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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
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
Ta
ble
1. T
he
su
mm
ary
of
es
sen
tia
l s
ign
ali
ng
mo
lecu
les
an
d g
row
th f
ac
tors
du
rin
g t
he
fra
ctu
re h
ea
lin
g c
as
ca
de
; th
eir
so
urc
e,
tim
e
of
exp
res
sio
n, a
nd
ma
jor
fun
cti
on
s.
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
Cyt
okin
es:
IL-1
, IL-
6, T
NF-α
Infla
mm
ator
y ce
lls,
MS
Cs
Day
s 1
to 3
and
dur
ing
bone
rem
odel
ling
Hem
atom
a fo
rmat
ion,
infla
mm
atio
n,
angi
ogen
esis
Kon
et a
l. 20
01,
Ai-A
ql e
t al.
2008
TGF-β
Deg
ranu
latin
g pl
atel
ets,
Infla
mm
ator
y ce
lls,
Ext
race
llula
r mat
rix
Day
s 1
to 2
1
(Pea
k in
day
7)
Ext
race
llula
r pro
tein
pro
duct
ion,
Cho
ndro
gene
sis,
End
ocho
ndra
l bon
e fo
rmat
ion
Sol
heim
199
8,
Cho
et a
l. 20
02,
Lieb
erm
an e
t al.
2002
PD
GF
Deg
ranu
latin
g pl
atel
ets,
Mac
roph
ages
,
Ost
eobl
asts
Day
s 1
to 3
S
timul
ator
for i
nfla
mm
ator
y ce
lls,
MS
Cs
and
oste
obla
sts
Sol
heim
199
8,
Cho
et a
l. 20
02,
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
ds
use
d i
n t
he
stu
die
s.
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)
Ana
lysi
s m
etho
ds
I C
SD
, lef
t fem
ur
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
efec
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
II C
SD
, lef
t fem
ur
Rat
Bio
activ
e gl
ass
(S53
P4)
Bio
activ
e gl
ass
(S53
P4)
Bio
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
aphy
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
/CS
30:
60:1
0
β-TC
P/C
S 9
0:10
Cem
-Ost
etic
CS
hem
ihyd
rate
,
CS
dih
ydra
te w
ith S
A,
Cor
resp
ondi
ng c
ontro
ls
3 m
g (im
preg
nate
d)
3 m
g (im
preg
nate
d)
3 m
g (im
preg
nate
d)
3 m
g (m
ixed
)
3 m
g (m
ixed
)
3 m
g (d
ry m
ixed
)
-
8 8 8 8 8 8
8 in
eac
h
3
Rad
iogr
aphy
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)
Ana
lysi
s m
etho
ds
IV
Hol
e de
fect
(0.2
83 c
c),
Hum
erus
and
fem
urs
She
ep
Cam
β-T
CP
gra
nule
s, w
ith P
EG
-GLY
and
SA
Cam
β-T
CP
gra
nule
s, w
ith P
EG
-GLY
and
SA
Cam
β-T
CP
gra
nule
s, w
ith P
EG
-GLY
and
SA
Cur
β-T
CP
mor
sels
, with
PE
G-G
LY a
nd S
A
Cur
β-T
CP
gra
nule
s, w
ith P
EG
-GLY
and
SA
Cur
β-T
CP
mor
sels
, with
PE
G-G
LY a
nd S
A
Cam
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
76
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
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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.
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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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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