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ISBN 978-951-42-9660-4 (Paperback)ISBN 978-951-42-9661-1 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)

U N I V E R S I TAT I S O U L U E N S I S

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Hanna T

ölli

OULU 2011

D 1136

Hanna Tölli

REINDEER-DERIVED BONE PROTEIN EXTRACT INTHE HEALING OF BONE DEFECTS

EVALUATION OF VARIOUS CARRIER MATERIALS AND DELIVERY SYSTEMS

UNIVERSITY OF OULU,FACULTY OF MEDICINE,INSTITUTE OF BIOMEDICINE,DEPARTMENT OF MEDICAL TECHNOLOGY,INSTITUTE OF CLINICAL MEDICINE,DEPARTMENT OF SURGERY,DIVISION OF ORTHOPAEDIC AND TRAUMA SURGERY

A C T A U N I V E R S I T A T I S O U L U E N S I SD M e d i c a 1 1 3 6

HANNA TÖLLI

REINDEER-DERIVED BONE PROTEIN EXTRACT IN THE HEALING OF BONE DEFECTSEvaluation of various carrier materials and delivery systems

Academic dissertation to be presented with the assent ofthe Faculty of Medicine of the University of Oulu forpublic defence in Auditorium 2 of Oulu UniversityHospital, on 9 December 2011, at 12 noon

UNIVERSITY OF OULU, OULU 2011

Copyright © 2011Acta Univ. Oul. D 1136, 2011

Supervised byProfessor Timo JämsäProfessor Pekka Jalovaara

Reviewed byProfessor Minna KellomäkiProfessor Jari Salo

ISBN 978-951-42-9660-4 (Paperback)ISBN 978-951-42-9661-1 (PDF)

ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)

Cover DesignRaimo Ahonen

JUVENES PRINTTAMPERE 2011

Tölli, Hanna, Reindeer-derived bone protein extract in the healing of bone defects.Evaluation of various carrier materials and delivery systemsUniversity of Oulu, Faculty of Medicine, Institute of Biomedicine, Department of MedicalTechnology, Institute of Clinical Medicine, Department of Surgery, Division of Orthopaedic andTrauma Surgery, P.O. Box 5000, FI-90014 University of Oulu, FinlandActa Univ. Oul. D 1136, 2011Oulu, Finland

Abstract

Various bone proteins and growth factors are needed during the bone healing cascade. If the bodycannot produce sufficient quantities of these factors, bone trauma healing can be improved withan implant that contains the required growth factors. However, an added bone protein extractneeds a suitable delivery system to protect the proteins from degradation and to release themgradually, promoting new bone formation. This study focused on evaluating and optimization ofthe bone forming capacity of various scaffold systems of reindeer bone protein extractformulations using different experimental models. The tested carrier systems for various reindeerbone protein extract doses were collagen sponge and bioactive glass granules in critical-size defectmodel of rat femur. Calcium salt compositions (beta-tricalcium phosphate (β-TCP),hydroxyapatite or calcium sulphate) in disc and compressed pellet forms were tested in the thighmuscle pouch model of mouse. Various β-TCP granules combined with polyethylene/glycerol andstearic acid gel were tested in a hole defect model of sheep femur and humerus. Control groupsinvolved carrier materials with no protein extract or untreated defects. In the sheep study,reference materials also included autograft and demineralized bone matrix (DBM). New boneformation, bone healing, and carrier resorption were evaluated based on radiographs, peripheralcomputerized tomography (pQCT), mechanical tests, histological examination, and micro-CT.New bone formation and bone union were markedly better in groups receiving higher doses of theextract and with follow-ups of six or more weeks, compared to empty defect or carrier withoutextract. Resorptions of carrier materials in active groups were faster and more active than in thecontrol groups. The greatest bone formation occurred in the groups that had the bone proteinextract readily available, which indicated that bone forming factors are required in sufficientconcentrations at an early stage. The micro-CT analysis showed that bone formation in the groupswith the extract was comparable to autograft, while the least bone formation was observed in theDBM and untreated groups. The present study indicated that the tested reindeer bone proteinextract can be used to improve bone formation with various carriers. The study suggests that aninorganic carrier material together with stearic acid is the one of most suitable carrier alternativesfor this extract. The developed medical device in paste form can be an alternative for autograft use.

Keywords: bone defects, bone extracts, calcium salts, carriers, fracture healing, growthfactors

Tölli, Hanna, Poron luuproteiiniuutteen käyttö luumurtumien hoitamisessa.Erilaisia kantaja-aine- ja implantointimahdollisuuksiaOulun yliopisto, Lääketieteellinen tiedekunta, Biolääketieteen laitos, Lääketieteen tekniikka,Kliinisen lääketieteen laitos, Kirurgia, Ortopedian ja traumatologian osastoryhmä, PL 5000,90014 Oulun yliopistoActa Univ. Oul. D 1136, 2011Oulu

Tiivistelmä

Luun paraneminen vaatii erilaisia proteiineja ja kasvutekijöitä. Jos elimistö ei pysty tuottamaanriittävää määrää näitä tekijöitä, luumurtuma ei parane luonnollisesti vaan vaatii hoitoa, jossamurtuma alueelle viedään tarvittavia kasvutekijöitä. Kasvutekijät kiinnitetään tarkoituksenmu-kaiseen kantaja-aineeseen, jonka avulla kasvutekijöiden vapautumista ja toimintaa voidaan sää-dellä. Tutkimuksen tavoitteena oli arvioida ja optimoida erilaisissa eläinmalleissa porosta eriste-tyn luuproteiiniuutteen kantaja-ainesysteemien toimivuutta. Testatut kantaja-ainemateriaalit oli-vat kollageenihuopa ja bioaktiivinen lasirae. Näitä testattiin useilla luuproteiiniannoksilla, jamallina oli rotan reisiluun kriittisen koon murtuma. Hiiren reisilihasmallilla testattiin kalsium-suolayhdistelmiä (beta trikalsiumfosfaatti (β-TCP), hydroksiapatiitti ja kalsiumsulfaatti) tabletti-na ja pellettinä. Lampaan reisi- ja olkaluiden reikämurtumamallilla testattiin erilaisia β-TCP-rakeita yhdistettynä polyetyleenistä, glyserolista ja steariinihaposta valmistettuun geeliin. Kont-rollisryhmistä toinen sisälsi kantaja-aineen ilman luuproteiiniuutetta, toisessa ryhmässä murtu-ma jätettiin kokonaan hoitamatta. Lammaskokeessa verrattiin lisäksi omaluusiirteen ja demine-ralisoidun luumateriaalin (DBM) toimivuutta verrattuna poron luuproteiiniuutteeseen. Uudis-luun muodostuminen, murtuman paraneminen ja kantaja-aineen hajoaminen arvioitiin natiivi-röntgenillä, perifeerisellä tietokonetomografialla (pQCT), mekaanisin testein, histologisesti jamikro-CT:llä. Luuproteiiniuutetta sisältäneillä ryhmillä oli luun paraneminen ja kantaja-aineenhajoaminen merkittävästi parempaa kuin uutetta sisältämättömillä ryhmillä. Uudisluun muodos-tuminen oli suurempaa korkeammilla annoksilla ja pitemmillä seuranta-ajoilla. Suurin uudis-luun muodostus mitattiin ryhmillä, joissa luuproteiiniuute oli heti käytettävissä implantoinninjälkeen. Tämä osoittaa, että varsinkin murtuman paranemisen alkuvaiheissa tarvitaan luuta muo-dostavia kasvutekijöitä riittävinä pitoisuuksina. Mikro-CT-analyysit osoittivat, että luuproteiini-uuttetta sisältäneet ryhmät olivat verrannollisia omaluusiirrehoidolle. Vähiten uudisluuta havait-tiin DBM ja tyhjäreikäryhmissä. Tutkimus osoittaa, että poron luuproteiiniuutetta erilaisten kan-tajamateriaalien kanssa voidaan käyttää parantamaan luun muodostumista. Erityisesti epäor-gaaninen kantajamateriaali steariinihapon kanssa on yksi soveltuvimmista vaihtoehdoista luu-uutteelle. Kehitelty pastamutoinen lääkinnällinen laite, joka sisälsi poron luuproteiiniuutteen jakalsiumsuolakantaja-aineen, osoittautui vaihtoehdoksi omaluusiirrehoidolle.

Asiasanat: kalsiumsuolat, kantaja-aineet, kasvutekijät, luu-uutteet, luupuutokset,murtuman paraneminen

7

Acknowledgements

This work was carried out in the Department of Medical Technology, Insitute of

Biomedicine, the Department of Surgery, Division of Orthopaedic and Trauma

Surgery, Insitute of Clinical Medicine, University of Oulu, and BBS-Bioactive

Bone Substitutes Oy during the years 2007-2011.

Several people have made a personal contribution to this work. I thank my

supervisors, Professor Timo Jämsä, PhD, and Professor (emeritus) Pekka

Jalovaara, MD, PhD, for your guidance and determined support of my study. You

have provided me excellent facilities for this thesis, and your experience has been

crucial in revising the studies and original papers. I want to thank my other co-

authors, Sauli Kujala, MD, PhD, Katri Merikanto (née Levonen), MSc (Pharm),

Kenneth Sandström, MSc (Eng), Professor Juhana Leppilahti, MD, PhD, and Elli

Birr, PhD, for their contribution to subworks of this thesis. Sauli, thanks for all

surgical advice and for helping with the surgical phases of rat and sheep work.

Katri, special thanks for your help with the bioactive glass implants. Kenneth, my

closest workmate, you have always given your help and support when I have

asked. Thanks for your knowledge and preparatory work with the implants, study

plans, and analysis. Juhana, thanks for encouraging and helping me during the

sheep study. Elli, your knowledge on histology and bone growth factors has been

very important during this study.

I am very grateful to the official referees of the thesis, Professor Minna

Kellomäki from Tampere University of Technology and Professor Jari Salo from

the University of Eastern Finland, for their valuable comments and revisions of

the thesis manuscript. I also wish to thank Anna Vuolteenaho (English), MA, and

Pirjo Kananen (Finnish), MA, for the revision of the languages of this thesis.

The crew at the Laboratory Animal Centre, Seija Seljänperä, RN, technical

researcher Veikko Lähteenmäki, Mrs Miia Vääräniemi, veterinarians Hanna-

Marja Voipio, PhD, and Sakari Laaksonen: Thank you for your expertise with

laboratory animals and your support during the study, also during the hard

moments.

Mr Risto Bloigu, from the Faculty of Medicine, is acknowledged for

statistical advice. I wish to thank my follow-up group members Professor Juha

Tuukkanen, and Professor Juhana Leppilahti for their support and for ideas

concerning this thesis.

I also want to thank my other workmates at BBS-Bioactive Bone Substitutes

Oy in Oulu, Akaa and Reisjärvi. You are experts on reindeer bone protein extract

8

and together we have solved many study problems and questions, and you have

supported me throughout these years that I have worked with you. Especially, I

want to thank the “lab girls”, Ms Heli Korkala, Ms Marja Juustila, Ms Matleena

Rentola, Ms Johanna Bräysy and Mrs Maria Riekki, for implant preparations and

for surgical and analytical help during this thesis. Furthermore, Heli and

Matleena, I really appreciate the “bed and breakfast and taxi services” that you

have given me when I have visited Oulu.

I wish to express my warmest gratitude to my parents Paula and Jouko, and to

my sisters and brothers and their families, and to my grandmother Annikki for

their love and support during my life. I want to dedicate this thesis to them.

Especially, dad, you have shown endless interest in and support for my studies.

Your expertise and help with big animals has encouraged me and our research

group to perform operations on sheep. I also want to thank my friends in the

Department of Medical Technology and outside academia for their support and

for the enjoyable times that you have given me without any research problem

worries.

I would also like to thank the Synthes Company and Mr Antero Enqvist for

providing Kirschner wires; and the Vivoxid Company and Ilkka Kangasniemi,

PhD, for providing bioactive glass for this study. The study was partly supported

by the Academy of Finland, the National Doctoral Programme of Musculoskeletal

Disorders and Biomaterials (TBDP), the Ahti Pekkala Foundation, and the

University of Oulu Support Foundation (Kerttu Saalasti Foundation). I wish to

thank all of these for supporting this study.

9

Abbreviations

BMP Bone morphogenetic protein

CS Calcium sulphate

CSD Critical-size defect

cDNA Complementary deoxyribonucleic acid

DBM Demineralized bone matrix

FDA Food and Drug Administration

FGF Fibroblast growth factor

GDF Growth and differentiation factor

GuHCl Guanidine hydrochloride

HA (HAP) Hydroxyapatite

HCl Hydrochloric acid

IGF Insulin-like growth factor

IL Interleukin

IM Intramuscular

IV Intravenous

MSC Mesenchymal stem cell

μCT Micro computerized tomography

OP-1 Osteogenic protein-1

ORIF Open reduction and internal fixation

PDGF Platelet-derived growth factor

PEG Polyethylene glycol

PGA Polyglycolic acid

PLA Polylactic acid

PLGA Polylactic-co-glycolic acid

PMMA Polymethyl methacrylate

pQCT Peripheral quantitative computerized tomography

rhBMP Recombinant human bone morphogenetic protein

SEM Scanning electron microscope

TCP Tricalcium phosphate

TGF-β Transforming growth factor beta

TNF-α Tumour necrosis factor alpha

VEGF Vascular-endothelial growth factor

10

11

List of original articles

This thesis is based on the following articles, which are referred to in the text by

their Roman numerals (I-IV).

I Tölli H, Kujala S, Jämsä T & Jalovaara P (2011) Reindeer bone extract can heal the critical-size rat femur defect. Int Orthop 35(4): 615–622.

II Tölli H, Kujala S, Levonen K, Jämsä T & Jalovaara P (2010) Bioglass as a carrier for reindeer bone protein extract in the healing of rat femur defect. J Mater Sci Mater Med 21(5): 1677–1684.

III Tölli H, Birr E, Sandström K, Jämsä T & Jalovaara P (2011) Calcium sulfate with stearic acid as an encouraging carrier for the reindeer bone protein extract. Materials 4(7): 1321–1332.

IV Tölli H, Kujala S, Birr E, Leppilahti J, Jämsä T & Jalovaara P (2011) Bone formation performance of reindeer bone protein extract formulations, autograft and demineralized bone matrix in sheep hole defect model. Manuscript.

12

13

Contents

Abstract

Tiivistelmä

Acknowledgements 7 Abbreviations 9 List of original articles 11 Contents 13 1 Introduction 15 2 Review of the literature 17

2.1 The principles of the fracture healing ..................................................... 17 2.2 Growth factors and signalling molecules in the bone healing

cascade .................................................................................................... 18 2.2.1 Bone morphogenetic proteins ....................................................... 22 2.2.2 Recombinant BMPs ...................................................................... 24

2.3 Bone grafting and bone-graft substitute .................................................. 25 2.3.1 Autograft and allograft ................................................................. 26 2.3.2 Demineralized bone matrix .......................................................... 27 2.3.3 Reindeer-derived bone protein extract .......................................... 28 2.3.4 Synthetic materials ....................................................................... 29

2.4 Carriers of bone protein extract ............................................................... 30 2.4.1 Criteria of ideal carrier ................................................................. 30 2.4.2 Collagen ....................................................................................... 31 2.4.3 Bioactive glass .............................................................................. 32 2.4.4 Calcium salts ................................................................................ 34 2.4.5 Carriers of reindeer bone protein extract ...................................... 36

2.5 Experimental models ............................................................................... 38 2.5.1 Rat segmental defect model .......................................................... 38 2.5.2 The model of ectopic bone formation ........................................... 40 2.5.3 Sheep hole defect model ............................................................... 41

3 Aims of the study 43 4 Materials and methods 45

4.1 Animals ................................................................................................... 48 4.2 The reindeer bone protein extract ........................................................... 48 4.3 Reference materials ................................................................................. 49 4.4 Carriers .................................................................................................... 49 4.5 Preparation of implants ........................................................................... 51

14

4.5.1 Reconstitution of collagen implants (I) ........................................ 51 4.5.2 Reconstitution of bioactive glass implants (II) ............................. 51 4.5.3 Reconstitution of calcium salt implants (III) ................................ 51 4.5.4 Reconstitution of β-TCP implants (IV) ........................................ 51 4.5.5 Reconstitution of autograft implants (IV) ..................................... 52 4.5.6 Control implants ........................................................................... 52

4.6 Surgical methods ..................................................................................... 52 4.6.1 Critical-size rat femur defect study (I, II) ..................................... 52 4.6.2 Mouse muscle pouch model (III) .................................................. 54 4.6.3 Sheep model study (IV) ................................................................ 55

4.7 Analysis methods .................................................................................... 56 4.7.1 Radiographic evaluation (I, II, III) ............................................... 57 4.7.2 Computed tomography (I, II) ........................................................ 57 4.7.3 Micro-CT (IV) .............................................................................. 57 4.7.4 Biomechanical testing (I, II) ......................................................... 58 4.7.5 Histological examination (I, III) ................................................... 58 4.7.6 Statistics ........................................................................................ 59

5 Results 61 5.1 Dosing of the reindeer bone protein extract (I, II)................................... 61 5.2 Collagen and bioactive glass as carrier materials (I, II) .......................... 65 5.3 Bone formation capacity with calcium salt carrier materials (III) ........... 66 5.4 Bone formation of the formulated bone protein extract in

inorganic carrier (IV) .............................................................................. 68 6 Discussion 73

6.1 Time and dose-dependent effects of the reindeer bone protein

extract ...................................................................................................... 73 6.2 Experimental models ............................................................................... 75 6.3 From carrier of the reindeer bone protein extract to an ideal

implant .................................................................................................... 76 6.4 Calcium salts as potential carrier candidates ........................................... 77

7 Conclusions 81 References 83 Original articles 107

15

1 Introduction

Native bone contains growth and differentiation factors and signalling molecules

such as bone morphogenetic proteins (BMPs) and other proteins that are

important in regeneration of bone and cartilage (Cho et al. 2002, Cheng et al. 2003, Xiao et al. 2007, Dimitriou et al. 2005). These factors and molecules and

their different concentrations are also needed in different phases during the whole

fracture healing process (Cho et al. 2002, Dimitriou et al. 2005). If the body

cannot produce sufficient quantities of these factors, bone trauma healing can be

improved with an implant that contains the required growth factors (Dimitriou et al. 2005, Pekkarinen et al. 2006, Baas et al. 2008). However, as a treatment of

bone fracture, added bone protein extract needs a suitable delivery system or

carrier to guarantee a gradual release during new bone formation (King et al. 1998).

The techniques used to repair bone defects are important, and when an area of

damaged bone is too large for self-repair, the repairing must be done by using

alternative materials. Autograft is the traditional method of bone repair

enhancement, but harvest site bleeding, pain, and infection are known issues

(Arrington et al. 1996, Finkemeier 2002). Autografts also have limited

availability, and many inorganic materials are used as an alternative (Betz 2002).

The quality of autograft in old people has been discussed. Calcium phosphates

such as hydroxyapatite and tricalcium phosphates and their variations are

commonly known materials (Moore et al. 1987, Li & Wozney 2001, Niedhart et al. 2003, Barrack 2005, Douglas et al. 2009). These materials provide an

osteoconductive scaffold to new bone formation (Carey et al. 2005, Peters et al. 2006, Baas et al. 2008). Bioactivity of inorganic materials can be increased by

adding osteogenic stimulus to the bone graft extender (Alam et al. 2001, Barrack

2005, Kim et al. 2005, Baas et al. 2008, Reichert et al. 2011).

A mixture of BMPs, growth factors and other bone proteins and molecules

has been extracted from the bone materials of a variety of animal species, humans

and bone tumours. Reindeer bone protein extract has been shown to be an

effective stimulator of new bone formation in a muscle pouch model in mice

(Jortikka et al. 1993a, Pekkarinen et al. 2003, 2004, 2005a,b,c). Furthermore, the

good healing capacity of reindeer bone extract in a segmental bone defect was

previously demonstrated in the rabbit (Pekkarinen et al. 2006).

Reindeer bone protein extract has high bone formation activity. However, in a

real bone healing situation, the extract cannot work without an appropriate carrier

16

that retains bone proteins and growth factors at the defect site for a prolonged

time frame, providing an initial support to which cells attach and form new bone

(Seeherman & Wozney 2005). The knowledge about the optimal formulation

system for bone protein extracts is limited. Despite modern surgical techniques,

segmental long bone defects are often difficult to manage. For this reason,

animals with segmental long bone defects similar to clinical defects have been

used as models for assaying the efficacy of bone protein extract and carrier

materials. The bone healing properties of reindeer bone extract have been

previously evaluated in the rabbit radius (Pekkarinen et al. 2006). However, the

time and dose-dependent effects of reindeer bone extract have not been evaluated

previously.

This study focused on evaluation and optimization of the bone forming

capacity of various organic and inorganic scaffold systems of reindeer bone

protein extract formulations. Bone healing capacity and new bone formation were

studied in various experimental models. The critical-size defect model of rat was

used to show trauma healing capacity of reindeer bone protein extract with

collagen sponge and bioactive glass granule carriers. Furthermore, various doses

of extract and follow-ups were tested in rat model studies. The muscle pouch

model in mice and the hole defect model in sheep were the methods used when

evaluating the suitability of calcium-based carrier materials with different

formulations of reindeer bone protein extract.

17

2 Review of the literature

2.1 The principles of the fracture healing

Fracture healing is a complex, but well orchestrated, physiological process.

During the repair process, injured bone can be reconstituted almost identically to

its original shape. The bone marrow, the cortex, the periosteum, and the external

soft tissues contribute in the healing process, but their role and activity depends

on the type of fracture, its location and multiple other parameters, such as growth

and signalling factors, pH, hormones as well as the way the fracture is treated

(Einhorn 1998, Dimitrou et al. 2005, Giannoudis et al. 2007).

The process of fracture healing can be divided into primary and secondary

healing. Primary cortical healing is typical when rigid internal fixation is used to

stabilize a fracture. This healing process means that bone-resorbing cells on one

side of the fracture provide tunnelling to the other side of the cortex, whereby

they re-establish new haversian systems by the formation of demodelling units,

known as cutting cones. New blood vessels are accompanied when

osteoprogenitor cells become osteoblasts. Little or no response of the bone

marrow, periosteum or external soft tissue is noted and thus, no callus formation

is seen. (Schatzker et al. 1989, Einhorn 1998, 2005). The majority of fractures

heal by secondary or indirect bone healing. This process involves responses in the

periosteum and external soft tissue, and it is thus a combination of

intramembranous and endochondral ossification that follows a specific time

sequence (Einhorn 1998, 2005).

A haemorrhage is typically seen in the first phase in the injured area because

of the rupture of blood vessels, which is soon followed by the formation of a clot.

This phase is also called inflammation stage as inflammatory cells invade the

injured area. These cells together with the haematoma are a source of signalling

molecules and cytokines that regulate the early events of the healing process

(McKibbin 1978, Leeson et al. 1988: 168–189, Einhorn 1998, 2005, Dimitrou et al. 2005). Clinically, this phase is associated with pain, swelling and heat and

lasts about two to three weeks (Slutsky & Herman 2005). A chondrogenesis

process is seen during the first 7 to 10 days of fracture healing. This leads to

cartilage formation or hard callus formation where intramembranous ossification

is taking place. The cells produce mainly type III collagen in this phase (Aro &

Kettunen 2010: 218-221). Two weeks after fracture, cell proliferation declines

18

and the mass of cartilage calcifies. The chondrocytes release phosphatases and

proteases enzymes. The phosphatases precipitate delivering of the calcium from

the mitochondria of the hypertrophic chondrocytes and form calcified cartilage.

The proteases control the rate of the mineralization process. Histologically, type II

collagen is mainly seen in this phase (Aro & Kettunen 2010: 281-221). This phase

corresponds to the time when clinical union is achieved (Slutsky & Herman

2005). It is called as soft callus stage and lasts until four to eight weeks after the

injury. Between four and eight weeks, the new bone begins to bridge the fracture.

This bony bridge can be also seen on x-rays. The resorption cells of the cartilage,

chondroclasts, become more active and degrade the calcified cartilage. This is

also a signal for the ingrowth of blood vessels that bring with them perivascular

mesechymal stem cells (MSCs). These cells differentiate into osteoprogenitor

cells and then into osteoblast cells to form new bone. This phase can be seen as

the expression of type I collagen (Aro & Kettunen 2010: 218-221). The creation

and mineralization of callus takes several weeks within new bone fills the

fracture. (Slutsky & Herman 2005). Beginning about 8 to 12 weeks after the

injury, osteoclast cells remodel and replace the callus with functionally competent

lamellar bone structure. The fracture site remodels itself, correcting any

deformities that may remain as a result of the injury. This final phase of the

fracture healing can last up to several years. (Einhorn 1998, 2005, Slutsky &

Herman 2005).

2.2 Growth factors and signalling molecules in the bone healing cascade

Native bone contains pro-inflammatory cytokines, the transforming growth factor

beta (TGF-β) superfamily and other growth factors, and angiogenic factors (Cho

et al. 2002, Gerstenfeld et al. 2003). These factors and molecules and their

specific concentrations are required during the various phases of skeletal tissue

formation and the entire fracture healing process (Cho et al. 2002, Dimitriou et al. 2005, Tsiridis et al. 2007) (Table 1).

19

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Dim

itrio

u et

al.

2005

BM

Ps:

BM

Ps

2-8,

GD

F-1,

-5, -

8, -1

0

Ext

race

llula

r mat

rix,

Ost

eobl

asts

,

Ost

eopr

ogen

itors

,

MS

Cs,

Cho

ndro

cyte

s

Day

s 1

to 2

1

(Tab

le 2

)

Diff

eren

tiatio

n of

MS

Cs

into

chon

droc

ytes

and

ost

eobl

asts

and

oste

opro

geni

tors

into

ost

eobl

asts

Cho

et a

l. 20

02,

Che

ng e

t al.

2003

,

Dim

itrio

u et

al.

2005

,

He

2005

FGFs

M

onoc

ytes

,

Mac

roph

ages

,

MS

Cs,

Cho

ndro

cyte

s

Day

s 1

to 2

1 A

ngio

gene

sis,

Mes

ench

ymal

cel

l mito

gene

sis

Sol

heim

199

8,

Hug

hes-

Fulfo

rd &

Li 2

011

20

Ess

entia

l sig

nalli

ng

mol

ecul

es a

nd g

row

th fa

ctor

s

Sou

rce

Tim

e of

exp

ress

ion

Maj

or fu

nctio

ns d

urin

g th

e fra

ctur

e

heal

ing

Ref

eren

ce

IGFs

:

IGF-

I, IG

F-II

Bon

e m

atrix

,

MS

Cs,

Ost

eobl

asts

Cho

ndro

cyte

s

IGF-

I: D

ays

1 to

21

IGF-

II: D

ays

7 to

14

Bon

e m

atrix

form

atio

n

End

ocho

ndra

l oss

ifica

tion

Pris

ell e

t al.

1993

,

Lieb

erm

an e

t al.

2002

,

Dim

itrio

u et

al.

2005

,

Met

allo

prot

eina

ses

Ext

race

llula

r mat

rix

Day

s 3

to 2

1 Th

e in

vasi

on o

f blo

od v

esse

ls

Ger

sten

feld

et a

l. 20

03,

Ai-A

ql e

t al.

2008

VE

GFs

M

SC

s D

ays

14 to

21

End

othe

lial c

ell p

rolif

erat

ion

Stre

et e

t al.

2002

,

Will

ems

et a

l. 20

11

Ang

iopo

ietin

-1,

Ang

iopo

ietin

-2

MS

Cs

Day

s 3

to 2

1 La

rger

ves

sel s

truct

ures

form

atio

n S

uri e

t al.

1998

,

Ger

sten

feld

et a

l. 20

03,

Dim

itrio

u et

al.

2005

IL =

Inte

rleuk

in, T

NF

= Tu

mou

r nec

rosi

s fa

ctor

, TG

F =

Tran

sfor

min

g gr

owth

fact

or, P

DG

F =

Pla

tele

t-der

ived

gro

wth

fact

or, M

SC

= M

esen

chym

al s

tem

cel

l,

BM

P =

Bon

e m

orph

ogen

etic

pro

tein

, GD

F =

Gro

wth

and

diff

eren

tiatio

n fa

ctor

, FG

F =

Fibr

obla

st g

row

th fa

ctor

, IG

F =

Insu

lin-li

ke g

row

th fa

ctor

, VE

GF

=

Vas

cula

r-en

doth

elia

l gro

wth

fact

or

21

Cytokines such as Interleukin-1 (IL-1), Interleukin-6 (IL-6) and tumour necrosis

factor-alpha (TNF-α) play a role especially in initiating the bone repair cascade.

Inflammatory cells, macrophages and cells of mesenchymal origin secret

cytokines during the first three days of the bone healing cascade as well as during

the bone remodelling (Kon et al. 2001, Dimitriou et al. 2005, Ai-Aql et al. 2008).

The most important tasks of the cytokines are chemotactic effect on other

inflammatory cells, stimulation of extracellular matrix synthesis, and

angiogenesis (Kon et al. 2001, Cho et al. 2002, Ai-Aql et al. 2008).

The TGF-β superfamily is a large group of various growth and differentiation

factors including bone morphogenetic proteins (BMPs), TGF-β, growth

differentiation factors (GDFs), activins and inhibins (Burt & Law 1994, Cho et al. 2002). These factors promote facture healing in many ways. TGF-β has various

roles from the inflammatory phase throughout the whole fracture healing process.

Its main sources are degranulating platelets, inflammatory cells, extracellular

matrix, chondrocytes and osteoblasts, and its main role is to work during

chondrogenesis and endochondral bone formation (Bonewald & Mundy 1990,

Solheim 1998, Cho et al. 2002). It induces production of extracellular proteins

such as collagen and alkaline phosphatase and enhances proliferation of MSCs,

chondrocytes and osteoblasts (Bonewald & Mundy 1990, Erlebacher et al. 1998,

Solheim 1998, Lieberman et al. 2002).

Platelet-derived growth factor (PDGF) is a dimer of two peptides and

released by degranulating platelets, macrophages, monocytes and osteoblasts at

the very early stages of fracture healing (Andrew et al. 1995, Solheim 1998).

PDGF is a stimulator for inflammatory cells and MSCs and osteoblasts

(Lieberman et al. 2002). During the early phases of bone healing, until osteoblast

formation, fibroblast growth factor (FGF) polypeptides play a critical role in

angiogenesis and mesenchymal cell mitogenesis (Hughes-Fulford & Li 2011,

Willems et al. 2011). Monocytes, macrophages, mesenchymal cells, osteoblasts

and chondrocytes synthesize these factors (Solheim 1998, Lieberman et al. 2002).

Furthermore, insulin-like growth factors (IGFs) are important factors having their

source in the bone matrix, endothelial and mesenchymal cells, osteoblasts and

non-hyperthropic chondrocytes (Solheim 1998). IGF-I is regulated by the growth

hormone and it promotes bone matrix formation throughout fracture healing

(Lieberman et al. 2002, Dimitriou et al. 2005). IGF-II stimulates type I collagen

production, cartilage matrix synthesis and cellular proliferation in the later stage

of endochondral bone formation (Prisell et al. 1993).

22

Optimal bone healing and regeneration requires adequate blood flow. Specific

metalloproteinases from the extracellular matrix degrade cartilage and bone,

allowing the invasion of blood vessels during the final stages of endochondral

ossification and bone remodelling (Gerstenfeld et al. 2003, Ai-Aql et al. 2008).

Vascular-endothelial growth factors (VEGFs) are essential stimulators of

endothelial cell proliferation and are expressed during endochondral formation

and bone formation (Street et al. 2002, Willems et al. 2011). Angiopoietin 1 and 2

are needed in formation of larger vessel structures and development of co-lateral

branches from existing vessels during the early stages throughout fracture healing

(Suri et al. 1998, Gerstenfeld et al. 2003, Dimitriou et al. 2005).

2.2.1 Bone morphogenetic proteins

In an osteoinduction process some osteoinductive agents or signals (bone graft

substitutes, generally proteins) stimulate primitive, undifferentiated and

pluripotent cells to develop a bone-forming cell lineage and to enable the

remodelling of new bone tissue (Parikh 2002). In 1938, Levander found that acid

alcohol-extracted bone induced heterotopic cartilage and bone growth when

injected intramuscularly. Decades later, in 1965, Urist confirmed that

demineralized bone matrix (DBM) has good osteoinductive capacity because this

matrix consists among others of bone morphogenetic proteins (BMPs) that are

growth factors and belong to the TGF-β superfamily. The pure form of BMPs is

not immunogenic and species-specific (Parikh 2002). BMPs enable skeletal tissue

formation during embryogenesis, growth, adulthood, and trauma healing (Kirker-

Head 2000). They constitute a large family of non-collagenous proteins, which

are osteoinductive but can also have effects outside of bone tissue (Jaatinen et al. 2002, Reddi 2005, Barrientos et al. 2008, Corradini et al. 2010). Therefore, BMPs

are also called body morphogenetic proteins (Reddi 2005).

More than twenty BMPs have now been described and about ten of them

have functions in different phases of bone healing (Wozney & Rosen 1998, Cheng

et al. 2003) (Table 2). Members of the BMP family are divided into different

subgroups depending on their primary amino acid sequence. Group one includes

BMP-2 and BMP-4, and group two consists of BMP-5, -6, and -7 (Celeste et al. 1990, Wozney 1992). The third group includes BMP-12 (or GDF-7), BMP-13

(GDF-6) and BMP-14 (GDF-5). Group four includes BMP-3 (or osteogenin) and

BMP-3b (GDF-10) (Sakou 1998, Dimitriou et al. 2005). BMP-1 is a member of

the astacin family and it plays a role in modulating BMP actions by binding and

23

antagonizing the actions of BMP-2 and BMP-4 (Wozney et al. 1990, Sarras

1996).

Table 2. Major functions of important BMPs during the fracture healing.

BMP Time of expression Major functions during the fracture healing Reference

GDF-8 Day 1 Negative regulator of skeletal muscle

growth

Cho et al. 2002

BMP-2 Days 1 to 21 Initiation of the healing cascade

Regulation of the expression of other

BMPs

Osteogenesis

Mineralization

Cheng et al. 2003

He 2005

Tsuji et al. 2006

Hughes-Fulford & Li

2011

BMP-3, -8 Days 14 to 21 Regulation of osteogenesis Cho et al. 2002

Cheng et al. 2003

BMP-4 6 h to day 5 Formation of callus Bostrom et al. 1996

Lammens et al. 2009

BMP-7 Days 14 to 21 Ossification Cho et al. 2002

Cheng et al. 2003

GDF-10, BMP-5 Days 3 to 21 Ossification Cho et al. 2002

BMP-6, BMP-9 Days 3 to 21 Ossification

Chondrocyte maturation

Cho et al. 2002

GDF-5 Days 7 to 14 Chondrogenesis Cho et al. 2002

The bone extracellular matrix, osteoblasts, osteoprogenitors, mesenchymal cells,

and chondrocytes are the main sources of BMPs. Even though BMPs are

structurally and functionally closely related, each of them has a unique role

during the fracture repair process. BMP-2 initiates fracture healing and regulates

the expression of other BMPs. It has a role in chondrogenesis and together with

BMP-6 and BMP-9 it induces osteoblast differentiation of MSCs (Cheng et al. 2003, He 2005). A second higher concentration of BMP-2 is seen during

osteogenesis or the phase of mineralization (Cheng et al. 2003, Tsuji et al. 2006,

Hughes-Fulford & Li 2011). The main difference between BMP-2 and BMP-9 is

seen in heterotopic bone formation: BMP-9 induces new bone only in damaged

24

soft tissue, whereas BMP-2 promotes bone formation in skeletal muscle (Leblanc

et al. 2011). BMP-3 and BMP-8 have a role in the regulation of osteogenesis at

the end of fracture healing (Cho et al. 2002, Cheng et al. 2003). BMP-4 has a

main role in the formation of callus at a very early stage in the bone healing

process (Bostrom et al. 1996, Lammens 2009). BMP-7 has a regulatory role both

in direct and indirect ossification, especially towards the end of these processes

(Cho et al. 2002, Cheng et al. 2003). BMP-5, BMP-6 and GDF-10 have the same

role as BMP-7 but during the entire fracture healing process (Cho et al. 2002).

BMP-6 can initiate chondrocyte maturation (Cho et al. 2002, Huang et al. 2005).

Expressions of GDF-5 and GDF-1 are seen during the chondrogenic phase

because they induce angiogenesis through chemotaxis of endothelial cells and

degrade matrix proteins (Cho et al. 2002). Furthermore, some BMPs can

stimulate the synthesis of other bone and angiogenic growth factors (Deckers et al. 2002).

2.2.2 Recombinant BMPs

The development in molecular biotechnology allowed the purification of bone-

inducing proteins (Sampath & Reddi 1981). The first three successfully isolated

and characterized polypeptides were named BMP-1, BMP-2 and BMP-3 (Wang et al. 1988). Transfection of the full-length complementary DNAs led to the

production of the first recombinant human bone morphogenetic proteins

(rhBMPs) (Wozney et al. 1988). Today, recombinant BMPs are usually produced

by two expression systems that are mammalian cells or bacteria (Bessa et al. 2008a). rhBMPs have been used to induce bone in many animal and clinical

experiments, and some commercial products have also been manufactured (de

Biase & Capanna 2005, McKay et al. 2007, White et al. 2007). Especially, the

bone healing capacity of rhBMP-2 has been well documented, and it has been

given approval by the European Union and the United States Food and Drug

Administration (FDA) (Govender et al. 2002, Burkus et al. 2002, Einhorn et al. 2003, Kim et al. 2005, Seeherman et al. 2006, Chu et al. 2007, McKay et al. 2007, Issa et al. 2008). Furthermore, rhBMP-7 (or ostegenic protein, OP-1) is

commercially available and both the European Union and FDA-approved for

repairing tibia fractures (White et al. 2007).

25

2.3 Bone grafting and bone-graft substitute

In the reconstruction of skeletal defects, it is common to transplant cancellous or

cortical bone to restore skeletal integrity and to enhance bone healing. Especially,

segmental bone defects due to infection or trauma are difficult to manage without

invoking the fracture healing cascade by transplanting bone to the defect. The

materials used in bone grafting can be broadly divided into autologous or

allogeneic cancellous or cortical bone, DBM, autologous bone marrow and

synthetic materials, and combinations of these (Bauer & Muschler 2000,

Finkemeier 2002, Van der Stok et al. 2011).

The differences between various bone grafts lie in their properties. Grafts can

be osteoinductive, osteoconductive, osteogenetic, or have two or more properties.

BMPs and other growth factors are primarily osteoinductive because they have

the capacity to stimulate development of new bone. The concept of

osteconduction means that bone grows on a surface. Osteoconductive material

itself has a weak capacity to form new bone; it is more like a support scaffold.

When this kind of material is placed in an osseous environment, living cells of the

host tissue can migrate into the scaffold, resulting in new bone formation and

promoting bone bridge formation in the defect. Synthetic materials are typically

osteoconductive. Combination of osteoconductive and osteoinductive is typical

for DBMs with carrier. DBMs have a weak osteoinduction capacity to new bone

formation alone but together with an osteoconductive carrier they are more

applicable for bone defect healing. Autografts and bone marrow cells are both

osteoconductive and osteogenetic materials with surviving pre-osteoblasts and

osteoblasts that have capacity to form new bone. (Bauer & Muschler 2000,

Finkemeier 2002, Schroeder & Mosheiff 2011).

However, there is no single method available to treat all kinds of bone healing

problems. All three abovementioned components (osteogenesis, osteoinduction

and osteoconduction) are needed in fracture healing (Schroeder & Mosheiff

2011). Furthermore, clinically, it is very important to take into account the injured

site in the skeleton (such as the spine, cranium, pelvis, ribs, limbs), fracture type,

form and size, stability of the fracture (Giannoudis et al. 2007), possible

infections in the trauma site, age of the patient, and patient-related factors

(smoking history, medications) when selecting the most suitable bone void filler

(Salo 2011).

26

2.3.1 Autograft and allograft

Bone-grafting with autologous cortical and cancellous bone harvested from the

iliac crest is the standard and traditional way to enhance bone repair (Pape et al. 2010). Autogenous grafts have been shown to be superior to allografts in many

studies, as in studies on bone remodelling and bone healing rate (Friedlaender

1987, Goldberg & Stevenson 1987, Gross et al. 1991, Kienapfel et al. 1992,

Johnson & Stein 1988, Virolainen et al. 1993, Athanasiou et al. 2010). Other

advantages of autografts are histocompatibility and low risk of transmitting

diseases. However, harvesting of bone grafts can lead to complications, such as

bleeding, pain, and infection (Arrington et al. 1996, Finkemeier 2002).

Furthermore, autografts have limited availability, especially in massive segmental

bone loss.

Limited availability of autografts has resulted in widespread use of allografts

with variable biologic properties. DBMs, morselized and cancellous chips,

cortical grafts, osteochondral and whole-bone segments are typical preparations

of allogeneic bone (Finkemeier 2002, Athanasiou et al. 2010). Bone banking

allows allograft bone to be widely used in clinical orthopaedics (Tomford &

Mankin 1999, Cornu et al. 2010). Allografts, DBMs and other bone extracts have

been shown to increase bone-healing capacity and to enhance integration in many

different studies (Einhorn et al. 1984, Peters et al. 2006, Pekkarinen et al. 2006,

Huffer et al. 2007, Baas et al. 2008, Freilich et al. 2009, Athanasiou et al. 2010).

Morselized and cancellous allografts are often preserved by freeze-drying and

vacuum-packing, which are associated with reduced immunogenicity, and in the

latter case the mechanical strength of the graft is decreased (Friedlaender 1983,

Pelker et al. 1984, Wolfe & Cook 1994, Cornu et al. 2010). Morselized and

cancellous allograft is typically used to fill larger defects and it provides some

mechanical support (Finkemeier 2002). Osteochondral and cortical allografts are

harvested from various regions of the skeleton and are available as whole-bone or

joint segments for limb salvage procedures and for filling bone defects

(Finkemeier 2002). These grafts are osteoconductive and provide good structural

support.

Allograft bone is also not without problems. It is associated with the risk of

viral diseases, and it may otherwise cause immunological reactions that interfere

with the bone healing process (Burchardt 1983, Stevenson 1987, Kinney et al. 2010). Thus, deep-frozen and sterilized allografts are more optimal than fresh

allografts (Finkemeier 2002, Cornu et al. 2010).

27

2.3.2 Demineralized bone matrix

Demineralized bone matrix (DBM) is prepared by an acid extraction process,

resulting in loss of most of mineralized component but retention of collagen, non-

collagenous proteins and other growth factors present in the extracellular matrix

(Urist & Dawson 1981, Reddi & Huggins 1972). Thus, DBM acts as an

osteoconductive material but it does not offer structural support. DBMs are

widely used to enhance fusion of the spine (Thalgott et al. 2001, Vaccaro et al. 2007, Weinzapfel et al. 2008). Furthermore, applications of DBM are grafting of

nonunion, ostelytic lesions around total joint implants, and benign bone cysts

(Whiteman et al. 1993, Tiedeman et al. 1995, Hass et al. 2007, Feng et al. 2011).

DBM is also typically used together with autologous bone as a volume extender

(Finkemeier 2002, Feng et al. 2011).

Various human DBM products are commercially available, both in Europe

and in the USA, as a bone graft substitute, bone graft extender, and bone void

filler in bony voids or gaps of the skeletal system (Finkemeier 2002, Barrack

2005, Engelhardt 2008). However, there are no studies on DBM use alone in

humans to show DBM as an excellent bone grafting material (Kinney et al. 2010).

Typical forms of human DBM products are a freeze-dried powder, crushed

granules or chips, gel and paste, and combined with other components such as

sodium hyaluronate, glycerol, or calcium phosphate (Peterson et al. 2004,

Reynolds et al. 2007, Athanisiou et al. 2010). There are some studies in the

literature in which bone-healing differences between various commercially

available DBM products have been found (Peterson et al. 2004, Wildemann et al. 2007). Furthermore, DBM has much lower bone forming effect compared with

the recombinant product (Bomback et al. 2004).

Animal DBMs have been also used in many animal and clinical experiments

(Einhorn et al. 1984, Wang et al. 1988, Solheim et al. 1992, Feighan et al. 1995,

Kloss et al. 2004, Kujala et al. 2004, Walboomers & Jansen 2005, Li et al. 2006,

Wang et al. 2007, Huffer et al. 2007, Baas et al. 2008, Zou et al. 2008, Murugan

et al. 2008, Athanasiou et al. 2010). Bovine DBM is the most frequently used in

experimental studies because of its good availability and bone forming activity

(Wang et al. 1988, Murugan et al. 2008, Athnasiou et al. 2010). Collos® DBM

products may be the most well-known bovine-derived DBMs (Kloss et al. 2004,

Walboomers et al. 2005, Li et al. 2005, Li et al. 2006). However, the risk of

disease transmission of bovine materials is an issue of discussion in clinical

applications. To overcome this problem, a similar other animal-derived DBMs are

28

also prepared such as equine DBM (Collos® E, by Ossacur AG) (Huffer et al. 2007, Baas et al. 2008, Nienhuijs et al. 2009) or a porcine DBM product (Altis

OBM) from Altis Biologics Ltd.

2.3.3 Reindeer-derived bone protein extract

The fracture healing cascade needs a mixture of different BMPs, growth and

signalling factors and other proteins (Dimitriou et al. 2005). This kind of natural

cocktail can be obtained from the bone by a specific extraction method. The

extraction process has many steps involving demineralization of bone material in

acid (typically HCl), extraction of non-collagenous proteins, filtration,

concentration, dialysis, freeze-drying and sterilization (Jortikka et al. 1993a). The

natural bone proteins have been extracted from the demineralized bone materials

of a variety of animal species as cow, pig, moose, sheep and reindeer, from

humans and from bone tumours and dentin matrix (Sampath & Reddi 1983,

Nilsson et al. 1986, Ko et al. 1990, Bessho et al. 1991a, Jortikka et al. 1993a,b,

Viljanen et al. 1996, Oksanen et al. 1998, Laitinen et al. 1997).

Reindeer bone protein extract has been shown to be an effective stimulator of

new bone formation in a muscle pouch model in mice (Jortikka et al. 1993a,

Pekkarinen et al. 2003, 2004, 2005a,b,c). Furthermore, the good healing capacity

of reindeer bone extract in a segmental bone defect was previously demonstrated

in the rabbit (Pekkarinen et al. 2006).

The ability of reindeer bone extract to heal various bone traumas has been

observed to be good, better than that of for example bovine or ostrich extract,

which has been suggested as being due to the fact that reindeer renew their antlers

annually (Jortikka et al. 1993b, Kapanen et al. 2002, Ulmanen et al. 2005).

Furthermore, it has been suggested that more of the protein material extracted

from reindeer bone is in monocomponent form compared with other species such

as cow, sheep or pig (Jortikka et al. 1993b). The manufacturing methods and

applications of reindeer bone protein extract are comparable to the other animal-

derived tissue extracts. The closest products are demineralized bone extracts made

from bovine and equine bone (Huffer et al. 2007, Murray et al. 2007, Baas et al. 2008, Nienhuijs et al. 2009). However, reindeer bone protein extract is more

purified compared to DBM, and bone proteins and growth factors may thus be

more available in the bone healing process than when using DBMs.

29

2.3.4 Synthetic materials

Numerous products including calcium salts, bioactive glasses, synthetic polymers

and their combinations are currently available in trauma and orthopaedic surgery.

For example, there are eighteen various bone substitutes in clinical use in the

Netherlands today (Van der Stok et al. 2011). Their biological, biomechanical and

structural properties determine their clinical application (Van der Stok et al. 2011). Common for synthetic materials is that they form an osteoconductive

scaffold to new bone formation (Carey et al. 2005, Peters et al. 2006, Baas et al. 2008, Alves et al. 2008). Thus, for repairing bone tissues, osteointegration means

the capability to make direct contact with living bone after implantation, and

bioactivity is a measure of the osteointegration rate (Moore et al. 2001, Carson &

Bostrom 2007, Ohtsuki et al. 2009). Calcium phosphates and their variations are

the most commonly known inorganic bone substitute materials (Moore et al. 1987, Li et al. 2001, Niedhart et al. 2003, Barrack 2005, Carey et al. 2005, Alves

et al. 2008, Douglas et al. 2009, Guda et al. 2011). Synthetic, biodegradable,

polymers have been also widely used in tissue-engineering. Polylactide (PLA),

polyglycolide (PGA) and their copolymers poly(lactic-co-glycolic acid) (PLGA)

are traditionally known as materials in bone fracture fixation such as self-

reinforcing screws and rods but also as carriers for antibiotics and rhBMPs

(Pyhältö et al. 2004, Carson & Bostrom 2007, Bessa et al. 2008b, Koort et al. 2008). Fiber technology has increased synthetic polymers and composition

materials use in bone tissue-engineering (Huttunen et al. 2008, Wang et al. 2010).

Especially, nanofibrous materials produced by electrospinning processes are

under developing (Jang et al. 2009). Furthermore, various bone cements as glass

ionomer cements and polymethyl methacrylate (PMMA) bone cements have long

history as orthopaedic applications (Moore et al. 2001, Provenzano et al. 2004,

Carson & Bostrom 2007).

Bioactivity of synthetic materials can be increased by adding osteogenic

stimulus to the bone graft extender (Moore et al. 1987, Alam et al. 2001, Barrack

2005, Kim et al. 2005, Baas et al. 2008, Reichert et al. 2011). Combinations of

bovine bone-derived growth factors in collagen and DBM or coralline-HA

carriers have been shown to be as good as iliac crest autografts when studying

fusion rates in spinal arthrodesis in rabbits and monkeys (Damien et al. 2002, Yee

et al. 2003, Urrutia et al. 2008, Dodds et al. 2010) and humans (Boden et al. 2004).

30

2.4 Carriers of bone protein extract

A mixture of bone proteins and growth factors extracted from long bones is

defined as bone extract. A preparation of this mixture with the carrier material is

named as an implant that can be used as a bone void filler or implanted into the

site of bone trauma (Pekkarinen et al. 2003, Dimitrou et al. 2005). The bone

proteins and other growth factors are soluble within biologic fluids, thus it must

be mixed with a carrier substance (Forslund & Aspenberg 1998). Otherwise, a

morphogenetic signal is needed in bone regeneration together with a respondent

cell population, as well as a matrix that delivers the signal and acts as a scaffold

for the new bone formation (cell recruitment, attachment, proliferation and

differentiation) (Ripamonti & Duneas 1998, King 2001).

2.4.1 Criteria of ideal carrier

An optimal carrier matrix should fulfil several criteria. The matrix should be

biocompatible, bioabsorbable, malleable, and sterilizable (Kirker-Head 2000, Li

& Wozney 2001, Lugienbuehl et al. 2004, Seeherman & Wozney 2005).

An ideal bone implant would be osteoconductive, osteoinductive, resorbable,

and radiolucent to allow easy radiographic evaluation of bone formation and

healing (Winn et al. 1999, Kirker-Head 2000, Saito & Takaoka 2003,

Lugienbuehl et al. 2004, Babis & Suocacos 2005). The bone graft substitute

should also avoid being rejected or inducing a foreign body response (Sciadini et al. 1997) and should retain growth and signalling factors at the implant site for a

period of time sufficient to induce bone formation (Takita et al. 2004). Porosity of

carrier is also essential because it affects the mechanical properties of bone

implant together with graft macro and micro architecture (Li et al. 2003, Zyman

et al. 2008). Thus, in the scaffold design it should be considered that it exceeds

the strength of the natural bone that it replaces and that adequate strength is

maintained throughout the bone remodelling phase (King 2001, Babis &

Suocacos 2005). DBMs, collagenous materials, synthetic polymers, calcium

phosphate materials (bioceramics), calcium sulphate materials, bioactive glasses

and metals are typical carrier devices (Kirker-Head 2000).

The carrier material can be in the form of blocks, granules, paste, solution,

and injectable or self-setting cement (Kamegai et al. 1994, Ohura et al. 1999,

Ruhé et al. 2004, Carey et al. 2005, Reynolds et al. 2007, Tas 2008, Alves et al. 2008). Collagen as organic material, and bioactive glass and calcium salts as

31

inorganic materials have been tested as suitable carriers in this study; the review

is thus focused on them.

2.4.2 Collagen

Collagen is the most well known of natural polymers that are widely used and

studied as carriers for bone treatment. Collagenous materials are superior in

compatibility, because collagen is the major protein component of hard and soft

tissues (Wozney & Rosen 1998). The major component of bone collagen is type I

collagen with traces of type III and V collagens (Keene et al. 1991, Gelse et al. 2003). Type I collagen defines various biomechanical properties in the bone such

as load bearing, tensile strength and torsional stiffness, particularly after

calcification (Ikada 2002: 2–3, Gelse et al. 2003). Collagen type II is the most

typical protein of cartilage together with types IX and X (Seyer et al. 1974,

Kandel et al. 1997, Gelse et al. 2003). Type IV collagen is found in basement

membranes, where it is the major structural component, providing a framework

for the binding of other basement membrane components and a substratum for

cells (Herbst et al. 1988, Olivero & Furcht 1993, Gelse et al. 2003, Khoshnoodi et al. 2008). In bone, this collagen type plays a role in embryonic cartilage and bone

induction (Paralkar et al. 1990, Gelse et al. 2003).

Collagen can be prepared in solution, membrane film, thread, sponge, gel or

tubed form for implantation (Kirker-Head 2000, Ikada 2002: 2–3, Geiger et al. 2003, Wallace & Rosenblatt 2003, Bessa et al. 2008b). Collagen sponges have the

advantage of being very versatile, easily manipulated and wettable. The

manufacture of collagen sponge carrier depends on several factors, including

sponge mass, sterilization methods, soaking time, protein concentration and

buffer composition (Geiger et al. 2003). They are the most common of bovine-

tendon-or skin-derived and commercially available products (Uludag et al. 1999).

Collagens (typically bovine-derived type I or IV collagens) have been tested and

evaluated in several animal models and clinical trials over the decades (Gao et al. 1993, Hollinger et al. 1998, Bessho et al. 1999, Kirker-Head 2000, Chen et al. 2002, Seeherman et al. 2002, Geiger et al. 2003, Barboza et al. 2004, Pekkarinen

et al. 2006, McKay et al. 2007, White et al. 2007, Miyazaki et al. 2009,

Moghaddam et al. 2010). Although a large variety of collagen and other carrier

formulations have been developed, BMPs and bone extracts have previously been

successfully used in the healing of fractures with absorbable collagen carrier of

type I or IV collagens alone (Hollinger et al. 1998, Bax et al. 1999, Bessho et al.

32

1999, Blokhuis et al. 2001, Chen et al. 2002, Govender et al. 2002, Einhorn et al. 2003, Pekkarinen et al. 2006, Issa et al. 2008, Moghaddam et al. 2010).

Collagen as a carrier is not structurally strong but easy to shape. Therefore,

collagen-coated frames have typically been used as composite carriers of BMPs

and DBMs. rhBMPs are reconstituted with water prior to injection and

impregnated the collagen sponge for several minutes before implantation (Geiger

et al. 2003, McKay et al. 2007). DBM is usually mixed together with collagen to

a form gel or putty (Coulson et al. 1999, Kirker-Head 2000). A typical composite

implant contains collagen together with some inorganic material, such as HA or

natural coral (Gao et al. 1996, 1997, Tuominen et al. 2000, Kujala et al. 2004,

Pekkarinen et al. 2005a, Kim et al. 2005, Yunoki et al. 2011, Wen et al. 2011).

2.4.3 Bioactive glass

Bioactive glasses are inorganic materials that have been used in granular form to

fill the bone defects. They meet many criteria of optimal carrier matrices

including biocompatibility, osteoconductivity, and biodegradability (Cao &

Hench 1996). Some bioactive glasses have already been used to repair bone

defects because their bioactivity allows them to achieve tight fixation resulting

from direct bonding to living bone. Thus, they are called surface-bioactive

ceramics (Oonishi et al. 1999, Silver et al. 2001, Pyhältö et al. 2004, Hench 2006,

Ghosh et al. 2008).

In the traditional bioactive glass (Na2O–CaO–SiO2–P2O5), SiO2 works as a

network former, with slow and incomplete resorption. Na2O, CaO, and P2O5 are

compounds found in bioglass granules, and the variation in their ratios can be

changed to adjust the dissolution rate (Hall et al. 1999, Griffon et al. 2001,

Livingston et al. 2002, Karageorgiour & Kaplan 2005). This material was the first

bioactive ceramic developed by Hench et al., and it is known as Bioglass (or 45S5

Bioglass®) and is FDA-approved (Hench et al. 1971, Cao & Hench 1996, Chen et al. 2006). This composition has been in clinical use since 1985. Today, there are

many variations on the original composition, such as sol-gel derived 58S and

S70C30 (Silver et al. 2001, Xia & Chang 2006, Chen et al. 2010, Mortazavi et al. 2010).

There is also a long research history with bioactive glasses in Finland.

Bioactive glass S53P4 was developed in Turku, Finland (Andersson & Karlsson

1991). In 1991, clinical tests started with this bioactive glass, especially on

craniomaxillary but also in spinal surgery (Aitasalo & Peltola 2004, Peltola et al.

33

2006, Frantzén et al. 2011). This kind of bioactive glass is a unique clinically

used bone graft, because it is also antibacterial (Zhang et al. 2010). It has been

successfully used in the treatment of osteomyelitis (Lindfors et al. 2010).

Bioactive glass S53P4 received European approval for orthopaedic use in 2006

(Frantzén et al. 2011).

The mechanisms that enable bioactive glasses to act as materials for bone

repair have been investigated and various inorganic reaction stages are commonly

thought to occur when bioactive glass is immersed in a physiological environment

(Ohtsuki et al. 2009). There is the ion exchange stage in which modifier cations

(mostly Na+) in the glass exchange with hydronium ions in the external solution.

In the hydrolysis Si-O-Si bridges are broken, forming Si-OH silanol groups, and

the glass network is disrupted. Then, in condensation of silanols, the disrupted

glass network changes its morphology to form a gel-like surface layer, depleted in

sodium and calcium ions. Precipitation is the reaction in which an amorphous

calcium phosphate layer is deposited on the gel, and in mineralization the calcium

phosphate layer gradually transforms into crystalline hydroxyapatite, which

mimics the mineral phase naturally contained with vertebrate bones. (Cao &

Hench 1996, Hench 2006, Ohtsuki et al. 2009). Furthermore, it has been

discovered that the morphology of the gel surface layer is a key component in

determining the bioactive response (Cao & Hench 1996, Zhong & Greespan

2000, Xia & Chang 2006). These kinds of glasses contain significantly higher

concentrations of SiO2 than traditional melt-derived bioactive glasses and still

maintain bioactivity (Saravanapavan et al. 2004).

Bioactive glasses have been used as carriers of BMPs, especially rhBMP

(Mahmood et al. 2001, Takita et al. 2004, Välimäki et al. 2005, Bergeron et al. 2007). There are many systems to prepare a BMP involved bioactive glass

implant: rhBMP was infused to the manually molded glass fiber balls (Mahmood

et al. 2001), the carrier and used dose of rhBMP had been mixed together before

implantation (Takita et al. 2004), and the defect site had been filled with bioactive

glass granules and growth factors have been injected there (Välimäki et al. 2005).

There is also studies where DBM cylinders contain bioactive glass rods to

accelerate bone formation (Pajamäki et al. 1993a,b). Some properties of bioactive

glass granules can affect the bone healing capacity of bone extracts, such as

porosity of granules, granule size, and degradability of glasses (Jones & Hench

2004, Jones et al. 2006). Bone bonding capacity is a significant advantage when

using bioglass as bone void filler (Hench 2006, Ohtsuki et al. 2009). The calcium

phosphate formation at the surface of bioactive glass occurs this strong bonding.

34

Bioactive glass bonds into bone when the surface between material and tissue is

mineralized (Andersson & Kangasniemi 1991). Variations in local pH levels also

strongly influence the osteoclastic activity and reactions of bioactive glass in bone

trauma (Cerruti et al. 2005).

2.4.4 Calcium salts

Inorganic materials fulfil many of ideal carrier requirements. Most of them are

structurally strong, immunologically inert, highly osteoconductive and variably

biodegradable (Kirker-Head 2000, Blom et al. 2002). Calcium salts have been

used for years in various variations because their composition is close to natural

bone composition and structure (Alam et al. 2001, Blom et al. 2002, Carey et al. 2005, Kroese-Deutman et al. 2005, Peters et al. 2006, dos Santos et al. 2008).

Calcium phosphate materials, including hydroxyapatite (HA), tricalcium

phosphate (TCP) and their composites, have been proposed as potential carrier

materials for BMPs and other bone protein extracts. Stoichiometric TCP

[Ca3(PO4)2] has Ca/P ratio of 1.5. There are both alpha and beta crystal forms of

TCP in available. The alpha state is formed at high temperatures and is more

soluble than a beta form (Bohner 2001, Yamada et al. 2007). Especially, β-TCP is

shown to be useful carrier for rhBMPs or DBM (Gao et al. 1996, Ohura et al. 1999, Blom et al. 2001, Niedhart et al. 2003, Kim et al. 2005, Huffer et al. 2007,

Baas et al. 2008). β-TCP has many positive features as carrier material. Its

resorption rate closely matches the course of normal cancellous bone remodelling,

it can bond directly to bone, and it is primarily osteoconductive in nature. β-TCP

is also more soluble than HA (Koerten & Meulen 1999, Bodde et al. 2007).

According to Moore et al. 2001, commercially available β-TCP granules dissolute

in 6-18 months whereas a commercially available HA granules reabsorb 1-2% per

year (Moore et al. 2001). β-TCP as hardenable calcium phosphate can be

prepared in a paste form that can be molded to a desired shape prior to setting

(Carey et al. 2005, Xu et al. 2006, Urban et al. 2007). Thus, an injectable form of

β-TCP combined with rhBMP-2 has been shown to successfully heal femoral

defects in rabbits (Seeherman et al. 2006).

HA [Ca10(PO4)6(OH)2] is fairly osteoconductive and has high protein-binding

capacity. High porosity and high surface implanted area are achieved because of

the continuous structure of HA-based material (den Boer et al. 2002, Itoh et al. 2006, Douglas et al. 2009, Schnettler et al. 2009). This property has been made

use of in surface coating of carrier with rhBMP-2 (Schnettler et al. 2009). It has

35

been suggested that the structural configuration of HA may be an important factor

in osteogenesis, and typical applications forms are powder, granule, disk, and

block (Magan & Ripamonti 1996, Tsuruga et al. 1997, Kuboki et al. 1998). A

porous form of HA is more susceptible to osteogenesis than non-porous particles

or solids (Rohanizadeh & Chung 2010, Roohani-Esfahani et al. 2010). HA has

been used as a carrier for BMPs in many studies (Takaoka et al. 1988, Horisaka et al. 1991, Kuboki et al. 1998, Kirker-Head 2000, Aebli et al. 2005, Rohanizadeh

& Chung 2010).

Porosity and particle size affect the degradation rate of the compound and the

biomechanical properties of the implant (Li et al. 2003, Galois & Mainard 2004,

Zyman et al. 2008, Ioku 2010, Roohani-Esfahani et al. 2010). There has been a

lot of discussion about optimal pore size and the role of micro- and

macroporosity. It has been found that the range of optimal size is 150–600

microns (Pratt et al. 2002, Roohani-Esfahani et al. 2010). HA is often combined

with β-TCP to form a more resorbable and porous carrier with a greater degree of

bone formation than HA alone. Thus, resorption kinetics of composite materials

can be adjusted by varying the rations of HA and β-TCP. This combination of

calcium phosphates has also been used as a carrier for rhBMPs and DBM. The

most typical methods have been that carrier granules have been mixed with DBM

and molded to form an implant or a used carrier has been coated or loaded with

the rhBMP-solution. (Alam et al. 2001, Pratt et al. 2002, Ruhé et al. 2004, Jung et al. 2008, Schopper et al. 2008).

Calcium sulphate (CS) [CaSO4] has been studied as a bone void filler for

more than a hundred years and it has many functions as part of a bone graft

composite. It acts as a binder to improve total bone contact and bone volume

around the implant (Turner et al. 2001, Ricci 2001: 1-10, Peters et al. 2006,

Schneiders et al. 2009). The hemihydrate form of CS [CaSO4·0.5H2O] is better

known as plaster of Paris, while the dehydrate form [CaSO4·2H2O] occurs

naturally as gypsum (Singh & Middendorf 2007, Thomas & Puleo 2009).

Depending on the method of dehydration of calcium, specific hemihydrates are

distinguished according to their crystal size as alpha-hemihydrate and beta-

hemihydrate (Singh & Middendorf 2007, Thomas & Puleo 2009). Alpha-

hemihydrate crystals are more prismatic than beta-hemihydrate crystals and, when

mixed with water, form a much stronger and harder superstructure (Thomas et al. 2005, Thomas & Puleo 2009). Pore size has an important role in bone ingrowth,

and increasing of pore size improves the bone healing effect of inorganic

materials such as calcium sulphates (Xu et al. 2006, Thomas & Puleo 2009). A

36

disadvantage of CS is that it resorbs fast, within a couple of weeks after

implantation. Therefore, it does not pride long-term mechanical support for the

healed bone, but it can work as carrier in the initial stage of the healing cascade

(Moore et al. 2001). Clinically, CS-based grafts are mainly used to fill bone

defects after tumour resection surgery (Van der Stok et al. 2011). CS has for many

years been used as carrier for rhBMPs and DBMs, also in clinical studies,

showing excellent biocompatibility. Preparation method of CS-based implant

depends on a form use. rhBMP-2 has been impregnated into the CS powder and

then formed by applying high pressure (Cui et al. 2008). Turner et al. 2001 filled

the defect site with DBM material and CS tablets (Turner et al. 2001). In the

study of Reynolds et al. 2007, the defects were filled with DBM and CS graft

binder (Reynolds et al. 2007).

2.4.5 Carriers of reindeer bone protein extract

A mixture of bone proteins and growth factors extracted from long bones is

defined as bone extract. A preparation of this mixture with the carrier material is

named as an implant that can be used as a bone void filler or implanted into the

site of bone trauma (Pekkarinen et al. 2003, Dimitrou et al. 2005). Reindeer bone

protein extract has high bone-forming activity, as seen in bioactivity and previous

tests, but in a real bone healing situation it cannot work without a carrier system

(Jortikka et al. 1993a, Pekkarinen et al. 2003, Ulmanen et al. 2005). The reindeer

bone protein extract sets its own limitations for the selection of carrier because of

its characteristics. The main limitation is that the extract is not water-soluble.

Thus, there are at least three different possibilities for implant preparations:

– The formulated bone extract suspension is impregnated into a porous matrix

(Fig. 1a).

– The extract and carrier are moulded together to form putty or compressed into

the discs (Fig 1b).

– The carrier discs or granules are surface-coated with the bone extract (Fig.

1c).

37

Fig. 1. There are three possibilities of preparing implants of reindeer bone protein

extract: Impregnation into a porous matrix (a), moulding to form a paste (b), and

surface coating of granules (c). (H Tölli, unpublished).

Pure collagen as a carrier has been tested in some of our previous studies

(Pekkarinen et al. 2003, 2005c,b, 2006). Lyophilized extract was mixed in water

and pipetted onto the collagen sponge, or the collagen sponge soaked in water and

then extract was bundled up to form an implant. The combinations of TCP, HA

and coral together with reindeer bone protein extract and collagen sponge have

been tested in mouse model (Pekkarinen et al. 2005a). Table 3 presents the used

carrier systems and evaluation methods of reindeer bone protein extract.

Table 3. Carrier systems and experimental models of reindeer bone protein extract.

Carrier (Source of collagen) Implant form Experimental model Reference

Collagen

(gelatine, type A, porcine skin)

Injectable Mice muscle pouch

model

Pekkarinen et al. 2003

Pekkarinen et al. 2005c

Collagen

(type IV, bovine)

Sponge Mice muscle pouch

model

Pekkarinen et al. 2004

Pekkarinen et al. 2005a

TCP/Collagen

HA/Collagen

TCP/HA/Collagen

Biocoral/Collagen

(type IV, bovine)

Composite

implants in form of

frames

Mice muscle pouch

model

Pekkarinen et al. 2005b

Gelatine

(type A, porcine skin)

Capsule Mice muscle pouch

model

Jortikka et al. 1993a

Pekkarinen et al. 2005c

Ulmanen et al. 2005

Collagen

(type IV, bovine)

Sponge Rabbit radius

segmental defect

Pekkarinen et al. 2006

38

2.5 Experimental models

The development of new biomaterials or treatment techniques requires

experimental and clinical testing to ensure biocompatibility, bioactivity and other

ideal system criteria (Liebschner 2004). Various animal species from mouse to

sheep, as well as various implantation sites and methods have been used in

biocompatibility and functional tests. Due to species-specific differences in bone

physiology, it is important to be able to choose a model that resembles the human

system as much as possible. (Nunamaker 1998).

Animal models with various fixation models are used to evaluate repair

materials of long bone fractures. Typical animals are sheep, rabbit, dog, primate

species and rat. The bones used as long bone defect models are typically ulna,

tibia, radius, femur or mandible (Liebschner 2004). A drill hole defect is one of

the model systems to test new methods and concepts. This model allows the

intraosseous implantation of various samples per animal without the need of an

extra fixation system (Nuss et al. 2006, Obenaus & Hayes 2011).

Other important study models are the non-union fracture or osteotomy model,

skull or cranial defect model and spinal fusion model. They have been widely

used in the evaluation of bone substitutes and trauma treatment (Nunamaker

1998, Burkus et al. 2003, Drespe et al. 2005, Seeherman et al. 2006, Cooper et al. 2010, Szpalski et al. 2010).

Muscle and subcutaneous tissues are the most common implantation sites for

studying osteoinductive properties of implants and bioassays. Mouse is the

typically used animal, but rats have also been used in osteoinductive bioassays

(Jortikka et al. 1993a, Pajamäki et al. 1994, Barr et al. 2010, Lee et al. 2011).

Segmental defect, hole defect, and muscle pouch models are used in the

present study, which is why the review focuses on them.

2.5.1 Rat segmental defect model

Rats have for a long time been popular animals in the study of fracture healing.

Animal costs are quite reasonable even in large group studies compared to rabbits

or some bigger animals (Nunamaker 1998). Solheim et al. (1992) have tested how

well the composite of demineralized bone mixed with polyorthoester can heal the

diaphyseal defect in the rat radius. Fracture healing has also been studied by

Chakkalakal et al. (1999) who evaluated the rigidity of the rat fibula, the mineral

content of the repair site, and the histological changes in the defect site.

39

A critical-size defect (CSD) is frequently used in the studies of segmental

bone defects models. It means the smallest size of the bone defect that does not

heal spontaneously or within the lifetime of the animal if it is left untreated for a

certain period of time (Schmitz & Hollinger 1986). The length of the CSD

depends on animal species and the defect place in the bone. It is typically twice

the diameter of long bones (Nilsson et al. 1986, Yasko et al. 1992, Hunt et al. 1996, Pekkarinen et al. 2006).

The femur is one of the longest bones and thus it is used in many segmental

bone defect studies as an experimental model for bone fractures and their

repairing methods. Additionally, the fractures of the femur are relatively common

and often associated with a major trauma, especially in humans. The most widely

used defect size in the rat has been 8 mm (Zart et al. 1993, Wolff et al. 1994,

Stevenson et al. 1994, 1997, Feighan et al. 1995). Einhorn et al. (1984) used the

criterion that the segment was about 20 per cent of the length of the femur and so

their defect size was 6 mm. The greatest defect size of rat femur used in

experiments has been 1 cm (Takagi & Urist 1982, Vögelin et al. 2005).

Segmental bone defect in the rat femur has been used as a model in both

biodegradable and non-biodegradable material studies. In the study of Yasko et al. (1992) the osteoinductive activity of rhBMP-2 in the osseous location of the rat

femur was tested with good results. Other rhBMP-2 studies also confirm the good

bone healing capacity of this protein together with inorganic and organic carrier

(Ohura et al. 1999, Vögelin et al. 2005). A load-bearing scaffold system was

manufactured from polypropylene fumarate and TCP. rhBMP-2 together with

dicalcium phosphate dehydrate was added to the scaffold prior to implantation to

the defect in rat femur. This did not increase bone mineral density, but the scaffold

system maintained bone length and allowed regeneration (Chu et al. 2007). BMP-

3 and BMP-7 have been tested in the rat femur model (Stevenson et al. 1994,

Chen et al. 2002, Little et al. 2005). The main finding was that BMP-7 can

maintain its osteoinductive capability in the presence of infection, and a sufficient

dose of BMP-7 is essential for callus formation in the infected defects. (Chen et al. 2002).

In 1982, Takagi and Urist used the rat femur defect model to show that

bovine extract could be used to heal large femoral diaphyseal defects, especially

together with autologous marrow (Takagi & Urist 1982). Bone marrow cells in

ceramic blocks or fibre metal implants were also effective in enhancing

osteogenesis and osteoconduction (Ohgushi et al. 1989, Wolff et al. 1994).

However, significantly more new bone was found in ceramic implants (Wolff et

40

al. 1994). The effect of DBMs or allografts with or without extra carriers as a

suitable alternative to autologous grafts has been studied for decades (Einhorn et al. 1984, Zart et al. 1993, Feighan et al. 1995, Stevenson et al. 1997). Hunt et al. (1996) have studied whether an extract from human osteosarcoma cells has the

ability to simulate the healing of the defect in the femoral diaphyses in rats. The

materials used in comparison have been collagen and autogenous bone graft. The

finding was that the cell extract was the most effective in bone forming,

promoting bone growth in major fracture. (Hunt et al. 1996).

Conservative fracture treatment includes the use of casts and splints. When

surgery must be used, internal or external fixation methods are commonly used to

treat the fractured bone (Park et al. 2003: 174–179). Finding the right bone

fixation system depends on the place and size of the defect and test animal

species. If internal fixation is selected, the skin is slit and muscles and periosteal

tissues are separated circumferentially from the diaphysis to expose the bone.

Next, a pre-drilled plate or other system is fixed to the bone with some fastening

techniques, after which the segmental defect is created using some burr-system

and saline solution (Yasko et al. 1992, Little et al. 2005). This shares the basic

elements of ORIF (open reduction and internal fixation) used in surgical treatment

of fractures (Nunamaker 1985, Giannoudis et al. 2007). An intramedullary nail is

also one alternative that can be used as a bone-internal fixation method. This

fixation system provides accurate fracture reduction and stabilization without soft

tissue dissection. (Hietaniemi et al. 1995, den Boer et al. 1999, 2003, Chu et al. 2007). A less rigid fixation system has been shown to promote endochondral

osteogenesis, while a more rigid fixation enhances intramembranous ossification

(Cullinane et al. 2002).

2.5.2 The model of ectopic bone formation

Subcutaneous or intramuscular implantation of scaffold-including osteogenic

material invokes bone formation at ectopic location (Reddi 1981). Embryonic

endochondral bone formation is the typical way of bone formation. This method

is also called the bioassay method (Pekkarinen et al. 2003). Mouse and rat are the

most typically used animal models.

Bioassay in the mouse hindquarter muscle is currently used as a standard

method. Ectopic new bone formation has been estimated after 10-21 days, usually

by radiology and histology (Jortikka et al. 1993a, Viljanen et al. 1993, Oksanen et al. 1998, Pekkarinen et al. 2004, Barr et al. 2010).

41

The rodent muscle pouch model has been used in many bone extract studies.

Various doses of extract have been tested derived from bovine (Bessho et al. 1991b, 1992, Bessho & Iizuka 1993, Gao et al. 1993, Jortikka et al. 1993b

Shigenobu et al. 1993, Kos et al. 2002), porcine (Wu & Hu 1988, Ko et al. 1990,

Jortikka et al. 1993b), rat (Begley et al. 1995), canine (Oksanen et al. 1998),

rabbit (Sato et al. 1993), bird (Ulmanen et al. 2005), moose (Viljanen et al. 1996,

Gao et al. 1996) and reindeer (Jortikka et al. 1993a,b, Pekkarinen et al. 2003,

2004, 2005a,b,c). Furthermore, the rodent muscle pouch model has been used in

many rhBMPs, DBMs or carrier material studies (Wang et al. 1990, Pajamäki et al. 1993a, 1994, Kusumoto et al. 1995, 1996, 1997, Volek-Smith & Urist 1996,

Cole et al. 1997, Alam et al. 2001, Zhang et al. 2004, Kim et al. 2005, Simon et al. 2006, Barr et al. 2010). The use of nude rats as s muscle pouch model has

increased in recent years (Qiu et al. 2009, Lee et al. 2011).

2.5.3 Sheep hole defect model

Sheep, goats and pigs are defined as big animal models because they are more

closely related to humans in size as well as bone and joint characteristics

(Nunamaker 1998). Sheep have typically been used to study fixation devices or

the healing effect of biomaterials with or without growth factors in segmental

defect model (Gerhart et al. 1993, Gao et al. 1996, 1997, Kirker-Head et al. 1998,

den Boer et al. 2003, Pluhar et al. 2006, Donati et al. 2008,) but due to the large

size of sheep there is often a risk of second fracture during the study (Gao et al. 1996). Therefore, the cancellous bone defect model or hole defect model in sheep

has less risk of a second fracture, but works well in testing the biocompatibility of

biodegradable materials and gives answers about material resorption and

substitution with new bone (Nuss et al. 2006).

The size of the drilled hole in sheep has varied, which is why the CSD criteria

in this model are not very clear. Typically, the diameter has been more than 5 mm

and depth more than 8 mm (Nuss et al. 2006, Bodde et al. 2007, Maus et al. 2008). Huffer et al. 2007 used the size 5 mm x 5 mm, but they found that this size

did not fulfil the CSD criteria (Huffer et al. 2007). Proximal and distal femur,

tibia and humerus are the most typical bones (Kessler et al. 2004, Worth et al. 2005, Nuss et al. 2006, Bodde et al. 2007, Huffer et al. 2007, Maus et al. 2008).

Nuss et al. (2006) presented a method for sheep hole defect surgery with eight

holes. They discovered that an important thing in the operation is to place the

holes correctly. Thus, spontaneous fracture is avoided and reliability of the study

42

results increases (Nuss et al. 2006). Various radiograhies, histological methods as

well as mechanical testing are the most used methods when analysing bone

regeneration and material biocompatibility. Histological methods typically give a

better view of the situation in the bone defect after follow-up than radiography

alone. (Aebli et al. 2005, Worth et al. 2005, Bodde et al. 2007, Huffer et al. 2007,

Maus et al. 2008).

The sheep hole defect model has been used in many material studies. Two

commercially available phosphate ceramics were found to be biocompatible and

osteoconductive in the study of Bodde et al. 2007. They used a model of two

holes drilled on the medial surface of the femur on both sides. The location of

defects was marked with three small titanium pins at each side of the drill holes.

(Bodde et al. 2007). Various growth factors have been used as composite of bone

replacement materials. Nuss et al. 2006 used various bone replacement materials

with or without parathyroid hormone, BMP-2, TGF-β and IGF-1 when they

developed and tested a sheep hole defect model with 8 holes (Nuss et al. 2006).

BMP-2 had been added with HA-coated implants or solvent-dehydrated bone

transplants. The result in both experiments was that BMP-2 accelerates bone

regeneration in the defects (Aebli et al. 2005, Kessler et al. 2004), whereas Maus

et al. 2008 found that an injection of hyaluronic acid-augmented BMP-2 did not

increase new bone formation compared to autologous bone or pure hyaluronic

acid (Maus et al. 2008). Xenoimplant, autograft and DBMs have been used in the

studies of Worth et al. 2005 and Huffer et al. 2007. The latter group found that

bovine or equine derived DBMs gave equivalent bone regeneration result as

autograft and better than sintered β-TCP ceramic (Huffer et al. 2007).

43

3 Aims of the study

The present study consists of experiments designed to evaluate the effects of

reindeer bone protein extract and its various formulation systems in the healing of

various animal bone defects and in bone forming in the ectopic muscle model.

The specific aims of this study were:

– to investigate the bone healing capacity of reindeer bone extract dosing with

bovine collagen sponge using a critical-size defect model of rat femur.

– to evaluate the bone healing effect of composite implants containing reindeer

bone extract combined with bioactive glass using a critical-size defect model

of rat femur.

– to evaluate the bone forming effect of reindeer bone extract formulations in

calcium salt carriers using a mouse muscle pouch model.

– to compare the bone formation performance of reindeer bone extract

formulation in inorganic carriers to autograft and commercial demineralized

bone matrix using a hole-defect model of sheep femur and humerus.

44

45

4 Materials and methods

The materials and methods used in the study series are summarized in Table 4.

Ta

ble

4. S

um

ma

ry o

f th

e m

ate

ria

ls a

nd

me

tho

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use

d i

n t

he

stu

die

s.

Stu

dy

Met

hods

C

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r (Ty

pe)

Dos

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bon

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Follo

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p

(wee

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lysi

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, lef

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Rat

Col

lage

n (ty

pe IV

, bov

ine)

Col

lage

n (ty

pe IV

, bov

ine)

Col

lage

n (ty

pe IV

, bov

ine)

Col

lage

n (ty

pe IV

, bov

ine)

Col

lage

n (ty

pe IV

, bov

ine)

Col

lage

n (ty

pe IV

, bov

ine)

Col

lage

n (ty

pe IV

, bov

ine)

Unt

reat

ed d

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t

2 m

g

5 m

g

15 m

g

20 m

g

50 m

g

30 μ

g rh

BM

P-2

- -

14

14

14

13

13

13

14

14

6

Rad

iogr

aphy

pQC

T

Mec

hani

cal t

estin

g

His

tolo

gy

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, lef

t fem

ur

Rat

Bio

activ

e gl

ass

(S53

P4)

Bio

activ

e gl

ass

(S53

P4)

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activ

e gl

ass

(S53

P4)

Bio

activ

e gl

ass

(S53

P4)

Bio

activ

e gl

ass

(S53

P4)

Unt

reat

ed d

efec

t

5 m

g

10 m

g

15 m

g

40 m

g

- -

9 9 8 8 8 8

8

8

8

10

8 8

Rad

iogr

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pQC

T

Mec

hani

cal t

estin

g

III

Mus

cle

pouc

h

mod

el, h

ind

legs

Mou

se

HA

/β-T

CP

/CS

60:

30:1

0

HA

/β-T

CP

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30:

60:1

0

β-TC

P/C

S 9

0:10

Cem

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ihyd

rate

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A,

Cor

resp

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3 m

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3 m

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3 m

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preg

nate

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3 m

g (m

ixed

)

3 m

g (m

ixed

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g (d

ry m

ixed

)

-

8 8 8 8 8 8

8 in

eac

h

3

Rad

iogr

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His

tolo

gy

46

Stu

dy

Met

hods

C

arrie

r (Ty

pe)

Dos

e of

bon

e pr

otei

n ex

tract

N

Follo

w-u

p

(wee

ks)

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lysi

s m

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ds

IV

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e de

fect

(0.2

83 c

c),

Hum

erus

and

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urs

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ep

Cam

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ith P

EG

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and

SA

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nule

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ith P

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and

SA

Cam

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nule

s, w

ith P

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and

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Cur

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mor

sels

, with

PE

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LY a

nd S

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nule

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ith P

EG

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and

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Cur

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mor

sels

, with

PE

G-G

LY a

nd S

A

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β-T

CP

gra

nule

s

Aut

ogra

ft

DB

M (h

uman

)

Unt

reat

ed d

efec

t

60 m

g / 3

cc

syrin

ge

30 m

g / 3

cc

syrin

ge

-

60 m

g / 3

cc

syrin

ge

60 m

g / 3

cc

syrin

ge

- - - - -

8 8 8 8 8 8 8 8 8 8

8

μCT

n =

num

ber

of s

tudy

cas

es,

CS

D =

cri

tica

l-si

ze d

efec

t, pQ

CT

= p

erip

hera

l qu

anti

tati

ve c

ompu

teri

zed

tom

ogra

phy,

rhB

MP

-2 =

rec

ombi

nant

hum

an b

one

mor

phog

enet

ic

prot

ein-

2, H

A =

hyd

roxy

apat

ite,

β-T

CP

= b

eta-

tric

alci

um p

hosp

hate

, C

S =

cal

cium

sul

phat

e, S

A =

ste

aric

aci

d, C

am =

Cam

bioc

eram

ics,

PE

G =

pol

yeth

ylen

e gl

ycol

, G

LY

=

glyc

erol

, Cur

= C

uras

an, D

BM

= d

emin

eral

ized

bon

e m

atri

x, μ

CT

= m

icro

-com

pute

rize

d to

mog

raph

y

47

48

4.1 Animals

All study protocols were carried out according to Finnish laws on animal welfare

and were approved by the institutional animal experiment and ethical committee.

The animals were supplied by the Laboratory Animal Centre, University of Oulu.

The numbers of test animals in the different research categories were as follows:

– Study I: One hundred and nine 2.5-month-old male Sprague-Dawley rats.

– Study II: Fifty 2.5-month-old male Sprague-Dawley rats.

– Study III: Forty-eight 7- to 12-week-old BALB/c mice.

– Study IV: Ten 3-year-old Finnish archipelago sheep.

4.2 The reindeer bone protein extract

The bone protein extract was extracted and purified from diaphyseal bone of the

reindeer as described previously (Jortikka et al. 1993a). In studies III and IV, the

obtained bone protein extract was freeze-dried at -20˚C degrees using excipients

(surfactant (Polysorbat 20, Fluka, Sigma-Aldrich), lyoprotectant (D-(+)-Trehalose

Dihydrate, Fluka, Sigma-Aldrich), bulking agent (Glycine, Riedel-de Haën,

Sigma-Aldrich) and buffer (D-Mannitol, Fluka, Sigma-Aldrich)).

The protein profile and the bioactivity of the freeze-dried bone protein extract

were evaluated by SDS-page (Ulmanen et al. 2005) and mouse muscle pouch

model study as described in study III (Fig. 2).

49

Fig. 2. Bone formation capacity of the 3 mg reindeer bone protein extract dose in

muscle pouch model after 3 weeks follow-up. Original magnification of histological

slice 10x. B = new Bone.

4.3 Reference materials

The reference materials used in this study were chosen according to known and

used materials in bone trauma surgery.

A commercial product of recombinant human bone morphogenetic protein-2

(InductOsTM kit, Wyeth Europa, Maidenhead/Berkshire, UK) was used as

comparison material in study I.

A commercial product of demineralized bone matrix (Grafton Plus®

Demineralized Bone Matrix 1cc (DBM) Paste, Osteotech Inc., Eatontown, New

Jersey, USA) was used as reference material in study IV.

Autograft is the most typical treatment device in difficult bone traumas. It

was used as reference material in study IV.

4.4 Carriers

Various carrier systems for reindeer bone protein extract were tested in this study.

Collagen was tested as an organic carrier. Bioactive glass and calcium salts were

tested as inorganic alternatives. The materials used were prepared for different

implant forms.

50

A commercially available, absorbable collagen sponge (Lyostypt®, compress

from a native collagen of bovine origin, mainly consisting of type IV collagen, B.

Braun, Tuttlingen, Germany) was used as a collagen carrier.

The distribution size of the bioactive glass granules used (Bioactive glass,

S53P4, Vivoxid, Turku, Finland) was 500-800 μm.

The carrier materials used and the study groups in study III were:

a) Porous discs that were 5 mm x 3 mm in size (Berkeley Advanced

Biomaterials Inc, USA), with a composition of 30% hydroxyapatite (HA),

60% beta-tricalcium phosphate (β-TCP), and 10% calcium sulfate (CS);

b) Cem-Ostetic porous discs that were 5 mm x 3 mm in size, with a composition

of 90% β-TCP and 10% CS;

c) Cem-Ostetic® (Berkeley Advanced Biomaterial Inc., USA) powder for putty;

d) CS hemihydrate (97%, Sigma-Aldrich) powder for putty;

e) Non-porous discs with a composition of 60% HA, 30% β-TCP and 10% CS;

and

f) CS dihydrate granules with stearic acid with a composition of stearic acid 50,

a mixture of fatty acids that consisted mainly of stearic acid and 40-60%

palmitic acid (Fluka, Sigma-Aldrich).

In the Study IV, the carrier materials used were:

1. Custom-made Cambioceramics β-TCP (Cambioceramics, Cam Bioceramics,

Leiden, The Netherlands), 300-500 μm of 1.24 g/cm3 density combined with

Polyethylene Glycol/Glycerol (PEG/GLY) (Clariant, Kemi Intressen, and

Croda, Kemi Intressen) matrix modified with stearic acid (Stearic acid 50,

mixture of fatty acids, consisting mainly of stearic acid and 40–60% palmitic

acid, Fluka, Sigma-Aldrich) (Study groups named: Paste 1, Paste 2, Paste 3, and Granule);

2. Curasan β-TCP Cerasorb M (Cerasorb® M Ortho, Curasan AG, Frankfurt,

Germany), 500-1000 μm of 0.61 g/cm3 density combined with PEG-GLY

matrix modified with stearic acid (Study groups named: Paste 4, and Paste 5);

3. Curasan β-TCP Cerasorb (Cerasorb®, Curasan AG, Frankfurt, Germany), 500-

1000 μm of 1.21 g/cm3 density combined with PEG-GLY matrix modified

with stearic acid (Study group named: Paste 6).

51

4.5 Preparation of implants

4.5.1 Reconstitution of collagen implants (I)

2 mg, 5 mg, 15 mg, 20 mg or 50 mg of the lyophilized reindeer bone extract was

sprinkled onto a collagen sponge (35 x 8 mm for small amounts and 25 x 25 mm

for large amounts, Lyostypt®). The collagen sponge was wetted in 200 μl sterile

water and the sponge with extract was bundled up to form an implant.

InductOsTM kit for implant contained 12 mg of rhBMP-2 and solvent. The

solvent was made according to manufacturer’s instructions just before the

operation. 20 μl of solvent, containing 30 μg of rhBMP-2, was pipetted on the

collagen sponge (Lyostypt®) to form the implant.

4.5.2 Reconstitution of bioactive glass implants (II)

A dose of 5 mg, 10 mg, 15 mg, or 40 mg of the reindeer bone extract was added

to carboxymethyl cellulose (CMC, Sigma-Aldrich) to obtain a 3.2%

(water/weight, w/w) gel. Bioactive glass granules (S53P4) were combined with

extract-CMC-gel, and the mixture was shaped into a rod (diameter 5 mm) and

lyophilized. Before drying, one 8-mm-wide implant contained 40% w/w of CMC-

gel with a propriety amount of bone extract, and 60% (w/w) bioactive glass

granules. The amount of CMC in the dried product was 2% (w/w).

4.5.3 Reconstitution of calcium salt implants (III)

The amount of 3 mg of the lyophilized reindeer bone extract was reconstituted in

0.9% physiological saline solution (Sodium chloride, Fagron, Tamro, Finland)

and impregnated into the porous discs (30% HA, 60% β-TCP, 10% CS and 90%

β-TCP, 10% CS) and the non-porous disc (60% HA, 30% β-TCP, 10% CS), or

mixed with the Cem-Ostetic powder (Cem-Ostetic®) or CS hemihydrate to form a

moulded disc, or dry-mixed with CS dihydrate granules and stearic acid to form a

compressed disc (Table 4).

4.5.4 Reconstitution of β-TCP implants (IV)

Polyethylene glycol 2000 (PEG), glycerol and stearic acid were heated until a

clear mixture was formed. The mixture was cooled under continuous mixing to

52

form an opalescent paste, after which the required amounts (60 mg or 30 mg) of

the lyophilized bone extract and β-TCP granules were added (Table 4). The

formulated paste was packed in syringes (including about 5.66 mg of reindeer

bone protein extract) and closed in aluminium foil pouches. All samples were

manufactured in a laminar flow cabin to reduce the bioburden, and then

terminally gamma-sterilized (15 kGy).

4.5.5 Reconstitution of autograft implants (IV)

In the autograft group the bone material removed from the test hole-sites of the

same sheep using chisel and trephane drill. The autograft material was preserved

in 0.9% physiological saline solution (Sodium chloride, Fagron, Tamro, Finland)

until implanting.

4.5.6 Control implants

The control implants were constructed in an identical fashion, but they contained

only carrier. In the mouse study (Study III) the right leg was used as a control

containing the respective carrier and excipients, excluding the bone extract.

Furthermore, studies I, II and IV involved the untreated group without any

implant.

4.6 Surgical methods

Bone healing capacity and new bone formation were studied in the various

experimental models, including the critical-size defect model in rat, the muscle

pouch model in mouse and the hole defect model in sheep.

4.6.1 Critical-size rat femur defect study (I, II)

Surgery was performed under general anaesthesia with a blend of fentanyl citrate

(80 µg/kg) - fluanisone (2.5 mg/kg) (Hypnorm®) and midazolam (1.25 mg/kg)

(Dormicum®). Preoperatively, the animals were administered cefuroxime

(Zinacef®, 20 mg/kg) subcutaneously. The pain medication after the operation

consisted of buprenorfin (Temgesic®) at 0.01-0.05 mg/kg subcutaneously. In cases

with respiratory problems, animals were treated with 1 mg of furosemid

53

(Furesis®). Eye gel (Viscotears®) was applied to avoid dehydration of eyes during

anaesthesia. A transverse skin incision was made over the posterior aspect of the

thigh after shaving the hair round the left hind limb. The muscles were elevated

circumferentially from the femoral diaphysis. A 23 mm x 4 mm x 2.5 mm

polyacetyl plaster plate was placed on the surface of the femur. The plate was

temporarily secured in place with a stainless steel holder, which was exclusively

designed and manufactured for this operation (Technical Services Unit,

University of Oulu) (Fig. 3). The plate was fixed using 0.8-mm threaded

Kirschner wires (K-wires), which have a 5-mm long threaded distal end (Synthes

Oy). The canal for the threaded K-wire was predrilled through the plate and

through both cortices of the femur using a 0.7 mm drill. The threaded K-wire was

bent so as to easily screw the threaded end of the wire through the plate and into

the bone. The correct depth for inserting the wire was determined by carefully

dissecting the other side of the bone with a probe to confirm that the wire

penetrated the distal cortex of the bone. A total of 6 threaded K-wires were used

for each plate, three on each side of the bone defect, each screwed through two

cortices. The excess wire was removed to the level of the plate using side-cutting

pliers.

An 8-mm diaphyseal critical-sized defect was created using a stainless steel

mini bur. After a thorough wash with saline, the implant was applied to the defect,

or the defect was left empty for controls. The muscles and skin were closed in two

layers using absorbable 4.0 sutures (Dexon®). The pain medication after the

operation consisted of buprenorfin (Temgesic®) at 0.01-0.05 mg/kg administered

subcutaneously. In case of respiratory problems during anaesthesia, animals were

treated with 1 mg of furosemid (Furesis®) subcutaneously. Eye gel (Viscotears®)

was applied to avoid eye dehydration during anaesthesia. The animals were

allowed full activity in their cages postoperatively. After the follow-ups, the rats

were killed in a chamber with carbon dioxide (CO2). The left leg of each rat was

dissected from the body, wrapped in a saline-wetted serviette, and frozen at -20 ºC

until biomechanical analysis.

54

Fig. 3. The plastic plate was temporarily secured in place with a stainless steel holder

which was exclusively designed and manufactured for this rat femur defect operation.

4.6.2 Mouse muscle pouch model (III)

Surgery was performed under general anaesthesia with a blend of fentanyl citrate

(80 µg/kg) - fluanisone (2.5 mg/kg) (Hypnorm®) and midazolam (1.25 mg/kg)

(Dormicum®). Both legs were cleaned and the eyes were treated with eye gel

against drying. The mouse was operated on the thermal mattress. Transverse skin

incisions were made near the spine at the site of the femurs. Then the implants

were introduced into both thigh muscle pouches in the bilateral hind legs (Fig. 4).

After the implantation, the muscles were closed with two sutures and the skin

with one suture.

The pain medication after the operation consisted of buprenorfin (Temgesic®)

at 0.01-0.05 mg/kg subcutaneously. The animals were allowed full activity in

their cages postoperatively. All animals were euthanized 21 days later, and the

hind legs were harvested.

55

Fig. 4. The mouse muscle pouch model was used to show ectopic bone formation of

various calcium salt implants. An active implant is on the left side and a control on the

right side.

4.6.3 Sheep model study (IV)

The operation was performed under general inhalation anaesthesia, induced by an

intravenous injection of propofol (5-7 mg/kg i.v.) and maintained with isoflurane

in 1-1.5% oxygen-air mixture. Before the anaesthesia the sheep were

premedicated with medetomidine by an intramuscular injection (0.015 ml/kg i.m.)

and intubated. The sheep were controlled with a heart monitor during the

operation.

A fentanyl (2 μg/kg/hour, Durogesic®) depot plaster was given preoperatively

for 72-h pain relief. Additionally, 2 ml of fentanyl (50 μg/ml i.m.) was injected

intramuscularly during the first 72 h after the operation. Then buprenorfin (0.3

mg/dose i.m.) was injected twice a day continuously for two days or more after

the operation when the depot plaster was removed.

Amoxycillin (15 mg/kg i.m., Betamox® Vet, 150 mg/ml) was injected

intramuscularly as antibiotic prophylaxis into the anterior half of the neck 24 h

preoperatively and once per day for two days postoperatively.

Hole defects were induced to the femoral and humeral distal and proximal

condyles of the sheep hind and front legs with a drill as described by Nuss et al. (2006). A hole (size 0.283 cc) with a diameter of 6 mm and a depth of 10 mm was

drilled (cordless drill, Bosch PSR12-2). Meanwhile, the location of the defect was

marked, using 1.0 mm dental, radiopaque, glass-fibre root canal posts. The posts

were cut to suitable length with a diamond blade. The drilled holes were filled

56

according to the randomization table with the test materials or left empty

(untreated controls) (Fig. 5). Finally, the subcutaneous tissues were closed in

layers with resorbable continuous 3-0 Polysorb sutures and skin with non-

resorbable 2-0 Monosof sutures.

After a predetermined time period of 8 weeks the animals were euthanized

and bone samples were taken for analysis. Euthanasia was performed with

pentobarbital (60mg/kg i.v. Mebunat®). Before this, the sheep were anaesthetized

intramuscularly with medetomidine (0.015 ml/kg Domitor® Vet, and 0.04 ml/kg

Ketalar®).

After euthanasia the bones were excised and preserved in ice. Then the bone

blocks were preserved in 4% buffered formalin for the first seven days and then in

70% ethanol. Some samples were broken in the excision phase and they were

removed from the analysis.

Fig. 5. Hole defects were induced to the femoral and humeral distal and proximal

condyles of the sheep hind and front legs with a drill as described by Nuss et al.

(2006). The drilled holes were marked with dental posts and filled with the test

materials.

4.7 Analysis methods

New bone formation, bone healing, and carrier material resorption were evaluated

based on radiographs, peripheral computerized tomography (pQCT), mechanical

tests, histological examination, and micro-CT.

57

4.7.1 Radiographic evaluation (I, II, III)

In studies I and II, radiographs of the left femur (26 kV, 9.00 mAs, 0.09 s/exp,

Mamex dc® ami, Orion Ltd., Soredex) were taken postoperatively, three weeks

and six weeks after the operation under neuroleptic analgesia (Hypnorm-

Dormicum®, 0.7 ml) and after sacrificing. Radiographs were evaluated with

Osiris 4.19 (Digital Imaging Unit, Geneva) software. The percentage of orthopic

new bone formation (BF) and the development of union or non-union (BU) of the

bone were estimated according to the scoring system presented in the study of

Sciadini et al. 1997.

In study III, radiographic evaluation (20 kV, 8.00 mAs, 0.32 s/exp, Mamex

dc® ami, Orion Ltd., Soredex) was used to evaluate formation of new bone and

resorption of the implant. New bone formation and resorption of implant was

evaluated by measuring the opalescent area in mm2 (Osiris 4.19 Digital Imaging

Unit, Geneva, software).

4.7.2 Computed tomography (I, II)

In studies I and II, all left femurs were scanned using a peripheral quantitative

computerized tomography (pQCT) device (Stratec XCT 960A, 5.21 software

version, Norland Stratec Medizintechnik GmbH, Birkenfeld, Germany). A voxel

size of 0.148 mm x 0.148 mm x 1 mm was used. One cross-sectional slice from

the middle of the defect in each sample was scanned in two directions: with

fixation system above the bone, and turned 90º axially. Average values of the two

scans were used in the statistical analysis. Cross-sectional bone areas (mm2) from

the scanned slices of the samples were recorded using the pQCT software, with a

threshold of 169 mg/cm3 to distinguish bone from surrounding soft tissue and a

threshold of 464 mg/cm3 for the inner surface of the bone.

4.7.3 Micro-CT (IV)

Samples were scanned by using micro-computerized tomography (micro-CT)

device (Magnification 15.54, Source 72 kV/138 µA, Rotation 180°, Rotation step

0.45°, Exposure 3.9 sec, Gain 1.0, Frame aceragin OFF, Flat field correction ON,

Random movement 20, Filter Al 0.25 mm, SkyScan 1072, x-ray microtomograph,

SkyScan, Belgium). Scanned samples were analysed using CTAn (SkyScan)

software. Resorption of carrier, the amount and quality of new bone and general

58

bone healing were visually evaluated from the images. Furthermore, new bone

volume (BV, mm3), bone surface to bone volume ratio (BS/BV, mm-1), trabecular

thickness (Tb.Th, mm), open porosity (Po(op), %), and closed porosity (Po(cl),

%) were measured from the defects.

4.7.4 Biomechanical testing (I, II)

In studies I and II, after pQCT imaging, the fixation system was removed from

the samples. The bone ends were embedded into the moulds of the sleeves with

dental stone (GC Fujirock, Improved Dental Stone). The torsional shaft was

adjusted to 2 cm. After the cast was hardened, the samples were placed in the

torque machine (Lepola et al. 1993), and torsionally loaded at a constant angular

speed of 6º per second until failure (Jämsä & Jalovaara 1996) (Fig. 6). Maximum

breaking load (Nm) and torsional stiffness (Nm/º) were recorded. Mechanically

unstable bones were not tested, and their values were considered to be zero in the

statistical analysis.

Fig. 6. Mechanically stable rat femurs were torsionally loaded after pQCT imaging by

the torque machine of Lepola et al. 1993.

4.7.5 Histological examination (I, III)

In studies I and III, two samples from every group were prepared for histology.

The specimens were fixed in 10% neutral-buffered formalin, decalcified in

EDTA-formalin solution (pH 7), processed in a tissue processor, and finally

embedded in paraffin. 4.5-μm-thick slices were prepared using a microtome and

stained with haematoxylin-eosin. The quality of new bone and inflammatory

59

response on the defect site were evaluated in histological analysis by light

microscopy (Nikon Eclipse, E200, Japan).

4.7.6 Statistics

Statistical analysis was performed using the SPSS for Windows statistical

package (SPSS Inc., version 15.0). The independent-samples t-test (study IV) or

the non-parametric Kruskall-Wallis test (study I–IV) was used to evaluate the

statistical differences between the groups. The Mann-Whitney test was used for

pairwise comparison between the reindeer bone protein extract-treated and the

control groups (study I-IV). The Benjamini-Hochberg procedure was used to

correct p-values for multiple comparisons (Study II). Values of p < 0.05 were

considered statistically significant.

60

61

5 Results

In study I, twelve rats died on the night after the operation, and two died later.

Seventeen rats had to be killed due to the failure of fixation before the study

endpoint. In study II, ten rats died within 24 hours of the operation. In study III,

five mice died or were sacrificed before the follow-up. No obvious cause was

found for the deaths of the rats. With mice, some deaths or sacrificing were

caused by walking or breathing problems. No direct relation to the reindeer bone

extract was found as some deaths occurred in the control groups as well.

All surviving animals were analysed after follow-ups. Reindeer bone protein

extract was able to heal the critical-size segmental bone defect in six weeks, and

significant bone formation was already observed after three weeks. However, the

healing effect depends on the doses and concentration of extract in the carrier.

The minimum dose for segmental defect healing in rat seems to be 5 mg.

Furthermore, study II showed for the first time that bioactive glass is a suitable

carrier for reindeer bone protein extract. In study III, inorganic carrier material

together with stearic acid had the best ectopic bone formation capacity. This kind

of carrier in paste form was used in study IV. It showed to be comparable to

autograft, and much better in bone formation than DBM.

5.1 Dosing of the reindeer bone protein extract (I, II)

Radiographical evaluation

In study I, bone formation (BF), measured by roentgenography, at the defect site

was greater at three and six weeks in all bone extract groups (p < 0.05) when

compared to the untreated and collagen groups, except for the group of 2 mg bone

extract in comparison with the untreated controls at three weeks. In this study

bone formation showed a clear dose-dependency, but only the difference between

the groups with 2 mg and 20 mg of bone extract reached the level of significance

(p = 0.030) (Table 5). Bone union (BU) at three weeks was better in the groups

receiving 5 mg, 20 mg and 50 mg of the extract than in the untreated or collagen

groups (p < 0.05). At six weeks, BU was better in the groups with 5 mg and 50

mg of the bone extract than in the untreated group (p < 0.05), and also better in

the groups with 5 mg, 15 mg and 50 mg of bone extract than in the collagen group

(p < 0.05). There were no significant differences in BU between the groups

62

receiving various amounts of the bone extract (Table 6). BF and BU were better

in the commercial rhBMP-2 group than in any of the other groups both three and

six weeks after the operation (p < 0.001) (Table 5 and 6).

In study II, all doses of the extract with bioactive glass increased BF observed

on radiographs compared with the bioactive glass alone and untreated groups

(p < 0.002 to p < 0.03). With the extract doses of 10-40 mg + bioactive glass,

bone formation was superior as soon as 3 weeks postoperatively when compared

to the plain bioactive glass and untreated groups (p < 0.03) (Table 5). BU at 6

weeks was better in the groups receiving bioactive glass + 10-40 mg of the extract

compared with the plain bioactive glass and untreated groups (p < 0.009 to

p < 0.03, respectively) (Table 6).

63

Ta

ble

5. B

on

e f

orm

ati

on

(B

F)

in t

he

de

fec

t a

rea

of

rat

fem

ur

thre

e t

o e

igh

t w

ee

ks a

fte

r th

e o

pe

rati

on

ev

alu

ate

d b

y r

ad

iog

rap

hic

an

aly

sis

us

ing

th

e s

co

rin

g s

ys

tem

pre

se

nte

d i

n t

he

stu

dy

of

Sc

iad

ini et

al.

(1

99

7).

Stu

dy

Car

rier

Dos

e of

ext

ract

B

F sc

ore

3 w

eeks

6 w

eeks

8

wee

ks

N

0 1

2 3

4 p

0 1

2 3

4 p

0 1

2 3

4 p

I C

olla

gen

2 m

g 10

5

3 2

0 0

0 5

3 0

0 c

Col

lage

n 5

mg

10

1 4

4 1

0 a

c

0

1 4

1 1

b c

Col

lage

n 15

mg

10

1 8

1 0

0 a

c

0

2 5

0 0

b c

Col

lage

n 20

mg

10

2 4

3 1

0 a

c

0

2 1

1 3

b c

d

Col

lage

n 50

mg

8 1

5 1

1 0

a c

0 2

3 1

1 a

c

Col

lage

n 30

μg

rhB

MP

-2

10

0 0

0 0

10

e

0

0 0

0 10

e

Col

lage

n 0

mg

10

10

0 0

0 0

8 2

0 0

0

Unt

reat

ed

- 10

8

2 0

0 0

5 4

1 0

0

II B

ioac

tive

glas

s 5

mg

6 6

0 0

0 0

1 4

1 0

0 g

j

0 5

1 0

0

Bio

activ

e gl

ass

10 m

g 6

2 4

0 0

0 f

i

0 5

1 0

0 h

k

0 0

5 1

0 g

k

Bio

activ

e gl

ass

15

mg

7 0

7 0

0 0

h k

0 1

5 1

0 h

k

0 0

2 5

1 g

k

Bio

activ

e gl

ass

40

mg

9 3

3 3

0 0

f i

0

3 4

2 0

h k

0

0 4

3 2

h k

Bio

activ

e gl

ass

0

mg

6 6

0 0

0 0

6 0

0 0

0

5 1

0 0

0

Unt

reat

ed

- 6

6 0

0 0

0

6

0 0

0 0

3

2 1

0 0

ap

< 0.

005,

bp

< 0.

001

vs. u

ntre

ated

, cp

< 0.

001

vs. c

olla

gen,

dp

< 0.

05 v

s. 2

mg

of b

one

extra

ct, e

p <

0.01

vs.

all

othe

r gro

ups

in s

tudy

I.

f p <

0.05

, gp

< 0.

01, h

p <

0.00

5 vs

. unt

reat

ed a

fter B

enja

min

i-Hoc

hber

g co

rrec

tions

i p

< 0.

05, j p

< 0.

01 ,

kp

< 0.

005

vs. b

ioac

tive

glas

s af

ter B

enja

min

i-Hoc

hber

g co

rrec

tions

64

Ta

ble

6. B

on

e u

nio

n (

BU

) in

th

e d

efe

ct

are

a o

f ra

t fe

mu

r th

ree

to

eig

ht

we

ek

s a

fte

r th

e o

pe

rati

on

ev

alu

ate

d b

y r

ad

iog

rap

hic

an

aly

sis

us

ing

th

e s

co

rin

g s

ys

tem

pre

se

nte

d i

n t

he

stu

dy

of

Sc

iad

ini et

al.

(1

99

7).

Stu

dy

Car

rier

Dos

e of

ext

ract

B

U S

core

3 w

eeks

6 w

eeks

8 w

eeks

N

0 1

2 3

p 0

1 2

3 p

0 1

2 3

p

I C

olla

gen

2 m

g 10

9

1 0

0

5

2 2

1

Col

lage

n 5

mg

10

6 4

0 0

a b

2 4

3 1

a b

Col

lage

n 15

mg

10

8 1

1 0

4 3

2 1

b

Col

lage

n 20

mg

10

6 3

1 0

a b

5 1

1 3

Col

lage

n 50

mg

8 5

2 1

0 a

b

3

2 2

1 a

b

Col

lage

n 30

μg

rhB

MP

-2

10

0 0

0 10

c

0 0

0 10

c

Col

lage

n 0

mg

10

10

0 0

0

8

2 0

0

Unt

reat

ed

- 10

10

0

0 0

8 1

1 0

II B

ioac

tive

glas

s 5

mg

6 6

0 0

0

5

1 0

0

5

1 0

0

Bio

activ

e gl

ass

10 m

g 6

6 0

0 0

2 4

0 0

d

1

5 0

0 e

Bio

activ

e gl

ass

15 m

g 7

6 1

0 0

2 5

0 0

d

1

2 4

0 e

Bio

activ

e gl

ass

40 m

g 9

6 3

0 0

1 4

4 0

e

0

3 3

3 f

Bio

activ

e gl

ass

0

mg

6 6

0 0

0

6

0 0

0

6

0 0

0

Unt

reat

ed

- 6

6 0

0 0

6 0

0 0

6 0

0 0

ap

< 0.

03 v

s. u

ntre

ated

, bp

< 0.

05 v

s. c

olla

gen,

cp

< 0.

001

vs. a

ll ot

her g

roup

s in

the

stud

y I.

dp

< 0.

05, e

p <

0.01

, f p <

0.00

5 vs

. unt

reat

ed a

nd b

ioac

tive

glas

s af

ter B

enja

min

i-Hoc

hber

g co

rrec

tions

.

65

Biomechanical evaluation

In study I, maximum torsional load of the bones (Nm) was significantly higher in

the groups receiving 2 mg, 20 mg, or 50 mg of the bone extract than in the

collagen group (p < 0.05). Stiffness of the bones (Nm/º) was significantly higher

in the groups receiving 2 mg, 15 mg, 20 mg, or 50 mg of the bone extract than in

the collagen group (p = 0.046 to p = 0.004). Maximum torsional load in the

rhBMP-2 group was the highest of all groups (p < 0.05). Stiffness of the bones in

the rhBMP-2 group was higher than in the control groups and in most bone

extract groups (p < 0.05).

In study II, torsional stiffness of the bone was marginally higher in the group

receiving bioactive glass + 15 mg of the extract compared with the untreated

group (p = 0.066).

Histological evaluation

In study I, descriptive histology examination showed no new bone formation,

only fibrotic tissue, in the specimens of the negative control groups. New bone

formation was seen in the specimens of the groups containing the reindeer bone

extract or rhBMP. The most abundant bone formation and bone union was seen in

the specimens of 20 mg to 50 mg extract and in rhBMP groups, which showed

hypertrophied and calcified chondrocytes, remodelling ectopic trabecular woven

bone and bone marrow. No inflammatory reaction was seen.

5.2 Collagen and bioactive glass as carrier materials (I, II)

Collagen sponge was used as carrier material for the reindeer bone protein extract

and as reference material (rhBMP-2) in study I. Computed tomography (pQCT)

showed cross-sectional bone areas to be greater in the groups that received 15 mg

to 50 mg extract with collagen than in the control groups without extract (Table 4)

after six weeks (p = 0.019 to p < 0.001). Cross-sectional bone area in the rhBMP-

2 group was the greatest compared to other groups (p < 0.001).

Bioactive glass material was used as a scaffold in study II. In this study,

pQCT showed cross-sectional bone areas to be greater in the bioactive glass + 15

mg of the extract group and in the plain bioactive glass group than in the

untreated group (p < 0.03). Bone density at the defect site was greater in all

groups that received the bone protein extract with bioactive glass and in the

66

bioactive glass alone group than in the untreated group (p < 0.004). There were no

significant differences in bone density between the plain bioactive glass group

and any of the extract groups.

5.3 Bone formation capacity with calcium salt carrier materials (III)

In study III, the radiographic evaluation of the HA/β-TCP/CS 30:60:10 discs

demonstrated some bone formation outside the active implant; however, the

control implants remained intact. The measurement area was significantly higher

for the active implants compared to the controls (p < 0.01). For the group β-

TCP/CS 90:10 discs, the radiographic evaluation displayed some bone formation

outside of the active implants; however, the control implants were nearly intact,

and there were no statistically significant differences between the active implants

and the controls. For the Cem-Ostetic group, the radiographic analysis displayed

no new visual bone formation; however, the measurement area was larger in the

active group than in the control group (p < 0.01). For the group CS hemihydrate

discs, the radiographic analysis revealed some new bone formation, and

significant differences (p < 0.01) were apparent between the active and control

groups (the control group had visibly resorbed). For the group HA/β-TCP/CS

60:30:10 discs, the radiographic evaluation showed some bone formation outside

of the implant on the active side and some in the control implants. Furthermore,

the active group had a larger measurement area than the control group (p =

0.001). For the group CS dihydrate-stearic acid compressed discs, the

radiographic analysis and harvesting analysis revealed new bone formation in the

active implant group. The difference between the active implants and the controls

was statistically significant (p < 0.01). (Fig. 7).

The comparison between all active groups revealed that the group CS

dihydrate-stearic acid compressed discs had the largest measurement area, as

determined by the radiographic analysis (p < 0.05). Furthermore, the groups Cem-

Ostetic and CS hemihydrate had significantly larger areas than the groups HA/β-

TCP/CS 30:60:10, β-TCP/CS 90:10 and HA/β-TCP/CS 60:30:10 (p < 0.01).

There was also a statistically significant difference between the active groups

HA/β-TCP/CS 30:60:10 and HA/β-TCP/CS 60:30:10 (p < 0.05). (Fig. 7).

67

Fig. 7. Radiographic analysis of active implant, containing the bone extract, and

controls after three weeks of follow-up (average opalescent area in mm2). Active group

is marked as dark grey and its control as light grey. The standard deviation is shown. ap < 0.01 vs. control, bp < 0.05 vs. other active groups, cp < 0.01 vs. HA/β-TCP/CS

30:60:10, β-TCP/CS 90:10 and HA/β-TCP/CS 60:30:10, dp < 0.01 vs. HA/β-TCP/CS

30:60:10.

In study III, for group a, the harvesting analysis indicated that this new formation

was bone-like. The histological evaluation demonstrated that endochondral bone

formation occurred in the active sample, but not in the control sample. For the

group β-TCP/CS 90:10, visual inspection during harvesting indicated bone-like

formations. The histological evaluation showed endochondral bone formation in

the active sample, but not in the control sample. For the groups Cem-Ostetic and

CS hemihydrate, harvesting and histological analysis confirmed that no new bone

was found in the samples. For the group HA/β-TCP/CS 60:30:10, the harvesting

analysis indicated that this new formation was bone-like. The histological

evaluation revealed endochondral bone formation and mature cartilage cells in the

active sample; however, none were found in the control sample. For the group CS

dihydrate and stearic acid, histological analysis revealed clear bone formation and

mature and calcified cartilage cells in the active sample (Fig. 8a). No visual bone

formation was apparent in the control sample (Fig. 8b).

0

10

20

30

40

50

60

70

80

90

100

110

120

130Ar

ea (m

m2 )

a

β-T

CP

/CS

90:

10

Cem

-Os

teti

c

a,c a,c

CS

he

mih

yd

rate

HA

/β-T

CP

/CS

60:

30:1

0

a,d

CS

dih

yd

rate

an

d S

A

a,b

HA

/β-T

CP

/CS

30:

60:1

0

68

Fig. 8. Histological analysis revealed clear bone formation, and mature and calcified

cartilage cells in the CS dihydrate + stearic acid carrier with the reindeer bone protein

extract (a), but no visual bone formation was apparent in the control sample without

the extract (b). C = calcified cartilage cells, B = bone, M = muscle, F = fibrotic tissue,

and I = implant carrier. (Original magnification 10x). (Reprinted with permission from

MDPI Publishing).

5.4 Bone formation of the formulated bone protein extract in inorganic carrier (IV)

In study IV, micro-CT analysis was used to show new bone formation, carrier

resorption, and to measure new bone volume (BV), bone surface to bone volume

ratio (BS/BV), trabecular thickness (Tb.Th), open porosity (Po(op)), and closed

porosity (Po(cl)) in the defect site. For the groups Paste 1 and Paste 2 (the bone

protein extract and Cambioceramics β-TCP in PEG-GLY matrix with stearic acid)

micro-CT evaluations showed good bone formation, bone union, and bone

remodelling, especially in the group Paste 1 (60 mg) in the defect area, even

though there were cortical areas without new bone or remnants of β-TCP

granules. Almost all β-TCP granules had resorbed during the follow-up. Paste 2

had half of the amount of bone protein extract (30 mg) and there were still some

remnants of β-TCP granules in the defects. In micro-CT analysis, Paste 1 and

Paste 2 had significantly greater BV and Po(cl) than the groups DBM or

Untreated (p < 0.05) (Fig. 9). They also had higher BS/BV than Autograft and

Untreated (Fig. 10). The Tb.Th was smaller than in the groups Autograft, DBM or

Untreated, but greater than in the groups including granules without the bone

protein extract (p < 0.05) (Table 7).

69

For the group Paste 4 (the bone protein extract and Cerasorb® M β-TCP in

PEG-GLY matrix with stearic acid) micro-CT evaluations showed good new bone

formation, bone union and bone remodelling in the defect area, although there

were cortical areas without new bone or remnants of β-TCP granules. β-TCP

granules had resorbed during the follow-up. Paste 4 had significantly greater BV

and Po(cl) than DBM or Untreated (p < 0.05) (Fig. 9). It also had higher BS/BV

than Autograft, DBM and Untreated (Fig. 10). The Tb.Th was smaller than in the

groups Autograft, DBM or Untreated, but greater than in the groups including β-

TCP granules without the bone protein extract (p < 0.05) (Table 7).

For the group Paste 6 (the bone protein extract and Cerasorb® β-TCP in PEG-

GLY matrix with stearic acid) micro-CT evaluations showed good new bone

formation and bone union in the defect area. Most of the β-TCP granules had

resorbed during the follow-up, but there were still some separate, unresorbed

granules in the defects. There was still the same problem as in the other groups

with PEG-GLY matrix and stearic acid that the implant (granules) had not filled

the whole defect and there were some empty areas, especially in the cortical site.

Similarly to other groups with the reindeer bone protein extract, also Paste 6 had

significantly greater BV than the groups DBM and Untreated (p < 0.05) (Fig. 9).

It also had higher BS/BV than autograft, DBM and Untreated (Fig. 10). Measured

Tb.Th was smaller than in the groups Autograft or Untreated, but greater than in

the groups including granules without the bone extract (Table 7). Although new

bone formation and bone volume were good, Paste 6 had the greatest proportional

Po(cl) amount (p < 0.05) (Table 7).

For the control groups Paste 3 and Paste 5 (Cambioceramics in PEG-GLY

matrix with stearic acid, without bone protein extract), micro-CT evaluations

showed that most β-TCP granules had not yet resorbed and they had packed as a

thick mass into the bottom of the defect. Unresorbed β-TCP granules could

clearly be seen, and they had packed as a thick mass at the bottom of the defect.

There was new bone formation in this granule mass, but only around the granules.

Bone union was not seen. Paste 3 and Paste 5 had statistically significantly greater

BV and Po(cl) than DBM and Untreated (p < 0.05) (Fig. 9). They also had higher

BS/BV than Autograft, Untreated and Paste 1 (p < 0.05) (Fig. 10). Measured

Tb.Th was smaller in these groups compared to the other control groups (except

the group Granule) or the groups with the reindeer bone protein extract (p < 0.05)

(Table 7).

For the other control group, Granule (pure Cambioceramics β-TCP), micro-

CT evaluations showed that implanted granules had filled the whole defect area

70

but the granules had not resorbed during the follow-up. There was some new bone

formation around the granules but no bone union. The Granule group also had

significantly greater BV and Po(cl) than DBM or Untreated (p < 0.05) (Fig. 9). It

also had greater Po(cl) than Autograft and higher BS/BV than Autograft,

Untreated and the groups including the reindeer bone extract (p < 0.05) (Fig. 10).

Measured Tb.Th was smaller in this group compared to the other control groups

(except Paste 5) and the groups with the bone protein extract (p < 0.05) (Table 7).

Furthermore, Autograft, DBM and Untreated defects were used as controls. For

the group Autograft, micro-CT evaluation showed clear new bone formation and

bone remodelling in the defect area. Empty areas in the cortical sites were not

seen, except in a couple of defects. In the DBM group (Grafton Plus® DBM) and

the Untreated group micro-CT evaluations showed no new bone formation in the

defect sites during the follow-up.

Fig. 9. Bone volumes (BV, mm3) were measured from the defect areas using micro-CT.

Mean value and standard deviation are shown. In the statistic comparisons all other

groups had significantly greater BV than DBM and untreated defects (p < 0.05). The

control groups have been marked as brick pattern.

BV

(mm3 )

0102030405060708090

DB

M

Unt

reat

ed

Pas

te 5

*

Pas

te 4

*

Pas

te 1

*

Pas

te 2

*

Pas

te 3

*

Gra

nule

*

Aut

ogra

ft*

Pas

te 6

*

71

Fig. 10. Bone surface to bone volume ratio (BS/BV, mm-1) was measured using micro-

CT analysis. It shows the amount of unresorbed granules after follow-up. Median

(quartiles, min and max values) is shown in box-plot figure. ´p < 0.05 vs. autograft,

DBM and untreated, ´´p < 0.05 vs. autograft, DBM, untreated or the groups with the

reindeer bone extract.

Table 7. Trabecular thickness (Tb.Th), open porosity (Po(op)), and closed porosity

(Po(cl)) were measured from the defect areas in imaging analysis of micro-CT results.

Mean and median are given. Values of p < 0.05 are considered statistically significant.

Group N Tb.Th (mm) median (quartiles)

Po(op) (%) mean (std. dev)

Po(cl) (%) median (quartiles)

Bone protein extract and β-TCP granules in PEG-GLY matrix Paste 1 8 0.16 (0.15, 0.17)a,b,c 60.60 (11.73)a,b 3.67 (1.76, 5.46)a,b Paste 2 8 0.16 (0.15, 0.17)b,c 59.38 (11.27)a,b 4.93 (2.77, 5.90)a,b,c Paste 4 7 0.15 (0.14, 0.17)b,c 61.46 (16.30)a,b 3.51 (1.77, 5.71)a,b Paste 6 8 0.15 (0.14, 0.18)b.c 52.77 (6.37)a,b 9.09 (4.84, 10.93)e

β-TCP granules in PEG-GLY matrix Paste 3 8 0.13 (0.13, 0.14)a,b,c,f 57.05 (18.61)a,b 2.95 (2.39, 4.45)a,b Paste 5 8 0.12 (0.11, 0.15)a,b,c,f 62.47 (18.49)a,b 3.06 (1.76, 5.00)a,b

β-TCP granules Granule 7 0.12 (0.11, 0.13)a,b,c,d,g 56.00 (7.54)a,b 4.14 (2.70, 4.33)a.b.c

Controls Autograft 8 0.18 (0.17, 0.18) 50.76 (10.80)a,b 2.04 (1.19, 3.33)a,b DBM 7 0.18 (0.17, 0.18) 89.75 (5.20) 0.54 (0.33, 0.85) Untreated 8 0.20 (0.17, 0.22) 79.40 (8.28) 0.75 (0.32, 1.25)

ap < 0.05 vs. DBM, bp < 0.05 vs. Untreated, cp < 0.05 vs. Autograft,dp < 0.05 vs. Paste 3, ep < 0.05 vs. all other groups, fp < 0.05 vs. groups including the reindeer bone extract

0

5

10

15

20

25

30

35

40

45

50

Pas

te 1

Pas

te 2

Pas

te 4

Pas

te 6

Past

e 3´

Past

e 5´

Gra

nule

´´

Auto

graf

t

DBM

Unt

reat

ed

BS/

BV (m

m-1)

*

*

72

73

6 Discussion

6.1 Time and dose-dependent effects of the reindeer bone protein

extract

The current results confirm previous results, which showed that the reindeer bone

extract has good osteoconductivity both in the mouse muscle pouch model and

the rabbit radius defect model (Pekkarinen et al. 2003, 2005c,b, 2006). It is

known that animal and fracture models, delivery systems and route of

administration influence the dose response in the bone healing studies

(Nunamaker 1998). Above a certain threshold, bone formation cannot be further

enhanced, as was seen in this study. The minimally effective doses of the reindeer

bone extract appear to be between 2 mg and 5 mg in this study and with an

organic scaffold model. Pekkarinen et al. (2006) used 5 mg and 10 mg of non-

sterilized reindeer bone extract in the rabbit radius. These amounts healed the

defect much better than the controls after six weeks of follow-up in the study with

collagen carrier and after eight weeks in the study with bioactive glass carrier.

The 10 mg bone extract group had significantly higher mean stiffness than the

collagen group (Pekkarinen et al. 2006).

New bone formation was seen in all specimens containing the reindeer bone

extract or rhBMP, but the most abundant bone formation and bone union was seen

in the specimens with 15 mg to 50 mg extract. Furthermore, the area of healed

new bone at the defect site increased at the doses of 15 mg of the extract or more,

which might support the use of the higher doses. On the other hand, in studies III

and IV, the formulated bone protein extract together with inorganic carrier gave

good bone formation with extract doses of 3 mg or lower. In study I, a larger

collagen sponge carrier was used with a higher amount of extract, and the

concentration was different compared to lower amounts. Perhaps the larger

sponge remained longer in the defect and gave better support for growth factors

and proteins to improve the bone-healing cascade. The bone protein extract

together with bioactive glass granules was moulded to the same implant size. The

concentration was thus greater and bone healing results were better with the

higher doses. This result was ensured with the ectopic bone formation model.

When bone proteins were not very well absorbed in the carrier or if the carrier

with extract had broken, new bone formation was greater. Thus, a minimum

effective dosage of extract is dependent on optimization of the extract

74

formulation, carrier material and form of implant. The greatest bone formation

occurred with the formulations that had the bone protein extract readily available.

This indicates that bone forming factors are required in sufficient concentrations

at the early stage.

More than twenty BMPs have now been described, and about ten of them

have functions in different phases of bone healing (Wozney & Rosen 1998, Cheng

et al. 2003). In study I, rhBMP-2 was included in the comparisons because the

bone healing capacity of rhBMP-2 has been well documented and it has been

approved by the EU and FDA for lower back spinal fusion and tibial fractures in

clinical use (Govender et al. 2002, Burkus et al. 2002, Einhorn et al. 2003, Kim et al. 2005, Seeherman et al. 2006, Chu et al. 2007, McKay et al. 2007, Issa et al. 2008). The bone formation capacity of rhBMP-2 in the rat femur segmental defect

model was superior compared to the negative control groups and to the reindeer

bone extract groups. However, in most cases of bone treated by 30 µg rhBMP-2,

the dose was too high, causing new bone area to overgrow. The optimal effective

dosage of rhBMP-2 is difficult to find because the amounts of rhBMP-2 used in

rat studies with collagen as a carrier have ranged from 0.5 µg to 200 µg (Yasko et al. 1992, Fujimura et al. 1995, King et al. 1998, Hyun et al. 2005, Kim et al. 2005). The used doses of bone extracts are milligrams compared to recombinant

products that are in micrograms. This is because the bone extracts and DBM

products involve a wide spectrum of all bone proteins and collagens while

recombinants have only one specific protein with a specific task in bone

regeneration (Sampath & Reddi 1983, Baas et al. 2006). Therefore, DBM

products are closer reference material for reindeer-derived extract than rhBMPs

(Huffer et al. 2007).

The resorption rate of carrier material is a factor that is important to consider

not only in planning a follow-up of an experimental study but also in choosing

clinical treatment (Van der Stok et al. 2011). Resorption times of synthetic bone

grafts vary from weeks to years (Van der Stok et al. 2011). Calcium sulphate has

the shortest reabsorption time (weeks) and bioactive glass the longest (years)

(Moore et al. 2001). The resorption rate of calcium phosphate is typically

between 6 and 24 months (Bohner 2001, Ioku 2010). In study II, we decided to

extend the follow-up time of the Bioactive glass + 40 mg extract group from 8 to

10 weeks to assess longer-term healing. The results of bone formation and bone

union in this group were significantly better after 10 weeks compared with all the

other groups after 8 weeks of follow-up. However, there was plenty of bioactive

glass material remaining even after 10 weeks’ follow-up when granule sizes of

75

500-800 µm were used. It is known that bioglass is radiopaque and can have

influence on the pQCT results, the density of bioactive glass being slightly higher

than that of bone (Griffon et al. 2001). This may explain why there were no

statistically significant differences in bone density between any of the implant

groups, although the bone extract groups displayed more bone formation than the

bioactive glass group (which displayed the highest density value). Thus, bioactive

glass may overcome the density of new bone. On the other hand, plain

radiographs showed signs of increased dissolution of bioactive glass in the bone

extract groups, which could decrease the density values.

6.2 Experimental models

For more than twenty years, our research group has been using the muscle pouch

model as a standard assay in evaluating the bone formation activity of reindeer

bone protein extract (Jortikka et al. 1993a, Pekkarinen et al. 2003). Furthermore,

our study group has experience of sheep long bone defect model and both dog and

rabbit ulnar defect models (Gao et al. 1996, 1997, Tuominen et al. 2000, 2001,

Pekkarinen et al. 2006). The rat femur defect model is also widely used in the

literature (Takagi & Urist 1982, Ohgushi et al. 1989, Yasko et al. 1992, Stevenson

et al. 1994, Wolff et al. 1994, Hunt et al. 1996, Chen et al. 2002, Vögelin et al. 2005). Thus, it was a surprise that the effect of reindeer bone extract in the rat

femur defect model was not as good as in the rabbit ulnar defect model owing to

differences in animal species and experimental design (Pekkarinen et al. 2006).

Nienhuijs et al. 2011 reported about same problem with their product (Collos® E)

when they used a rat subcutaneous implantation model (Nienhuijs et al. 2011). It

could imply that animal-derived extracts can be immunogenic in the rat model

(Sampath & Reddi 1983, Viljanen & Lindholm 1993, Bessho et al. 1999). In

some studies nude rats have been used to obviate problems of immunogenic

reactions to bone protein extracts (Ripamonti et al. 1991, Edwards et al. 1998,

Bomback et al. 2004, Wang et al. 2007, Qiu et al. 2009, Lee et al. 2011). It was

also surprising that DBM did not work in study IV although there were no

infections or other problems with the sheep. It could be that sheep as a model is

not suitable for Grafton® or other human DBM products compared to DBM

products of animal origin, such as Collos®, which has given superior healing

results in sheep and dog models (Huffer et al. 2007, Baas et al. 2008). However,

the Grafton® product had also been tested in the muscle pouch model of our study

group, but no signs of bone formation were seen within 21 days either

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roentgenographically or histologically (data not shown). Furthermore, there are

some studies in the literature in which bone healing differences have been found

between different commercially available DBM products (Peterson et al. 2004,

Wildemann et al. 2007, Wang et al. 2007, Athanasiou et al. 2010). There are not

many animal-derived products that used in the clinical trials but not also reported

adverse effects (Bai et al. 1996, Kujala et al. 2004, 2008).

6.3 From carrier of the reindeer bone protein extract to an ideal implant

Ideally, bone formation and scaffold degradation follow one another until the

defect area has been completely replaced by new bone. As long as bone formation

is not extensive enough to supply mechanical strength, the scaffold material

should degrade so slowly that support characteristics are not exposed (Kroese-

Deutman et al. 2005). Reindeer bone protein extract has high bone formation

activity, as seen in bioactivity and previous tests (Jortikka et al. 1993b,

Pekkarinen et al. 2003, Ulmanen et al. 2005); however, in a real bone-healing

situation, the extract cannot work without a scaffold system. Pure collagen as

carrier has been tested in some of our previous studies (Pekkarinen et al. 2004,

2005b,c, 2006) and in study I. Lyophilized extract was mixed in water and

pipetted onto the collagen sponge, or the collagen sponge, soaked in water and

then in extract, was bundled up to form an implant. The results showed good bone

formation in the mouse pouch model and also in the segmental defect model.

However, it seems that collagen alone is not an adequate scaffold and carrier

system for bone proteins. Kim et al. 2005 found in their study that β-TCP with

rhBMP-2 was more effective in ectopic bone formation than collagen with

rhBMP-2. They supposed that β-TCP provided more space and support for bone

formation (Kim et al. 2005). Thus, an inorganic scaffold would be a better choice

to support bone healing with extract as well.

Study II showed for the first time that bioactive glass is a candidate carrier for

the reindeer bone protein extract when used in a rat femur defect model. The

results suggest that the bone extract enhances the healing of bone, compared with

bioactive glass alone. Although bioactive glass is used alone in bone surgery, its

healing effect is increased when it is combined with bone extract or BMP

products (Kotani et al. 1992, Pajamäki et al. 1993a,b, Mahmood et al. 2001,

Takita et al. 2004, Välimäki et al. 2005, Bergeron et al. 2007).

77

Granulometry is influenced by the biological properties of bioactive glass

(Wheeler et al. 1998, Oonishi et al. 1999, Mahmood et al. 2001, Granito et al. 2009). The size of the bioactive glass granules used in this study was 500-800

μm, which appeared to be a good size because the granules could be impacted

firmly in the bone defect and because granules of this size are not resorbed too

quickly. In our preliminary bioactive glass study (results not shown) we used

smaller bioactive glass granule size (< 300 μm), but after 3 weeks the granules

had resorbed, and no bone formation could be seen even after 8 weeks. Different

ratios of the compounds of bioactive glass granules could have been used to

adjust the degradation process, allowing more time for osteoconduction and a

larger pore size would also have yielded slower resorption of the bioactive glass;

thus pore sizes > 300 μm are recommended (Wheeler 1998, Hall et al. 1999,

Lindfors & Aho 2003, Karageorgiour & Kaplan 2005, Granito et al. 2009).

6.4 Calcium salts as potential carrier candidates

Study III was aimed to find a suitable inorganic carrier candidate for the reindeer

bone protein extract. The lyophilized extract was absorbed into pores in the β-

TCP/CS 90:10 group, partly surface-coated and partly absorbed in the HA/β-

TCP/CS 30:60:10 group, surface-coated in the HA/β-TCP/CS 60:30:10 group,

blended into the carrier material in the CS hemihydrate and the Cem-Ostetic

groups, and dry-blended without re-suspending the lyophilized extract in the CS

dihydrate-stearic acid group. Because the combination of the lyophilized

formulation and the carrier was different in each of the study groups, the

distribution and availability of the extract was also different and statistical

comparison between carrier groups is not very valid. The greatest bone formation

was seen in the groups that had the bone extract readily available, which indicates

that the bone forming factors are needed at the early stage at sufficient

concentration. This was seen especially in the groups HA/β-TCP/CS 60:30:10 and

CS dihydrate-stearic acid. In the groups β-TCP/CS 90:10, Cem-Ostetic putty and

CS hemihydrate a difference was seen between active implants and controls, and

they worked as implant with bone protein mixture coating. The lowest bone

formation was found in the group HA/β-TCP/CS 30:60:10. This may indicate that

the bone extract was absorbed deep into the scaffold during implant preparation,

and the releasing amount of bone proteins was too low to induce new bone

formation. Our results support previous studies that formation of new bone

depends on a ceramic content with high HA/TCP ratio and high dose of bone

78

proteins (Alam et al. 2001, Baas et al. 2008). Furthermore, this study confirms

that the presence of bioactive components reduces fibrous tissue formation and

increases bone formation around inorganic scaffolds (Wolff et al. 1994, Chen et al. 2007, Baas et al. 2008, Ioku 2010). However, the amount and availability of

bone proteins should be in balance with the bone healing and forming cascade

(Dimitriou et al. 2005).

In study III, it was found that stearic acid had a positive effect, enhancing

bone ingrowth and formation in the environment of calcium sulphate carrier.

Stearic acid has been widely used as excipient in tablet manufacturing because

adding stearic acid decreases the viscosity of ceramic suspension while increasing

the microstructural uniformity of particle packing (Tseng et al. 1999). Wright

Medical Technology Inc. has used stearic acid as a tabletting aid in their calcium

sulphate products, such as Osteoset®, and noticed good bone healing capacity, as

we found in this study (Urban et al. 2003, Turner et al. 2003). Therefore, we think

that calcium stearate has not only tabletting aid properties but also supports bone

formation, like carboxymethyl cellulose does (Reynolds et al. 2007).

Study IV supports earlier findings showing that the presence of bioactive

components reduces fibrous tissue formation and increases bone formation

around inorganic scaffolds (Baas et al. 2008). The implantation in study IV was

easy to perform and no extra mixing of product was needed on the operation

table, which is a recommendable criterion for implants in trauma surgery.

However, the analysis showed that the amount of granules used was not enough

to fill the whole defect area after the matrix was dissolved, so that the cortical site

of the defect was usually empty without new bone or traces of granules. This can

have deleterious effects on bone healing (Lindfors et al. 2010). This was

compared to the pure β-TCP group in which double the amount of granules was

used and the whole defect area was full of granules after the follow-up as well.

Thus, the effect of bone healing cannot be compared only by the difference in

bone formation between the study groups; optimizing of the PEG-GLY matrix

with stearic acid and granules is needed.

Ideally, bone formation and scaffold degradation follow one another until the

defect area has been completely replaced by new bone. As long as bone formation

is not extensive enough to supply mechanical strength, the scaffold material

should degrade so slowly that support characteristics are not exposed (Kroese-

Deutman et al. 2006). Evaluating of micro-CT images showed that granule

resorption was the fastest in the group with the bone protein extract, but there was

also good new bone formation seen and bone remodelling was ongoing. The

79

resorption of granules was not observed in the group that involved only granules

or granules with PEG-GLY matrix and stearic acid. Bone formation was seen, but

usually only around the granules. Analysis of micro-CT images confirmed the

evaluation. The highest bone surface to bone volume ratio value but the smallest

trabecular thickness was seen in the control groups with no reindeer bone protein

extract. This might mean that the granules block normal bone formation or that

the granules give enough load. Therefore, thicker new bone lamellas are not

needed before the granules are resorbed in the defect. However, the follow-up

time used was too short to confirm these consequences. Traditional micro-CT

analysis is not the most optimal analysis method to show details in bone healing

because of β-TCP granule remnants in the defects. However, all results of micro-

CT supported each other. In future, histology and scanned electron microscopy

(SEM) imaging could confirm our micro-CT results.

It has been found that the form, shape and micro- and nanostructures of the

scaffold affect both bone formation and scaffold resorption properties (Li et al. 2003, Zyman et al. 2008, Roohani-Esfahani et al. 2010). In the group involving

morsel form of β-TCP and the bone protein extract, the granules had resorbed

during the follow-up. New bone formation and bone union was seen and the

healing of the defect was very similar with that of autograft. The highest bone

volume was seen in the group that involved a smaller and spherical β-TCP

granule form. However, the measured closed porosity was also greatest in this

group, while open porosity was almost lowest. Thus, the quality of new bone may

not be similar to the quality of old bone in this group. This was also seen in

trabecular thickness. Autograft, DBM and even empty defects had the thickest

new trabeculars. The groups that had been treated with the reindeer bone protein

extract had very similar trabecular thickness. The smallest thickness was seen in

the groups with granules with no bone extract. This means that synthetic

osteoconductive scaffold provides adequate support to new bone formation in the

injury site, but lack of biological activity slows down resorption of granules, and

new bone lamellae have no space to increase their thickness (Moore et al. 2001,

Kim et al. 2005). Autograft, DBM and reindeer-derived bone protein extract have

osteoinductive stimuli that improve the bone-healing cascade, increase carrier

resorption and form space for new bone in the defect.

In study IV, one aim was to compare ceramic implants containing the reindeer

bone protein extract with autograft. Micro-CT evaluation showed that the results

of new bone formation and trabecular thickness were similar in the autograft

group and the groups with the reindeer bone extract. This was an encouraging

80

result in terms of finding a substitute for autograft use. Previous results with

DBM materials support our results (Huffer et al. 2007). Although the paste form

implant containing the reindeer bone protein extract with β-TCP-carrier needs still

minor optimization, clinical application has been reached and studies on the

reindeer bone protein extract can be focused on clinical trials.

81

7 Conclusions

The present study indicates that the tested reindeer bone protein extract can be

used to improve bone formation with various carriers. The study suggests that an

inorganic carrier material together with stearic acid is the one of most suitable

carrier alternatives for the reindeer bone protein extract. The developed medical

device in a paste form can be an alternative for autograft use. Based on the current

findings it can be concluded that

– Reindeer bone extract improved bone formation in critical-size defect model

in a six-week follow-up. The needed dose of bone protein extract depends on

the formulation and carrier systems and the trauma being treated.

– Bioactive glass used in this study can be used as a carrier for reindeer protein

extract. The long-term stability of the selected form of bioactive glass can

interfere with results.

– The greatest bone formation and carrier resorption were seen in the groups

that had the bone extract readily available near the surface of the implant. The

combination of β-TCP or CS and stearic acid appears to be the most ideal

carrier alternative for reindeer bone protein extract.

– β-TCP granules in the PEG-GLY matrix with stearic acid provide a functional

scaffold system for reindeer bone protein extract, but the proportional amount

of granules in the matrix must be still optimized. Bone formation effect was

significantly better with the reindeer-derived bone extract than with a

commercially available DBM product. The tested medical device containing

the reindeer bone protein extract and β-TCP formulation can be a suitable

alternative for autograft use.

82

83

References

Aebli N, Stich H, Schawalder P, Theis JC & Krebs J (2005) Effects of bone morphogenetic protein-2 and hyaluronic acid on the osseointegration of hydroxyapatite-coated implants: an experimental study in sheep. J Biomed Mater Res A 73(3): 295–302.

Ai-Aql ZS, Alagl AS, Graves DT, Gerstenfeld LC & Einhorn TA (2008) Molecular mechanism controlling bone formation during fracture healing and distraction osteogenesis. J Dent Res 87(2): 107–118.

Aitasalo K & Peltola M (2004) Bioaktiivinen lasi pään ja kaulan alueen kirurgiassa. Lääketieteellinen Aikakauskirja Duodecim 120(16): 1986–1993.

Alam I, Asahina I, Ohmamiuda K., Takahashi K, Yokota S & Enomoto S (2001) Evaluation of ceramics composed of different hydroxyapatite to tricalcium phosphate ratios as carriers for rhBMP-2. Biomaterials 22: 1643–1651.

Alves HLR, dos Santos LA & Bergmann CP (2008) Injectability evaluation of tricalcium phosphate bone cement. J Mater Sci Mater Med 19(5): 2241–2246.

Andersson OH & Karlsson KH (1991) On the bioactivity of bioactive glass-ceramic materials to bone and muscle. J Non-Cryst Solids 129: 145–151.

Andersson OH & Kangasniemi I (1991) Calcium phosphate formation at the surface of bioactive glass in vitro. J Biomed Mater Res 25(8): 1019–1030.

Andrew JG, Hoyland JA, Freemont AJ & Marsh DR (1995) Platelet-derived growth factor expression in normally healing human fractures. Bone 16(4): 455–460.

Aro H & Kettunen J (2010) Luunmurtumat. In Kröger H, Aro H, Böstman O, Lassus J & Salo J (eds) Traumatologia. Helsinki, Kandidaattikustannus Oy: 218–221.

Arrington ED, Smith WJ, Chambers HG, Bucknell AL & Davino NA (1996) Complications of iliac crest bone graft harvesting. Clin Orthop Relat Res 329: 300–309.

Athanasiou V, Papachristou DJ, Panagopoulos A, Saridis A, Scopa CD & Megas P (2010) Histological comparison of autograft, allograft-DBM, xenograft, and synthetic grafts in a trabecular bone defect: An experimental study in rabbits. Med Sci Monit 16(1): BR24–BR31.

Baas J, Lamberg A, Jensen TB, Elmengaard B & Søballe K (2006) The bovine bone protein lyophilisate Colloss improves fixation of allografted implants – an experimental study in dogs. Acta Orthop 77(5): 791–798.

Baas J, Elmengaard B, Bechtold J, Chen X & Søballe K (2008) Ceramic bone graft substitute with equine bone protein extract is comparable to allograft in terms of implant fixation: a study in dogs. Acta Orthop 79(6): 841–850.

Babis GC & Suocacos PN (2005) Bone scaffolds: The role of mechanical stability and instrumentation. Injury 36S: S38–S44.

Bai MH, Liu XY, Ge BF, Yallag C & Chen DA (1996) An implant of a composite of bovine bone morphogenetic protein and plaster of paris for treatment of femoral shaft nonunions. Int Surg 81(4): 390–392.

84

Barboza EP, Caúla AL, Caúla Fde O, de Souza RO, Geolás Neto L, Sorensen RG, Li XJ & Wikesjö UM (2004) Effect of recombinant human bone morphogenetic protein-2 in an absorbable collagen sponge with space-providing biomaterials on the augmentation of chronic alveolar ridge defects. J Periodontol 75(5): 702–708.

Barr T, McNamara AJ, Sándor GK, Clokie CM & Peel SA (2010) Comparison of the osteoinductivity of bioimplants containing recombinant human bone morphogenetic proteins 2 (Infuse) and 7 (OP-1). Oral Surg Oral Med Oral Pathol Oral Radiol Endod 109(4): 531–540.

Barrack RL (2005) Bone graft extenders, substitutes, and osteogenic proteins. J Arthroplasty 20(4S): 94–97.

Barrientos S, Stojadinovic O, Golinko MS, Brem H & Tomic-Canic M (2008) Growth factors and cytokines in would healing. Wound Rep Reg 16: 585–601.

Bauer TW & Muschler GF (2000) Bone graft materials: An overview of the basic science. Clin Orthop Relat Res 371: 10–27.

Bax BE, Wozney JM & Ashhurst DE (1999) Bone morphogenetic protein-2 increases the rate of callus formation after fracture of the rabbit tibia. Calcif Tissue Int 65(1): 83–89.

Begley CT, Doherty MJ, Mollan RA & Wilson DJ (1995) Comparative study of the osteoinductive properties of bioceramic, coral and processed bone graft substitutes. Biomaterials 16(15): 1181–1185.

Bergeron E, Marquis ME, Chrétien I & Faucheux N (2007) Differentiation of preosteoblasts using a delivery system with BMPs and bioactive glass microspheres. J Mater Sci Mater Med 18(2): 255–263.

Bessa PC, Casal M & Reis RL (2008a) Bone morphogenetic proteins in tissue engineering: the road from the laboratory to the clinic, part I (basic concepts). J Tissue Eng Regen Med 2(2-3): 1–13.

Bessa PC, Casal M & Reis RL (2008b) Bone morphogenetic proteins in tissue engineering: the road from laboratory to clinic, part II (BMP delivery). J Tissue Eng Regen Med 2(2-3): 81–96.

Bessho K, Tanaka N, Matsumoto J, Tagawa T & Murata M (1991a) Human dentin-matrix-derived bone morphogenetic protein. J Dent Res 70(3): 171–175.

Bessho K, Tagawa T & Murata M (1991b) Analysis of bone morphogenetic protein (BMP) derived from human and bovine bone matrix. Clin Orthop Relat Res 268: 226–234.

Bessho K, Tagawa T & Murata M (1992) Comparison of bone matrix-derived bone morphogenetic proteins from various animals. J Oral Maxillofac Surg 50(5): 496–501.

Bessho K & Iizuka T (1993) Changes in bone inducing activity of bone morphogenetic protein with aging. Ann Chir Gynaecol Suppl 207: 49–53.

Bessho K, Kusumoto K, Fujimura K, Konishi Y, Ogawa Y, Tani Y & Iizuka T (1999) Comparison of recombinant and purified human bone morphogenetic protein. Br J Oral Maxillofac Surg 37(1): 2–5.

Betz RR (2002) Limitations of autograft and allograft: new synthetic solutions. Orthopedics 25(5S): S561–S570.

85

Blokhuis TJ, den Boer FC, Bramer JAM, den Boer FC, Bakker FC, Patka P & Haarman HJ (2001) Biomechanical and histological aspects of fracture healing stimulated with osteogenic protein-1. Biomaterials 22(7): 725–730.

Blom EJ, Klein-Nulend J, Yin L, van Waas MA & Burger EH (2001) Transforming growth factor-beta1 incorporated in calcium phosphate cement stimulates osteotransductivity in rat calvarial bone defects. Clin Oral Implants Res 12(6): 609–616.

Blom EJ, Klein-Nulend J, Wolke JGC, van Waas MAJ, Driessens FCM & Burger EH (2002) Transforming growth factor-β1 incorporation in a calcium phosphate bone cement: Material properties and release characteristics. J Biomed Mater Res 59(2): 265–272.

Bodde EWH, Wolke JGC, Kowalski RSZ & Jansen JA (2007) Bone regeneration of porous β-tricalcium phosphate (ConduitTM TCP) and of biphasic calcium phosphate ceramic (Biosel®) in trabecular defects in sheep. J Biomed Mater Res 82(3): 711–722.

Boden SD, Grob D & Damien C (2004) Ne-Osteo bone growth factor for posterolateral lumbar spine fusion: results from a nonhuman primate study and a prospective human clinical pilot study. Spine 29(5): 504–514.

den Boer FC, Patka P, Bakker FC, Wippermann BW, van Lingen A, Vink GQM, Boshuizen K & Haarman HJ (1999) New segmental long bone defect model in sheep: quantitative analysis of healing with dual energy x-ray absorptiometry. J Orthop Res 17: 654–660.

den Boer FC, Bramer JA, Blokhuis TJ, Van Soest EJ, Jenner JM, Patka P, Bakker FC, Burger EH & Haarman HJ (2002) Effect of recombinant human osteogenic protein-1 on the healing of a freshly closed diaphyseal fracture. Bone 31(1): 158–164.

den Boer FC, Wippermann BW, Blokhuis TJ, Patka P, Bakker FC & Haarman HJTHM (2003) Healing of segmental bone defects with granular porous hydroxyapatite augmented with recombinant human osteogenic protein-1 or autologous bone marrow. J Orthop Res 21(3): 521–528.

Bohner M (2001) Physical and chemical aspects of calcium phosphates used in spinal surgery. Eur Spine J 10(S2): S114–S121.

Bomback DA, Grauer JN, Lugo R, Troiano N, Patel TCh & Friedlaender GE (2004) Comparison of posterolateral lumbar fusion rates of Grafton Putty and OP-1 Putty in an athymic rat model. Spine 29(15): 1612–1617.

Bonewald LF & Mundy GR (1990) Role of transforming growth factor-beta in bone remodeling. Clin Orthop Relat Res 250: 261–276.

Bostrom M, Lane JM, Tomin E, Browne M, Berberian W, Turek T, Smith J, Wozney J & Schildhauer T (1996) Use of bone morphogenetic protein-2 in the rabbit ulnar nonunion model. Clin Orthop Relat Res 327: 272–282.

Burchardt H (1983) The biology of bone graft repair. Clin Orthop Relat Res 174: 28–42. Burkus JK, Transfeldt EE, Kitchel SH, Watkins RG & Balderston RA (2002) Clinical and

radiographic outcomes of anterior lumbar interbody fusion using recombinant human bone morphogenetic protein-2. Spine 27(21): 2396–2408.

86

Burkus JK, Dorchak JD & Sanders DL (2003) Radiographic assessment of interbody fusion using recombinant human bone morphogenetic protein type-2. Spine 28(4): 372–377.

Burt DW & Law AS (1994) Evolution of the transforming growth factor-beta superfamily. Prog Growth Factor Res 5(1): 99–118.

Cao W & Hench LL (1996) Bioactive materials. Ceram Int 22: 493–507. Carey LE, Xu HHK, Simon CG, Takagi S & Chow LC (2005) Premixed rapid-setting

calcium phosphate composites for bone repair. Biomaterials 26(24): 5002–5014. Carson JS & Bostrom MPG (2007) Synthetic bone scaffolds and fracture repair. Injury

38(S1): S33–S37. Celeste AJ, Iannazzi JA, Taylor RC, Hewick RM, Rosen V, Wang EA & Wozney JM

(1990) Identification of transforming growth factor β family members present in bone-inductive protein purified from bovine bone. Proc Natl Acad Sci 87: 9843–9847.

Cerruti M, Greespan D & Powers K (2005) Effect of pH and ionic strength on the reactivity of Bioglass® 45S5. Biomaterials 26: 1665–1674.

Chakkalakal DA, Strates BS, Mashoof AA, Garvin KL, Novak JR, Fritz ED, Mollner TJ & McGuire MH (1999) Repair of segmental bone defects in the rat: An experimental model of human fracture healing. Bone 25(3): 321–332.

Chen X, Kidder LS & Lew WD (2002) Osteogenic protein-1 induced bone formation in an infected segmental defect in the rat femur. J Orthop Res 20(1): 142–150.

Chen QZ, Thompson ID & Boccaccini AR (2006) 45S5 Bioglass® – derived glass-ceramic scaffolds for bone tissue engineering. Biomaterials 27: 2414–2425.

Chen QZ, Li Y, Jin LY, Quinn JM & Komesaroff PA (2010) A new sol-gel process for producing Na(2)O-containing bioactive glass ceramics. Acta Biomater 6(10): 4143–4153.

Cheng H, Jiang W, Phillips FM, Haydon RC, Peng Y, Zhou L, Luu HH, An N, Breyer B, Vanichakarn P, Szatkowski JP, Park JY & He Tc (2003) Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg 85-A(8): 1544–1552.

Cho TJ, Gerstenfeld L & Einhorn TA (2002) Differential temporal expression of members of the transforming growth factor β Superfamily during murine fracture healing. J Bone Miner Res 17(3): 513–520.

Chu TM, Warden SJ, Turner CH & Stewart RL (2007) Segmental bone regeneration using a load-bearing biodegradable carrier of bone morphogenetic protein-2. Biomaterials 28(3): 459–467.

Cole BJ, Bostrom MP, Pritchard TL, Sumner DR, Tomin E, Lane JM & Weiland AJ (1997) Use of bone morphogenetic protein 2 on ectopic porous coated implants in the rat. Clin Orthop Relat Res 345: 219–228.

Cornu O, Boquet J, Nonclercq O, Docquier PL, Van Tomme J, Delloye C & Banse X (In Press) Synergetic effect of freeze-drying and gamma irradiation on the mechanical properties of human cancellous bone. Cell Tissue Bank.

87

Corradini E, Schmidt PJ, Meynard D, Garuti C, Montosi G, Chen S, Vukicevic S, Pietrangelo A, Lin HY & Babitt JL (2010) BMP6 treatment compensates for the molecular defect and ameliorates hemochromatosis in Hfe knockout mice. Gastroenterology 139(5): 1721–1729.

Cooper GM, Mooney MP, Gosain AK, Campbell PG, Losee JE & Huard J (2010) Testing the critical size in calvarial bone defects: Revisiting the concept of a critical-size defect. Plast Reconstr Surg 125: 1685–1692.

Coulson R, Clokie CML, Peel S (1999) Collagen and a thermally reversible poloxamer deliver demineralised bone matrix (DBM) and biologically active proteins to sites of bone regeneration. Proc Portland Bone Symposium 619–637.

Cui X, Zhang B, Wang Y & Gao Y (2008) Effects of chitosan-coated pressed calcium sulfate pellet combined with recombinant human bone morphogenetic protein 2 on restoration of segmental bone defect. J Craniofac Surg 19(2): 459–465.

Cullinane DM, Fredrick A, Eisenberg SR, Pacicca D, Elman MV, Lee C, Salisbury K, Gerstenfeld LC & Einhorn TA (2002) Induction of a neoarthrosis by precisely controlled motion in an experimental mid-femoral defect. J Orthop Res 20: 579–586.

Damien CJ, Grob D, Boden SD & Benedict JJ (2002) Purified bovine BMP extract and collagen for spine arthrodesis: preclinical safety and efficacy. Spine 27(16 S1): S50–S58.

de Biase P & Capanna R (2005) Clinical applications of BMPs. Injury 36(S3): S43–S46. Deckers MML, van Bezooijen RL, van der Horst G, Hoogendam J, van der Bent C,

Papapoulos SE & Löwik CWGM (2002) Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor A. Endocrinology 143(4): 1545–1553.

Dimitriou R, Tsiridis E & Giannoudis PV (2005) Current concepts of molecular aspects of bone healing. Injury 36: 1392–1404.

Dodds RA, York-Ely AM, Zhukauskas R, Arola T, Howell J, Hartill C, Cobb RR & Fox C (2010) Biomechanical and radiographic comparison of demineralised bone matrix, and a coralline hydroxyapatite in a rabbit spinal fusion model. J Biomater Appl 25(3): 195–215.

Donati D, Di Bella C, Lucarelli E, Dozza B, Frisoni T, Nicoli Aldini N & Giardino R (2008) OP-1 application in bone allograft integration: preliminary results in sheep experimental surgery. Injury 39(S2): S65–S72.

Douglas T, Pamula E, Hauk D, Wiltfang J, Sivananthan S, Sherry E & Warnke PH (2009) Porous polymer/hydroxyapatite scaffolds: characterization and biocompatibility investigations. J Mater Sci Mater Med 20(9): 1909–1915.

Drespe IH, Polzhofer GK, Turner AS & Grauer JN (2005) Animal models for spinal fusion. Spine J 5(6S): 209S–216S.

Edwards JT, Diegmann MH & Scarborough NL (1998) Osteoinduction of human demineralised bone: characterization in a rat model. Clin Orthop Relat Res 357: 219–228.

88

Einhorn TA, Lane JM, Burstein AH, Kopman CR & Vigorita VJ (1984) The healing of segmental bone defects induced by demineralised bone matrix. A radiographic and biomechanical study. J Bone Joint Surg 66-A(2): 274–279.

Einhorn TA (1998) The cell and molecular biology of fracture healing. Clin Orthop Relat Res 355S: S7–S21.

Einhorn TA, Lane JM, Burstein AH, Kopman Einhorn TA, Majeska RJ, Mohaideen A, Kagel EM, Bouxsein ML, Turek TJ & Wozney JM (2003) A single percutaneous injection of recombinant human bone morphogenetic protein-2 accelerates fracture repair. J Bone Joint Surg 85-A(8): 1425–1435.

Einhorn TA (2005) The science of fracture healing. J Orthop Trauma 19(10S): S4–S6. Engelhardt SA (2008) Bone graft materials in Orthopaedics: 50+ Companies vie for a

piece of the $1.9BB pie. International Orthopaedic Product News 52: 51–61. Erlebacher A, Filvaroff EH, Ye JQ & Derynck R (1998) Osteoblastic responses to TGF-β

during bone remodeling. Mol Biol Cell 9(7): 1903–1918. Feighan JE, Davy D, Prewett AB & Stevenson S (1995) Induction of bone by a

demineralized bone matrix gel: A study in a rat femoral defect model. J Ortop Res 13: 881–891.

Feng Y, Wang S, Jin D, Sheng J, Chen S, Cheng X & Zhang C (2011) Free vascularised fibular grafting with OsteoSet®2 demineralised bone matrix versus autograft for large osteonecrotic lesions of the femoral head. Int Orthop 35(4): 475–481.

Finkemeier CG (2002) Bone-grafting and bone-graft substitutes. J Bone Joint Surg 84-A(3): 454–464.

Forslund C & Aspenberg P (1998) OP-1 has more effect than mechanical signals in the control of tissue differentiation in healing rat tendons. Acta Orthop Scand 69(6): 622–626.

Frantzén J, Rantakokko J, Aro HT, Heinänen J, Kajander S, Gullichsen E, Kotilainen E & Lindfors NC (2011) Instrumented spondylodesis in degenerative spondylolisthesis with bioactive glass and autologous bone. J Spinal Disord Tech 24(7): 455–461.

Freilich M, Shafer D, Wei M, Kompalli R, Adams D & Kuhn L (2009) Implant system for guiding a new layer of bone. Computed microtomography and histomorphometric analysis in the rabbit mandible. Clin Oral Implants Res 20(2): 201–207.

Friedlaender GE (1983) Immune responses to osteochondral allografts. Current knowledge and future directions. Clin Orthop Relat Res 174: 58–68.

Friedlaender GE (1987) Bone grafts. J Bone Joint Surg 69-A(5): 786–790. Fujimura K, Bessho K, Kusumoto K, Ogawa Y & Iizuka T (1995) Experimental Studies on

Bone Inducing Activity of Composites of Atelopeptide Type I Collagen as a Carrier for Ectopic Osteoinduction by rhBMP-2. Biochem Biophys Res Commun 208(1): 316–322.

Galois L & Mainard D (2004) Bone ingrowth into two porous ceramics with different pore sizes: an experimental study. Acta Orthop Belg 70(6): 598–603.

Gao TJ, Lindholm TS, Marttinen A & Puolakka T (1993) Bone inductive potential and dose-dependent response of bovine bone morphogenetic protein combined with type IV collagen carrier. Ann Chir Gynaecol Suppl 207: 77–84.

89

Gao TJ, Lindholm TS, Kommonen B, Ragni P, Paronzini A, Lindholm TC, Jämsä T & Jalovaara P (1996) Enhanced healing of segmental tibial defects in sheep by a composite bone substitute composed of tricalcium phosphate cylinder, bone morphogenetic protein, and type IV collagen. J Biomed Mater Res 32(4): 505–512.

Gao TJ, Lindholm TS, Kommonen B, Ragni P, Paronzini A, Lindholm TC, Jalovaara P & Urist MR (1997) The use of a coral composite implant containing bone morphogenetic protein to repair a segmental tibial defect in sheep. Int Orthop 21: 194–200.

Geiger M, Li RH & Friess W (2003) Collagen sponges for bone regeneration with rhBMP-2. Adv Drug Deliv Rev 55(12): 1613–1629.

Gelse K, Pöschl E & Aigner T (2003) Collagens-structure, function, and biosynthesis. Adv Drug Deliv Rev 55: 1531–1546.

Gerhart TN, Kirker-Head CA, Kriz MJ, Holtrop ME, Hennig GE, Hipp J, Schelling SH & Wang E (1993) Healing segmental femoral defects in sheep using recombinant human bone morphogenetic protein. Clin Orthop Relat Res 293: 317–326.

Gerstenfeld LC, Cullinane DM, Darnes GL, Graves DT & Einhorn TA (2003) Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J Cell Biochem 88(5): 873–884.

Ghosh SK, Nandi SK, Kundu B, Datta S, De DK, Roy SK & Basu D (2008) In vivo response of porous hydroxyapatite and β-tricalcium phosphate prepared by aqueous solution combustion method and comparison with bioglass scaffolds. J Biomed Mater Res B Appl Biomater 86(1): 217–227.

Giannoudis PV, Einhorn TA & Marsh D (2007) Fracture healing: The diamond concept. Injury 38(S4): S3–S6.

Goldberg VM & Stevenson S (1987) Natural history of autografts and allografts. Clin Orthop Relat Res 225: 7–16.

Govender S, Csimma C, Genant HK, Valentin-Opran A, Amit Y, Arbel R, Aro H, Atar D, Bishay M, Börner MG, Chiron P, Choong P, Cinats J, Courtenay B, Feibel R, Geulette B, Gravel C, Haas N, Raschke M, Hammacher E, van der Velde D, Hardy P, Holt M, Josten C, Ketterl RL, Lindeque B, Lob G, Mathevon H, McCoy G, Marsh D, Miller R, Munting E, Oevre S, Nordsletten L, Patel A, Pohl A, Rennie W, Reynders P, Rommens PM, Rondia J, Rossouw WC, Daneel PJ, Ruff S, Rüter A, Santavirta S, Schildhauer TA, Gekle C, Schnettler R, Segal D, Seiler H, Snowdowne RB, Stapert J, Taglang G, Verdonk R, Vogels L, Weckbach A, Wentzensen A, Wisniewski T & BMP-2 Evaluation in Surgery for Tibial Trauma (BESTT) Study Group (2002) Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures: a prospective, controlled, randomized study of four hundred and fifty patients. J Bone Joint Surg 84-A(12): 2123–2134.

Granito RN, Ribeiro DA, Rennó AC, Ravagnani C, Bossini PS, Peitl-Filho O, Zanotto ED, Parizotto NA & Oishi J (2009) Effects of biosilicate and bioglass 45S5 on tibial bone consolidation on rats: a biomechanical and a histological study. J Mater Sci Mater Med 20(12): 2521–2526.

90

Griffon DJ, Dunlop DG, Howie CR, Gilchrist T, Salter DM & Healy DM (2001) Early dissolution of a morsellised impacted silicate-free bioactive glass in metaphyseal defects. J Biomed Mater Res 58(6): 638–644.

Gross TP, Jinnah RH, Clarke HJ & Cox QG (1991) The biology of bone grafting. Orthopedics 14(5): 563–568.

Guda T, Walker JA, Pollot BE, Appleford MR, Oh S, Ong J & Wenke JC (2011) In vivo performance of bilayer hydroxyapatite scaffolds for bone tissue regeneration in the rabbit radius. J Mater Sci Mater Med 22(3): 647–656.

Hall EE, Meffert RM, Hermann JS, Mellonig JT & Cochran DL (1999) Comparison of bioactive glass to demineralised freeze-dried bone allograft in the treatment of intrabony defects around implants in the canine mandible. J Periodontol 70(5): 526– 535.

Hass HJ, Krause H, Kroker S, Wagemann W & Meyer F (2007) Bone formation using human demineralised bone matrix (Grafton) for the treatment of bone cysts in children. Eur J Pediatr Surg 17(1): 45–49.

He TC (2005) Distinct osteogenic activity of BMPs and their orthopaedic applications. J Musculoskelel Neuronal Interact 5(4): 363–366.

Hench LL, Splinter RJ, Allen WC & Greenlee TK (1971) Bonding mechanism at the interface of ceramics prosthetic materials. J Biomed Mater Res Symp 2: 117–141.

Hench LL (2006) The story of Bioglass. J Mater Sci Mater Med 17(11): 967–978. Herbst TJ, McCarthy JB, Tsilibary EC & Furcht LT (1988) Differential effects of laminin,

intact type IV collagen, and specific domains of type IV collagen on endothelial cell adhesion and migration. J Cell Biol 106: 1365–1373.

Hietaniemi K, Peltonen J & Paavolainen P (1995) An experimental model for non-union in rats. Injury 26(10): 681–686.

Hollinger JO, Schmitt JM, Buck DC, Shannon R, Joh SP, Zegzula HD & Wozney J (1998) Recombinant human bone morphogenetic protein-2 and collagen for bone regeneration. J Biomed Mater Res 43(4): 356–364.

Horisaka Y, Okamoto Y, Matsumoto N, Yoshimura Y, Kawada J, Yamashita K & Takagi T (1991) Subperiosteal implantation of bone morphogenetic protein adsorbed to hydroxyapatite. Clin Orthop Relat Res 268: 303–312.

Huang KK, Shen C, Chiang CY, Hsieh YD & Fu E (2005) Effects of bone morphogenetic protein-6 on periodontal wound healing in a fenestration defect of rats. J Periodontal Res 40: 1–10.

Huffer WE, Benedict JJ, Turner AS, Briest A, Rettenmaier R, Springer M & Walboomers XF (2007) Repair of sheep long bone cortical defects filled with COLLOSS, COLLOSS E, OSSAPLAST, and fresh iliac crest autograft. J Biomed Mater Res Part B Appl Biomater 82(2): 460–470.

Hughes-Fulford M & Li CF (2011) The role of FGF-2 and BMP-2 in regulation of gene induction, cell proliferation and mineralization. J Orthop Surg Res 6:8.

Hunt TR, Schwappach JR & Anderson HC (1996) Healing of a segmental defect in the rat femur with use of an extract from a cultured human osteosarcoma cell-line (Saos-2). A preliminary report. J Bone Joint Surg 78-A(1): 41–48.

91

Huttunen M, Törmälä P, Godinho P & Kellomäki M (2008) Fiber-reinforced bioactive and bioabsorbable hybrid composites. Biomed Mater 3(3): 034106.

Hyun SJ, Han DK, Choi SH, Chai JK, Cho KS, Kim CK & Kim CS (2005) Effect of recombinant human bone morphogenetic protein-2, -4, and –7 on bone formation in rat calvarial defects. J Periodontol 76(10): 1667–1674.

Ikada Y (2002) Biological Materials. In: Barbucci R (ed): Integrated Biomaterials Science. New York, Kluwer Academic: 2–3.

Ioku K (2010) Tailored bioceramics of calcium phosphates for regenerative medicine. J Cer Soc Japan 118(9): 775–783.

Issa JPM, do Nascimento C, Iyomasa MM, Siéssere S, Regalo SCH, Defino HLA & Sebald W (2008) Bone healing process in critical-sized defects by rhBMP-2 using poloxamer gel and collagen sponge as carriers. Micron 39(4): 373–379.

Itoh S, Nakamura S, Kobayashi T, Shinomiya K, Yamashita K & Itoh S (2006) Effect of electrical polarization of hydroxyapatite ceramics on new bone formation. Calcif Tissue Int 78: 133–142.

Jaatinen R, Bondestam J, Raivio T, Hildén K, Dunkel L, Groome N & Ritvos O (2002) Activation of the bone morphogenetic protein signalling pathway induces inhibin βB-subunit mRNA and secreted inhibin B levels in cultured human granulosa-luteal cells. J Clin Endocrinol Metab 87(3): 1254–1261.

Jang JH, Castano O & Kim HW (2009) Electrospun materials as potential platforms for bone tissue engineering. Adv Drug Deliv Rev 61(12): 1065–1083.

Jämsä T & Jalovaara P (1996) Technical Note: A cost-effective, accurate machine for testing the torsional strength of sheep long bones. Med Eng Phys 18(5): 433–435.

Johnson AL & Stein LE (1988) Morphologic comparison of healing patterns in ethylene oxide-sterilized cortical allografts and untreated cortical autografts in the dog. Am J Vet Res 49(1): 101–105.

Jones JR & Hench LL (2004) Factors affecting the structure and properties of bioactive foam scaffolds for tissue engineering. J Biomed Mater Res Part B Appl Biomater 68(1): 36–44.

Jones JR, Ehrenfried LM & Hench LL (2006) Optimising bioactive glass scaffolds for bone tissue engineering. Biomaterials 27: 964–973.

Jortikka L, Marttinen A & Lindholm TS (1993a) High yield of osteoinductivity can be derived from demineralised bone matrix using collagenase digestion. Ann Chir Gynaecol 82: 31–36.

Jortikka L, Marttinen A & Lindholm TS (1993b) Partially purified reindeer (Rangifer Tarandus) bone morphogenetic protein has a high bone-forming activity compared with some other artiodactylis. Clin Orthop Relat Res 297: 33–37.

Jung RE, Weber FE, Thoma DS, Ehrbar M, Cochran & Hämmerle CHF (2008) Bone morphogenetic protein-2 enhances bone formation when delivered by a synthetic matrix containing hydroxyapatite/tricalciumphosphate. Clin Oral Impl Res 19: 188–195.

92

Kamegai A, Shimamura N, Naitou K, Nagahara K, Kanematsu N & Mori M (1994) Bone formation under the influence of bone morphogenetic protein/self-setting apatite cement composite as a delivery system. Biomed Mater Eng 4(4): 291–307.

Kandel RA, Boyle J, Gibson G, Cruz T & Speagle M (1997) In vitro formation of mineralized cartilaginous tissue by articular chondrocytes. In Vitro Cell Dev Biol Anim 33(3): 174–181.

Kapanen A, Ryhänen J, Birr E, Väänänen HK & Tuukkanen J (2002) Bone morphogenetic protein 3b expressing reindeer antler. J Biomed Mater Res 59(1): 78–83.

Karageorgiour V & Kaplan D (2005) Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 26: 5474–5491.

Keene DR, Sakai LY & Burgeson RE (1991) Human bone contains type III collagen, type VI collagen, and fibrillin: type III collagen is present on specific fibers that may mediate attachment of tendons, ligaments, and periosteum to calcified bone cortex. J Histochem Cytochem 39(1): 59–69.

Kessler S, Koepp HE, Mayr-Wohlfart U, Ignatius A, Claes I, Puhl W & Günther KP (2004) Bone morphogenetic protein 2 accelerates osteointegration and remodelling of solvent-dehydrated bone substitutes. Arch Orthop Trauma Surg 124: 410–414.

Khoshnoodi J, Pedchenko V & Hudson BG (2008) Mammalian collagen IV. Microsc Res Tech 71: 357–370.

Kienapfel H, Sumner DR, Turner TM, Urban RM & Galante JO (1992) Efficacy of autograft and freeze-dried allograft to enhance fixation of porous coated implants in the presence of interface gaps. J Orthop Res 10(3): 423–433.

Kim CS, Kim JI, Kim J, Choi SH, Chai JK, Kim CK & Cho KS (2005) Ectopic bone formations associated with recombinant human bone morphogenetic proteins-2 using absorbable collagen sponge and beta tricalcium phosphate as carriers. Biomaterials 26: 2501–2507.

King GN, King N & Hughes FJ (1998) Effect of two delivery systems for recombinant human bone morphogenetic protein-2 on periodontal regeneration in vivo. J Periodontal Res 33(4): 226–236.

King GN (2001) The importance of drug delivery to optimize the effects of bone morphogenetic proteins during periodontal regeneration. Curr Pharm Biotechnol 2(2): 131–142.

Kinney RC, Ziran BH, Hirshorn K, Schlatterer D & Ganey T (2010) Demineralized bone matrix for fracture healing: fact or fiction? J Orthop Trauma 24: S52–S55.

Kirker-Head CA, Gerhart TN, Armstrong R, Schelling SH & Carmel LA (1998) Healing bone using recombinant human bone morphogenetic protein 2 and copolymer. Clin Orthop Relat Res 349: 205–217.

Kirker-Head CA (2000) Potential applications and delivery strategies for bone morphogenetic proteins. Adv Drug Deliv Rev 43(1): 65–92.

Kloss FR, Schlegel KA, Felszeghy E, Falk S & Wiltfang J (2004) Applying an osteoinductive protein complex for regeneration of osseous defects (abstract). Mund Kiefer Gesichtschir 8(1): 12–17.

93

Ko L, Ma GX & Gao HL (1990) Purification and chemical modification of porcine bone morphogenetic protein. Clin Orthop Relat Res 256: 229–237.

Koerten HK & van der Meulen J (1999) Degradation of calcium phosphate ceramics. J Biomed Mater Res 44(1): 78–86.

Kon T, Cho TJ, Aizawa T, Yamazaki M, Nooh N, Graves D, Gerstenfeld LC & Einhorn TA (2001) Expression of osteoprotegerin, receptor activator of NF-κB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing. J Bone Miner Res 16(6): 1004–1014.

Koort JK, Mäkinen TJ, Suokas E, Veiranto M, Jalava J, Tömälä P, Aro HT (2008) Sustained release of ciprofloxacin from an osteoconductive poly(DL)-lactide implant. Acta Orthop 79(2): 295–301.

Kos M, Luczak K, Matusiewicz M, Krzystek-Korpacka M & Rapala M (2002) Perspectives of clinical application of bone morphogenetic proteins in children. Eur J Pediatr Surg 12(4): 272–277.

Kotani S, Yamamuro T, Nakamura T, Kitsugi T, Fujita Y, Kawanabe K & Kokubo T (1992) Enhancement of bone bonding to bioactive ceramics by demineralized bone powder. Clin Orthop Relat Res 278: 226–234.

Kroese-Deutman HC, Wolke JGC, Spauwen PHM & Jansen JA (2006) Closing capacity of cranial bone defects using porous calcium phosphate cement implants in a rabbit animal model. J Biomed Mater Res A 79(3): 503–511.

Kuboki Y, Takita H, Kobayashi D, Tsuruga E, Inoue M, Murata M, Nagai N, Dohi Y & Ohgushi H (1998) BMP-induced osteogenesis on the surface of hydroxyapatite with geometrically feasible and nonfeasible structures: topology of osteogenesis. J Biomed Mater Res 39(2): 190–199.

Kujala S, Raatikainen T, Ryhänen J, Kaarela O & Jalovaara P (2004) Composite implant of native bovine bone morphogenetic protein (BMP), collagen carrier and biocoral in the treatment of resistant ulnar nonunions: report of five preliminary cases. Arch Orthop Trauma Surg 124: 26–30.

Kujala S, Vähäsarja V, Serlo W & Jalovaara P (2008) Treatment of congenital pseudarthrosis of the tibia with native bovine BMP: a case report. Acta Orthop Belg 74(1): 132–136.

Kusumoto K, Bessho K, Fujimura K, Konishi Y, Oqawa Y & Iizuka T (1995) Comparative study of bone marrow induced by puriefied BMP and recombinant human BMP-2. Biochem Biophys Res Commun 215(1): 205–211.

Kusumoto K, Bessho K, Fujimura K, Konishi Y, Oqawa Y & Iizuka T (1996) Self-regenerating bone implant: ectopic osteoinduction following intramuscular implantation of a combination of rhBMP-2, atelopeptide type I collagen and porous hydroxyapatite. J Craniomaxillofac Surg 24(6): 360–365.

Kusumoto K, Bessho K, Fujimura K, Akioka J, Oqawa Y & Iizuka T (1997) Comparison of ectopic osteoinduction in vivo by recombinant human BMP-2 and recombinant Xenopus BMP-4/7. Biochem Biophys Res Commun 239(2): 575–579.

94

Laitinen M, Jortikka L, Halttunen T, Nevalainen J, Aho AJ, Marttinen A & Lindholm TS (1997) Measurement of total and local bone morphogenetic protein concentration in bone tumours. Int Orthop 21(3): 188–193.

Lammens J, Liu Z & Luyten F (2009) Bone morphogenetic protein signalling in the murine distraction osteogenesis model. Acta Orthop Belg 75(1): 94–102.

Leblanc E, Trensz F, Haroun S, Drouin G, Bergeron É, Penton CM, Montanaro F, Roux S, Faucheux N & Grenier G (2011) BMP-9-induced muscle heterotopic ossification requires changes to the skeletal muscle microenvironment. J Bone Miner Res 26(6): 1166–1177.

Lee JH, Lee KM, Baek HR, Jang SJ, Lee JH & Ryu HS (2011) Combined effects of porous hydroxyapatite and demineralised bone matrix on bone induction: in vitro and in vivo study using a nude rat model. Biomed Mater 6(1): 015008.

Leeson TS, Leeson CR & Paparo AA (1988) Specialized connective tissue: Cartilage and bone. In: Wonsiewicz (ed) Text/Atlas of Histology. Pennsylvania, W.B. Saunders Company: 168–189.

Lepola V, Väänänen K & Jalovaara P (1993) The effect of immobilization on the torsional strength of the rat tibia. Clin Orthop Relat Res 297: 55–61.

Levander G (1938) A study of bone regeneration. Surg Gynecol Obstet 67: 705–714. Li RH & Wozney JM (2001) Delivering on the promise of bone morphogenetic proteins.

Trends Biotechnol 19(7): 255–265. Li S, De Wijn JR, Li J, Layrolle P & De Groot K (2003) Macroporous biphasic calcium

phosphate scaffold with high permeability/porosity ratio. Tissue Eng 9(3): 535-548. Li H, Zou X, Woo C, Ding M, Lind M & Bünger C (2005) Experimental anterior lumbar

interbody fusion with an osteoinductive bovine bone collagen extract. Spine 30(8): 890–896.

Li H, Springer M, Zou X, Briest A & Bünger C (2006) Ectopic bone induction by equine bone protein extract. Adv Exp Med Biol 585: 393–402.

Lieberman JR, Daluiski A & Einhorn TA (2002) The role of growth factors in the repair of bone. J Bone Joint Surg 84-A(6): 1032–1044.

Liebschner MAK (2004) Biomechanical considerations of animal models used in tissue engineering of bone. Biomaterials 25: 1697–1714.

Lindfors NC & Aho AJ (2003) Granule size and composition of bioactive glasses affect osteoconduction in rabbit. J Mater Sci Mater Med 14(4): 365–372.

Lindfors NC, Hyvönen P, Nyyssönen M, Kirjavainen M, Kankare J, Gullichsen E & Salo J (2010) Bioactive glass S53P4 as bone graft substitute in treatment of osteomyelitis. Bone 47(2): 212–218.

Little DG, McDonald M, Bransford R, Godfrey CB & Amanat N (2005) Manipulation of the anabolic and catabolic responses with OP-1 and zoledronic acid in a rat critical defect model. J Bone Miner Res 20(11): 2044–2052.

Livingston T, Ducheyne P & Garino J (2002) In vivo evaluation of a bioactive scaffold for bone tissue engineering. J Biomed Mater Res 62(1): 1–13.

Luginbuehl V, Meinel L, Merkle HP & Gander B (2004) Localized delivery of growth factors for bone repair. Eur J Pharm Biopharm 58(2): 197–208.

95

Magan A & Ripamonti U (1996) Geometry of porous hydroxyapatite implants influences osteogenesis in baboons (Papio ursinus). J Craniofac Surg 7(1): 71–78.

Mahmood J, Takita H, Ojima Y, Kobayashi M, Kohgo T & Kuboki Y (2001) Geometric effect of matrix upon cell differentiation: BMP-induced osteogenesis using a new bioglass with a feasible structure. J Biochem 129(1): 163–171.

Maus U, Andereya S, Gravius S, Siebert CH, Ohnsorge JAK & Niedhart C (2008) Lack of effect on bone healing of injectable BMP-2 augmented hyaluronic acid. Arch Orthop Trauma Surg 128(12): 1461–1466.

McKay WF, Peckham SM & Badura JM (2007) A comprehensive clinical review of recombinant human bone morphogenetic protein-2 (INFUSE® Bone Graft). Int Orthop 31(6): 729–734.

McKibbin B (1978) The biology of fracture healing in long bones. J Bone Joint Surg 60-B(2): 150–162.

Miyazaki M, Morishita Y, He W, Hu M, Sintuu C, Hymanson HJ, Falakassa J, Tsumura H & Wang JC (2009) A porcine collagen-derived matrix as a carrier for recombinant human bone morphogenetic protein-2 enhances spinal fusion in rats. Spine J 9(1): 22–30.

Moghaddam A, Elleser C, Biglari B, Wentzensen A & Zimmermann G (2010) Clinical application of BMP 7 in long bone non-unions. Arch Orthop Trauma Surg 130: 71–76.

Moore DC, Chapman MW & Manske D (1987) The evaluation of a biphasic calcium phosphate ceramic for use in grafting long-bone diaphyseal defects. J Orthop Res 5(3): 356–365.

Moore WR, Graves SE & Bain GI (2001) Synthetic bone graft substitutes. ANZ J Surg 71(6): 354–361.

Mortazavi V, Nahrkhalaji MM, Fathi MH, Mousavi SB & Esfahani BM (2010) Antibacterial effects of sol-gel-derived bioactive glass nanoparticle on aerobic bacteria. J Biomed Mater Res A 94(1): 160–168.

Murray SS, Brochmann EJ, Harker JO, King E, Lollis RJ & Khaliq SA (2007) A Statistical model to allow the phasing out of the animal testing of demineralised bone matrix products. Altern Lab Anim 35(4): 405–409.

Murugan R, Ramakrishna S & Rao KP (2008) Analysis of bovine-derived demineralized bone extracts. J Mater Sci Mater Med 19(6): 2423–2426.

Niedhart C, Maus U, Redmann E, Schmidt-Rohlfing B, Niethard FU & Siebert CH (2003) Stimulation of bone formation with an in situ setting tricalcium phosphate/rhBMP-2 composite in rats. J Biomed Mater Res A 65(1): 17–23.

Nienhuijs ME, Poulsen K, van der Zande M, Briest A, Merkx MA, Stoelinga PJ, Jensen ON, Jansen JA & Walboomers XF (2009) Analytical assessment of the osteoinductive material COLLOS E. J Biomed Mater Res B Appl Biomater 89(2): 300–305.

Nilsson OS, Urist MR, Dawson EG, Schmalzried TP & Finerman GAM (1986) Bone repair induced by bone morphogenetic protein in ulnar defects in dogs. J Bone Joint Surg 68-B(4): 635–642.

96

Nunamaker DM (1985) Methods of internal fixation. In: Newton CD & Nunamaker DM (eds) Textbook of small animal orthopaedics. New York, USA, IVIS. URI: http://www.ivis.org/special_books/ortho/chapter_16/ivis.pdf. Cited 2011/10/13.

Nunamaker DM (1998) Experimental models of fracture repair. Clin Orthop Relat Res 355S: S56–S65.

Nuss KM, Auer JA, Boos A & von Rechenberg B (2006) An animal model in sheep for biocompatibility testing of biomaterials in cancellous bone. BMC Musculoskelet Disord 7:67.

Obenaus A & Hayes P (2011) Drill hole defects: induction, imaging, and analysis in the rodent. Methods Mol Biol 690: 301–314.

Ohgushi H, Goldberg VM & Caplan AI (1989) Repair of bone defects with marrow cells and porous ceramic, experiments in rats. Acta Orthop Scand 60(3): 334–339.

Ohtsuki C, Kamitakahara M & Miyazaki T (2009) Bioactive ceramic-based materials with designed reactivity for bone tissue regeneration. J R Soc Interface 6: S349–S360.

Ohura K, Hamanishi C, Tanaka S & Matsuda N (1999) Healing of segmental bone defects in rats induced by a β-TCP-MCPM cement combined with rhBMP-2. J Biomed Mater Res 44(2): 168–175.

Oksanen J, Marttinen A, Paatsama S & Lindholm TS (1998) Extraction and characterization of native canine bone morphogenetic protein (cBMP) qualified with osteoinductivity. Acta Vet Scand 39(2): 165–171.

Olivero DK & Furcht LT (1993) Type IV collagen, laminin, and fibronectin promote the adhesion and migration of rabbit lens epithelial cells in vitro. Invest Ophthalmol Vis Sci 34: 2825–2834.

Oonishi H, Hench LL, Wilson J, Sugihara F, Tsuji E, Kushitani S & Iwaki H (1999) Comparative bone growth behaviour in granules of bioceramic materials of various sizes. J Biomed Mater Res 44(1): 31–43.

Pajamäki KJ, Andersson OH, Lindholm TS, Karlsson KH, Yli-Urpo A & Happonen RP (1993a) Effect of bovine bone morphogenetic protein and bioactive glass on demineralised bone matrix grafts in the rat muscular pouch. Ann Chir Gynaecol Suppl 207: 155–161.

Pajamäki KJ, Andersson OH, Lindholm TS, Karlsson KH & Yli-Urpo A (1993b) Induction of new bone allogeneic demineralised bone matrix combined to bioactive glass composite in the rat. Ann Chir Gynaecol Suppl 207: 137–143.

Pajamäki KJ, Andersson OH, Lindholm TS, Karlsson KH, Yli-Urpo A, Laippala P & Happonen RP (1994) Effect of glass bioactivity on new bone development induced by demineralised bone matrix in a rat extraskeletal site. Arch Orthop Trauma Surg 113(4): 210–214.

Pape HC, Evans A & Kobbe P (2010) Autologous bone graft: properties and techniques. J Orthop Trauma 24: S36–S40.

Paralkar VM, Nandedkar AK, Pointer RH, Kleinman HK & Reddi AH (1990) Interaction of osteogenin, a heparin binding bone morphogenetic protein, with type IV collagen. J Biol Chem 265(28): 17281–17284.

97

Parikh SN (2002) Bone graft substitutes: past, present, future. J Postgrad Med 48(2): 142–148.

Park SH, Llinás A, Goel VK & Keller JC (2003) Hard tissue replacement. In Park JB & Bronzino JD (eds): Biomaterials Principles and Applications. Boca Raton, CRC Press LLC: 174–179.

Pekkarinen T, Lindholm TS, Hietala O & Jalovaara P (2003) New bone formation induced by injection of native reindeer BMP. Scand J Surg 92: 227–230.

Pekkarinen T, Lindholm TS, Hietala O & Jalovaara P (2004) Influence of ethylene oxide sterilization on the activity of native reindeer bone morphogenetic protein. Int Orthop 28(2): 97–101.

Pekkarinen T, Lindholm TS, Hietala O & Jalovaara P (2005a) The effect of different mineral frames on the ectopic bone formation in mouse hind leg muscles induced by native reindeer bone morphogenetic protein. Arch Orthop Trauma Surg 125: 10–15.

Pekkarinen T, Hietala O, Jämsä T & Jalovaara P (2005b) Effect of gamma irradiation on the osteoinductivity of native reindeer bone morphogenetic protein extract. Acta Orthop 6(2): 231–236.

Pekkarinen T, Hietala O, Jämsä T & Jalovaara P (2005c) Gamma irradiation and ethylene oxide in the sterilization of native reindeer bone morphogenetic protein extract. Scand J Surg 94(1): 67–70.

Pekkarinen T, Jämsä T, Määttä M, Hietala O & Jalovaara P (2006) Reindeer BMP extract in the healing of critical-size bone defects in the radius of the rabbit. Acta Orthop 77(6): 952–959.

Pelker RR, Friedlaender GE, Markham TC, Panjabi MM & Moen CJ (1984) Effects of freezing and freeze-drying on the biomechanical properties of rat bone. J Orthop Res 1(4): 405–411.

Peltola M, Aitasalo K, Suonpää J, Varpula M & Yli-Urpo A (2006) Bioactive glass S53P4 in frontal sinus obliteration: a long-term clinical experience. Head Neck 28(9): 834–841.

Peters CL, Hines JL, Bachus KN, Craig MA & Bloebaum RD (2006) Biological effects of calcium sulfate as a bone graft substitute in ovine metaphyseal defects. J Biomed Mater Res A 76(3): 456–462.

Peterson B, Whang PG, Iqlesias R, Wang JC & Lieberman JR (2004) Osteoinductivity of commercially available demineralized bone matrix. Preparations in a spine fusion model. J Bone Joint Surg 86-A(10): 2243–2250.

Pluhar GE, Turnes AS, Pierce AR, Toth CA & Wheeler DL (2006) A comparison of two biomaterial carriers for osteogenic protein-1 (BMP-7) in an ovine critical defect model. J Bone Joint Surg 88-B(7): 960–966.

Pratt JNJ, Griffon DJ, Dunlop DG, Smith N & Howie CR (2002) Impaction grafting with morsellised allograft and tricalcium phosphate–hydroxyapatite: incorporation within ovine metaphyseal bone defects. Biomaterials 23: 3309–3317.

Prisell PT, Edwall D, Lindblad JB, Levinovitz A & Norstedt G (1993) Expression of insulin-like growth factors during bone induction in rat. Calcif Tissue Int 53(3): 201–205.

98

Provenzano MJ, Murphy KPJ & Riley LH (2004) Bone cements: Review of their physiochemical and biochemical properties in percutaneous vertebroplasty. Am J Neuroradiol 25: 1286–1290.

Pyhältö T, Lapinsuo M, Pätiälä H, Rokkanen P, Niiranen H & Törmälä P (2004) Fixation of distal femoral osteotomies with self-reinforced poly(L/DL)lactide 70:30/bioactive glass composite rods. An experimental study on rats. J Mater Sci Mater Med 15(3): 275–281.

Qiu QQ, Liu XH & Connos J (2009) Effects of e-beam radiation, storage, and hydration on osteoinductivity of DBM/AM composite. J Biomed Mater Res B Appl Biomater 91(1): 401–408.

Reddi AH (1981) Cell biology and biochemistry of endochondral bone development. Coll Relat Res 1(2): 209–226.

Reddi AH (2005) BMPs: from bone morphogenetic proteins to body morphogenetic proteins. Cytokine Growth Factor Rev 16(3): 249–250.

Reddi AH & Huggins C (1972) Biochemical sequences in the transformation of normal fibroblasts in adolescent rats. Proc Nat Acad Sci USA 69(6): 1601–1605.

Reichert JC, Wullschleger ME, Cipitria A, Lienau J, Cheng TK, Schütz MA, Duda GN, Nöth U, Eulert J & Hutmacher DW (2011) Custom-made composite scaffolds for segmental defect repair in long bones. Int Orthop 35(8): 1229–1236.

Reynolds MA, Aichelmann-Reidy ME, Kassolis JD, Prasad HS & Rohrer MD (2007) Calcium Sulfate–Carboxymethylcellulose Bone Graft Binder: Histologic and Morphometric Evaluation in a Critical Size Defect. J Biomed Mater Res B Appl Biomater 83(2): 451–458.

Ricci JL (2001) Material characteristics of calcium sulphate cement. In Pecora G & Leonardis D (eds.): Use of calcium sulphate cement for dental and maxillofacial bone repair. Rome, Elite Publishing: 1–10.

Ripamonti U, Magan A, Ma S, van den Heever B, Moehl T & Reddi AH (1991) Xenogeneic osteogenin, a bone morphogenetic protein and demineralised bone matrices, including human, induce bone differentiation in athymic rats and baboons. Matrix 11(6): 404–411.

Ripamonti U & Duneas N (1998) Tissue morphogenesis and regeneration by bone morphogenetic proteins. Plast Reconst Surg 101(1): 227–239.

Rohanizadeh R & Chung K (In press) Hydroxyapatite as a carrier for bone morphogenetic protein. J Oral Implantol.

Roohani-Esfahani SI, Nouri-Khorasani S, Lu Z, Appleyard R & Zreiqat H (2010) The influence hydroxyapatite nanoparticle shape and size on the properties of biphasic calcium phosphate scaffolds coated with hydroxyapatite - PCL composites. Biomaterials 31: 5498–5509.

Ruhé PQ, Kroese-Deutman HC, Wolke JGC, Spauwen PHM & Jansen JA (2004) Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants in cranial defects in rabbits. Biomaterials 25: 2123–2132.

Saito N & Takaoka K (2003) New synthetic biodegradable polymers as BMP carriers for bone tissue engineering. Biomaterials 24: 2287–2293.

99

Sakou T (1998) Bone morphogenetic proteins: from basic studies to clinical approaches. Bone 22(6): 591–603.

Salo J (2011) Bone substitutes in clinical work. In: Bentley G (ed.) European Instructional Lectures. 12th EFFORT Congress, Copenhagen, Denmark European Instructional Lectures 11: 3–7.

Sampath TK & Reddi AH (1981) Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation. Proc Natl Acad Sci USA 78(12): 7599–7603.

Sampath TK & Reddi AH (1983) Homology of bone-inductive proteins from human, monkey, bovine and rat extracellular matrix. Proc Natl Acad Sci USA 80(21): 6591–6595.

dos Santos EA, Farina M, Soares GA & Anselme K (2008) Surface energy of hydroxyapatite and β-tricalcium phosphate ceramics driving serum protein adsorption and osteoblast adhesion. J Mater Sci Mater Med 19(6): 2307–2316.

Saravanapavan P, Jones JR, Verrier S, Beilby R, Shirtliff VJ, Hench LL & Polak JM (2004) Binary CaO-SiO2 gel-glasses for biomedical applications. Biomed Mater Eng 14: 467–486.

Sarras MP (1996) BMP-1 and the astacin family of metalloproteinases: a potential link between the extracellular matrix, growth factors and pattern formation. Bioessays 18(6): 439–442.

Sato T, Iwata H, Takahashi M & Miura T (1993) Heat tolerance of activity toward ectopic bone formation by rabbit bone matrix protein. Ann Chir Gynaecol Suppl 207: 37–40.

Schatzker J, Waddell J & Stoll JE (1989) The effects of motion on the healing of cancellous bone. Clin Orthop Relat Res 245: 282–287.

Schmitz JP & Hollinger JO (1986) The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop Relat Res 205: 299–308.

Schneiders W, Reinstorf A, Biewener A, Serra A, Grass R, Kinschner M, Heineck J, Rehberg S, Zwipp H & Rammelt S (2009) In vivo effects of modification of hydroxyapatite/collagen composites with and without chondroitin sulphate on bone remodelling in the sheep tibia. J Orthop Res 27(1): 15–21.

Schnettler R, Knöss PD, Heiss C, Stahl JP, Meyer C, Kilian O, Wenisch S & Alt V (2009) Enhancement of bone formation in hydroxyapatite implants by rhBMP-2 coating. J Biomed Mater Res B Appl Biomater 90(1): 75–81.

Schopper C, Moser D, Spassova E, Goriwoda W, Lagogiannis G, Hoering B, Ewers R, Redl H (2008) Bone regeneration using a naturally grown HA/TCP carrier loaded with rhBMP-2 is independent of barrier-membrane effects. J Biomed Mater Res A 85(4): 954–963.

Schroeder JE & Mosheiff R (2011) Tissue engineering approaches for bone repair: Concepts and evidence. Injury 42: 609–613.

Sciadini MF, Dawson JM & Johnson KD (1997) Bovine-derived bone protein as a bone graft substitute in a canine segmental defect model. J Orthop Trauma 11(7): 496–508.

Seeherman H, Wozney J & Li R (2002) Bone morphogenetic protein delivery systems. Spine 27(16 S1): S16–S23.

100

Seeherman H & Wozney JM (2005) Delivery of bone morphogenetic proteins for orthopaedic tissue regeneration. Cytokine Growth Factor Rev 16(3): 329–345.

Seeherrman H, Li R, Bouxsein M, Kim H, Li J, Smith-Adaline EA, Aiolova M & Wozney JM (2006) rhBMP-2/Calcium phosphate matrix accelerates osteotomy-site healing in a nonhuman primate model at multiple treatment times and concentrations. J Bone Joint Surg 88-A(1): 144–160.

Seyer JM, Brickley DM & Glimecher (1974) The isolation of two types of collagen from embryonic bovine epiphyseal cartilage. Calcif Tiss Res 17: 25–41.

Shigenobu K, Kaneda K, Nagai N & Kuboki Y (1993) Localization of bone morphogenetic protein-induced bone and cartilage formation on a new carrier: fibrous collagen membrane. Ann Chir Gynaecol Suppl 207: 85–90.

Silver IA, Deas J & Erecińska M (2001) Interactions of bioactive glasses with osteoblasts in vitro: effects of 45S5 Bioglass®, and 58S and 77S bioactive glasses on metabolism, intracellular ion concentrations and cell viability. Biomaterials 22: 175–185.

Simon Z, Deporter DA, Pilliar RM & Clokie CM (2006) Heterotopic bone formation around sintered porous-surfaced Ti-6Al-4V implants coated with native bone morphogenetic proteins. Implant Dent 15(3): 265–274.

Singh NB & Middendorf B (2007) Calcium sulphate hemihydrate hydration leading to gypsum crystallization. Prog Cryst Growth Charact Mater 53(1): 57–77.

Slutsky DJ & Herman M (2005) Rehabilitation of distal radius fractures: A biomechanical guide. Hand Clin 21: 455–468.

Solheim E, Pinholt EM, Andersen R, Bang G & Sudmann E (1992) The effect of a composite of polyorthoester and demineralized bone on the healing of large segmental defects of the radius in rats. J Bone Joint Surg 74-A(10): 1456–1463.

Solheim E (1998) Growth factors in bone. Int Orthop 22(6): 410–416. Stevenson S (1987) The immune response to osteochondral allografts in dogs. J Bone Joint

Surg 69-A(4): 573–582. Stevenson S, Cunningham N, Toth J, Davy D & Reddi AH (1994) The effect of osteogenin

(a bone morphogenetic protein) on the formation of bone in orthotopic segmental defects in rats. J Bone Joint Surg 76-A(11): 1676–1687.

Stevenson S, Li XQ, Davy DT, Klein L & Goldberg VM (1997) Critical biological determinants of incorporation of non-vascularized cortical bone grafts. Quantification of a complex process and structure. J Bone Joint Surg 79-A(1): 1–16.

Van der Stok J, Van Lieshout EMM, El-Massoudi Y, Van Kralingen GH & Patka P (2011) Bone substitutes in the Netherlands – A systematic literature review. Acta Biomater 7(2): 739–750.

Street J, Bao M, deGuzman L, Bunting S, Peale FV, Ferrara N, Steinmetz H, Hoeffel J, Cleland JL, Daugherty A, van Bruggen N, Redmond HP, Carano RAD & Filvaroff EH (2002) Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci USA 99(15): 9656–9661.

Suri C, McClain J, Thurston G, McDonald DM, Zhou H, Oldmixon EH, Sato TN & Yancopoulos GD (1998) Increased vascularization in mice overexpressing angiopoietin-1. Science 282(5388): 468–471.

101

Szpalski C, Barr J, Wetterau M, Saadeh PB & Warren SM (2010) Cranial bone defects: current and future strategies. Neurosurg Focus 29(6):E8.

Takagi K & Urist MR (1982) The role of bone marrow in bone morphogenetic protein-induced repair of femoral massive diaphyseal defects. Clin Orthop Relat Res 171: 224–231.

Takaoka K, Nakahara H, Yoshikawa H, Masuhara K, Tsuda T & Ono K (1988) Ectopic bone induction on and in porous hydroxyapatite combined with collagen and bone morphogenetic protein. Clin Orthop Relat Res 234: 250–254.

Takita H, Vehof JWM, Jansen JA, Yamamoto M, Tabata Y, Tamura M & Kuboki Y (2004) Carrier dependent cell differentiation of bone morphogenetic protein-2 induced osteogenesis and chondrogenesis during the early implantation stage in rats. J Biomed Mater Res A 71(1): 181–189.

Tas AC (2008) Preparation of porous apatite granules from calcium phosphate cement. J Mater Sci Mater Med 19(5): 2231–2239.

Thalgott JS, Giuffre JM, Fritts K, Timlin M & Klezi Z (2001) Instrumented posterolateral lumbar fusion using coralline hydroxyapatite with or without demineralised bone matrix, as an adjunct to autologous bone. Spine J 1(2): 131–137.

Thomas MV, Puleo DA & Al-Sabbagh M (2005) Calcium sulfate: a review. J Long Term Eff Med Implants 15(6): 599–607.

Thomas MV & Puleo DA (2009) Calcium sulfate: properties and clinical applications. J Biomed Mater Res B Appl Biomater 88(2): 597–610.

Tiedeman JJ, Garvin KL, Kile TA & Connolly JF (1995) The role of a composite, demineralised bone matrix and bone marrow in the treatment of osseous defects. Orthopaedics 18(12): 1153–1158.

Tomford WW & Mankin HJ (1999) Bone banking. Update on methods and materials. Orthop Clin North Am 30(4): 565–570.

Tseng WJ, Liu DM & Hsu CK (1999) Influence of stearic acid on suspension structure and green microstructure of injection-molded zirconia ceramics. Ceramics Int 25: 191–195.

Tsiridis E, Upadhyay N & Giannoudis P (2007) Molecular aspects of fracture healing: which are the important molecules? Injury 38(S1): S11–S25.

Tsuji K, Bandyopadhyay A, Harfe BD, Cox K, Kakar S, Gerstenfeld L, Einhorn T, Tabin CJ & Rosen V (2006) BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing. Nat Genet 38(12): 1429–1429.

Tsuruga E, Takita H, Itoh H, Wakisaka Y & Kuboki Y (1997) Pore size of porous hydroxyapatite as the cell-substratum controls BMP-induced osteogenesis. J Biochem 121(2): 317–324.

Tuominen T, Jämsä T, Tuukkanen J, Nieminen P, Lindholm TC, Lindholm TS & Jalovaara P (2000) Native bovine bone morphogenetic protein improves the potential of biocoral to heal segmental canine ulnar defects. Int Orthop 24: 289–294.

102

Tuominen T, Jämsä T, Oksanen J, Tuukkanen J, Gao TJ, Lindholm TS & Jalovaara P (2001) Composite implant composed of hydroxyapatite and bone morphogenetic protein in the healing of a canine ulnar defect. Ann Chir Gynaecol Suppl 90(1): 32–36.

Turner TM, Urban RM, Gitelis S, Kuo KN & Andersson GBJ (2001) Radiographic and histologic assessment of calcium sulphate in experimental animal models and clinical use as a resorbable bone-graft substitute, a bone-graft expander, and a method for local antibiotic delivery. J Bone Joint Surg 83A-Suppl2 (PT1): 8–18.

Turner TM, Urban RM, Gitelis S, Haggard WO & Richelsoph K (2003) Resorption evaluation of a large bolus of calcium sulfate in a canine medullary defect. Orthopedics 26: S577–S579.

Ulmanen MS, Pekkarinen T, Hietala OA, Birr EA & Jalovaara P (2005) Osteoinductivity of partially purified native ostrich (Struthio camelus) bone morphogenetic protein: Comparison with mammalian species. Life Sci 77: 2425–2437.

Uludag H, Augusta DD, Palmer R, Timony G & Wozney J (1999) Characterization of rhBMP-2 pharmacokinetics implanted with biomaterial carriers in the rat ectopic model. J Biomed Mater Res 46: 193–202.

Urban RM, Turner TM, Hall DJ, Infanger SI, Cheema N & Lim TH (2003) Healing of large defects treated with calcium sulfate pellets containing demineralized bone matrix particles. Orthopedics 26: S581–S585.

Urban RM, Turner TM, Hall DJ, Inoue N & Gitelis S (2007) Increased bone formation using a calcium sulfate and calcium phosphate composite graft. Clin Orthop Relat Res 459: 110–117.

Urist MR (1965) Bone formation by autoinduction. Science 150: 893–899. Urist MR & Dawson E (1981) Intertransverse process fusion with the aid of

chemosterilized autolyzed antigen-extracted allogeneic (AAA) bone. Clin Orthop Relat Res 154: 97–113.

Urrutia J, Thumm N, Apablaza D, Pizarro F, Zylberberg A & Quezada F (2008) Autograft versus allograft with or without demineralized bone matrix in posterolateral lumbar fusion in rabbits. J Neurosurg Spine 9: 84–89.

Vaccaro AR, Stubbs HA & Block JE (2007) Demineralized bone matrix composite grafting for posterolateral spinal fusion. Orthopedics 30(7): 567–570.

Välimäki VV, Yrjans JJ, Vuorio E & Aro HT (2005) Combined effect of BMP-2 gene transfer and bioactive glass microspheres on enhancement of new bone formation. J Biomed Mater Res A 75(3): 501–509.

Viljanen VV & Lindholm TS (1993) Heterotopic osteoinduction in a rat membrane-isolated latissimus dorsi island flap. A pilot study. Ann Chir Gynaecol Suppl 207: 55–62.

Viljanen VV, Gao TJ, Marttinen A & Lindholm TS (1996) Partial purification and characterization of bone morphogenetic protein from bone matrix of the premature moose (Alces alces): degradation of bone-inducing activity during storage. Eur Surg Res 28(6): 447–460.

103

Virolainen P, Vuorio E & Aro HT (1993) Gene expression at graft-host interfaces of cortical bone allografts and autografts. Clin Orthop Relat Res 297: 144–149.

Vögelin E, Jones NF, Huang JI, Brekke JH & Lieberman JR (2005) Healing of a critical-sized defect in the rat femur with use of a vascularized periosteal flap, a biodegradable matrix, and bone morphogenetic protein. J Bone Joint Surg 87-A(6): 1323–1331.

Volek-Smith H & Urist MR (1996) Recombinant human bone morphogenetic protein (rhBMP) induced heterotopic bone development in vivo and in vitro. Proc Soc Exp Biol Med 211(3): 265–272.

Walboomers XF & Jansen JA (2005) Bone tissue induction, using a COLLOSS-filled titanium fibre mesh-scaffolding material. Biomaterials 26(23): 4779–4785.

Wallace FG & Rosenblatt J (2003) Collagen gel systems for sustained delivery and tissue engineering. Adv Drug Deliv Review 55: 1631–1649.

Wang EA, Rose V, Cordes P, Hewick RM, Kriz MJ, Luxenberg DP, Sibley BS & Wozney JM (1988) Purification and characterization of other distinct bone-inducing factors. Proc Natl Acad Sci USA 85(24): 9484–9488.

Wang EA, Rosen V, D’Alessandro JS, Bauduy M, Cordes P, Harada T, Israel DI, Hewick RM, Kerns KM, Lapan P, Luxenberg DP, McQuaid D, Moutsatsos IK, Nove J & Wozney JM (1990) Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci USA 87(6): 2220–2224.

Wang JC, Alanay A, Mark D, Kanim LE, Campbell PA, Dawson EG & Lieberman JR (2007) A comparison of commercially available demineralised bone matrix for spinal fusion. Eur Spine J 16(8): 1233–1240.

Wang J, Valmikinathan CM, Liu W, Laurencin CT & Yu X (2010) Spiral-structured, nanofibrous, 3D scaffolds for bone tissue engineering. J Biomed Mater Res A 93(2): 753–762.

Weinzapfel B, Son-Hing JP, Armstrong DG, Blakemore LC, Poe-Kochert C & Thompson GH (2008) Fusion rates after thoracoscopic release and bone graft substitutes in idiopathic scoliosis. Spine 33(10): 1079–1083.

Wen B, Karl M, Pendrys D, Shafer D, Freilich M & Kuhn L (2011) An evaluation of BMP-2 delivery from scaffolds with miniaturized dental implants in a novel rat mandible model. J Biomed Mater Res B Appl Biomater 97(2): 315–326.

Wheeler DL, Stokes KE, Hoellrich RG, Chamberland DL & McLoughlin SW (1998) Effect of bioactive glass particle size on osseous regeneration of cancellous defects. J Biomed Mater Res A 41(4): 527–533.

White AP, Vaccaro AR, Hall JA, Whang PG, Friel BC & McKee MD (2007) Clinical applications of BMP-7/OP-1 in fractures, nonunions and spinal fusion. Int Orthop 31(6): 735–741.

Whiteman D, Gropper PT, Wirtz P & Monk P (1993) Demineralized bone powder. Clinical applications for bone defects of the hand. J Hand Surg Br 18(4): 487–490.

Wildemann B, Kadow-Romacker A, Haas NP & Schmidmaier G (2007) Quantification of various growth factors in different demineralised bone matrix preparations. J Biomed Mater Res A 81(2): 437–442.

104

Willems WF, Larsen M, Giusti G, Friedrich PF & Bishop AT (2011) Revascularization and bone remodeling of frozen allografts stimulated by intramedullary sustained delivery of FGF-2 and VEGF. J Orthop Res 29(9): 1431–1436.

Winn SR, Uludag H & Hollinger JO (1999) Carrier systems for bone morphogenetic proteins. Clin Orthop Relat Res 367S: S95–S106.

Wolfe MW & Cook SD (1994) Use of osteoinductive implants in the treatment of bone defects. Med Prog Technol 20(3-4): 155–168.

Wolff D, Goldberg VM & Stevenson S (1994) Histomorphometric analysis of the repair of a segmental diaphyseal defect with ceramic and titanium fibermetal implants: Effects of bone marrow. J Orthop Res 12(3): 439–446.

Worth A, Mucalo M, Horne G, Bruce W & Burbidge H (2005) The evaluation of processed cancellous bovine bone as a bone graft substitute. Clin Oral Impl Res 16: 379–389.

Wozney JM (1992) The bone morphogenetic protein family and osteogenesis. Mol Reprod Dev 32(2): 160–167.

Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM & Wang EA (1988) Novel regulations of bone formation: molecular clones and activities. Science 242(4885): 1528–1534.

Wozney JM, Rosen V, Byrne M, Celeste AJ, Moutsatsos I & Wang EA (1990) Growth factors influencing bone development. J Cell Sci Suppl 13: 149–156.

Wozney JM & Rosen V (1998) Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair. Clin Orthop Relat Res 346: 26–37.

Wu ZY & Hu XB (1988) Separation and purification of porcine bone morphogenetic protein. Clin Orthop Relat Res 230: 229–236.

Xia W & Chang J (2006) Well-ordered mesoporous bioactive glasses (MBG): A promising bioactive drug delivery system. J Control Release 110(3): 522–530.

Xiao YT, Xiang LX & Shao JZ (2007) Bone morphogenetic protein. Biochem Biophys Res Commun 362(3): 550–553.

Xu HHK, Weir MD, Burguera EF & Fraser AM (2006) Injectable and macroporous calcium phosphate cement scaffold. Biomaterials 27: 4279–4287.

Yamada M, Shiota M, Yamashita Y & Kasugai S (2007) Histological and histomorphometrical comparative study of the degradation and osteoconductive characteristics of alpha- and beta-tricalcium phosphate in block grafts. J Biomed Mater Res B Appl Biomater 82(1): 139–148.

Yasko AW, Lane JM, Fellinger EJ, Rosen V, Wozney JM & Wang EA (1992) The healing of segmental bone defects, induced by recombinant human bone morphogenetic protein (rhBMP-2). J Bone Joint Surg 74-A(5): 659–670.

Yee AJM, Bae HW, Friess D, Robbin M, Johnstone B & Yoo JU (2003) Augmentation of rabbit posterolateral spondylodesis using a novel demineralized bone matrix-hyaluronan putty. Spine 28(21): 2435–2440.

105

Yunoki S, Sugiura H, Ikoma T, Kondo E, Yasuda K & Tanaka J (2011) Effects of increased collagen-matrix density on the mechanical properties and in vivo absorbability of hydroxyapatite-collagen composites as artificial bone materials. Biomed Mater 6(1): 015012.

Zart DJ, Miya L, Wolff DA, Makley JT & Stevenson S (1993) The effects of cisplatin on the incorporation of fresh syngeneic and frozen allogeneic cortical bone grafts. J Orthop Res 11(2): 240–249.

Zhang R, Xu D, Landeryou T, Toth C, Dimaano N, Berry J, Evans J & Hawkins M (2004) Ectopic bone formation using osteogenic protein-1 carried by a solution precipitated hydroxyapatite. J Biomed Mater Res A 71(3): 412–418.

Zhang D, Leppäranta O, Munukka E, Ylänen H, Viljanen MK, Eerola E, Hupa M & Hupa L (2010) Antibacteial effects and dissolution behaviour of six bioactive glasses. J Biomed Mater Res A 93(2): 475–483.

Zhong J & Greespan DC (2000) Processing and properties of sol-gel bioactive glasses. J Biomed Mater Res 53(6): 694–701.

Zou X, Zou L, Foldager C, Bendtsen M, Feng W & Bünger CE (2008) Different mechanism of spinal fusion using equine bone protein extract, rhBMP-2 and autograft during the process of anterior lumbar interbody fusion. Biomaterials 30(6): 991–1004.

Zyman Z, Glushko V, Dedukh N, Malyshkina S & Ashukina N (2008) Porous calcium phosphate ceramic granules and their behaviour in differently loaded areas of skeleton. J Mater Sci Mater Med 19(5): 2197–2205.

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107

Original articles

I Tölli H, Kujala S, Jämsä T & Jalovaara P (2011) Reindeer bone extract can heal the critical-size rat femur defect. Int Orthop 35(4): 615–622.

II Tölli H, Kujala S, Levonen K, Jämsä T & Jalovaara P (2010) Bioglass as a carrier for reindeer bone protein extract in the healing of rat femur defect. J Mater Sci Mater Med 21(5): 1677–1684.

III Tölli H, Birr E, Sandström K, Jämsä T & Jalovaara P (2011) Calcium sulfate with stearic acid as an encouraging carrier for the reindeer bone protein extract. Materials 4(7): 1321–1332.

IV Tölli H, Kujala S, Birr E, Leppilahti J, Jämsä T & Jalovaara P (2011) Bone formation performance of reindeer bone protein extract formulations, autograft and demineralized bone matrix in sheep hole defect model. Manuscript.

Reprinted with kind permissions from Springer Science & Business Media (I, II),

and MDPI Publishing (III).

The original publications are not included in the electronic version of the

dissertation.

108

A C T A U N I V E R S I T A T I S O U L U E N S I S

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1119. Mikkola, Ilona (2011) Prevalence of metabolic syndrome and changes in bodycomposition, physical fitness and cardiovascular risk factors during militaryservice

1120. Venhola, Mika (2011) Vesicoureteral reflux in children

1121. Naillat, Florence (2011) Roles of Wnt4/5a in germ cell differentiation and gonaddevelopment & ErbB4 in polarity of kidney epithelium

1122. Nurmenniemi, Sini (2011) Analysis of cancer cell invasion with novel in vitromethods based on human tissues

1123. Kangas-Kontio, Tiia (2011) Genetic background of HDL-cholesterol andatherosclerosis : Linkage and case-control studies in the Northern Finnishpopulation

1124. Cederberg, Henna (2011) Relationship of physical activity, unacylated ghrelin andgene variation with changes in cardiovascular risk factors during military service

1125. Miller, Brian (2011) Paternal age, psychosis, and mortality : The Northern Finland1966 Birth Cohort, Helsinki 1951–1960 Schizophrenia Cohort, and FinnishNonaffective Psychosis Cohort

1126. Tolvanen, Mimmi (2011) Changes in adolescents’ oral health-related knowledge,attitudes and behavior in response to extensive health promotion

1127. Pirilä-Parkkinen, Kirsi (2011) Childhood sleep-disordered breathing – dentofacialand pharyngeal characteristics

1128. Thevenot, Jérôme (2011) Biomechanical assessment of hip fracture : developmentof finite element models to predict fractures

1129. Moilanen, Kristiina (2011) Diagnostics and determinants of schizophrenia : TheNorthern Finland 1966 Birth Cohort Study

1130. Haapsamo, Mervi (2011) Low-dose aspirin therapy in IVF and ICSI patients

ABCDEFG

UNIVERS ITY OF OULU P.O.B . 7500 F I -90014 UNIVERS ITY OF OULU F INLAND

A C T A U N I V E R S I T A T I S O U L U E N S I S

S E R I E S E D I T O R S

SCIENTIAE RERUM NATURALIUM

HUMANIORA

TECHNICA

MEDICA

SCIENTIAE RERUM SOCIALIUM

SCRIPTA ACADEMICA

OECONOMICA

EDITOR IN CHIEF

PUBLICATIONS EDITOR

Senior Assistant Jorma Arhippainen

Lecturer Santeri Palviainen

Professor Hannu Heusala

Professor Olli Vuolteenaho

Senior Researcher Eila Estola

Director Sinikka Eskelinen

Professor Jari Juga

Professor Olli Vuolteenaho

Publications Editor Kirsti Nurkkala

ISBN 978-951-42-9660-4 (Paperback)ISBN 978-951-42-9661-1 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)

U N I V E R S I TAT I S O U L U E N S I S

MEDICA

ACTAD

D 1136

ACTA

Hanna T

ölli

OULU 2011

D 1136

Hanna Tölli

REINDEER-DERIVED BONE PROTEIN EXTRACT INTHE HEALING OF BONE DEFECTS

EVALUATION OF VARIOUS CARRIER MATERIALS AND DELIVERY SYSTEMS

UNIVERSITY OF OULU,FACULTY OF MEDICINE,INSTITUTE OF BIOMEDICINE,DEPARTMENT OF MEDICAL TECHNOLOGY,INSTITUTE OF CLINICAL MEDICINE,DEPARTMENT OF SURGERY,DIVISION OF ORTHOPAEDIC AND TRAUMA SURGERY