u t o c l a v e dg r a f t s( 1 . 2 8± 0 . 0 4μm)( P＜ 0 . 0 0 5 ) . Co r r e s p o n d i n g l y ,t h e r ewa sas i g n i fi c a n td ie r e n c ei n ± t h eo s t e o b l a s tn u mb e rp e rμm i nf r o z e n( 0 . 1 6 0 . 0 1 6 ) a n dp a s t e u r i z e d( 0 . 1 9± 0 . 0 0 2 )g r a f t swh e nc o mp a r e dwi t h a u t o c l a v e dg r a f t s( 0 . 0 7 5± 0 . 0 0 1 )( P＜ 0 . 0 0 5 ) .

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Fi g.3 Th et i s s u ea n dc e l l u l a re v e n t sd u r i n gt h eh e a l i n gp r o c e s si n d u c e db y3t y p e so fb o n ea l l o g r a f t sa t4we e k sa f t e rt r a n s p l a n t a t i o n ( t o l u i d i nb l u e ,2 0 0×) .Pa s t e u r i z e da l l o g r a f t p a r t i c l e sr e s i d u e s( p b p ) ,n e wb o n e( n e wb )( A) ;f r o z e na l l o g r a f t p a r t i c l e s( f o p )a r ea l mo s t c o mp l e t e l yr e s o r b e d ,n e wl yf o r me dwo v e nb o n e( n e wb )( B) ;a u t o c l a v e da l l o g r a f t b o n ep a r t i c l e sa r es t i l lu n r e s o r b e d( a b p )( C) ;p a s t e u r i z e d ↓) a l l o g r a f t a d v a n c e dr e mo d e l i n go nt h eh o s tb o n ee d g e( ,c o n t i n u i t ywi t hn e wl yf o r me db o n e( n e wb )i nd e f e c t( D) ;f r o z e na l l o g r a f t a d v a n c e d ↓) r e mo d e l i n go nt h eh o s tb o n ee d g e( ,c o n t i n u i t ywi t hn e wl yf o r me dwo v e nb o n e( n e wb )i nd e f e c t( E) ;a u t o c l a v e da l l o g r a f t t h eb e g i n n i n go f ↓) r e mo d e l i n go nt h eb o n ee d g e( ,wi t h o u tc o n t i n u i t ywi t hn e wl yf o r me db o n e( n e wb )i nd e f e c t( F) .

Ta b l e1

Th er e s u l t so fn u me r i c a lr a t i n go ft h eh e a l i n gp r o c e s si n d u c e db yi mp l a n a t i o no fd ie r e n t i a l l yp r e p a r e db o n ea l l o g r a f t s

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2we e k sa f t e rt r a n s p l a n t a t i o n

4we e k sa f t e rt r a n s p l a n t a t i o n

Au t o c l a v e da l l o g r a f t s Fr o z e na l l o g r a f t s Pa s t e u r i z e da l l o g r a f t s

4 . 4± 0 . 2 4 1 8 . 4± 0 . 6 7 7 . 2± 0 . 3 4

7 . 2± 8 . 0 4 1 2 . 4± 0 . 7 8 1 1 . 3± 0 . 9 2

Th ea s s e s s me n twa sp e r f o r me db y2i n d e p e n d e n to b s e r v e r s .Th er e s u l t sa r ee x p r e s s e da sme a n± s t a n d a r de r r o ro ft h eme a n(s t a t i s t i c a l l y s i g n i fi c a n td ie r e n c ev s.a u t o c l a v e da l l o g r a f t s ,P＜ 0 . 0 0 5 ,St u d e n tst t e s t ) .


6


June2002


Di s c us s i on Be c a u s et h e r ei sah i g hr i s ko fHI Va n do t h e rv i r a l ［6 t r a n s mi s s i o nt h r o u g hb o n ea l l o g r a f tt r a n s p l a n t a t i o n ,7 , ］,i 8 ,1 0 ti sn e c e s s a r yt od e v e l o pa na d e q u a t ep r o c e d u r e t h a tc a na c h i e v ev i r a li n a c t i v a t i o n ,b u td o e sn o tc o mp r o mi s et h eb i o l o g i c a lo s t e o i n d u c t i v ep r o p e r t i e so fb o n e ［2 ］.I a l l o g r a f t s ,1 6 th a sb e e ns h o wnt h a tp a s t e u r i z a t i o n ］a ( 5 6° Cf o r2 0mi n )s u c c e s s f u l l ye l i mi n a t e sHI V［1 7 n d , i mp o r t a n t l y ,t h a tt h et h e r ma le n e r g yo fp a s t e u r i z a t i o n d o e sn o tae c tt h ea c t i v i t yo ft h eg r o wt hf a c t o r si nt h e ［］ b o n ema t r i xr e s p o n s i b l ef o ro s t e o i n d u c t i o n 1 6.I n a c c o r d a n c ewi t ht h e s er e s u l t s ,o u rs t u d yh a sd e mo n s t r a t e dt h a tt r a n s p l a n t a t i o no fp a s t e u r i z e db o n ea l l o g r a f t s i n d u c e st h es a mer a t eo fb o n ef o r ma t i o nwi t h i nt h eb o n e d e f e c ta sf r o z e na l l o g r a f t s .Be c a u s ef r o z e nb o n ea l l o g r a f t s ［1 ］ p e r f o r ms a t i s f a c t o r yi nb o n et r a n s p l a n t a t i o ns u r g e r y 3 wec a nc o n c l u d et h a tp a s t e u r i z e db o n ea l l o g r a f t sma yb e u s e df o rt h es a mep u r p o s e .Att h es a met i me ,p a s t e u r i z a t i o nd o e sp r e v e n tv i r a lt r a n s mi s s i o n ,wh i c hi sc r u c i a lt o t o d a yst r a n s p l a n t a t i o ns u r g e r i e s .Th i ss t u d yi st h efi r s t r e p o r to ft h ee c a c yo fp a s t e u r i z e db o n ea l l o g r a f t si n i n d u c i n gb o n ef o r ma t i o ni ne x p e r i me n t a lb o n ed e f e c t si n r a b b i t s . Du r i n gh i s t o l o g i c a la n a l y s i s ,i tb e c a mea p p a r e n tt h a t t h e r ea r ec e r t a i nh i s t o l o g i c a l f e a t u r e sa n ds t r u c t u r e swi t h i n b o n ed e f e c t st h a tmu s tb ec o n s i d e r e da n di n c l u d e di n e v a l u a t i n gt h es u c c e s so fb o n ea l l o g r a f t s ,a st h e ys t r o n g l y r e fl e c tt h ei n t e n s i t yo ft h eb o n e i n d u c t i o np r o c e s swi t h i n t h es i t eo fa l l o g r a f tt r a n s p l a n t a t i o n .Th efi r s th i s t o l o g i c a l s t r u c t u r eo fi n t e r e s twa st h ep a r t i c l e so ft h et r a n s p l a n t e d a l l o g r a f t s .I twa sf o u n dt h a tt h ema j o r i t yo fp a r t i c l e si n t h ep a s t e u r i z e da l l o g r a f t s ,a swi t ht h ef r o z e na l l o g r a f t s , h a db e e na l mo s tc o mp l e t e l yr e s o r b e da n di n c o r p o r a t e d wi t h i n4we e k so ft r a n s p l a n t a t i o n ,wh i l emo s to ft h e a u t o c l a v e db o n ep a r t i c l e swe r es t i l li n t a c ta n du n r e s o r b e d . Th e s er e s u l t ss u g g e s tt h a tp a s t e u r i z e db o n ea l l o g r a f t p a r t i c l e sa c tr a p i d l yi nt h eb o n e i n d u c t i o np r o c e s s ,a sd o f r o z e no n e s .I nt h e s ea l l o g r a f t s ,i n c o r p o r a t i o no fp a r t i c l e s wa sf o u n dt ob ec o n n e c t e dwi t hn e wb o n ef o r ma t i o n , wh i c hwa st h es e c o n ds t r u c t u r eo fi n t e r e s t .Ne wl yf o r me d b o n ewa sf o u n di nd ie r e n ts t a g e so fd e v e l o p me n t .Mo s t o ft h i sn e wl yf o r me db o n ewa swo v e nb o n e ,a c c o r d i n gt o t h ec l a s s i fi c a t i o no fb o n et i s s u e ,a n dr a r e l yl a me l l a rb o n e t i s s u e .I nt h ec a s eo fa u t o c l a v e db o n ea l l o g r a f t s ,h o we v e r ,n e wl yf o r me dwo v e nb o n ewa so b s e r v e do n l y r a r e l y .Wi t h i n4we e k s ,n e wl yf o r me db o n et i s s u ei n d u c e db yp a s t e u r i z e da n df r o z e na l l o g r a f t sh a de v e n


b r i d g e do v e rs o mep a r t so ft h eb o n ed e f e c t .Th en e wl y f o r me db o n et i s s u ee s t a b l i s h e dc o n t i n u i t ywi t hh o s t b o n e e d g e s ,a n di nt h e s ec a s e st h eb o n e i n d u c t i o np r o c e s s e s we r emo r ep r o n o u n c e d . I na d d i t i o n ,h i s t o l o g yr e v e a l e dn e we n d o s t e a l ,p e r i o s t e a l ,a n dp e r i v a s c u l a rb o n ef o r ma t i o ni nb o t hp a s t e u r i z e d a n df r o z e nb o n eg r a f t s , wi t h mi n o r mo r p h o l o g i c d ie r e n c e sa f t e r2we e k sa n ds t r o n g e rr e a c t i o n sa f t e r4 we e k s . Ev e nt h o u g hh i s t o l o g yc l e a r l yr e v e a l e dd ie r e n c e si n t i s s u er e a c t i o na f t e rt r a n s p l a n t a t i o no ft h ed ie r e n ta l l o g r a f t s ,i nwa sn o tp o s s i b l et oc o mp a r et h es t a t u so f d ie r e n ts p e c i me n so nt h eb a s i so fd e s c r i p t i v eh i s t o l o g y . Toe s t a b l i s ht h ed ie r e n c e sb e t we e nb o n ei n d u c t i o n s i n d u c e db yd ie r e n tt y p e so fa l l o g r a f tt r a n s p l a n t a t i o nwi t h mo r ea c c u r a c y ,ap r o p o s e ds e mi q u a n t i t a t i v es y s t e mwa s a p p l i e dwi t hn u me r i c a lr a t e sf o re a c ho ft h ek e ymo r p h o l o g i c a ls t r u c t u r e sd e s c r i b e da b o v e .He i p l eh a sd e ［3 ］ s c r i b e das i mi l a rme t h o do fn u me r i c a le v a l u a t i o n ,b u t a sh i se x p e r i me n td ie r e df r o mo u r s ,wemo d i fi e dt h e p a r a me t e r si n c l u d e di nt h ee v a l u a t i o na n dd e s i g n e dan e w o r i g i n a ls c a l es y s t e m. Th en u me r i c a lr a t i n gs c a l eu s e df o rq u a n t i fi c a t i o no f h i s t o l o g i c a ls t r u c t u r e si mp o r t a n tt ot h eb o n e i n d u c t i o n p r o c e s s ,d e s i g n e de x p r e s s l yf o rt h i ss t u d y .Th ep r o p o s e d s c a l es y s t e mp r o v e dt ob eu s e f u la n dr e v e a l e dt h es u p e r i o r i t yo ft h ef r o z e na l l o g r a f t si nb o n ei n d u c t i o n .Th e n u me r i c a lv a l u eo fp a s t e u r i z e da l l o g r a f t sd i dn o td ie r s i g n i fi c a n t l y f r o m t h o s eo ft h ef r o z e n a l l o g r a f t s , c o n fi r mi n ga g a i nt h a tp a s t e u r i z e db o n ea l l o g r a f t sd on o t l o s et h e i ro s t e o i n d u c t i v ep r o p e r t i e s . Hi s t o mo r p h o me t r i cp a r a me t e r s-o s t e o i dt h i c k n e s sa n d o s t e o b l a s tn u mb e rd i dn o td ie rs i g n i fi c a n t l yb e t we e n f r o z e na n dp a s t e u r i z e dg r o u p sb u twe r es i g n i fi c a n t l y h i g h e ri nr e l a t i o nt ot h ea u t o c l a v e dg r o u p .Th eh i g h e r o s t e o b l a s tn u mb e r si nt h e s e2g r o u p sc l e a r l yi n d i c a t e st h a t t r a n s p l a n t a t i o no ft h e s ea l l o g r a f t si n d u c e sh i g h e rc e l l u l a r ］ i n fi l t r a t i o na n do s t e o b l a s td ie r e n t i a t i o n［1 9 . Th e s e fi n d i n g so n c emo r ei n d i c a t et h a tp a s t e u r i z e da l l o g r a f t s h a v et h es a meo s t e o i n d u c t i v ep r o p e r t i e sa sf r o z e nb o n e . Th e s e3me t h o d so fe v a l u a t i o n( h i s t o l o g y ,t h en u me r i c a lr a t i n gs y s t e m,a n dh i s t o mo r p h o me t r y )a l lg a v et h e s a mer e s u l t sr e g a r d i n gp a s t e u r i z e da n df r o z e nb o n e , l e a d i n gt ot h ec o n c l u s i o nt h a tp a s t e u r i z a t i o no fb o n e a l l o g r a f t sd o e sn o ta l t e rt h eb i o l o g i c a lv a l i d i t ya n dt h a t t r a n s p l a n t a t i o no fp a s t e u r i z e da l l o g r a f t si n d u c e ss a t i s f a c t o r yn e wb o n ef o r ma t i o n . Th er e s u l t sr e g a r d i n gt h ee c a c yo ft h ed ie r e n tt y p e s

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o fb o n ea l l o g r a f t sa n dt h e i rd ie r e n c e s we r ea l s o c o n fi r me db yar a d i o l o g i c a lme t h o d .Ho we v e r ,a st h i s me t h o dp r o v i d e so n l yag e n e r a lp e r s p e c t i v er e g a r d i n gt h e b o n ed e f e c th e a l i n gp r o c e s s ,wed i dn o tu s ei tf o rf u r t h e r ［3 ］ e v a l u a t i o n ,n o rd i dHe i p l ei nh i ss t u d y . Ac e r t a i na c t i v a t i o no fb o n ec e l l sl e a d i n gt oab o n e h e a l i n gr e s p o n s ewa sa l s or e v a l e di nt h ea u t o c l a v e dg r o u p . Th es i g n i fi c a n t l yf e we ra c t i v eo s t e o b l a s t st h a twe r e r e v e a l e di nt h ea u t o c l a v e db o n eg r a f t ,h o we v e r ,wa s p r o b a b l yd u et oo s t e o c o n d u c t i v ep r o p e r t i e s ,a swek n o w t h a th i g ht e mp e r a t u r e s( 8 0° C)i n a c t i v a t eg r o wt hf a c t o r s a n dt h a tt h e r ei st h u sn of u r t h e rs t i mu l a t i o no f ［1 ］.Th o s t e o g e n e s i s 5 ea b o v eo n l yc o n fi r m wh ya u t o c l a v e db o n eg r a f t sh a v eb e e na b a n d o n e di nb o n et r a n s p l a n ［1 ］. t a t i o ns u r g e r y ,3 Be c a u s et h ev a l u ef o ra u t o c l a v e da n df r o z e nb o n e a l l o g r a f t si nt r a n s p l a n t a t i o ns u r g e r ya r ek n o wn ,weu s e d t h e s ea l l o g r a f t sa sc o n t r o l sf o re s t i ma t i o no ft h ee c a c yo f p a s t e u r i z e db o n eg r a f t s . I nt h a tr e s p e c t ,wec a nc o n c l u d et h a th e a tp r e t r e a t me n ta t5 6° Cf o r2 0mi nd o e sn o ta l t e rt h eb i o l o g i c a l q u a l i t i e so fp a s t e u r i z e db o n ea l l o g r a f t s .Si n c ei ti sk n o wn ［1 ］, t h a tp a s t e u r i z a t i o np r e v e n t sHI Vt r a n s mi s s i o n 1 ,1 7 wes u g g e s tt h a ts u c hp r e t r e a t e db o n ea l l o g r a f t sf r o ma b o n eb a n kc o u l db es a f e t ya n ds u c c e s s f u l l yu s e df o rb o n e t r a n s p l a n t a t i o n . Ac k n o wl e d g me n t s .Th i ss t u d ywa sp e r f o r me du n d e rap r o j e c tf u n d e db y t h eCr o a t i a nMi n i s t r yo fSc i e n c ea n dTe c h n o l o g y .

Re f e r e nc e s -7 1 . So l o mo nL:Bo n eg r a f t s .JBo n eJ o i n tSu r gBr( 1 9 9 1 ) ,7 0 6 0 7 . 2 . Fr i e d l a e n d e rGE:Bo n ea l l o g r a f t s :Th eb i o l o g i c a lc o n s e q u e n c e so f -1 i mmu n o l o g i c a le v e n t s .JBo n eJ o i n tSu r gAm( 1 9 9 1 ) ,1 1 1 9 1 2 2 . 3 . He i p l eKG,Ch a s eSW a n dHe r n d o nCH:Ac o mp a r a t i v es t u d yo ft h e h e a l i n gp r o c e s sf o l l o wi n gd ie r e n tt y p e so fb o n et r a n s p l a n t a t i o n .J -1 Bo n eJ o i n tSu r gAm ( 1 9 6 3 ) ,1 5 9 3 6 1 6 . 4 . Ne mz e kJ A,Ar n o c z k ySPa n dSwe n s o nCL:Re t r o v i r a lt r a n s mi s s i o nb y t h et r a n s p l a n t a t i o no fc o n n e c t i v e t i s s u ea l l o g r a f t s .Ane x p e r i me n t a l


-1 s t u d y .JBo n eJ o i n tSu r gAm ( 1 9 9 4 ) ,1 0 3 6 0 4 1 . 5 . Ne mz e kJ A,Ar n o c z k ySPa n dSwe n s o nCL:Re t r o v i r a lt a n s mi s s i o ni n b o n ea l l o t r a n s p l a n t a t i o n .Th eee c t so ft i s s u ep r o c e s s i n g .Cl i nOr t h o p ( 1 9 9 6 ) ,2 7 5 2 8 2 . 6 . Si mo n d sRJ ,Ho l mb e r gSD,Hu r wi t zRL,Co l e ma nTR,Bo t t e n fi e l dS, Co n l e yLJ ,Ko h l e n b e r gSH,Ca s t r oKG,Da h a nBA,Sc h a b l eCA, Ray fiel d MA and Roger s MF:Tr ans mi s s i on of human i mmu n o d e fi c i e n c yv i r u st y p eIf r o m as e r o n e g a t i v eo r g a na n dt i s s u e d o n o r .NEn g lJMe d( 1 9 9 2 ) ,7 2 6 7 3 2 . 7 . Sc h r a t tHE,Re g e lG,Lo b e n h oe rPa n dTs c h e r n eH:Or g a n i s a t i o no f ab o n ea n dt i s s u eb a n k .Co n s e q u e n c e sf o ro r g a n i z a t i o no fb o n ea n d t i s s u eb a n k sa f t e rHI Va n dh e p a t i t i sCi n f e c t i o n s .Un f a l l c h i r u r g( 1 9 9 6 ) -8 ,8 8 0 8 8 . 8 . Be t t i nD,De t h l o M,Po l s t e rJ ,St e i n b e c kJa n dFi e d l e rR:HI Vs c r e e n i n gi nb o n et a n s p l a n t a t i o n .Un f a l l c h i r u r g( 1 9 9 3 ) ,6 3 6 6 4 0 . 9 . Kn a e p l e rH,v o nGa r r e lT,Se i p pHM a n dAs c h e r lR:Ex p e r i me n t a l s t u d i e so ft h e r ma ld i s i n f e c t i o na n ds t e r i l i z a t i o no fa l l o g e n e i cb o n e t r a n s p l a n t sa n dt h e i re f f e c t so nb i o l o g i c a lv i a b i l i t y . Un f a l l c h i r u r g -4 ( 1 9 9 2 ) ,4 7 7 8 4 . 1 0 . No r ma n Ta y l o rFHa n dVi l l a rRN:Bo n ea l l o g r a f t :Ac a u s ef o rc o n c e r n ?JBo n eJ o i n tSu r gBr( 1 9 9 7 ) ,1 7 8 1 8 0 . 1 1 . Sp i r eB,Do r mo n tD,Ba r r e Si n o u s s iF,Mo n t a g n i e rLa n dCh e r ma n n J C:I n a c t i v a t i o no fl i mp h a d e n o p a t h y a s s o c i a t e dv i r u sb yh e a t ,g a mma r a y s ,a n du l t r a v i o l e tl i g h t .La n c e t( 1 9 8 5 ) ,1 8 8 1 8 9 . 1 2 . To mf o r dWW:Tr a n s mi s s i o no fd i s e a s et h r o u g ht r a n s p l a n t a t i o no f mu s c u l o s k e l e t a la l l o g r a f t s .JBo n eJ o i n tSu r gAm ( 1 9 9 5 ) ,1 7 4 2 1 7 5 4 . 1 3 . To mf o r dWW,Pl o e t zJ Ea n dMa n k i nHJ :Bo n ea l l o g r a f t so ff e mo r a l h e a d s :Pr o c u r e me n ta n ds t o r a g e .JBo n eJ o i n tSu r gAm ( 1 9 8 6 ) , -5 5 3 4 3 7 . 1 4 . As p e n b e r gP,J o h n s s o nEa n dTh o r n g r e nKG:Do s e d e p e n d e n tr e d u c t i o no fb o n ei n d u c t i v ep r o p e r t i e sb ye t h y l e n eo x i d e .JBo n eJ o i n tSu r g -1 Br( 1 9 9 0 ) ,1 0 3 6 0 3 7 . 1 5 . Kn a e p l e rH,Ko c hFa n dBu g a n yH:St u d i e so nHI Vi n a c t i v a t i o ni n a l l o g e n e i cb o n et r a n s p l a n t su s i n gc h e mi c a l d i s i n f e c t i o na n dr a d i o a c t i v e -6 i r r a d i a t i o n .Un f a l l c h i r u r g( 1 9 9 2 ) ,1 . 1 6 . St e v e n s o nSa n dHo r o wi t zM:Th er e s p o n s et ob o n ea l l o g r a f t s .JBo n e -9 J o i n tSu r gAm ( 1 9 9 2 ) ,9 3 9 5 0 . 1 7 . Ue mu r aY,Ya n gYHJ ,He l d e b r a n tCM,Ta k e c h iKa n dYo k o y a maK: I n a c t i v a t i o na n de l i mi n a t i o no fv i r u s e sd u r i n gp r e p a r a t i o no fh u ma n -2 i n t r a v e n o u si mmu n o g l o b u l i n .Vo xSa n g( 1 9 9 4 ) ,2 4 6 5 4 . 1 8 . Sp i r eB,Ba r r e Si n o u s s iF,Mo n t a g n i e rLa n dCh e r ma n nJ C:I n a c t i v a t i o no fl y mp h a d e n o p a t h ya s s o c i a t e dv i r u sb yc h e mi c a ld i s i n f e c t a n t s . -9 La n c e t( 1 9 8 4 ) ,8 9 9 0 1 . 1 9 . Pa r fi t tAM:Bo n eh i s t o mo r p h o me t r y :St a n d a r d i z a t i o no fn o me n c l a t u r e , s y mb o l sa n du n i t s .Su mma r yo fp r o p o s e ds y s t e m.Bo n eMi n e r( 1 9 8 8 ) -5 ,1 .

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