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저작자표시-비영리-변경금지 2.0 대한민국 이용자는 아래의 조건을 따르는 경우에 한하여 자유롭게 l 이 저작물을 복제, 배포, 전송, 전시, 공연 및 방송할 수 있습니다. 다음과 같은 조건을 따라야 합니다: l 귀하는, 이 저작물의 재이용이나 배포의 경우, 이 저작물에 적용된 이용허락조건 을 명확하게 나타내어야 합니다. l 저작권자로부터 별도의 허가를 받으면 이러한 조건들은 적용되지 않습니다. 저작권법에 따른 이용자의 권리는 위의 내용에 의하여 영향을 받지 않습니다. 이것은 이용허락규약(Legal Code)을 이해하기 쉽게 요약한 것입니다. Disclaimer 저작자표시. 귀하는 원저작자를 표시하여야 합니다. 비영리. 귀하는 이 저작물을 영리 목적으로 이용할 수 없습니다. 변경금지. 귀하는 이 저작물을 개작, 변형 또는 가공할 수 없습니다.

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A Doctoral Dissertation

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2011.06.

Thisthesishasbeenexaminedandapproved.

Thesisdirector,Kyoung-KapLee,Prof.ofDept.ofVeterinaryMedicine

JongtaeCheong,Prof.ofDept.ofVeterinaryMedicine

Nam-SooKim,Prof.ofDept.ofVeterinaryMedicine

Jae-HoonKim,Prof.ofDept.ofVeterinaryMedicine Joo-MyoungLee,Prof.ofDept.ofVeterinaryMedicine

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CONTENTS

ABSTRACT --- 1

LIST OF ABBREVIATIONS ---3

LIST OF TABLES--- 4

LIST OF FIGURES --- 5

GENERAL INTRODUCTION --- 7

CHAPTER Ⅰ.

Evaluation of the Biocompatibility of Cuttlebone in Mouse

Ⅰ-1INTRODUCTION ---11

Ⅰ-2MATERIALS AND METHODS ---13

Ⅰ-3 RESULTS --- 18

Ⅰ-4DISCUSSION --- 22

CHAPTER Ⅱ.

Evaluation of the Bone Defect Regeneration

after Implantation with Cuttlebone in Rabbit

Ⅱ-1INTRODUCTION ---26

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Ⅱ-3 RESULTS --- 35

Ⅱ-4DISCUSSION --- 43

CONCLUSIONS --- 47

REFERENCES --- 48

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SangcheolWon

(Supervisedby professorJongtaeCheong)

DepartmentofVeterinary Medicine GraduateSchool,Jeju NationalUniversity

Jeju,Korea

Abst

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act

Bone grafting is widely used to bridge majorbone defects orto promote bone union.Naturalcalcium carbonate has been used as a bone substitute materialand used to scaffold for bone morphogenetic protein (BMP).The aims ofthis study were to evaluate the biocompatibility ofcuttlebone (CB) and hydroxyapatite(HA)from CB (CBHA),and to evaluatethebonedefect regeneration ofCB,CBHA,and HA from coral(CHA).Each materialwas shaped into disks (5 ㎜ in diameter and 2 ㎜ in thickness). To test biocompatibility,thediskswereimplantedintothedorsalsubcutaneoustissue in mice. Fibrous capsule thickness around each disk was evaluated histologically at2and 4 weeksafterimplantation.In theevaluation ofbone defect regeneration,5 ㎜-diameter defects were created in rabbit calvaria. Concerning biocompatibility, fibrous capsule thickness of CBHA was significantly thinnerthan thatofCB and CHA (p<0.05)at2 and 4 weeks

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afterimplantation.Concerning 12-week totalchangesofradiologicGray-level histogram, CBHA was significantly higher than CHA (p<0.05). In the evaluation of bone defect regeneration, bone formation of CHA was significantly higher than that of CB and CBHA (p<0.05).Based on the clinicalandhistologicalresults,CBHA wouldbeasafematerialforuseinside the body and has more effective osteoconduction than CB.Itis suggested thatCBHA isavaluablebonegraftmaterial.

Key words;BoneGraft,Calcium Carbonate,Cuttlebone,Hydroxyapatite, Scaffold

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BMP Bonemorphogeneticprotein CB Cuttlebone

CB1 Cuttleboneafterdefattingandfreeze-drying CB1bmp CB1withrhBMP-2

CB2 Cuttleboneafterremovingorganiccomponents CBHA Hydroxyapatitefrom cuttlebone2

CC Calcium carbonate

CHA Hydroxyapatitefrom coral FGF Fibroblastgrowthfactor HA Hydroxyapatite

IGF Insulin-likegrowthfactor PDGF Plateletderivedgrowthfactor

rhBMP-2 Recombinanthumanbonemorphogeneticprotein2 TGF-β Transforminggrowthfactor-beta

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Li

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Table 1.Experimentaldesign forthe assessmentofbiocompatibility ofthe implants in mice --- 16

Table 2.Experimentaldesign for the bone defect regeneration in rabbits --- 30

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Fig 1.Preparation ofCB1forimplantation --- 14

Fig 2.Preparation ofCB2forimplantation --- 14

Fig 3.CB1,CB2,CBHA,and CHA implants were shaped cylindricaldisks about5㎜ in diameterand 2㎜ in thickness--- 15

Fig 4.Schematicdrawing forappliedimplantsin subcutaneoustissueinmice (a)andphotograph ofappliedimplants(b)--- 16

Fig 5.X-ray diffraction patternsofHA (JCPDS #09-0432)(a)and products preparedby hydrothermalreaction ofCB2(b)--- 18

Fig 6.Thicknessoffibrouscapsulesurrounding CB1,CB2,CBHA,and CHA implantsmeasuredat2and 4weeksafterimplantation in mice ---20

Fig7.Granulationtissueformation(arrowheads)aroundgraftmaterials(G)in group CB2 (a) and CBHA (b) at 2 weeks after implantation --- 21

Fig8.Granulationtissueformation(arrowheads)aroundgraftmaterials(G)in group CB2 (a) and CBHA (b) at 4 weeks after implantation --- 21 Fig 9.Photographs of rabbit calvarialdefect (∅5 ㎜) formation (a) and calvarialdefectsfilledwithCB1,CB1bmp,andCHA (b)---31

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Fig 10.Photographs of rabbit calvarialdefect formation (a) and calvarial defectsfilledwithCB2andCBHA (b)---31 Fig 11.Histogram processing ofradiograph by Photoshop program --- 33 Fig 12. Measurement of bone defect regeneration in rabbit calvaria by histomorphometricparameters--- 34 Fig 13.Total12-week changes ofGray-levelhistogram in each specimen --- 37 Fig 14.The rate of bone defect regeneration group CB1,CB2,CB1bmp, CBHA,and CHA specimens--- 38 Fig 15.Histologic sections ofgroup CB1 (a),CB1bmp (b),CB2 (c),CBHA (d),and CHA (e)at4weeksafterimplantation --- 40 Fig 16.Histologic sections ofgroup CB1 (a),CB1bmp (b),CB2 (c),CBHA (d),and CHA (e)at12weeksafterimplantation --- 41 Fig 17.Histologicfeaturesofnew boneformation ingroupCB1(a),CB1bmp (b),CB2(c),andCBHA (d)at12weeksafterimplantation--- 42

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GENERAL I

NTRODUCTI

ON

A bonegraftmay bethecriticalfactorbetween successfulboneunion and catastrophicfailure(Millisand Martinez,2002).Bonegrafting iswidely-used tobridgemajordefectsortoestablishthecontinuity ofthelong bone,toaid in fusion ofjoints,to fillcavities ordefects,and to promote bone union in delayed union and nonunion fractures(Finkemeier,2002;Millisand Martinez, 2002;Piermatteiand Flo,1997).Depending on the type of bone graft,a particular graftmay provide a source oflive bone cells or osteoinductive factors,givemechanicalstability,oractas a scaffold fornew bonegrowth (MillisandMartinez,2002;PiermatteiandFlo,1997).

Bone grafts orbone graftsubstitutes are named according to theirorigin and composition (Johnson and Hulse, 2002; Millis and Martinez, 2002). Autograftbone is transplanted from one siteto anotherin thesameanimal (Johnson and Hulse,2002).Thecurrentgold standard forbonegraftsisthe autogenous bone graft (Carson and Bostrom, 2007). Such grafts are histocompatible with host immune systems and willnot initiate rejection responses (Millis and Martinez,2002;Stevenson,1987).Allograft bone is transplanted from one animalto anotherofthe same species (Johnson and Hulse,2002).Cellularantigensofthesegrafts may berecognized asforeign body by thehost's immunesystems,resulting in graftrejection (Millisand Martinez,2002;Stevenson,1987).Xenograftbone is transplanted from one animaltoanotherofadifferentspecies(JohnsonandHulse,2002).Itpresents similarproblem totheallograft(Kim,2008;MillisandMartinez,2002).

Bone graft substitutes contain many types of materials, including biomaterials,ceramics,and calcium ceramics.Biomaterials consistofmetals (Kim,1998),polymers (Kim et al., 2008a),and ceramics:bioactive glass (Froum etal.,1998;Macedoetal.,2004;Ryu etal.,2000).Calcium ceramics

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consistofcalcium sulfate(Walsh etal.,2003),calcium phosphate(Leeetal.,

2007b;LeGeros,2002;Tadic and Epple,2004),and calcium carbonate (CC; CaCO3) (Lee et al., 2008a; Vuola et al., 2000).In ceramic biomaterials,

bioabsorbable,andbioactivesubstancesareabletophysicallybonddirectlyto the hostbed,whereas bioinertsubstances neveractually bond to the bone (CarsonandBostrom,2007).

The composition ofbone graftsmay includecancellous bone,corticalbone, corticocancellous bone, bone marrow, or bone and articular cartilage: osteochondral(Millis and Martinez,2002).Cancellous bone grafts consistof highly cellular trabecular bone removed from the medullary cavity oflong bone metaphyseal regions (Millis and Martinez, 2002). Their primary advantageistostimulateand producenew bone(Millisand Martinez,2002). Corticalbone grafts consistofthe dense outer corticalbone thatprovides structuralsupport(Millis and Martinez,2002).Corticocancellous grafts are a combination ofboth corticaland cancellousbone(Millisand Martinez,2002). Bone marrow is used to provide live undifferentiated mesenchymal cells (Millis and Martinez,2002).A composite graftis one in which cancellous bone orbone marrow is added to a preserved corticalallograft(Millis and Martinez,2002).An osteochondralbone graftconsists ofarticular cartilage and associated subchondral bone; the intended use of this graft is to resurfacejoints,butsuccessfullong-term application remainsa problem and itisnotcommonlyused(MillisandMartinez,2002).

Theidealscaffold should providean initialsupportforosteoprogenitorcells todepositmineralized bonematrix;itshouldbeslowly resorbedatthesame timethenewly formed bonetissuegrowsinsidethescaffold (Mastrogiacomo

etal.,2005).A highporosityandahighdegreeofinterconnectionamong the poresareabsoluterequirementsforthevascularizationoftheimplantandfor new boneformation(Mastrogiacomoetal.,2005).Inaddition,idealbonegraft

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a nonviable scaffolding conductive to bone ingrowth;osteoinductive factors, which are the chemical agents that induce the various stages of bone regeneration and repair; osteogenic cells, which have the potential to differentiate and facilitate the various stages of bone regeneration; and structuralintegrity(Gazdagetal.,1995;IlanandLadd,2003).

NaturalCC such as coral(Guillemin etal.,1987;Vuola etal.,1996,1998, 2000),eggshell(Dupoirieux etal.,1995,1999,2001a;Durmuşetal.,2007;Lee

et al., 2008a) has been used as a bone substitute material.Through a hydrothermalreaction,theCaCO3skeletonofCC ischangedtohydroxyapatite

(HA:Ca10(P04)6(OH)2).HA hasbeen isolated from coral(CHA)(Chou etal.,

2007;Holmesetal.,1984;LeGeros,2002;Roy andLinnehan,1974;Sivakumar

etal.,1996;Xu etal.,2001),eggshell(Park etal.,2008;Park etal.,2009), andcuttlebone(CBHA)(Ivankovicetal.,2009,2010;Kim etal.,2008b;Wang etal.,2001;Xing etal.,2007).Thebasicdifferencebetween HA and CC is thatCC is biodegradable and HA is notoronly very little (Vuola etal.,

1996).Porous HA is biocompatible and osteoinductive (Chiroffetal.,1975). HA hasattracted a greatdealofinterestas a biomaterialforimplantsand bone augmentation since its chemicalcomposition is close to thatofbone (Kim etal.,2008b).Cuttlebone(CB),which iscomposedofCC hasaporous structurewithallporeinterconnectedthroughouttheskeletonandCB.

The purpose ofthis study was to evaluate the biocompatibility ofCB in mousemodeland toevaluatethebonedefectregeneration ofCB in arabbit calvarialdefectmodel.

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CHAPTER Ⅰ

Evaluation of the Biocompatibility of Cuttlebone

in Mouse

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Ⅰ-1.I

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The development of new biomedicaldevices from various materials has received a great dealof attention recently (Butler et al.,2001).When a materialisintendedforsafeuseinsidethebody,itsinvivoperformanceand biocompatibilitymustbescrupulouslyverified(Ryhänenetal.,1998).

Measuring thethicknessofan encapsuling membranearound theimplantis abasictoolforestimating biocompatibility (Ryhänen etal.,1998).Utilization ofthe thickness ofthe scarcapsulearound an implantaloneis problematic because there are factors other than the material itself that can affect capsular thickness (Ryhänen et al., 1998). The fibrous tissue includes inflammatory components such as macrophages, fibroblasts, neutrophils, collagen, and numerous blood vessels (Butler et al., 2001), and capsule formation depends on various factors, including implant size (Aalto and Heppleston,1984),shape(Matlaga etal.,1976),surfacetexture(Behling and Spector,1986),surfacechemistry (Clark etal.,1976),poresize(Whiteetal.,

1981),and implantation site (Bakkeretal.,1988).Subcutaneous implantation ofbiomaterialsinducesacuteand chronicinflammatory reactionsresulting in fibrous tissue formation around the device (Butler etal.,2001).Thickness appears to directly correspond with theothercellularcomponents presentin the fibrous tissue matrix (Butler et al., 2001). The tissue and cellular responsesto implantsarescreened on thebasisofmorphologicobservations onroutinehistologicevaluation(Butleretal.,2001).

Commonly, biocompatibility of bone graft substitutes and scaffolds are investigated by two types ofin vivo tests:subcutaneous implantin mice (Kim etal.,2004;Leeetal.,2006;Yeom etal.,2007)orrats(Butleretal.,

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employing rabbits(Durmuşetal.,2007;Guetal.,2009;Kim etal.,2008a)or

rats (Bosch etal.,1995;Dupoirieux etal.,2001b;Lee etal.,2008a,2008b;

Park etal.,2008).Typically,beforetheimplant,biomaterialsareprepared to eliminatetheimmuneresponse.

The purpose of this study was to compare tissue responses after implantation ofCB in a mouse model.Furthermore,thethickness offibrous capsulesurroundingspecimenswasmeasuredbyhistologicanalysis.

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Ⅰ-2.Mat

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Ⅰ-2-1. Fabrication of implants

Three kinds ofimplants (CB1,CB2 and CBHA)were prepared from the same genus of cuttle bone (Sepia esculenta). The CHA implants used consisted of HA from coral(BoneMedikⓇ,Metabiomed,Korea).The CB1 implants were processed in severalsteps that included defatting,freezing, drying,and sterilization (Fig 1,Choietal.,2003;Choiand Lee,1998).The CB2 implants were processed by removing organic components,washing, drying,andsterilizing(Fig2,Kim etal.,2008b).

CBHA implantswereprocessed in hydrothermalsynthesis:CB2 wasputin 2M(NH4)2HPO4in a Teflon® lined hydrothermalbomb (HydrothermalReactor

SystemⓇ,HanwoulEngineering,Korea)and heated for16h at180℃.Then, the block was immersed in 2M (NH4)2HPO4and treated at200℃ for24 h

hydrothermally.Afterthoroughly washing with distilled water,theblock was dried at90℃ (Kim etal.,2008b).AfterX-ray diffraction (XRD)examination of the block,it was used as CBHA.These implants were shaped into cylindricaldisks about5 ㎜ in diameterand 2 ㎜ in thickness (Fig 3)and weresterilizedbyethyleneoxidegas.

Ⅰ-2-2. Experimental animals and surgical procedure

Twenty 9-week-old,22 ± 0.2 g male BALB/c mice were used in the experiments.They were housed undera standard light-dark schedule,were fedastockdietandhadaccesstotapwateradlibitum.

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Fig 1. Preparation of CB1 for implantation. The CB1 implants were

processed through defatting, freezing, drying, and sterilization.

Fig 2. Preparation of CB2 for implantation. The CB2 implants were

processed through removing organic components, washing, drying,

and sterilization.

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Fig 3. CB1, CB2, CBHA, and CHA implants were shaped cylindrical

disks about 5 ㎜ in diameter and 2 ㎜ in thickness and were

sterilized by ethylene oxide gas.

The experimental protocol was approved by the Animal Care and Use Committee,Jeju NationalUniversity (approvalnumber2010-0042).The mice were divided into four experimentalgroups as shown in Table 1.Aseptic surgicaltechnique was applied during the surgicalprocedure.Mice were anesthetized by theintramuscularinjection ofa doseoftiletamine/zolazepam (Zoletil50Ⓡ,Virbac,France).Afterthe anesthesia,the middle ofthe back of each mouse was shaved.The incision sites were washed with 70% ethanol and scrubbed with 10% povidone iodine,and a skin incision with 1.5 ㎝ in length was made.Sterilized implants were inserted subcutaneously through the incision site (Fig 4) and the wound was closed with 4-0 nylon. Immediately following implantation,the mice were injected subcutaneously with a dose of gentamicin sulfate (Gentamicin 5% Inj.Ⓡ, Daesung MicrobiologicalLabs.,Korea)for3days.

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Mice Group

Application

Site Type of implants n = 10

CB1 L. Dorsal back SQ Cuttlebone 1 CHA R. Dorsal back SQ HA from coral n = 10

CB2 L. Dorsal back SQ Cuttlebone 2 CBHA R. Dorsal back SQ HA from cuttlebone

Table 1. Experimental design for the assessment of biocompatibility

of the implants in mice

L.: Left, R.: Right, n: number of experimental animal, CB:

Cuttlebone, HA: Hydroxyapatite, SQ: Subcutis

Fig 4. Schematic drawing for applied implants in subcutaneous

tissue in mice (a). Photograph of applied implants (b). Sterilized

implants were inserted subcutaneously through the incision site.

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Ⅰ-2-3. Histologic investigations and analysis of fibrous capsular membrane thickness

Tenmicewereeuthanizedat2and 4weeksaftersurgery.Implanteddisks and thesurrounding tissuewereremoved asasinglemassand immediately immersed in 10% phosphate-buffered formalin for 3 days.The mass was decalcified for at least 7 days using 5% formic acid. Implants were dehydrated in an ethanol series, embedded in paraffin, and cut into 6 mm-thick sections.The sections were stained with hematoxylin and eosin (H&E)stain and Masson's trichrome stain.The fibrous capsularmembrane thickness around the implants was determined with a CCD camera-based digital image analysis system. The system consisted of a microscope (Olympus BX41;Japan)and an Olympus DP20 video camera.The fibrous capsularmembranethicknesswasdetermined ateach pointofthehorizontal andverticallines.Thefibrouscapsularmembranethicknesswasexpressedas ameanvalueofeighthits.

Ⅰ-2-4. Statistical analysis

TheStatisticalPackagefortheSocialScienceversion 17.0software(SPSS, USA) was used for data analysis.Mann-Whitney's u-test was used to evaluate differences between each group.A p value ≤ 0.05 was considered statisticallysignificant.

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Ⅰ-3.Resul

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Ⅰ-3-1. Characterization CBHA

The typical X-ray diffraction (XRD) patterns of product prepared by hydrothermalreaction ofCB2 at200℃ for24 h are shown in Fig 5.The XRD patternsoftheCB wereconfirmed asHA on thebasicofJCPDS card #09-0432. They were completely transformed into HA by hydrothermal reaction.

Fig 5. X-ray diffraction patterns of HA (JCPDS # 09-0432) (a) and

products prepared by hydrothermal reaction of CB2 (b). Arrows

indicate the picks of HA.

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Ⅰ-3-2. Analysis of fibrous capsular membrane thickness

Theanalyticalresultsoffibrouscapsularmembranethicknessareshown in Fig 6.At2 weeks,the thickness offibrous capsule in group CBHA was significantly lowerthan thatofothergroups.Groups CB1,CB2,and CHA displayed nosignificantdifferences.At4weeks,thethicknessofthefibrous capsulein group CB1 wassignificantly higherthan thatofothergroups.In group CBHA,the thickness ofthe fibrous capsule was significantly lower than thatofothergroups.Therewasnosignificantdifferencebetween group CB2 and CHA.In allgroups,the thickness offibrous capsules at4 weeks wasthinnerthanat2weeks.

Ⅰ-3-3. Histologic evaluation

At 2 weeks after implantation,allgraft materials in the subcutis were surrounded by mild to moderatedensefibrousstromacomposed ofabundant collagenousfiberthanstainedblueuponMasson'strichromestaining (Fig 7). Variablenumbersofinflammatorycellssuchasneutrophils,macrophages,and foreignbodygiantcellshadinfiltratedaroundthegraftmaterials.Someblood vessels in the subcutis showed marked congestion. At 4 weeks after implantation,fibrousstromalreaction and inflammatory reaction around graft materials had gradually decreased compared to the mice observed 2 weeks (Fig 8).Occasionally,new formedcapillarieswereobservedintheproliferated fibrousstroma.

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Fig 6. Thickness of fibrous capsule surrounding CB1, CB2, CBHA,

and CHA implants measured at 2 and 4 weeks after implantation in

mice. Values are expressed as mean ± SE.

l

Significantly lower than other groups at 2 and 4 weeks after

implantation (p<0.05).

Significantly higher than other groups at 4 weeks after

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Fig 7. Granulation tissue formation (arrowheads) around graft

materials (G) in group CB2 (a) and CBHA (b) at 2 weeks after

implantation. Group CB2 showed more thick fibrous tissues and

inflammatory reactions than group CBHA. The sections were

stained using Masson’s trichrome.

Fig 8. Granulation tissue formation (arrowheads) around graft

materials (G) in group CB2 (a) and CBHA (b) at 4 weeks after

implantation. Group CB2 still showed thicker fibrous tissues than

group CBHA. The sections were stained using Masson’s trichrome.

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Ⅰ-4.Di

scussi

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Transformation ofCB into CBHA through a hydrothermalreaction process has been widely-reported, and CBHA has been confirmed by X-ray diffractometry analysis (Ivankovic et al., 2009, 2010; Kim et al., 2008b;

Sivakumaretal.,1996;Wang etal.,2001;Xing etal.,2007).Ivankovicetal.

(2010)reportedthatCB wascompletelytransformedintoCBHA after48hat 200℃.In the presentstudy,CB was completely transformed into the same CBHA,although theconditionsoftimeand temperatureweredifferent.Itis suggested thatthe same resultcan be obtained overthe criticaltime and temperature.

Tissue reactions thatare importantfrom the standpointofbiocompatibility mainly relate to an inflammatory reaction (Ryhänen et al.,1998).In this study,inflammatory cells were observed during a 4-week period.Salthouse (1984)reported the response ofmacrophages to implants;within 24 hours, macrophages were found in close contactwith the implantsurface.Then, fibroblasts and connective tissue proliferated and,finally,the implant was encapsulated.Butleretal.(2001)reportedthatthethicknessoffibroustissue waswidelyvariableandthefibroustissuesurrounding subcutaneousimplants wasthinnerthan thatsurrounding intraperitonealceramic.In thisexperiment, implantation was inserted subcutaneously.Previous tests ofbiocompatibility established that more biocompatible implants had thinner surrounding connectivetissue(Dupoirieux etal.,2001a;Hwang etal.,1999;Jacob etal.,

1998;Lim etal.,2007).Kim etal.(2004) reported the same result from mouseabdominalconnectivetissue.Presently,significantly thinnerconnective

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tissue wasobserved in theCBHA group (p<0.05).CBHA was confirmed to have higher biocompatibility than CB1, CB2, and CHA. According to Jacob-LaBarre et al.(1994) and Dupoirieux et al.(2001a),rough-textured surfaces generate thicker capsules,and smooth surfaces usually generate thinner capsules. Superior tissue compatibility should be associated with smooth,well-contoured implantswith noacuteangles(Salthouse,1984).The long-term biologicalresponseclearly implicatesmacrophagesasthedominant celltypeatimplantsurfacesandthatimplantstabilitydependslargelyonthe dynamic behavior of macrophages (Jacob-LaBarre et al., 1994). These observations suggestthatthe CHA surface is rougherthan thatofCBHA. This was grossly evident in the present study.Furthermore,group CB2 displayed thinnerconnective tissue than thatofgroup CB1 2 and 4 weeks later.TheseresultsindicatethatCB2 preparation ismoreefficientthan that of CB1,albeit no significant difference between them.The thickness of connective tissues tended to be thinner in allgroups at 4 weeks.Many researchers have been trying to find out the ideal conditions in the preparation of allograft or xenograft materials. The most generally-used methods are demineralization (Bigham et al., 2008; Begley et al., 1995;

Dahnersand Jacobs,1985;Jung etal.,2006;Tuliand Singh,1978;Um and Him,1993),freezing (Leeetal.,2007a),freeze-drying (Choung,1996;Leeet

al., 2007a),defat-freezing (Lee et al., 2007a; Song and Lee, 2007),and freeze-drying afterdefatting (Choiand Lee,1998;Choung,1996;Lee etal.,

2007a;Song and Lee,2007).Freezing afterdefatting confers a higherbone neogenesis effect than freeze-drying after defatting (Choi and Lee,1998; Choung,1996;Lee etal.,2007a;Song and Lee,2007).Specimens processed with freezing after defatting,however,are more limited with respect to transportand storage than freeze-drying afterdefatting (Lee etal.,2007a).

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Also,as described above,the preparation methods associated with allograft and xenograftuniquely involvematerialsthatcontain BMP.Boneallograftor xenograftmaterials containing BMP are reported as being stable when they are processed with freeze-drying afterdefatting (Choiand Lee,1998).But, CB is a naturalCC ceramic that is different from the implant materials applied in the aforementioned preparation methods.CB1 was processed with freeze-drying after defatting (Fig 1)and CB2 was processed as described previously(Kim etal.,2008b)(Fig2).

According totheresultsofLietal.(1999),theratioofcellcomponentsof fibrouscapsuleschangeswith thetimeafterimplantation.In theirstudy,the population of fibroblasts in fibrous capsules decreased gradually and the populationoffibrocytesincreasedgraduallyafterimplantation(Lietal.,1999). Presently,the population changes of fibroblasts and fibrocytes showed a similartrendat4weeksafterimplantation.

Presently, CBHA was the most biocompatible materials among the experimentalimplants.However,furtherexperimentsarenecessarytofindout moredetailsaboutthesuitablepreparationofCB.

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CHAPTER Ⅱ

Evaluation of the Bone Defect Regeneration

after Implantation with Cuttlebone in Rabbit

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Ⅱ-1.I

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i

on

Bone regeneration initiated by autogenous cancellous bone occurs by three major steps.First,the undifferentiated osteoprogenitor cells are recruited. Then,by osteoinduction,these cells differentiate to give rise to osteoblasts and chondrocytes.Finally,asuitablescaffold on which activeosteoprogenitor cellscanproducenew boneisestablished(Gazdagetal.,1995).However,HA and calcium ceramic are not intrinsically osteoinductive (Rose and Oreffo, 2002).Osteoinduction is mediated by numerous growth factors provided by the bone matrix itself.BMP is the typical osteoinductive material. Low molecular weight proteins that initiate endochondral bone formation, presumably by stimulating localprogenitor cells of osteoblast lineage and enhancingbonecollagensynthesis(Gazdagetal.,1995).

Senn (1889)noted thatdecalcified bonecan inducehealing ofbonedefects. Urist(1965)described ectopicboneinduction in intramuscularimplantation of decalcified bone.Isolation ofthe bone-inducing substance revealed a protein thatwasnamedBMP (UristandStrates,1971).

BMPs are members ofthe TGF-β superfamily,a large family ofsecreted factors.The principalactivity ofBMP is the whole process ofembryonic endochondralossification,butitisectopicboneinduction during postfetallife (Urist,1965).Recombinanthuman BMP-2 (rhBMP-2)wasthefirstmolecule studiedindetail,being producedusing Chinesehamsterovary cells(Wang et

al.,1990);theauthorssuggested thatthis factorwould beusefulin healing bonydefects(Wang,1993;Wozney,1998).Moreover,rhBMP-2increasedbone density,bone formation,and callus volume (Bax etal.,1999;Chee etal.,

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diffused (Uludag et al., 2001), and that the osteoinductive potency of rhBMP-2 is significantly increased when itis implanted with a biomaterial scaffold (Uludag etal.,1999;Yokota etal.,2001).Although many potential scaffoldsforBMP havebeen evaluated,suitablescaffoldsforBMP havenot yetbeenrealized(RoseandOreffo,2002;Saitoetal.,2001).

Weibrich etal.(1998,2000)compared thesurfaceareaof12otherimplants and suggested thatthe surface area ofdemineralized bovine bone was 79.7 m2/g.This was five times higher than the surface area of other bone regeneration materials (Weibrich etal.,2000).But,the surface area ofCB (386 m2/g)was four times higher than thatofdemineralized bovine bone. BufferingcapacityofCB showedthesamepatternasthatofCC (Kim etal.,

2000).CB iscomposed of53.25% Ca,26.78% O,14.90% Na,4.37% Cl,0.50% Sr,0.06% P,0.06% S,and 0.05% Si(Kim etal.,2000).Mostofcalcium in CB waspresentin aform ofCC.Highly interconnectiveporositiesserveas channelsforinternalgrowth ofbloodvesselsand increasebone(Weibrich et

al., 2000) and bone containing macro- and micro-pores, which are interconnectedtoallow necessary body nutrientsandfluidstobetransported, making bone an extremely complex structure (Ben-Nissan,2003).Scanning electronmicroscopyhasrevealedthemultiplayerstructureofCB (Birchalland Thomas,1983;Florek etal.,2009;Kim etal.,2000;Sherrard,2000;Tiseanu

etal,2005).The minimum pore diameter required for bone ingrowth and angiogenesisinto a scaffold isgenerally considered to be100 ㎛ (Lu etal.,

1999;Nadeetal.,1983;Okiietal.,2001).Theporousstructurediameterof CB isusually between200and600㎛ (BirchallandThomas,1983),whichis similartocancellousbone(Guetal.,2009).Theoptimalosteoconductivepore sizeforceramicsappearstobebetween150and500㎛ (Flatleyetal.,1983). Kim etal.(2008b),Lin (1993),andOkumuşandYildirim (2005)reportedthat CB representa new xenograftmaterialin cancellousbone.Untilnow,there havebeennoreportsonthepotencyofCB asaxenograftbonesubstitutein

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compactbonestudies.

The purpose ofthis study was to compare bone defectregeneration after implantation of CB in a rabbit calvarial defects model. Bone defect regeneration was measured by radiologic,histologic,and histomorphometric methods.

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Ⅱ-2.Mat

er

i

al

sandMet

hods

Ⅱ-2-1. Fabrication of implants

The CB1,CB2,CBHA,and CHA implants were processed as described in Chapter Ⅰ. CB1bmp implant was CB1 with rhBMP-2. The rhBMP-2 (rhBMP2Ⓡ,Korea Bone Bank,Korea)was produced by a Chinese hamster ovarian cell expression system to yield a glycosylated 26 kilodalton homodimer.rhBMP-2 (100 ㎍/㎝3)(Itoh etal.,1998)was instilled into CB1 withuseofasyringe(fineneedleinfiltration)atthetimeoftheoperation. Ⅱ-2-2. Experimental animals and surgical procedure

Twenty seven 9-month-old,3.2 ± 0.5 ㎏ male New Zealand white rabbits were keptundera standard light-dark schedule and were fed a stock diet and had access to tap water ad libitum.The experimentalprotocolwas approved by the AnimalCare and Use Committee,Jeju NationalUniversity (approvalnumber 2010-0042).The rabbits were divided into six groups as shown inTable2.Asepticsurgicaltechniquewasappliedduring thesurgical procedure.They wereanesthetized with an intramuscularinjection ofadose of tiletamine/zolazepam (Zoletil50Ⓡ, Virbac, France). After the anesthesia, incision sites were shaved.The incision sites were then washed with 70% ethanoland scrubbed with 10% povidoneiodine.Incision siteswere exposed withasagittalincisionthroughtheskinandtheperiosteum atthemidlineof the calvaria.With the use ofa medicaltrephine burr(TPHB-B5Ⓡ,Osung Mnd,Korea),fourstandardized full-thickness 5 ㎜-diameterosseous defects werecreatedinthecalvarium ofeachrabbitundersalineirrigation(Figs9-a, 10-a).In group CB1,CB1bmp,and CHA,the leftrostraldefectwas filled with CB1disk,theleftcaudaldefectwasfilled with CB1bmpdisk,theright

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Rabbits Group

Application

Site Types of implants

n = 18

Blank R. Ros. Calvaria Blank CHA R. Cau. Calvaria CHA

CB1 L. Ros. Calvaria CB1

CB1bmp L. Cau. Calvaria CB1 + rhBMP-2

n = 9

CB2

R. Ros. Calvaria Cuttlebone 2 L. Ros. Calvaria Cuttlebone 2 CBHA

R. Cau. Calvaria HA from cuttlebone L. Cau. Calvaria HA from cuttlebone caudaldefectwasfilledwithCHA disk,andtherightrostraldefectwaskept andusedasanegativecontrol(Fig9-b).

IngroupsCB2andCBHA,bothsidesoftherostralandcaudaldefectswere filled with CB2and CBHA disks(Fig 10-b).Theskin wasclosed with 3-0 nylon. Immediately following implantation, the rabbits were injected subcutaneouslywithadoseofpenicillinG procaine(CombimycinInj.Ⓡ,Green CrossVeterinaryProducts,Korea)for3days.

Table 2. Experimental design for the bone defect regeneration in

rabbits

L: Left, R: Right, Ros: Rostral, Cau: Caudal, n: number of

experimental animal, CB: Cuttlebone, CHA: Hydroxyapatite from

coral, rhBMP-2: Recombinant human bone morphogenetic protein-2

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Fig 9. Photographs of rabbit calvarial defect (∅5 ㎜) formation (a)

and calvarial defects filled with CB1, CB1bmp, and CHA (b).

Fig 10. Photographs of rabbit calvarial defect formation (a) and

calvarial defects filled with CB2 and CBHA (b).

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Ⅱ-2-3. Radigraphic interpretation and radiologic Gray-level histogram (brightness) evaluation

Nine rabbits were euthanized at4,8,and 12 weeks postoperatively.The implants and the surrounding calvarial bone were removed. The defect regionswassubmittedtocontactradiographyinadorso-ventralplanewitha Kodak Direct View CR975 X-ray System (Eastman Kodak,USA) at an exposure of 42 kVp and 6.4 mAs. Bone formation was evaluated by Gray-level histogram in radiograph processing (Fig 11) using AdobeⓇ PhotoshopⓇ 7.0(AdobeSystems,USA).Thehistogram ofeach implantwas measuredbeforeimplantation tocalibratetheradiologicGray-levelhistogram. Then,the histogram ofthe implants were measured at4,8,and 12 weeks afterimplantation,and the changes ofthe histogram were recorded on the basisofacalibratedhistogram.

Ⅱ-2-4. Histologic investigations and histomorphometric analysis

Removed calvarialbone was immersed in 10% phosphate-buffered formalin for 3 days and decalcified for 7 days using 5% formic acid. It was dehydrated in an ethanol series, embedded in paraffin, and cut into 6 mm-thick sections.The sections were stained with H&E stain.The defect closure was determined with a CCD camera-based digitalimage analysis system (Fig 12).Therateofbonedefectregeneration wasdetermined by a histomorphometric method:rate ofbone defectregeneration (%)= [(original defectwidth,㎜)-(remaineddefectwidth,㎜)]/(originaldefectwidth,㎜)× 100(Jungetal.,2007;Leeetal.,2008b;Sohnetal.,2010).

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Fig 11. Histogram processing of radiograph by Photoshop program.

Radiologic Gray-level histogram was measured to evaluate of bone

defect regeneration of each implant 4, 8, and 12 weeks after

implantation.

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Fig 12. Measurement of bone defect regeneration in rabbit calvaria

by histomorphometric parameters. The measurement was performed

4, 8, and 12 weeks after implantation.

Ⅱ-2-5. Statistical analysis

TheStatisticalPackagefortheSocialScience,version 17.0software(SPSS, USA) was used for data analysis.Mann-Whitney's u-test was used to evaluate differences between each group.A p value ≤ 0.05 was considered statisticallysignificant.

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Ⅱ-3.Resul

t

s

Ⅱ-3-1. Radiologic evaluation

At4weeks,in group CB1and CB1bmp,theradiopaqueimplantsiteswere observed.IngroupCHA,theimplantsitewasmoreradiopaquethanthesites in group CB1 and CB1bmp.In group CB2,partialbone regeneration was observedandtheradiopaqueimplantsitewasdelineatedfrom thesurrounding bone by a radiolucentborder.In group CBHA,bone regeneration processing towards the centre ofthe defectwas observed and radiopaque implantsite wasdelineatedfrom thesurroundingbonebyaradiolucentborder.

At 8 weeks,the radiopaque implantsite in group CB1 revealed a more enlargedcirclethangroupCB1bmpandCHA.IngroupCB1bmp,aradiopaque implantsite was observed and the radiopaque implantsite was delineated from thesurrounding boneby aradiolucentborder.In group CHA,themore radiopaqueimplantsitewasobservedthangroupCB1andCB1bmp.In group CB2,partialbone regeneration was observed and the more enlarged circle was observed than group CBHA. In group CBHA, bone regeneration processing towardsthecentreofthedefectwasobservedandtheradiopaque implant site was delineated partially from the surrounding bone by a radiolucentborder.

At12weeks,in group CB1and CB1bmp,partially radiopaqueimplantsites wereobserved.In group CHA,amoreradiopaqueimplantsitewasobserved than in groups CB1 and CB1bmp.In group CB2,a radiopaque implantsite was observed and the circle was more enlarged than in group CBHA.In group CBHA,boneregeneration processing towardsthecentreofthedefects was observed.At12 weeks,bone regeneration strikingly advanced in group CBHA comparingwiththegroupCB1,CB2,andCB1bmp.

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Ⅱ-3-2. Total changes of radiologic Gray-level histogram (brightness) during 12 weeks after implantation

In group CBHA,totalchanges ofthe radiologic Gray-levelhistogram was significantly higher than that of the group CHA during 12 weeks after implantation (p<0.05),butthere was no significantdifference compared with thatofthe group CB1,CB2,and CB1bmp (Fig 13).In group CHA,total changesofradiologicGray-levelhistogram during12weeksafterimplantation was significantly lowerthan thatofthe othergroups (p<0.05)(Fig 13).In group CB1, CB2, CB1bmp, and CBHA, total changes of the radiologic Gray-levelhistogramsweresimilarly changedduring the12weeks,butthere was no significantdifference compared with thatofthe othergroups (Fig 13).At12weeks,groupCHA wassignificantly lowerthan thatoftheother group (p<0.05) (Fig 13).This evaluation was calibrated as the numerical value.

Ⅱ-3-3. Histomorphometric analysis for bone defect regeneration The rate ofbone defectregeneration ofthe implanted site in group CHA was significantly higherthan thatofothergroups at4 and 8 weeks after implantation (p<0.05) (Fig 14). At 12 weeks, the rate of bone defect regeneration in group CHA wassignificantly higherthan thatofgroup CB1, CB2, and CB1bmp (p<0.05). But, there were no significant differences betweengroupCHA andCBHA (Fig14).

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Fig 13. Total 12-week changes of radiologic Gray-level histogram

in each specimen. The change of Gray-level histogram in group

CBHA was the highest after implantation, but there were no

significant differences between group CB1 and CB1bmp. Values

expressed as mean ± SE.

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Fig 14. The rate of bone defect regeneration group CB1, CB2,

CB1bmp, CBHA, and CHA specimens. Values are expressed as

mean ± SE.

Significantly higher than other groups at 4, 8, and 12 weeks

after implantation (p<0.05).

Significantly higher than group CBHA at 8 weeks after

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Ⅱ-3-4. Histologic evaluation

At4weeksafterimplantation,allgraftmaterialsin thecalvarialdefectarea weresurrounded by moderatetoseveredensefibrouscollagenousfibers(Fig 15).Multifocaltodiffuseinflammation composed ofneutrophils,macrophages, and foreign body giantcells were scattered in the adjacentarea offibrous tissues.In the CHA group,proliferated collagenous fibers were invaginated intothegraftmaterials.

At8weeksafterimplantation,overallfibrousstromalreactionineachgroup wasgradually increased,ascomparedto4weeks.However,theinflammatory reaction wasdramatically decreased.Compared with othergroups,mostgraft materialswerereplacedbyinwardgrowingnew boneingroupCHA.

At12weeksafterimplantation,variableextentsofbonegrowth intodefect areaswereobservedinallexperimentalgroups(Fig 16).New boneformation extended into graftmaterials in group CBHA and CHA (Fig 17).However, fibrous stromaland inflammatory reactions stillremained in groups CB1, CB1bmp,CB2,butlittleremainedin groupsCBHA andCHA.IngroupCHA, anobviouscontinuityofcorticalbonefrom thecalvariawasobservedinboth sides ofthe bone defect.A marked increased number ofosteoblasts were presentatthe surface ofbonny spicules.Multifocalbone marrow formation alsoexistedincorticalbone(Figs16,17).

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Fig 15. Histologic sections of group CB1 (a), CB1bmp (b), CB2 (c),

CBHA (d), and CHA (e) at 4 weeks after implantation (black

arrows:

defect

margins).

Group

CB1

and

CB1bmp

were

encapsulated by fibrous connective tissue (a, b: red arrows). Bone

defect regeneration was observed at defect margins in group CBHA

and CHA (d, e: blue arrows). The sections were stained using

H&E.

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Fig 16. Histologic sections of group CB1 (a), CB1bmp (b), CB2 (c),

CBHA (d), and CHA (e) at 12 weeks after implantation (black

arrows: defect margins). CB1 and CB1bmp implants were still

encapsulated by thick fibrous connective tissue (a, b: red arrows).

New bone formation was observed in group CBHA (d: blue arrow).

The sections were stained using H&E.

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Fig 17. Histologic features of new bone formation in group CB1

(a), CB1bmp (b), CB2 (c), and CBHA (d) at 12 weeks after

implantation. New bone formation (asterisks) was observed around

the graft material (G) in group CB1 or CB1bmp and inside of graft

material in group CBHA. The sections were stained using H&E.

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Ⅱ-4.Di

scussi

on

Sohnetal.(2010)reportedthatthecriticalsizedefectwasnotsuggestedin a rabbitcalvarialmodel.Huh etal.(2005)suggested thatthe criticalsize defectdependson thepresenceoftheperiosteum.A defectwith a diameter of5㎜ isacommonly usedsizearatmodels(Leeetal.,2008a;Dupoirieux

etal.,1995).Presently,considering theconvenienceofoperation,defectswith adiameterof5㎜ werecreatedinthecalvariaofrabbitsandtheperiosteum surroundingtheimplantwaswidely-removed.

Interconnecting pores are the pathway for new vessels, which is an advantage fornew bone formation (Okiietal.,2001).Blood supply to the cancellous bone is much betterthan to the compactbone.Forthis reason, bone regeneration in cancellous bone is faster than in compact bone. Nevertheless,this study was performed to the compact bone,in order to directlyobservethebonedefectregenerationwithoutcartilageformation.

Itoh et al.(1998) reported that the lowest dose ofrhBMP-2 needed for defecthealing was between 40 and 160 ㎍/㎝3and suggested a 100 ㎍/㎝3 dose,basedonresultsincanineulnardefectmodel.Inthisstudy,thedoseof rhBMP-2 was 100 ㎍/㎝3 (Itoh etal.,1998).From the results,we decided thatthedoseofrhBMP-2inCB wassuitableornot,becausethisstudywas notfocusedonthedeterminationofdoseofrhBMP-2.Butthedoseof100㎍ /㎝3waseffectiveinbonedefectregeneration.

Inbonedefectregeneration,radiologicanalysisiscommonlyusedtocompare thefeasibility ofbonegraftusein differentstudies(Dupoirieux etal.,1995;

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CHA,a more radiopaque implantsite was observed than apparentin the surrounding boneand theothergroupsat12weeksafterimplantation.Bone regeneration was determined by changes of radiodensity.For this reason, bone regeneration processing ofCHA implantcould notbe observed during 12 weeks after implantation.But,8 and 12 weeks after implantation,an ill-defined change was apparentbetween bone and implant,indicating that boneremodelingoccurredintheCHA group.Thisfindingissameresultwith otherstudies(Dupoirieuxetal.,1999;Durmuşetal,.2007).IngroupCB1,the sizeofdefectwasenlargedat8weeksafterimplantation.

This osteolysis is thought to be immune responses.In this study,this foreign body reaction can be found in the biocompatibility testofCB1.A similarobservation wasreportedby Durmuşetal.(2007).Inthelatterstudy, anexcessiveforeignbodyreactioncharacteristicofencapsulationwasevident. Karaismailoğlu et al. (2002), suggested that xenografts still have some disadvantagesin termsofgraftincorporation,resorption,mechanicalstrength, and other problems linked to immunologic rejection and microbiologic contamination.Presently,groupsCB1and CB1bmp wereencapsulated with a dense fibrous tissue with inflammatory cells such as neutrophils, macrophages,and foreign body giant cells at 4,8,and 12 weeks after implantation.Forthisinflammatory reaction,freeze-drying afterdefatting was notsuitableforCB.

In group CBHA,themostvisibleboneregeneration processing towardsthe centreofthedefectwasobserved.Thebiocompatibility ofCBHA echoesthe findingsofanotherstudy(Chiroffetal.,1975).

In thestudy ofZegzula etal.(1997),Gray-leveldensity wasmeasured on thebasisofthestandard radiographs,and thepercentageofradiopacity was determinedwithacomputeralgorithm thatdividestheradiopaqueareaofthe

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defectby the totalarea ofthe measuring frame.Presently,the radiologic Gray-levelhistogram wasmeasuredforobjectiveassessmentoftheradiologic findingsusing ssimilarprinciple.TheGray-levelhistogram changein group CBHA during 12weekswasthehighest,indicating thatboneregeneration in this group is most active,corroborating the results ofradiologic analysis. However,there was no significant difference in the Gray-levelhistogram changes between groups CB1 and CB1bmp during 12 weeks.Evaluation of bone defectregeneration was measured objectively by Gray-levelhistogram. Theresultsupportedthatofapreviousstudy(Zegzula,1997).

Toconfirm theboneregeneration capacity,thedefectclosureofeach group was measured 4,8,and 12 weeks afterimplantation.The measurementof defectclosure has been used before (Jung etal.,2007;Lee etal.,2008b). According totheresultsofKim etal.(2008a),theamountofnewly-matured bone in the implanted materialwith rhBMP-2 was more than thatevident without rhBMP-2.In this study,after 8 weeks,group CB1bmp showed significantly higherresultthan group CBHA (p<0.05).Itis considered that the rhBMP-2 played a criticalrole in bone regeneration,butCB1 did not participate as the scaffold of rhBMP-2, considering the thickness of connectivetissue.ItisconsideredthatrhBMP-2containedin CB1wasfreed tothesurrounding bonetissuewhereitpromoted theboneregeneration,but rhBMP-2didnotspreadtotheotherimplantsites.Regardingoverallchanges ofdefectclosure,the bone regeneration capacity ofCHA was the highest, followed by CBHA.Becausethismeasurementofbonedefectregeneration is invasive,the radiologic Gray-levelhistogram willbe a valuable method in clinicaluse.Advantages of CB and CBHA include their read availability, inexpensivecost,unlimited quantity and soft-textured surface.However,they sufferfrom weak strength.Conversely,CB and CBHA are easy to process

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and are suitable for use in irregular bone defects like sealing bone wax. Considering thebiocompatibility ofCBHA,thepossibility thatBMP actsasa scaffold is expected.Furthermore,CBHA willbeableto beused by carrier orscaffold in stem cells.Itwasinconvenientnottohaveastudy ofCB2+ rhBMP-2.

Idealbonesubstitutesshould beresorbed with timeand replaced by newly formedbone(Parketal.,2008).In thisstudy,newly formedbonesandbone resorption were observed inside ofgraftmaterialin group CBHA 12 weeks after implantation.But,there was no new bone formation in groups CB1, CB1bmp,and CB2.Itisconsidered thatthe CBHA is a morebiocompatible materialin compactbone defectregeneration than CB1,CB1bmp,and CB2. However,furtherexperimentsarenecessary to find outin moredetailabout thesuitableabsorptivityofCBHA.

To apply the most effective scaffold of bone graft substitute, various preparations ofCB and differenttypes (such as powder,paste,granule)of CBHA implantationshouldbestudied.

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CONCLUSI

ONS

This study was conducted to evaluate the biocompatibility ofCB1,CB2, CBHA,andCHA using amousesubcutaneousimplantmodelandbonedefect regeneration with CB1,CB2,CB1bmp,CBHA,and CHA in arabbitcalvarial defectsmodel.Theefficiency wascomparedwithothercommonlyusedCHA. The findings ofthis study indicate thatCBHA is a safermaterialforuse insidethebody than CHA.CBHA hasamoreeffectiveosteoconduction than CB1,CB1bmp,and CB2.CBHA isavaluablebonegraftmaterialin compact bonedefectmodels.

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골대체재로써의 오적골에 대한 평가

(지도교수:정 종 태) 원 상 철 제주대학교 대학원 수의학과 골대체재는 지연유합이나 유합부전 그리고 골절술과 관절고정술 시 골편의 연 속성 확립이 필요한 경우 골절의 주요 결손부위를 채우는데 주로 활용되고 있다. 자가골을 대체할 수 있는 천연 골이식재의 대표적인 것이 calcium carbonate (CC)로 구성된 coral이며,갑오징어의 오적골(Cuttlebone,CB)또한 coral과 같은 천연 CC로 이루어져 있다.지금까지 오적골은 몇몇 연구자들에 의해 이종골이식 재로써의 가능성이 제시되어왔으나,대부분의 연구가 해면질골에서의 실험이었으 며,체계적인 생체적합성 검사 및 편평골에서의 골형성에 관한 보고는 없었다. 본 연구에서는 오적골의 다양한 전처리 후 직경 5㎜ 두께 2㎜의 형태로 가공하 여 생체적합성 검사와 rhBMP-2의 담체로써 골전도력과 골유도력을 평가하고자 하였다. 결합조직의 두께는 2,4주차 모두 CBHA군에서 가장 유의성 있게 얇았다 (p<0.05).RadiologicGray-levelhistogram의 측정에서는 4주차에서 CBHA군이 CHA군보다 유의성 있게 높게 나타났으며 (p<0.05),12주차에서는 CHA군의 변 화율이 가장 적었다.전체 12주 동안의 변화율에서는 CBHA가 가장 많은 변화를

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보였다.폐쇄율에 있어서는 4,8,12주차 모두 CHA군이 다른 군에 비해 유의성 있게 높게 나타났으며 (p<0.05),8주차에서는 CB1bmp군이 CBHA군보다 유의성 있게 높게 나타났다 (p<0.05). 이상의 결과들은 CBHA가 생체 내에 적용하는 골대체재로서 생체적합성이 매 우 높은 것으로 나타났으며,CB1,CB1bmp,CB2보다 골유도능력에서도 그 효과 가 우수한 것으로 나타났다.따라서 CBHA는 편평골에 있어 생체적합성이 뛰어 난 골대체재로 그 가치가 있는 것으로 생각되며,수의 임상에 있어서 활용성이 매우 높을 것으로 사료된다. 중심어;골대체제,담체,수산화인회석,오적골,탄산칼슘

수치

Tabl e 1.Expe r i ment alde s i gn f ort he as se s sme ntofbi oc ompat i bi l i t y oft he i mpl ant s i n mi c e ----------------------------------------------- 16
Fig  2.  Preparation  of  CB2  for  implantation.  The  CB2  implants  were  processed  through  removing  organic  components,  washing,  drying,  and  sterilization.
Fig  3.  CB1,  CB2,  CBHA,  and  CHA  implants  were  shaped  cylindrical  disks  about  5  ㎜  in  diameter  and  2  ㎜  in  thickness  and  were  sterilized  by  ethylene  oxide  gas.
Table  1.  Experimental  design  for  the  assessment  of  biocompatibility  of  the  implants  in  mice
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