PHD Geert Vertenten Definitieve Versie

Evaluation of potential bone
substitutes in a goat model
Geert Vertenten
Proefschrift ter verkrijging van de graad van
Doctor in de Diergeneeskundige Wetenschappen (PhD)
aan de Faculteit Diergeneeskunde, Universiteit Gent
februari 2010
1
Promotoren : Prof. Dr. L. Vlaminck
1
Prof. Dr. F. Gasthuys
2
Prof. Dr. M. Cornelissen
1
Vakgroep Heelkunde en Anesthesie van de Huisdieren
Faculteit Diergeneeskunde, Salisburylaan 133, B-9820 Merelbeke
2
Vakgroep Medische Basiswetenschappen
Faculteit Geneeskunde en Gezondheidswetenschappen
De Pintelaan 185 B3, B-9000 Gent
ISBN: 9789058641946
Printed by DCL Print & Sign, Zelzate, Belgium. www.dclsigns.be
Evaluation of potential bone substitutes in a goat model

Geert Vertenten
Vakgroep Heelkunde en Anesthesie van de Huisdieren
Faculteit Diergeneeskunde
Universiteit Gent
ISBN/EAN-NUMBER: 9789058641946
Cover Photo: Jozef Kusters, Schootseweg 50, 2381 Weelde

CONTENTS
LIST OF ABBREVIATIONS
PREFACE 1
CHAPTER 1 INTRODUCTION 3
ENHANCING BONE HEALING/REGENERATION:
PRESENT AND FUTURE PERSPECTIVES IN
VETERINARY ORTHOPAEDICS
Indications for use of enhanced bone regeneration
techniques 6
Different substitutes to enhance bone healing/
regeneration 8
Reports of enhanced bone regeneration techniques in
veterinary clinical cases 16
Current research focuses 24
CHAPTER 2 AIMS OF THE STUDY 39
CHAPTER 3 EVALUATION OF BONE SUBSTITUTES 43

3.1 EVALUATION OF AN INJECTABLE,
PHOTOPOLYMERIZABLE THREE-
DIMENSIONAL SCAFFOLD BASED ON D,L-
LACTIDE AND ε-CAPROLACTONE IN A TIBIAL
GOAT MODEL 45
3.2 EVALUATION OF AN INJECTABLE,

PHOTOPOLYMERIZABLE, AND THREE-
DIMENSIONAL SCAFFOLD BASED ON
METHACRYLATE-ENDCAPPED POLY(D,L-
LACTIDE-CO-ε-CAPROLACTONE) COMBINED
WITH AUTOLOGOUS MESENCHYMAL STEM
CELLS IN A GOAT TIBIAL UNICORTICAL
DEFECT MODEL 75
3.3 EVALUATION OF BONE REGENERATION WITH

AN INJECTABLE IN SITU POLYMERIZABLE
PLURONIC® F127 HYDROGEL DERIVATIVE
COMBINED WITH AUTOLOGOUS
MESENCHYMAL STEM CELLS IN A GOAT
TIBIAL DEFECT MODEL 113
Contents
CHAPTER 4 ADAPTED ANALYTICAL METHODS 147

4.1 IMMUNOHISTOCHEMICAL ANALYSIS OF LOW-
TEMPERATURE METHYLMETHACRYLATE
RESIN-EMBEDDED GOAT TISSUES 149
4.2 AGREEMENT BETWEEN MICRO-COMPUTED
TOMOGRAPHY AND HISTOMORPHOMETRY
FOR EVALUATION OF NEW BONE FORMATION
IN A TISSUE-ENGINEERED CORTICAL TIBIAL
GOAT MODEL 173
GENERAL DISCUSSION 201

Development of an in vivo model 201
Evaluation of experimental injectable bone enhancing
products 206
Optimalisation of the evaluation techniques for bone
healing 218
Future perspectives 226
APPENDIX 241
SUMMARY 245
SAMENVATTING 251
CURRICULUM VITAE 257
BIBLIOGRAPHY 259
DANKWOORD 265
LIST OF ABBREVIATIONS
ACD-A Anticoagulant Citrate Dextrose Solution Formula A

Ad-BMP Adenoviral Bone Morphogenetic Protein
α-TCP alfa TriCalcium Phosphate
AP Alkaline Phosphatase
BM Bone Marrow
BMP Bone Morphogenetic Protein
BMSC Bone marrow-derived Mesenchymal Stem Cells
BS Bone Surface
BSD Bone Surface Density
BSS Bone-Specific Surface
BV Bone Volume
BVD Bone Volume Density
Cbfa-1 Core binding factor alpha1
CD Cluster of Differentiation
cDNA complementary DNA
CT Computed Tomography
DBM Demineralized Bone Matrix
ECM ExtraCellular Matrix
EDTA EthyleneDiamineTetraacetic Acid
FBS Fetal Bovine Serum
FDA Food and Drug Administration
H&E Hematoxylin and Eosin
HA HydroxyApatite
HEMA HydroxyEthylMethAcrylate
Histo Histology
HRP HorseRadish Peroxidase
MEM Minimum Essential Medium
µCT micro-Computed Tomography
MMA Methyl MethAcrylate
MSC Mesenchymal Stem Cell
List of abbreviations
MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazoliumsalt
no number
OC Osteocalcin
PBS Phosphate-Buffered Saline
PLU Pluronic
PSF Polysulfone
rhBMP recombinant human Bone Morphogenetic Protein
rpm revolutions per minute
SD Standard Deviation
TGFβ Tumor Growth Factor Beta
TS Trabecular Separation
TT Trabecular Thickness
TV Total Volume
UGCT Centre for X-ray Tomography at the Ghent University
UVA UltraViolet A
Vim Vimentin
VITO Flemish Institute for Technological Research
VOI Volume of Interest
“De Weg is Wijzer dan de
Wegwijzer”
Prof. dr. Ulrich Libbrecht

PREFACE
The self-healing capacity of bone is widely used for the repair of

small fractures. However, if segments of bone are lost or damaged so
severely that they have to be removed, a bony union is not possible
and bone grafts are required to achieve an acceptable healing.
The clinical practice of bone grafts to repair, replace or supplement

the bone stock has a long history, dating back to McEwen in 1881. It
was demonstrated that frozen preserved allograft was superior in
performance to fresh allogeneic bone, so a more extensive use of
bone grafts became possible.
Newer techniques involve the use of natural and synthetic bone

grafts. The ultimate goal is to combine the strength of polymers with
the osteoconductivity, or preferably osteoinductivity of other materials
resulting in an ideal bioactive composite implant with a suitable
hardness, strength and modulus corresponding to the biomechanical
properties of bone.
The increasing popularity of arthroscopic procedures in

orthopaedics and the requirement to bridge large and irregular bone
defects resulted in great interest in fixation materials that are
injectable, in situ forming and biodegradable. Several injectable
materials have been used as osteogenic bone substitutes but none
has gained universal acceptance up to now.
In this PhD thesis, several potential ‘home made’ injectable bone

enhancing composites, which already showed their bone stimulating
properties in vitro, were evaluated in a newly developed in vivo animal
model. Furthermore, the evaluation of the bone substitute in this
model by immunohistochemistry and micro computed tomography is
highlighted and refined.
1
CHAPTER 1
Introduction
Enhancing bone healing/regeneration:
present and future perspectives in
veterinary orthopaedics
Adapted from: G. Vertenten, F. Gasthuys, M. Cornelissen, E. Schacht, L. Vlaminck

(2010). Enhancing bone healing/regeneration: present and future
perspectives in veterinary orthopaedics. Veterinary and
Comparative Orthopaedics and Traumatology.

Enhancing bone healing/regeneration
Fracture healing typically results in restoration of the original

structure and function of the bone tissue unlike muscle or skin tissue
that are not able to regenerate without scar tissue formation. In
fracture repair, proper reduction and immobilisation are essential to
achieve optimal bone healing. This can be accomplished through the
use of specific reduction techniques, surgical instruments and
orthopaedic implants (Brinker et al., 1990).
Intimate contact of the fracture fragments is required for secondary

osteons to progress from one fragment to another, although smaller
defects will also heal spontaneously without the need for additional
‘bone healing enhancers’. Larger bone defects, specifically those
defined as ‘critical sized defects’ will show no spontaneous closure
and represent a huge challenge in both human and veterinary
orthopaedics, requiring additional means to enhance bony union
(Gartner & Hiatt, 2001). Traditional techniques are mainly based on
the transplantation of autologous bone tissue which is known to be
incorporated more rapidly than any other type of graft. Despite
development of better surgical techniques, human literature still
reports substantial morbidity associated with bone graft donor sites
specifically for posterior iliac crest graft harvest (Younger & Chapman,
1989, Schwartz et al., 2009). Comparable morbidity has not been
reported in veterinary literature. However, the quantity of available
bone graft tissue is often limited in small-sized patients dealing with
large bone defects encouraging the use of allografts, xenografts and
different alloplasts as substitutes. The use of ‘foreign’ substances to
replace bone deficits carriers its specific risks depending on the
characteristics of the applicated bone substitute. Consequently, the
search for the ‘ideal bone graft’ is still ongoing which should deliver
osteogenic cells either directly (osteogenesis) or by stimulating
differentiation of bone cells from undifferentiated mesenchymal cells
(osteoinduction), and provide a matrix as a scaffold for new bone
ingrowth (osteoconductive) and to support the bony column during the
healing process. Especially in larger bone graft constructs, the
4
CHAPTER 1
generation of an adequate blood supply (angiogenesis) is mandatory

to provide the graft with necessary nutrients enabling long-term
incorporation. Remodelling of the graft tissue allows the bone to
counteract possible infection and to receive circulating factors and
nutrition (Kanczler & Oreffo, 2008). Most commercially available bone
grafts only carry one or more of these properties when incorporated
into host tissue. The final selection which bone graft material to use is
subsequently based on the specific requirements for the encountered
clinical situation (Millis & Martinez, 2003).
In the last decade, the search for the ‘ideal bone graft’ has lead to
the development of multiple alternatives.
5
INDICATIONS FOR USE OF ENHANCED BONE

REGENERATION TECHNIQUES
The use of bone enhancing grafts can be indicated in different

surgical disciplines including surgery of the head, dentistry, long bone
and joint surgery. Its use in veterinary surgery is rather limited but
promising results in human studies might create similar and new
surgical techniques and opportunities for veterinary indications in the
near future.
Surgery of the head and dentistry
Cleft palate deformity is a relatively common congenital

abnormality of the head in human and animals. Bone enhancement
techniques play important roles in cleft repair (Tollefson et al., 2008).
Head trauma represents a common pathology encountered both in
small and large animal practice (Legendre, 2005). It most often results
in fractures amenable to classic osteosynthesis techniques for repair
but can sometimes lead to substantial bone loss. The reconstruction
of large bone defects in the cranio-maxillo-facial area still represents a
major surgical challenge despite considerable progress made in the
field of enhanced bone regeneration.
Dentistry related bone graft application in man focuses on repair of

alveolar bone defects caused by periodontal and peri-implant related
bone destruction and alveolar ridge height preservation for esthetical
purposes and to provide a basis for future implant placement (Callan,
2001). An edentulous upper jaw is a frequent handicap mainly in
human and domestic small animals. Loss of teeth and aging induce
bone resorption resulting into progressive atrophy of the maxillary
bone. Rehabilitation of this atrophic maxilla with dental implants is
impossible without bone grafting. This is routinely achieved in the
posterior maxilla by using a sinus floor elevation procedure in man
6
CHAPTER 1: Indications
whereby the thickness of the maxillary sinus floor is increased with a

suitable bone substitute (Bettega et al., 2009).
Application of bone substitutes in veterinary dentistry has been

advocated in dogs and cats to preserve the alveolar bone height or
provide jaw stability following specific tooth extractions (Bellows,
2004, Legendre, 1997, Marretta, 2002).
Long bone and joint surgery
Bone enhanced healing is an essential but not always obvious part

of the surgical treatment of many orthopaedic conditions. Bone grafts
can be used to bridge major defects or to establish the continuity of a
long bone (e.g. after trauma or tumor resection). Even more, these
grafts are indicated in the procedures for fusion of joints, for filling
cavities or defects, and to promote bony union in delayed union or
nonunion fractures (Millis & Martinez, 2003). The aetiology of a non-
union may be induced by multiple factors. A poor blood supply to the
affected area together with a poor general nutritional status can
predispose to a non-union fracture. Even more, poor apposition of the
fractured bone ends, pathological fractures, presence of foreign
bodies, large quantities of necrotic bone, infections or non-justified
corticosteroid therapy have also been reported as possible
aetiological factors (Stevens & Lowe, 1995). Enhanced bone
regeneration is justified in cases of non-unions, not only to provide
support and fill existing lacunae but also to enhance biological repair
when the skeletal defect reaches the so-called critical size (Olivier et
al., 2004). Bone enhancement techniques are a real challenge in the
treatment of critical size defects which have been defined as that
defect size whereby normal complete calcification of the defect will not
occur during the remaining lifetime of the animal or man (Arnold,
2001).
7
DIFFERENT SUBSTITUTES TO ENHANCE BONE

HEALING/REGENERATION
Bone grafts harvested from a donor site site can be transplanted to

the patient’s defect to stimulate bone formation or man-made
biomaterials can be placed in a defect site as a bone substitute. In this
chapter an overview is given of the downfalls and attractions of these
materials (Tabel 1).
Auto-, allo- and xenografts
Autografts still represent the “gold standard” material for enhanced

bone regeneration because these grafts contain all the essential
components to promote bone formation, including osteoprogenitor
cells, matrix, and bone morphogenetic proteins. Philip von Walther
has been cited as having performed, in 1820, the first clinically
successful autogenous bone transfer in man (Chase & Herndon,
1955). However ten years earlier, Merrem had already achieved good
results with bone-graft experiments in animals (Hutchinson, 1952).
The use of cancellous and cortical bone autografts in veterinary

orthopaedic surgery has also become very popular and is well
documented (Schena, 1983). Cancellous bone grafts are typically
used to provide live cells and growth factors that stimulate the
production of new bone. Because little support is provided by these
cancellous grafts, the addition or use of cortical bone is more justified
when structural support is of major importance (Millis & Martinez,
2003).
Autografts are still preferred over the use of allo- and xenografts
although these latter two obviate donor morbidity encountered during
autograft retrieval and can serve as an osteoconductive and -inductive
tool to enhance bone healing (Mahendra & Maclean, 2007, Blokhuis &
8
CHAPTER 1: Different substitutes
Lindner, 2008). On the other hand, both graft substances possess

considerable less capacity for osteoinduction and osteoconduction
compared to autografts. Their resorption rate is often mismatched
compared to the rate of new bone formation increasing the chance for
non-integration of the graft. Moreover, the antigenic response elicited
by the presence of ‘foreign’ material increases the likelihood of graft
rejection like encountered more specifically using pure bone
xenografts.
Demineralized bone matrix (DBM) is a good option as an allograft

material. By reducing the mineral phase, growth factors become more
available thus increasing its osteoinductive properties (Kao & Scott,
2007). However, since no structural strength is provided, its primary
use is limited to a structurally stable environment. Several excipients
like hydroxyapatite, autografts or even bone marrow aspirate can be
included into DBM to improve its handling characteristics and
mechanical properties (Mahendra & Maclean, 2007). DBM is available
for human use in a variety of forms including fibres, flex, mouldable
gels, putties, as well as an injectable version. Because DBM lacks
structural properties, it is recommended only as a gap filler in non-
weight bearing areas (Hoffer et al., 2008).
Deproteinized bovine bone is the most widely used xenograft

substance (Kao & Scott, 2007). Heat-treated bovine cortical bone has
also been proposed as a xenograft alternative to bone grafts and
synthetic alloplasts because it combines the advantages of allografts
including a high stiffness and acceptable strength, and of synthetic
materials, which are characterized by an abundant supply and a
reduced risk of rejection and disease transfer (Berglundh & Lindhe,
1997).
Alternative xenografts originate from the exoskeleton of

crustaceans (chitosan) (Kim et al., 2008).
9
Synthetic and natural bone substitutes
Many synthetic and natural materials are available to the surgeon,

including ceramics and/or ceramics-collagen composites, natural
corals, coralline hydroxyapatite, and resorbable polymers in different
forms (sponges, microfibers, foils, porous membranes). They have
been experimentally used together with titanium implants to enhance
bone healing. Good bone healing properties were reported in sheep
(Marcacci et al., 1999, Mastrogiacomo et al., 2006, Meinig, 2002,
Teixeira et al., 2007), goats (Meinig, 2002), rats (Saadeh et al., 1998,
Wolff et al., 1994), dogs (Johnson et al., 1996), rabbits (Fujibayashi et
al., 2003, Wefer et al., 2000, Meinig, 2002, Wheeler et al., 2000), pigs
(Meinig, 2002) and cats (Dorea et al., 2005).
Bone marrow - stem cells
Bone marrow contains osteoprogenitor stem cells that are able to

form bone when combined with various elements incorporated into an
osseous matrix (Tiedeman et al., 1991). Although several
investigations have indicated that bone marrow is certainly capable of
promoting new-bone formation, techniques for enriching the potential
bone forming active component of bone marrow – namely
mesenchymal stem cells (BMSC) – are of primary importance,
because these cells constitute only 0.01% of all marrow cells (Devine
et al., 2002). Even if osteogenic cells at the site of a fracture are
working at full capacity, the defect will not heal if too few cells are
present, nor will any drugs directed at enhancing bone formation be
effective (Bruder et al., 1994). The use of pure bone marrow has
yielded inconsistent results in the promotion of bone formation
(Tshamala & van Bree, 2006).
Molecules enhancing bone healing
Several growth-promoting substances involved in local regulation

of bone healing have been identified at fracture sites. These
substances can be divided into two groups namely the peptide
10
signalling molecules (generally referred to as growth factors) and

immunomodulatory cytokines, such as interleukin 1 and 6 (Einhorn,
1995).
Growth factors exert multiple effects on cells at both local and

systemic levels. These factors include bone morphogenetic proteins,
transforming growth factor-β, platelet-derived growth factors 1 and 2,
osteogenic growth peptide and a variety of hematopoietic factors such
as lymphokines and monokines (Kirkerhead, 1995). Recombinant
technology has allowed isolation, production and application of these
synthesized molecules for osteoinduction and osteoconduction
purposes required for healing of bone defects (Simpson et al., 2006).
Urist (1965) noted that demineralized bone matrix (DBM) could

induce de novo formation of cartilage and bone when implanted in
extraskeletal sites. Further investigations identified the active
component of the demineralised bone matrix (DBM) as proteinaceous
and demonstrated that it could be extracted from the bone matrix
(Urist et al., 1979). The proteinaceous and osteoinductive component
was named “bone morphogenetic protein” (BMP) and up to now, over
20 types of BMP have been identified, each having a variety of
systemic functions (Helm et al., 2002). BMP-2, 4 and 7 and more
recently BMP-6 and 9 were demonstrated to have osteoinductive
potential (Schmitt et al., 1999, Cheng et al., 2003, Nakase et al., 1994,
Yoshimura et al., 2001).
Early studies used BMP purified from bone, while growth factors
are currently produced as recombinant proteins by synthesis from
microbiological agents (e.g. Escherichia coli) transfected with a growth
factor gene (e.g. human BMP-2 gene). The resulting recombinant
human BMP-2 (rhBMP-2) is purified and tested for its biological
activity before in vivo application. Local application of rhBMP-2 in
multiple critical sized defect experiments resulted in production of
structurally sound orthotopic bone in rats (Yasko et al., 1992), sheep
(Kirkerhead et al., 1995), rabbits (Zegzula et al., 1997) and dogs
11
(Sciadini & Johnson, 2000). Interspecies amino acid sequence

homology for rhBMP-2 is 100% present in most mammalian species,
allowing its use in all species commonly treated by veterinarians.
However, BMP derived from the animal species has been shown to
result in better bone formation at lower doses compared with the use
of recombinant BMP from another species (Schmitt et al., 1999).
Development of an optimal delivery system for BMP use is still of

major concern. They can be administered systemically with possible
risk of unintended adverse effects. Gene transfer technology can be
used to deliver growth factor genes (cDNA) to specific cells located at
the fracture site using a viral or non-viral vector and in vivo or ex vivo
methods (Niyibizi et al., 1998). These genes are then expressed by
cells at the fracture site achieving sustained high concentrations of
biologically more active growth factors compared to ex vivo
synthetized BMP’s. As cDNA is a stable molecule with a long shelf
time storage and manufacturing may be less expensive than
synthetizing recombinant proteins, this molecule offers positive
perspectives for use in gene therapy. Delivery of the BMP genes to
the fracture site using gene therapy has been evaluated in laboratory
animal models using non-union fractures with promising results
(Southwood et al., 2004). Nevertheless, further research is needed to
counter the multiple drawbacks still encountered. Unexpected
cartilage formation was observed after single injections of adenovirus
carrying BMP-2 in 50% of created femoral defects (Betz et al., 2006)
and after mesenchymal stem cell (MSC)-mediated gene delivery of
BMP-2 in an articular fracture model (Zachos et al., 2007) both in rats.
A last delivery method consists of implanting BMP’s with a carrier

matrix. In this modality, 2 different BMP’s are currently available for
clinical human applications, rhBMP-2 (InductOs® in Europe and
INFUSE Bone Graft® in USA, Canada and Australia) and rhBMP-7
(Osigraft® in Europe and OP1 Implant® in USA, Canada and
Australia). Both are manufactured by a process involving mammalian
12
cell expression. Non-union, open tibial fractures, spinal fusions and

certain oral and maxillofacial bone grafting procedures are conditions
for which a clinical approval has been granted for the use of BMP’s
(Bishop & Einhorn, 2007, McKay et al., 2007, Kirker-Head et al.,
2007).
Table 1. Overview of some frequently used bone grafts and bone
substitutes used to facilitate the bone repair process, with their
attractive characteristics and drawbacks (adapted from Lippens,
2009).
Pros Cons
Autograft Osteoconductive Donor site morbidity
Osteoinductive Limited availability
Residing cells Longer operation time
No immune response Additional operation site & scar
No risk for disease
transmission
Minimal ethical concerns
Allograft Osteoconductive Risk of disease transfer

Higher availabitlity Immunogenicity of fresh
Avoids donor site morbidity allografts
Variable quality
Limited mechanical stability
Faster resorption rate
No to limited osteoinduction
although debatable
Xenograft Osteoconductive Not osteoinductive (except

Graft extender DBM), although debatable
Readily available Ethical/religious concerns
Available in large quantities Low mechanical support
Risk for disease transfer
Batch difference
Calcium Osteoconductive filling Not osteoinductive (although

phosphate material some reports claim the opposite)
Not inflammatory, well Brittle and poor tensile strength
tolerated No mechanical strength
Calcium sulphate Bone filler Not osteoconductive

Not inflammatory Not osteoinductive
Fast resorption
13
Bioactive glass Osteoconductive No structural support, although

Graft extender higher mechanical strength then
Able to bound to soft and bone calcium phosphates
tissue Slow resorption rate (12-16
Actively aids the bone months)
formation process
Bone marrow Osteoinductive Low concentration of active

component
Inconsistent results
No mechanical support
Stem cells Osteoinductive Sufficient amount needed
Demineralized Osteoinductive Low concentration of active

bone matrix component
Bone Different extraction methods Best result if derived from same
morphogenetic possible animal species
protein Osteoinductive Inconsistent results
Commercially available for No mechanical support
clinical applications
The positive characteristics of the several bone substitutes and

enhancers can be combined by mixing materials. Finally, alternative
ways can stimulate new bone formation.
Composites
Non-solid bone enhancing substances as bone marrow have been

reported to be washed easily out of the fracture site. Many authors
have studied the positive effects of composite grafts formed by
combining bone-graft substitutes (e.g. demineralized bone matrix,
ceramics) and autologous bone marrow which enhances the practical
use of the products and possibly also its bone regeneration properties
(Jackson et al., 1981, Lindholm & Urist, 1980, Green et al., 1986,
Ohgushi et al., 1989, Connolly et al., 1991).
‘Bone tissue engineering’ has become a new approach to enhance

bone regeneration. In this field, it is believed that by combining a
synthetic 3D porous template (scaffold) with an osteogenic potent cell
population, it will be possible to develop bone tissue equivalents that
can induce total regeneration of a large affected area. This ideal cell
population should posses a high osteogenic potential while the cells
should be easily expandable and can be maintained in cultures for
14
prolonged periods. MSC’s are considered highly suitable to fulfil the

requirements for such a cell population (Salgado et al., 2006). MSC’s
seeded on scaffolds have been used to repair experimentally induced
critical sized bone defects in rats (Kadiyala et al., 1996), mice
(Krebsbach et al., 1998), dogs (Bruder et al., 1998) and sheep (Kon et
al., 2000). In large animal models, a significant advantage in the
healing of segmental bone defects was observed after delivery of a
MSC’s loaded bioceramic scaffold in a mechanically stable
environment (Bruder et al., 1998, Kon et al., 2000). Finally,
angiogenesis in a tissue-engineered device may be induced by
incorporating growth factors (e.g., vascular endothelial growth factor),
genetically modified cells, and/or vascular cells (Barralet et al., 2009).
Alternative ways to enhance bone healing
Yasuda reported in 1953 that new bone was formed around a

negative electrode (cathode) while bone resorption occurred at the
positive electrode (anode) if both electrodes are placed directly on the
bone (Yasuda, 1977). Several forms of electrostimulation currently
exist to enhance bone healing including direct current implants,
external pulsed electromagnetic field systems, capacitively coupled
electrical stimulation and surface interferential stimulation (Briggs et
al., 2004, Nawrocki et al., 2006). More than 80% of human non-unions
treated with electrostimulation successfully progress towards a bony
union (Ducharme & Nixon, 1996).
Extracorporeal shock wave therapy has also been used for the
treatment of a number of musculoskeletal conditions and has shown
promising results in attempts to improve fracture healing and delayed
union in general (Schaden et al., 2001). The rationale underlying
explanation of this treatment is the stimulation of bone growth and
vessels by the production of nitric oxide (Ciampa et al., 2005).
15
REPORTS OF ENHANCED BONE REGENERATION

TECHNIQUES IN VETERINARY CLINICAL CASES
Enhanced bone regeneration has been mainly applied in human

medicine and experimental animals. The application of enhanced
bone regeneration in veterinary medicine is relatively limited to
experimental studies using animal models for human purposes, apart
from few case reports and a small number of clinical trials. However,
bone grafting and enhanced bone regeneration are an interesting but
often underused part of the surgical treatment of many orthopaedic
conditions in domestic animals.
Dogs and cats
Autologous cancellous grafts have been used in dogs and cats as

treatment of highly comminuted fractures for stimulation of bone union
before implant failure (Olds, 1973), in patients with a poor osteogenic
potential (older, debilitated or small and toy-breed patients) (Millis &
Martinez, 2003), in non-union fractures (Schwarz et al., 1991), to fill
bone defects created by aneurismal bone cysts (Dowdle et al., 2003),
or after performing surgical curettage of bone (Duval et al., 1995),
following tooth extraction (Kim et al., 2005), and to enhance healing
following ventral stabilization procedures in the cervical spine (Voss et
al., 2006, Ozak et al., 2006) or after joint arthrodesis (Shani et al.,
2006, Johnson & Bellenger, 1980).
Frozen allogeneic cancellous bone graft is commercially available

since several years as cancellous bone chips (Osteoallograft®,
Veterinary Transplant Service, Kent, WA). When used in the primary
repair of fractures and for carpal and scapulohumeral arthrodesis in
dogs, these grafts are effectively incorporated (Kerwin et al., 1996).
The commercial chips can be mixed with autogenous cancellous bone
graft to increase the volume of graft for application into a cortical
16
CHAPTER 1: Reports of veterinary clinical cases
defect (Millis & Martinez, 2003). Although a delayed sequence in all

aspects of the repair process and some bone resorption are initially
observed, cancellous allografts are successfully incorporated in
canine ulnar defects after a longer period of time (Heiple et al., 1987).
Cortical and cortico-cancellous bone grafts (auto- and allografts)

are primarily used in small animals to provide structural support and
osteoconduction in areas devoid of portions of the bony column, such
as a highly comminuted fracture or after bone removal required for
tumour resection (Fitch et al., 1997, Liptak et al., 2006). Less frequent
indications for the application of these bone grafts are arthrodesis of
joints, lengthening of bones, correction of cleft palates, mal- and non-
unions (Sinibaldi, 1989, Boudrieau et al., 1994, Ishikawa et al., 1994,
Hildreth & Johnson, 2007). Recently, the strength of allogeneic
cortical bone pins has been evaluated for use as biodegradable
fixation devices in fracture fixation (Liptak et al., 2008).
A cancellous bovine bone xenograft was also successfully used

together with autogenous cancellous bone (at a ratio of 4:1) to fill a
curetted osteolytic lesion of the distal radius in a single dog (Worth et
al., 2007).
Use of DBM as a substitute or adjunct for autogenous cancellous

bone graft has been described in a retrospective and case-matched
study of seventy-five dogs that had orthopaedic procedures
(comminuted fractures, tibial plateau leveling osteotomies where
correction for tibial rotation created an osteotomy gap, arthrodeses,
open corrective osteotomies). Mean healing time (± standard
deviation) for orthopaedic surgeries with DBM augmentation were 15
± 6.97 weeks and complication rate was 19% (14 dogs). Dogs with a
tibial plateau leveling osteotomy gap filled with DBM were allowed to
return to normal exercise 2 weeks earlier than dogs with a well-
apposed tibial plateau leveling osteotomy site. Radiographic healing,
duration of exercise restriction, and timing of destabilization were
similar in dogs undergoing carpal and tarsal arthrodesis whether they
17
received DBM, autogenous graft, or both (Hoffer et al., 2008). The use
of DBM has been evaluated experimentally in cats for human
purposes (Toombs & Wallace, 1985). DBM gel (Grafton Flowable Gel,
Osteotech, NJ) enhanced spinal fusion in an experimental study with
dogs, either alone or in combination with autograft material. The gel
formulation of DBM has better handling properties and is able to
spread into the irregular contours of the surgical defects. The mixture
of autograft with DBM diminishes the required quantities of the
autograft material, appears to facilitate a more rapid incorporation of
autograft, and induces an excellent repair response (Frenkel et al.,
1992).
Despite the extensive and frequent use of ceramics, natural corals,

coralline hydroxyapatite, resorbable polymers in combination with
titanium implants in human medicine and dentistry, the application in
veterinary medicine is almost exclusively restricted to experimental
procedures (Fuller et al., 1996, Gauthier et al., 1999, Damron, 2007).
Clinically, different ceramics have been used successfully in dogs or
cats for different indications including excision of a tumour using
calcium phosphate (Gauthier et al., 2000), after arthrodeses with β-
tricalciumphosphate (Hauschild et al., 2007) or hydroxyapatite (Dorea
et al., 2007), non-unions treated with β-tricalciumphosphate (Franch et
al., 2006, Hauschild et al., 2007), long-bone fractures or chronic
osteomyelitis and osteochondrosis using dentine hydroxyapatite
(Oktar et al., 2005) and β-tricalciumphosphate (Hauschild et al.,
2007), and alveolar supplementation following canine extraction
(Legendre, 1996, Legendre, 1997).
Bone marrow has been used to enhance bone regeneration in

skeletal long bone defects and non-unions in dogs (Tiedeman et al.,
1991, Grundel et al., 1991). Clinically, supplementation of ceramic
substances with bone marrow improves the handling characteristics of
the graft material and accelerates radiographic healing (Grundel et al.,
1991). Bone marrow graft added to macroporous biphasic calcium
18
phosphate is an appropriate material in dogs to fill bone defects in

irradiated tissue and can be used after bone removal for oncologic
obligations (Malard et al., 2005).
Six non-union fractures (five radius/ulna, one tibia) and four

fractures (one femur, one metatarsal bone, two radius/ulna)
associated with critical sized bone defects as a result of bone
resorption were treated in dogs by percutaneous injection of
autologous bone marrow derived stromal cells. Complete bone
healing was achieved in 7/10 cases. The failure of the therapy in three
dogs can be attributed to resorption of an extremely large segment of
the bone, excessive instability and chronicity of the disease
(Zamprogno et al., 2008). Despite multiple positive animal
experiments and the successful application in man, reports of clinical
use of BMP in veterinary patients are rare. One report described the
successful treatment of a 4-year-old Pomeranian with a 2-year history
of a femoral non-union fracture with a revision surgery and adjunctive
use of rhBMP-2 (Itoh et al., 1998). Additionally, the use of
nonglycosylated BMP-2 in a fibrin matrix delivery vehicle was reported
for the management of long-bone atrophic nonunions in 5 cats and 3
dogs with an uncomplicated outcome in 6 cases. The implant was
administered through a stab incision into the fracture gap (Schmokel
et al., 2004). Four dogs with delayed- or non-unions after long bone
fractures, osteotomy or arthrodesis were treated with either minimally
invasive, fluoroscopically guided, percutaneous administration or
direct surgical application of rhBMP-2. A rapid radiographic union was
noticed in all dogs with an excellent long-term outcome. Adverse
effects included transient worsening of lameness after percutaneous
administration of rhBMP-2 (Milovancev et al., 2007). A rhBMP-2
solution impregnated on a commercial collagen sponge (InductOs®)
was placed along the diaphysis of an atrophic radius in an Italian
Greyhound, with a history of recurring fractures. Two months after
rhBMP-2 treatment, new mineralized bone was present, which
significantly increased the diameter of the radius and allowed the
19
removal of the external skeletal fixator (Bernard et al., 2008). Finally,

rhBMP-2 delivered from an absorbable collagen sponge containing
tricalcium phosphate and hydroxyapatite was also clinically
successfully used in dogs as a graft substitute in reconstruction of
large mandibular defects (Boudrieau et al., 2004, Lewis et al., 2008,
Spector et al., 2007).
The application of bone tissue engineering in dogs and cats to

enhance bone regeneration is up to now limited to experimental
studies (Kraus & Kirker-Head, 2006).
The use of electric current to enhance bone regeneration has not

yet gained widespread use in dogs and cats. No clinical studies have
been published, although the majority of the original research was
done in small animals (Clark, 1987). Although the use of
extracorporeal shock wave therapy in dogs and cats is gaining in
popularity, no studies have objectively evaluated the efficacy
associated with the application of this technique to musculoskeletal
tissue (Danova & Muir, 2003).
Ruminants
Although dogs are still outnumbered for orthopaedic research

compared to sheep and goats, the numbers of small ruminants used
for bone research substantially increased over the last decade
(Pearce et al., 2007). Large animal models were developed to verify
the practicability of bone enhancing products closer to the realistic
clinical situations. Most studies use large segmental long bone defects
to investigate a wide scala of different bone substitutes that enhance
bone healing. The studies differ with regard to animal model (sheep,
goat), treated bone (femur, tibia, mandible), as well as chemical
composition, geometry and resorbability of the used bone enhancing
product (Cancedda et al., 2007). Small ruminants have also been
used as model for denstistry related research (Vlaminck et al., 2008),
as well as for cranio-facial (Chim & Gosain, 2007, Nolff et al., 2009),
spinal (Kobayashi et al., 2009) and joint research (Takahata et al.,
20
2005). In contrast, no clinical reports are available on the use of bone

enhancing materials in sheep and goats.
The use of bone grafts in cattle is also limited, mostly because of

pure economic considerations. Autogenous cancellous bone grafts
were successfully used for treatment of osteolytic defects in the
phalanges of cattle (Kasari et al., 1992). Septic physitis of the
metacarpal or metatarsal bones were treated in young animals using
homologous cancellous bone grafts (Barneveld, 1994).
Horses
Autogenous cancellous bone graft techniques have been

described in horses to enhance the treatment of primary fracture
repair (Henninger et al., 1991), delayed or non-union fractures
(Ducharme & Nixon, 1996), bone cysts (Kold & Hickman, 1983,
Jackson et al., 2000), osteomyelitis (Honnas et al., 1995), joint
arthrodesis (Lescun et al., 2004, Archer et al., 1988, Richardson et al.,
1987, Zubrod & Schneider, 2005, Bertone et al., 1989) and cervical
vertebral interbody fusion (DeBowes et al., 1984). Autogenous cortical
bone grafts have also been used occasionally in horses (Kirkerhead,
1996). Autologous cortico-cancellous rib grafts have been used for
correction of wry nose (Schumacher et al., 2008) and a cortico-
cancellous graft was used to fill a mandibular bone cyst after surgical
debridement (Jackman & Baxter, 1992).
Although rarely used in horses (Markel, 1996), a full cortical

allograft was successfully used to repair a metatarsal fracture in
addition to external coaptation in a foal (Cassotis et al., 1997).
Xenografts are also rarely applied in horses mainly because of
technical difficulties and costs (Kirkerhead, 1996), although successful
incorporation of bovine xenografts has been recorded in a cervical
spinal fusion procedure (DeBowes et al., 1984).
The use of tricalcium phosphate has been reported in circular

metacarpal/metatarsal defects in horses for the purpose of enhancing
21
bone healing (Rose et al., 1988). It was concluded that tricalcium

phosphate was effective as a synthetic bone-grafting material in
horses. However, no additional advantage was gained by the use of
tricalcium phosphate because control defects healed similarly as
healed defects implanted with autogenous cancellous bone combined
with tricalcium phosphate on a 50/50 weight basis.
It has been shown that MSC’s derived from sternal bone marrow
aspirates or subcutaneous adipose tissue from foals and horses show
potential for use in tissue engineering applications (Vidal et al., 2006,
Vidal et al., 2007). Yet, no clinical applications of bone marrow and/or
stem cells to enhance bone healing in horses have been described so
far. Controlled, well-designed studies of the basic biologic
characteristics and properties of these cells are needed to stimulate
this new equine research field. Stem cell research in the horse has
exciting perspectives that will most likely benefit the health of horses
(Koch et al., 2008, Van Haver et al., 2008).
Some experimental studies have evaluated the effect of bone

morphogenetic proteins on bone healing in horses. Injection of
rhBMP-2/calcium phosphate into surgically induced osteotomies and
ostectomies of the accessory metatarsal bones accelerated early
bone healing in an equine model (Perrier et al., 2008). Ishihara et al.
(2008) evaluated healing of equine metatarsal osteotomies and
ostectomies in response to percutaneous injection of adenoviral (Ad)
BMP-2, Ad-BMP-6, or beta-galactosidase protein vector control
administered 14 days after surgery. This study demonstrated a
greater relative potency of Ad-BMP-2 over Ad-BMP-6 in accelerating
osteotomy healing when administered in this regimen, although both
genes were effective at increasing bone at both osteotomy and
ostectomy sites (Ishihara et al., 2008). Adequate gene transfer may
be achieved by use of an adenovirus vector in equine cells. High
vector doses can be used in equine cells because of relative
resistance to cytotoxic effects in those cells. Greater permissiveness
22
and sustained expression of transgenes in BMSCs make them a

preferential cell target for gene therapy in horses (Ishihara et al.,
2006). Few clinical reports demonstrate the positive effect of rhBMP-
2 in horses to enhance bone healing. rhBMP-2 was used in a delayed
union of a comminuted first phalangeal fracture in an adult Hanover
mare (Kirkerhead, 1996), while nonglycosylated rhBMP-2 was applied
after arthrodesis in a subadult Warmblood with severe degenerative
joint disease of the pastern joint (Lippold et al., 2004).
Bone tissue engineering holds great promise for its therapeutic use
in horses, but so far no experimental or clinical studies have been
described in literature.
Electrostimulation has generally yielded unfavourable results in

horses thus requiring further evaluation (Bramlage et al., 1985, Collier
et al., 1985b, Collier et al., 1985a). On the other hand, the use of
shock-wave therapy in horses has roughly mirrored its use in humans.
Shock-wave therapy has proven to stimulate bone remodelling in
horses, especially in stress fractures (McClure & Merritt, 2003).
23
CURRENT RESEARCH FOCUSES
Several biomaterials for tissue engineering and regeneration are

supplemented by either cells or genes and are designed to improve
the complicated biological event of tissue repair. Ideally, the scaffold
should have the following characteristics (Hutmacher et al., 2001): be
highly porous with an interconnected pore network for cell growth and
flow transport of nutrients and metabolic waste; be biocompatible and
bioresorbable with controllable degradation and resorption rates to
match tissue replacement; have surface chemistry suitable for cell
attachment, proliferation and differentiation and finally have
mechanical properties to match those of the tissues at the site of
implantation (Thomson et al., 1995). The ideal “tissue engineered
bone substitute” has not yet been found so far. Researchers in
different fields including organic chemistry must continue to design
and fabricate a synthetic scaffold to transform the ultimate dream of a
“tissue-engineered bone substitute” into reality.
Two alternative routes of bone repair using biomaterials are

presently under investigation: tissue engineering by preformed
scaffolds and in situ scaffold formation using injectable materials
(Hench & Polak, 2002). The use of preformed scaffolds in bone tissue
engineering is based on in vitro seeding of the 3D scaffolds with
osteogenic cells. The cell-seeded constructs are cultured in
bioreactors and implanted afterwards into the place of injury. With the
growing popularity of non-invasive arthroscopic procedures and the
requirement to bridge large and irregular bone defects, injectable
materials that harden in situ are particularly promising for bone
regeneration. Several injectable materials have been investigated as
osteogenic bone substitutes, although none has delivered satisfying
results. Prior to injection, the material may be a solution, a paste,
micro- or nanoparticles, beads or thread-like material. They can be
24
CHAPTER 1: Current research
cell-free systems or cell and/or bioactive molecule suspension

systems (Hou et al., 2004).
As the ideal bone substitute has not yet been founded, the search
for the ideal treatment for bone defects is ongoing to optimise the
bone repair process. Especially the field of bone tissue engineering
receives a lot of attention.
25
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38
CHAPTER 2
Aims of the study
CHAPTER 2: Scientific aims
General aim : To evaluate potential bone substitutes and bone

enhancing products in an in vivo tibial goat model
Several ‘home-made’ in situ crosslinkable and biodegradable

polymers showed attractive characteristics to stimulate healing of
bone defects in standardized in vitro tests. The step from in vitro to in
vivo remains important in the validation of a potential bone substitute,
therefore the in vivo properties of those polymers will be investigated
in an in vivo goat tibial model.
Evaluation of bone substitutes
First, the biocompatibility and bone healing properties of the in situ

crosslinkable, biodegradable, methacrylate-endcapped porous bone
scaffold composed of D,L-lactide, ε-caprolactone and 1,6-hexanediol,
in which crosslinkage is achieved by photo-initiators, will be evaluated.
Secondly, different combinations of the methacrylate-endcapped

poly(D,L-lactide-co-ε-caprolactone) with BMSC seeded on gelatin
CultiSpher-S® microcarriers and α-tricalcium phosphate will be
applied and evaluated in the same goat model.
Finally, a chemically modified form of the Pluronic® F127 hydrogel

mixed with autologous BMSC seeded on gelatin CultiSpher-S®
microcarriers and hydroxyapatite tubular carriers will be evaluated.
Optimalization of evaluation techniques
During the experiments, several evaluation techniques will be

used. However, immunohistochemistry is not totally adapted to
evaluate bone healing on goat tissue embedded in a low-temperature
methacrylate resin. Also, micro tomographical analysis offers
promising perspectives to evaluate bone healing. Therefore, both
techniques will be optimised for their use on goat bony tissue
embedded in a low-temperature methacrylate resin.
41
CHAPTER 3
Evaluation of bone substitutes
3.1
Evaluation of an injectable,
photopolymerizable three-dimensional
scaffold based on D,L-lactide and ε-
caprolactone in a tibial goat model.
Adapted from: G. Vertenten, L. Vlaminck, T. Gorski, E. Schreurs, W. Van Den
Broeck, L. Duchateau, E. Schacht, F. Gasthuys (2008). Evaluation

of an injectable, photopolymerizable three-dimensional scaffold
based on D,L-lactide and ε-caprolactone in a tibial goat model.
Journal of Materials Science: Materials in Medicine: doi:

10.1007/s10856-008-3404-7
PART 3.1: Summary
SUMMARY
An in situ crosslinkable, biodegradable, methacrylate-

endcapped porous bone scaffold composed of D,L-lactide, -
caprolactone and 1,6-hexanediol, in which crosslinkage is
achieved by photo-initiators, was developed for bone tissue
regeneration. Three different polymer mixtures (pure polymer
and 30% bioactive glass or α-tricalcium phosphate added) were
tested in a uni-cortical tibial defect model in eight goats. The
polymers were randomly applicated in one of four (6.0 mm
diameter) defects leaving a fourth defect unfilled.
Biocompatibility and bone healing properties were evaluated by
serial radiographies, histology and histomorphometry. The pure
polymer clearly showed excellent biocompatibility and moderate
osteoconductive properties. The addition of α-TCP increased the
latter characteristics. This product offers potentials as a carrier
for bone healing promoter substances.
47
Evaluation of an injectable scaffold
INTRODUCTION
Large bone defects in man and animals are a challenge for

reconstructive surgery. Traditional techniques are based on the
transplantation of homologous bone tissue (Gross et al., 2002, Malloy
& Hilibrand, 2002, Paprosky & Martin, 2002, Slooff et al., 1996).
However, the supply of adequate bone is often limited and the
collection is painful with risk of haemorrhage, infection, nerve damage,
cosmetic disability and loss of function (Damien & Parsons, 1991).
Newer techniques involve the use of natural and synthetic bone

grafts. The main recognised bone substitute groups are: calcium
phosphates (bone-derived, synthetic ceramics, coralline
hydroxyapatites, hydroxyapatite-composites, tricalcium phosphates),
calcium carbonates (natural coral), calcium sulphate (plaster of Paris),
glass and glass-ceramics, polymers, metals, bone and bone-derived
materials (autograft, allograft, xenograft, demineralised bone matrix)
and osteoinductive growth factors (BMPs and TGFβ-family). Most of
the substitutes can be used for filler-reconstruction of moderate-sized
(1–4 cm of diameter) cystic lesions in skeleton, but only a few can be
used as a replacement for a weight bearing part of the skeleton. The
ultimate goal is to combine the strength of metals and polymers with
the osteoconductivity, or preferably osteoinductivity of other types of
materials resulting in an ideal bioactive composite implant with a
suitable hardness, strength and modulus corresponding to the
biomechanical properties of bone (Aho & Heikkila, 2005).
The increasing popularity of arthroscopic procedures in

orthopaedics and the requirement to bridge large and irregular bone
defects resulted in great interest in fixation materials that are
48
PART 3.1: Introduction
injectable, in situ forming and biodegradable. Several injectable

materials have been used as osteogenic bone substitutes. However,
none has gained universal acceptance. The most commonly used
injectable bone material polymethylmethacrylate is not biodegradable
and polymerizes with production of high temperatures. If the
polymerization reaction occurs outside the body, heat is not generated
during implantation but the polymer often does not fit completely in
irregular and large bone defects. Most composite polymers are not
biodegradable (Temenoff & Mikos, 2000). Injectable scaffolds
generally necessitate to solidify their constituent precursors or
macromonomers into a three-dimensional matrix. Typical solidification
mechanisms during the scaffold formation include: calcium phosphate
setting, thermally or photochemically activated radical polymerization
or crosslinking, chemical crosslinking, enzymatic crosslinking, thermal
gelation, ionic gelation, ionic crosslinking, Michael-type addition
reactions, and self-assembly mechanisms (Hou et al., 2004). In situ
formation of these scaffolds in the bone defect provides many
advantages over ex vivo preparation and production of a correctly
sized graft including improved contact between the scaffold and
surrounding tissue (Hou et al., 2004).
The development of injectable biodegradable orthopaedic

biomaterials which polymerize under controled conditions can provide
an alternative to current treatments for debilitating orthopaedic
conditions (Burdick & Anseth, 2002). This type of injectable material
should be able to polymerize in situ in a relatively short period of time
without negative effects on the surrounding tissue. It must be
biocompatible, promote formation of new bone tissue, have
appropriate viscosity before and efficient mechanical properties after
setting, and should be sterilizable (Temenoff & Mikos, 2000).
Recently, tissue engineering offered potential solutions for functional
and structural restoration of damaged or lost tissue. Tissue
engineering of bone requires a suitable osteoconductive and
49
osteoinductive matrix (Hench & Polak, 2002, Hutmacher, 2000, Rose

& Oreffo, 2002), and additional sources of osteogenic cells.
A new in situ crosslinkable, biodegradable, methacrylate-

endcapped bone scaffold composed of D,L-lactide, ε-caprolactone
and 1,6-hexanediol, in which crosslinkage is achieved by photo-
initiators, was recently developed for bone tissue regeneration. By
adding precise amounts of gelatine particles of selected size, a
scaffold can be obtained with controlled porosity, pore size and pore
connectivity. In addition, calcium phosphates, other osteoconductive
materials or demineralised bone can be added to promote
osteoconduction. The in vitro bioresorbable and osteoconductive
properties of this new polymer have been described (Declercq et al.,
2005).
The objective of this study was to investigate biocompatibility of

this bone substitute in a goat model and to evaluate its effect on bone
regeneration in a uni-cortical tibial defect study.
50
PART 3.1: Materials and Methods
MATERIALS AND METHODS
The study was approved by the Ethical Committee of the Faculty of

Veterinary Medicine of the University of Ghent (EC 2004/86).
Preparation of scaffolds
Three different scaffolds were used in the study. Composite no. 1

was purely composed of poly-(D,L-lactide-co-ε-caprolactone) with
15 wt% 2-hydroxyethyl methacrylate polyester (Declercq et al., 2005).
This basis was further mixed with 30% bioactive glass and 30% α-
tricalcium phosphate (α-TCP) in composite no. 2 and 3, respectively.
In order to create scaffolds with 70% porosity, an appropriate amount
of gelatine particles (size of 250–355 µm) was added in all three of
them.
All materials were sterilized by ethylene oxide (12 h, 37°C, 48 h

degassing) and mixed under sterile conditions immediately before
implantation.
Surgical procedure
Eight adult female goats with a mean age of 2.22 ± 0.55 years and
a mean body weight of 53.2 ± 4.9 kg were used. The goats were
housed in groups and had continuously access to food and water.
The goats were deprived of food for 48 h and received sodium

®
ceftiofur (Excenel , Pfizer Animal Health) (0.2 g IM) and flunixine
®
(Finadyne , Schering Plough Animal Health) (200 mg IM) 6 h before
®
surgery. After sedation with xylazine (Xyl-M , VMD) (0.2 mg/kg IM),
®
anaesthesia was induced with midazolam (Dormicum , Roche) and
®
ketamine (Anesketin , Eurovet NV) (respectively 0.011 mg/kg,
51
®
2.2 mg/kg IV) and maintained with isoflurane (IsoFlo , Abbott) in
oxygen using a routine monitoring protocol (ECG, pulsoximetry,
capnography, direct blood pressure and arterial blood gasses).
Ringer’s lactate solution (5 ml/kg/h) was administered during the
anaesthetic period.
The animals were placed in dorsal recumbency with both hindlegs

separately suspended. After surgical preparation, a 10 cm longitudinal
skin and periosteal incision was made midway and medial to each
tibia. The periosteum was elevated and four holes (6.0 mm diameter)
were drilled in the medial diaphyseal cortex of the tibia using a
®
trephine burr (3I , Implant Innovations). Sterile physiologic saline was
used for cooling during burring. The centre of the most proximal defect
was drilled at 2.25 cm proximal to the predetermined midpoint of the
tibia. Each additional hole was drilled 1.5 cm more distally using a
sterile plastic template. Hemostasis was provided by use of
epinephrine soaked gauzes pushed in the defects prior to application
of the bone substitute. In each leg, the three composites were
randomly assigned to a hole whereas the fourth hole was left empty to
serve as a control. Each composite was firstly placed on the borders
2
and bottom of the defect and photopolymerized for 40 s (500 mW/cm
TM
blue light, 3M Unitek Visible Light Curing Unit ). After setting, the
remaining defect was further filled with a second layer of additional
composite that was also photopolymerized prior to wound closure,
The surgical incision was closed in three layers using continuous
suture patterns and resorbable sutures. Postoperatively, the animals
received sodium ceftiofur for 7 days (0.2 g IM) and flunixine
meglumine for 3 days (200 mg IM). Two goats were euthanized
4 weeks and two other goats 8 weeks after surgery. One goat was
further euthanized at 12, 18, 24 and 36 weeks after surgery.
52
Clinical and radiographic follow-up
During the study period, goats were daily evaluated for healing of
the surgical site and development of complications related to the
surgical intervention.
Immediately following surgery, cranio-caudal and latero-medial

radiographic projections of each tibia were taken. Both digital as well
as conventional radiographs were obtained. At 4 weeks intervals,
bone healing was further radiographically evaluated up to 18 weeks
postoperatively. A final radiographic evaluation was done in one goat
at 24 and at 36 weeks after surgery in another animal.
The conventional radiographs were blindly evaluated for defect

density, periosteal reaction and soft tissue reaction using the criteria
of Dorea et al. (2005) by two investigators (Table 1).
Digital radiographs were used to measure grey scale densities at

the level of the bone defects (Image J 1.34s).
Histological evaluation
After euthanasia, all soft tissue surrounding both tibias was

removed. The tibial bones were split longitudinaly and the bone
marrow was removed. Each defect site was separately isolated and
fixed in formol 10% for 12 h. The samples were rinced with tap water
and dehydrated at 4°C using an ethanol gradient (48 h in 50, 75 and
96% and 72 h in 100% ethanol). Afterwards samples were defatted in
xylene for 48 h at 4°C and embedded in destabilised Technovit
®
9100 New (Heraeus Kulzer) (polymerization for 24 h at 0°C). Four
µm sections were cut with a microtome (SM2500, Leica
Microsystems), stretched with 70% ethanol on a slide and dried for
12 h at 60°C.
The sections were stained with haematoxylin & eosin, Von Kossa
and Toluidineblue stain. All samples were blindly evaluated under the
53
microscope by the same investigator. They were evaluated for the

tissue type, presence of residual graft material within the defect, the
quality of bone healing and the presence of inflammatory reactions.
Histomorphometric analysis (AnalySIS) was performed on the Von

Kossa stained sections obtained until 12 weeks after surgery using a
4× magnification (Olympus BX61 microscope). The volume of Von
Kossa positive (black-brown), Von Kossa negative (violet) and
colourless stained material were measured and expressed as a
percentage of the total defect area.
Table 1. Scores for defect density, periosteal reaction and callus formation
and soft tissue reaction (Dorea et al., 2005).
Density Size
0 radiolucent 0 none
-1 more radiolucent 1 small
1 radiopaque 2 moderate
2 mildly increased radiopacity 3 abundant
3 moderately increased radiopacity 4 exaggerated
4 extensively increased radiopacity
Distribution Soft tissue

0 none 0 no soft tissue reaction
1 regular and in the defect site 1 moderate soft tissue
reaction
2 irregular but in the defects site 2 severe soft tissue reaction
3 regular but out of the defect site
4 irregular and out of the defect site
Density = density of the defect; Size = periosteal reaction and callus formation
around each defect graded by size; Distribution = periosteal reaction and callus
formation around each defect graded by distribution; Soft tissue = soft tissue
reaction around the defects.
54
Statistical analysis
The conventional radiograph scores (average of the two

investigators) were compared between the three composites and the
control defect by the Friedman test with tibia and time as block factor
at the 5% global significance level. The four composites were pairwise
compared by the stratified Wilcoxon rank sum test using Bonferroni’s
multiple comparisons adjustment technique.
The digital radiograph density assessments were analysed by a

mixed model with tibia as random effect and composite, time, position
and the interaction between composite and time as categorical fixed
effects at the 5% global significance level.
Histological bone healing assessments were analysed by a mixed

model with tibia as random effect and composite, time and the
interaction between composite and time as categorical fixed effects at
the 5% global significance level.
If a test of significance gives a P-value (Probability value) lower

than the significance level, the results of this test are informally
referred to as 'statistically significant'
Pairwise comparisons in the mixed model were based on

Bonferroni’s multiple comparisons adjustment technique.
55
RESULTS
Profuse bleeding was encountered during the creation of 25 uni-

cortical bone defects which could be stopped by epinephrine soaked
gauzes providing an acceptable hemostasis. All of the graft materials
were easily implanted into the tibial defects and were considered
stable prior to wound closure. None of the goats showed signs of pain
or lameness during the study period. Except for discrete
subcutaneous fluid accumulation in five animals at 4 weeks after
surgery no other clinically visible adverse tissue reactions were
observed.
Conventional radiographs
A significant difference between the composites was found for the

density of each defect over time (P < 0.0001), with significant pairwise
comparisons between the control and composite no. 1 (P = 0.0002)
and between composite no. 1 and no. 3 (P = 0.0008) (Fig. 1). The size
of the periostal reaction and callus formation around each defect
(P = 0.0018) differed significantly between the composites, with
significant pairwise comparisons between the control and composite
no. 1 (P = 0.0007) and between composite no. 1 and no. 3
(P = 0.0046) (Fig. 2). No significant differences were found between
the composites with respect to the distribution of the periosteal
reaction and callus formation (P = 0.17) and the soft tissue reaction
(P = 0.179) (Figs. 3 and 4).
56
PART 3.1: Results
4
3,5
3
Mean density
2,5
2
1,5 Control
Composite1
1
Composite2
0,5 Composite3
0
0 4 8 12 18 24 32
Time post surgery (weeks)
Figure 1. Evolution of the mean densities of unicortical tibial defects treated
with different composites based on serial conventional radiographic evaluation
in eight goats.
3,5
Mean periosteal reaction and
Control
3 Composite1
Composite2
callus formation
2,5 Composite3
1,5
1
0,5
0
0 4 8 12 18 24 32
Figure 2. Evolution of the mean periosteal reaction and callus formation
around unicortical tibial defects treated with different composites based on
serial conventional radiographic evaluation in eight goats.
57
Mean distribution of periosteal 2,5
2
reaction
1,5
1 Control
Composite1
0,5 Composite2
Composite3
0
0 4 8 12 18 24 32
Figure 3. Evolution of the mean distribution of periosteal reaction around tibial
defects treated with different composites based on serial conventional
radiographic evaluation in eight goats.
0,6
Control
Mean soft tissue reaction
Composite1
0,5
Composite2
Composite3
0,4
0,3
0,2
0,1
0
0 4 8 12 18 24 32
Figure 4. Evolution of the mean soft tissue reaction around tibial defects
treated with different composites based on serial conventional radiographic
evaluation in eight goats.
58
PART 3.1: Results
Digital radiographs
The mean density of the control defect and the defects with
composites no. 1, no. 2 and no. 3 were respectively 79.5 ± 2.9,
76.0 ± 2.9, 77.6 ± 2.9 and 77.9 ± 2.9. No significant differences were
found between the composites (P = 0.64), nor was there a significant
interaction between composite and time (P = 0.93). Densities however
differed significantly between radiographic projections (P < 0.0001).
An overall mean density of 89.68 was calculated on latero-medial
projections compared to 65.83 on cranio-caudal projections.
New bone formation was most pronounced in the control defects

causing bridging of the defect by bone tissue as soon as 8 weeks after
surgery (Fig. 5). Independent of the type of composite used, the new
bone formation was most pronounced at the periosteal and endosteal
sides of the defects (Fig. 6) which specifically resulted in the
development of pronounced callus tissue on the periosteal side
(Fig. 7).
Figure 5. Photomicrograph of a
tibial control defect 8 weeks
postoperative in a goat. A bridge
of cancellous primary bone
covers more than 50% of the
original cortical defect. (1:
periost; 2: normal corticalis
surrounding defect; 3: medulla)
(Von Kossa).
59
Figure 6. Photomicrograph of a tibial defect treated with composite no. 3,

8 weeks postoperatively. The new bone formation is most pronounced at the
periosteal and endosteal side of the defect. There is a slight ingrowth of bone
at the periphery of the composite material (4) (1: periost; 2: normal cortex
surrounding the defect; 3: medulla) (Von Kossa).

8 weeks postoperatively. A big bony callus is visible at the periosteal side of
the defect. There is a slight ingrowth of bone at the periphery of the composite
material (4) (1: periost; 2: normal cortex surrounding the defect; 3: medulla)
(Von Kossa).
60
PART 3.1: Results
All composite treated defects were filled with a mixture of

composite and both fibrous and bone tissue. Fibrous tissue was
localised in the periphery and in the centre of the different composite
materials. No bone precursors such as cartilage were observed.
There was a slight ingrowth of bone at the periphery of the composite
materials visible (Figs. 6 and 7). The quantity of composite material
gradually decreased over time to completely disappear no sooner
than 32 weeks after surgery (Fig. 8). None of the defects showed
signs of inflammatory or immunologic reactions.

32 weeks postoperatively. Almost no composite left anymore. (1: periost; 2:
normal cortex surrounding the defect; 3: medulla) (Von Kossa).
61
Histomorphometry (Fig. 9)
The percentages of Von Kossa positive staining were significantly

(P = 0.0016) influenced by the type of material used. Control defects
contained significantly more Von Kossa positive material (mean
31.1% ± 4.4) compared to composites no. 1 (P = 0.0012) and 2
(P = 0.0211) (mean values of 13.4% ± 4.4 and 18.2% ± 4.4,
respectively). No differences were found between control defects and
defects containing composite no. 3 (23.9% ± 4.4, P = 0.3096), nor
between the three composites.
70
Control
60 Composite 1
Mean percentage
50 Composite 2
40 Composite 3
30
20
10
0
Von Kossa Von Kossa Polymer and
positive negative empty space
Figure 9. Mean percentage (+standard deviation) of each tibial defect for Von
Kossa positive, Von Kossa negative and empty space (only for goats that
lived no longer then 12 weeks post-surgery).
The percentages of Von Kossa negative staining did not differ

between the defects (P = 0.366). The mean percentages of control
defects and defects filled with composites no. 1, 2 and 3 were
49.5% ± 4.9, 48.3% ± 5.0, 55.1% ± 5.0 and 48.7% ± 5.0, respectively.
Significant differences were found between the four groups

(P = 0.0036) when evaluating the percentages of the colourless
material. Empty defects (19.44% ± 3.4) contained significantly
62
PART 3.1: Results
(P = 0.002) less colourless material compared to defects filled with

composite no. 1 (37.6% ± 3.5). The other composites did not differ
significantly (mean of composite no. 2: 25.99% ± 3.5; mean of
composite no. 3: 26.82% ± 3.5).
63
DISCUSSION
A new in situ crosslinkable, biodegradable, methacrylate-

endcapped bone scaffold composed of D,L-lactide, ε-caprolactone
and 1,6-hexanediol was recently developed for bone tissue
regeneration. Solidification is done by photopolymerization, a
commonly used technique in dental restorative procedures (Tarle et
al., 1998). Photopolymerization has several advantages over
conventional polymerization techniques including spatial and temporal
control over polymerization, fast curing rates (less than a second to a
few minutes) at room or physiological temperature, and minimal heat
production (Nguyen & West, 2002). Gelatine particles were included in
the polymer to create porous scaffolds facilitating tissue ingrowth and
vascularization. These characteristics have been demonstrated by
Declercq et al. (2005) who compared the osteoconductivity of
scaffolds with different apparent porosities (50, 60 and 70) and
different porosigens (gelatine, sodium chloride, and sugar) by
scanning electron microscopy and histological analysis. The same
authors further proved the bioresorbable and osteoconductive
properties of the polymer in an in vitro setting using osteoblast cell
cultures. In the present study these same properties were tested in an
in vivo setting using the pure scaffold and combinations with bone
substitutes (α-TCP and bioactive glass) that have proven their
influence on bone healing in other studies (Fleming et al., 2000,
Virolainen et al., 1997).
Goats were used in the present study because of their easiness in

handling and housing and their frequent use as experimental animals
in orthopaedic and bone substitute research (Buma et al., 2004, Dai et
al., 2005, Li et al., 2006, Meinig, 2002, van der Donk et al., 2001, Xu
et al., 2005). The use of a critical sized defect model, as has been
described in small laboratory animals (Frame, 1980, Hollinger &
64
PART 3.1: Discussion
Kleinschmidt, 1990, Hopp et al., 1989, Lewandrowski et al., 2000,

Liljensten et al., 2000, Saadeh et al., 2001, Shermak et al., 2000, Stal
et al., 2001, Zegzula et al., 1997) as well as sheep (Kirker-Head et al.,
1998, Lewandrowski et al., 2001a, Lewandrowski et al., 2001b,
Liebschner, 2004, Shang et al., 2001), was considered too invasive in
the current stadium of the ongoing research. The use of uni-cortical
cylindrical defects has been reported for experimental work on bone
substitutes and bone healing (Dalkyz et al., 2000, Dorea et al., 2005,
Griffon et al., 1996, MacNeill et al., 1999, Melo et al., 2005). The
medial diaphyseal cortex of the tibia was preferred for creation of
cortical defects because it can be easily approached for surgical
intervention with minimal dissection. Although none of the animals in
the present study developed important postoperative complications
related to the surgical intervention, specific care should be taken to
prevent excessive weight-bearing and possible tibial fracture as has
been observed in comparable experiments using sheep (Vertenten,
personal communication).
During the surgical interventions profuse bleeding from the bone

marrow hindered the static positioning of the bone substitutes before
setting as this experimental substance did not stick to bone in humid
environments. This problem was easily overcome by using
epinephrine soaked gauzes to provide hemostasis. To avoid
inadvertent application of the composite in the medullary cavity,
photopolymerisation was performed in two steps. This further assured
complete polymerisation of the entire volume of applicated bone
substitute.
Bone healing is routinely analysed by histomorphological,

histometrical and immunohistochemical techniques as means of
assessing the differentiation status of bone deposition and growth.
Currently, few embedding resins exist for which both morphological
and immunohistochemical analyses can be performed on mineralised
tissue. Paraffin, the standard embedding medium for bone enzyme
65
and immunohistochemistry is only suitable for use with demineralised

tissue that often shows badly preserved cancellous structure (Yang et
al., 2003). Methyl methacrylate (MMA) which is the first choice
embedding resin for histological examination of undecalcified bone
precludes immunohistochemal analysis because of its exothermic
polymerisation reaction destroying both enzyme activity and tissue
®
antigenicity. Technovit 9100 New is a low temperature MMA
embedding resin that has been reported to significantly improve tissue
antigenicity preservation as it allows polymerisation at −20°C (Yang et
al., 2003). Therefore, this resin was chosen to allow optimal
immunohistochemical analysis of the obtained bone samples in this
study. A supplementary difficulty of MMA embedded tissue is the
difficulty to make thin sections. Sections are usually cut between
50 µm and 500 µm (Chistolini et al., 1999, Gugala & Gogolewski,
1999, Jallot et al., 1999, Kessler et al., 2001) and sometimes grinded
to 10–40 µm (Blokhuis et al., 2000, Cunin et al., 2000, Lamghari et al.,
1999). With the SM2500 microtome undecalcified sections of 4 µm
could be made. Those thin sections were very fragile, so gentle
manipulation was needed. Nevertheless thin sections make it easier
to evaluate the tissues as there is less superposition of several tissue
layers. As grinding of the section is unnecessary, more sections can
be made and tissue can be evaluated at more levels and by more
different stainings.
Bone healing was evaluated by both conventional as well as digital

radiographs on fixed time intervals. The conventional radiographs
were interpreted blindly by two persons in a similar way as Dorea
et al. (2005) using categorical variables what increases the likelihood
of finding significant differences in contrast to the use of continual
variables as were produced in density measurements on digital
radiographies. The highest overall mean density was observed in the
control defect followed by composites no. 3, no. 2 and no. 1,
respectively. The observed differences in densities between the
different projections can be explained by the superposition of the
66
lateral cortex on the latero-medial projections causing a larger density

than on cranio-caudal projections.
The observed density differences between composites and the

more pronounced periosteal reaction of composite no. 3 based on
conventional radiographs suggest an advantage of adding α-TCP to
the polymer. The absence of distribution differences of the periosteal
reaction, callus formation and soft tissue reactions might indicate a
comparable immunologic reaction to the different bone substitutes.
The present study clearly illustrated the excellent biocompatibility

of the different polymer mixtures illustrated by the absence of
inflammatory signs on histological examination.
Von Kossa positive material corresponds with phosphate and

carbonate, the anions that bind calcium in tissues. On the bone
sections in the present study, this corresponded with mineralized bone
as well as the presence of bioactive glass and α-TCP particles. Von
Kossa negative material mainly represents the presence of fibrous
tissue whereas colourless zones indicated the presence of the pure
polymer as well as artifacts. No histomorphometric evaluation of bone
samples obtained after more than 12 weeks postoperatively was
performed as it was impossible to identify the original defects in these
cases because of pronounced periosteal reactions.
Histomorphometrical evaluation demonstrated slower bone healing
and remodelling in the presence of composites in comparison with
control defects. This was not a surprising result as tissue ingrowth and
gradual degradation of bone substitutes is more time consuming than
the rate of new bone formation in the absence of foreign material.
Polymer degradation was not optimal as was illustrated by the
formation of fibrous tissue in and around the composite material. The
polymer did not induce fibrous tissue formation as no differences in
terms of percentage were seen between control and treated defects.
The lack of new bone formation in the centre of the different
composite treated defects reflects a lack of osteoconductive
67
properties despite the porous architecture of the used polymers. This

problem might be improved by the addition of cellular bone precursors
or osteoconduction inducing substances.
68
PART 3.1: Conclusion
CONCLUSION
This study demonstrated the excellent biocompatible results of the

examined polymer poly(D,L-lactide-co-ε-caprolactone) + 15 wt% 2-
hydroxyethyl methacrylate but was unable to show osteoconductive
properties after application in non critical sized defects in goats tibias.
The addition of α-TCP had a positive influence on bone healing.
Further biochemical enhancement is needed to optimize porous
architecture and degradation properties. It can be concluded that this
material offers potentials as a carrier for other bone healing promoting
substances.
69
ACKNOWLEDGEMENTS
The authors thank Cindy De Baere, Bart De Pauw, Liliana

Standaert and Lobke De Bels for technical assistance.
70
PART 3.1: References
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74
3.2
Evaluation of an injectable,
photopolymerizable, and three-
dimensional scaffold based on
methacrylate-endcapped poly(D,L-
lactide-co-epsilon-caprolactone)
combined with autologous
mesenchymal stem cells in a goat tibial
unicortical defect model
Adapted from: G. Vertenten, E. Lippens, J. Gironès, T. Gorski, H. Declercq, J.

Saunders, W. Van den Broeck, K. Chiers, L. Duchateau, E.
Schacht, M. Cornelissen, F. Gasthuys, L. Vlaminck (2009).

Evaluation of an injectable, photopolymerizable, and three-
dimensional scaffold based on methacrylate-endcapped poly(D,L-
lactide-co-epsilon-caprolactone) combined with autologous

mesenchymal stem cells in a goat tibial unicortical defect model.
Tissue Engineering part A: doi: 10.1089/ten.tea.2008.0367
PART 3.2: Summary
SUMMARY
An in situ crosslinkable, biodegradable, methacrylate-

endcapped poly(D,L-lactide-co-ε-caprolactone) in which
crosslinkage is achieved by photo-initiators was developed for
bone tissue regeneration. Different combinations of the polymer
with bone marrow–derived mesenchymal stem cells (BMSCs) and
α-tricalciumphosphate (α-TCP) were tested in a unicortical tibial
defect model in eight goats. The polymers were randomly applied
in one of three defects (6.0 mm diameter) using a fourth unfilled
defect as control. Biocompatibility and bone-healing
characteristics were evaluated by serial radiographies, histology,
histomorphometry, and immunohistochemistry. The results
demonstrated cell survival and proliferation in the polymer-
substituted bone defects. The addition of α-TCP was associated
with less expansion and growth of the BMSCs than other
polymer composites.
77
Evaluation of an injectable scaffold with stem cells
INTRODUCTION
Large bone defects in humans and animals generally do not heal

spontaneously and require, in most cases, a specific surgical
intervention for the reconstruction of the defect. One of the
possibilities is the use of an autologous cancellous graft. The inherent
drawback of an autologous graft, however, is that the grafts have to
be harvested from another place in the body, resulting in donor-site
morbidity (Damien & Parsons, 1991). A possible alternative is the use
of allogenic bone collected from a donor of the same species. These
grafts were proven to have a lower osteogenic capacity, a higher
resorption rate, a larger immunogenic response, and less extensive
revascularization than autologous grafts. Further, there are justified
concerns about possible viral contamination of the graft material,
including transmission of live virus to the recipient (Kakaiya et al.,
1991).
Bone regeneration using tissue engineering techniques has

emerged as an alternative approach in the treatment of malfunctioning
or depleted bone. In this approach, a biomaterial scaffold can be
implanted in the bone defect. This scaffold serves as an adhesive
substrate for seeded cells and assures a sufficient physical support to
guide the formation of the new bone-related extracellular matrix. The
use of autologous cell sources has received widespread attention
because of the potential benefits in the tissue engineering process
(Bianco & Robey, 2001, Jiang et al., 2002, Murphy et al., 2002).
The design criteria for polymeric scaffolds for bone tissue support
include a high porosity, structural integrity, and degradability at a rate
commensurate with the production of new extracellular matrix by cells
seeded on the scaffold. A highly porous scaffold is desirable to allow
uniform cell migration throughout the material and to optimize
transport to and from implanted cells. Pore size and interconnectivity
78
play a major role in tissue ingrowth, creating an internal surface area

available for cell attachment, spreading, and expansion. The
mechanical properties of the scaffold are also of major importance,
especially with respect to hard tissues such as bone, to transmit
mechanical force and manage mineralization requirements (Thomson
et al., 2000).
We previously reported that the in situ crosslinkable methacrylate-

endcapped porous bone scaffold composed of D,L-lactide, ε-
caprolactone and 1,6-hexanediol could be used as a substrate for
bone tissue engineering (Vertenten et al., 2008b). This polymer
showed excellent biocompatibility and moderate osteoconductive
properties in vivo. The addition of α-tricalcium phosphate (α-TCP)
increased the latter characteristics (Vertenten et al., 2008b). In other
parallel experimental work, mouse embryonic stem cells were
cultivated on commercially available biodegradable macroporous
microcarriers, and osteogenic differentiation was initiated in an
adapted medium for a period of 2 weeks. Encapsulation of the cell-
loaded microcarriers in the experimental scaffold, using either triacetin
or hydroxyethylmethacrylate (HEMA), as solvent and with or without
gelatine, as porogen, resulted in a homogeneous distribution of the
microcarriers in the polymer. However, viability of the cells was
estimated by transmission electronic microscopy to be optimal when
gelatine was omitted, and triacetin was added instead of HEMA
(Tielens et al., 2007).
The objective of the present study was to evaluate the effect of

bone marrow derived mesenchymal stem cells loaded on
microcarriers in combination with the crosslinkable methacrylate-
endcapped porous polymer scaffold including α-TCP on bone
regeneration in a unicortical tibial defect experimental design in a goat
model. We firstly hypothesized that the implanted cells would easily
survive and proliferate, and would demonstrate bone-forming
characteristics inside the polymer scaffold. We secondly anticipated
79
an improvement in bone healing characteristics of the treated cortical

defects compared to earlier reported results concerning a comparable
experiment without the use of stem cells (Vertenten et al., 2008b).
80
The study was approved by the Ethics Committee of the Faculty of

Veterinary Medicine, Ghent University (EC 2006/097). Eight adult
female goats with a mean age of 46.3 ± 22.9 months and a mean
body weight of 54.1 ± 7.5kg were used. The goats were housed in
2
groups (groups of four in boxes of 16m ) and had free access to food
and water. All surgical procedures were performed under general
anesthesia respecting all aspects of the well-being of the animals
(including starvation before surgery, as well as antibiotics and
analgesics before, during, and after surgery) (Vertenten et al., 2008b).
Isolation and culture of bone marrow–derived cells
Bone marrow (BM) was aspirated by a Jamshidi needle (11 gauge,

10 cm; DMS, Hampshire, United Kingdom) under sterile conditions
from the iliac crest of the anesthetized goats. The BM was collected in
a 10 mL lithium heparin tube (Venoject, Leuven, Belgium) in which 1
mL of ACD-A anticoagulant (Anticoagulant Citrate Dextrose Solution
Formula A; Baxter, Brussels, Belgium) was added. The needle was
abundantly flushed with ACD-A anticoagulant and lithium heparin
before the aspiration of the BM. After removal of blood clots, the BM
was thoroughly washed with Minimum Essential Medium (MEM)-α
medium containing 10 vol% fetal bovine serum (FBS), 0.5 vol%
penicillin–streptomycin, and 1 vol% Funigizone Amphotericin B
(Invitrogen, Merelbeke, Belgium), and centrifuged (10 min, 1000 rpm).
The cell pellet was resuspended in osteogenic differentiation medium,
that is, MEM-α medium supplemented with 10 vol% FBS, 0.5 vol%
penicillin–streptomycin, 1 vol% Fungizone Amphotericin B, 100 µM L-
ascorbic acid 2-phosphate (Sigma-Aldrich NV/SA, Bornem, Belgium),
and 10 nM dexamethasone (Sigma-Aldrich NV/SA), and seeded in
81
five T75 tissue culture dishes. The culture dishes were placed in a
humidified incubator (37°C, 5% CO2/95% air), and the medium was
renewed after 2 days, leading to the removal of the nonadherent cells
(mainly hematopoietic cells). Stromal cells were kept in culture for 2
weeks (renewal of the medium twice a week) until they reached
confluence. Afterward trypsinized, the cells were brought in
suspension, stained with trypan blue, and counted in a Bürcker cell
chamber. CultiSpher-S microcarriers (diameter 130–380 µm) (Percell
Biolytica AB, Åstorp, Sweden) were prepared and sterilized according
to the manufacturer's instruction. About 0.09 g hydrated carriers were
divided over 5 wells of a 12-well suspension plate in osteogenic
medium. Approximately 400,000 cells were added to each well,
depending on the amount of harvested cells. Cells were allowed to
adhere under static conditions for 48 h. Afterward, the cell–carrier
constructs were carefully transferred to a dynamic culture system
(stirring speed 55 rpm) as has been described by Declercq et al.
(2005). The constructs were cultured for an additional 2–3 weeks in
osteogenic differentiation medium, with the addition of β-
glycerophosphate after 1-week colonization at 37°C (5% CO2). Before
implantation, cell colonization on the carriers was evaluated by 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazoliumsalt (MTS) analysis (100 µL MTS solution added to 100
µL CultiSpher carriers in 500 µL phenol-red–free medium). After 4-h
incubation in the dark at 37°C, the reduction of the tetrazolium salt into
formazan in living cells was determined spectrophotometrically by
measuring the absorbance of the coloured formazan at a wavelength
of 480 nm. The cell-loaded constructs were mixed with the polymer
and implanted in the tibial defects.
Preparation of scaffolds
The synthesis and characterization of the methacrylate-endcapped

polymers (D,L-lactide-co-caprolactone) have been described in depth
82
earlier (Declercq et al., 2005, Tielens et al., 2007, Vertenten et al.,

2008b). Composition of the scaffolds was selected based on the
results previously reported (Declercq et al., 2005, Tielens et al., 2007).
No extra porogen was included in the composition of the scaffolds.
Preceding experiments showed that porosity could be induced by the
leaching of the plastizer and as a result of the degradation of the
polyester and the CultiSpher microcarrier particles. Further, during the
mixing of the viscous crosslinkable polyester and the cell-seeded
microcarriers, air bubbles were trapped and contributed as
macroporous pockets. In vitro experiments with cells grown on
microcarriers and immobilized in the crosslinkable polyester proved to
maintain an acceptable viability.
Three different preparations of the scaffold were used in the

present study. Composite no. 1 consisted of pure methacrylate-
endcapped polymer with a triacetin solution containing the photo-
initiators as plasticizers. In composite no. 2, this mixture was
supplemented with microcarriers loaded with autologous bone marrow
derived mesenchymal stem cells differentiated in the osteogenic
lineage. In composite no. 3, 30% w/w α-TCP was further added to the
polymer–triacetin–microcarrier mixture.
All polymers were sterilized by ethylene oxide for 12 h at 37°C and

subsequently aerated for 48 h. The cell-loaded CultiSpher-S carriers
were thoroughly rinsed with physiological solution and shortly
dehydrated by pipetting the constructs on filter paper. Two hundred
and fifty microliters of these carriers were added to the
catalyst/triacetin/polymer/(α-TCP) paste and carefully mixed.
Surgical procedure
The surgery was performed as described earlier (Vertenten et al.,

2008b). Briefly, four noncritical sized defects (6.0 mm diameter) were
drilled in the medial diaphyseal cortex of each tibia using a trephine
83
burr (3I; Implant Innovations, Copenhagen, Denmark). In each leg, the

three composites were randomly assigned to a hole, whereas the
fourth hole was left empty to serve as a control. Each composite was
first placed on the borders and bottom of the defect, and
2
photopolymerized for 40 s (500 mW/cm blue light, Visible Light
Curing Unit™; 3M Unitek, Diegem, Belgium). Afterward, the remaining
defect was further filled with a second layer of additional polymer
before the standard wound closure.
During the study period, goats were daily evaluated for healing of
the surgical site and development of complications related to the
surgical intervention.
At, respectively, 2, 4, 8, and 12 weeks after surgery, two goats

were euthanized and tibial defects were harvested for histological and
immunohistochemical analysis of bone healing.
During the complete study period, goats were daily clinically

evaluated for healing of the surgical site and development of possible
complications related to the surgical intervention.
Immediately after surgery, standardized craniocaudal and

mediolateral digital radiographs (Digivex 40 15 kW high-frequency
generator; Medex Loncin S.A., Loncin, Belgium) of each tibia were
taken. At 2 weeks' interval, healing of the tibial defects was
radiographically evaluated in each goat until the time of euthanasia,
when further histological and immunohistochemical analysis of the
bone defects was performed.
All radiographs were blindly evaluated for defect radiographic

density, periosteal reaction, and soft tissue reaction using the criteria
described by Dorea et al. (2005). Gray-scale densities at the level of
the bone defects were further objectively evaluated using a standard
84
imaging software packet (Image J 1.34S; National Institutes of Health,

Bethesda, Maryland).
Histological and immunohistochemical analysis
After euthanasia, the different bone defect sites were harvested

from the tibias. Because it was one of the aims to perform a von
Kossa staining on the histological sections, undemineralized samples
were embedded in destabilized Technovit 9100 New (Heraeus Kulzer,
Wehrheim, Germany) as described earlier (Vertenten et al., 2008b).
Four-micrometer sections were cut with a heavy duty microtome
(SM2500; Leica Microsystems, Wetzlar, Germany), stretched with
70% ethanol on a slide, and dried for 12 h at 60°C.
The sections were stained with hematoxylin and eosin (H&E), von
Kossa, and Toluidine blue stain. All samples were qualitatively
evaluated using a standard light microscope by the same investigator
blinded to treatment. Evaluation criteria included the tissue type,
presence of residual graft material within the defect, the quality of
bone healing, and the presence of inflammatory reaction.
Histomorphometric analysis was performed with a 2× magnification

(Olympus BX61 Microscope; Olympus Soft Imaging Solutions GmbH,
Münster, Germany) on the von Kossa–stained sections using a
specific software program (Analysis 5.0; Olympus Soft Imaging
Solutions GMbH). The surface area of von Kossa–positive (black-
brown colouration representing calcified tissue), von Kossa–negative
(violet colouration representing connective tissue), and colourless
(representing polymer and artifacts) stained materials were measured
and expressed as a percentage of the total defect area.
All sections were stained immunohistochemically for cluster of

differentiation 3 (CD3) (lymphocytes), MAC387 (macrophages),
vimentin (mesenchymal tissue), and osteocalcin (Vertenten et al.,
2008a), and evaluated for the presence and localization of positive
85
staining. Before immunohistochemical staining, sections were

deacrylized. Antigen retrieval was performed using proteinase K
(Dako, Heverlee, Belgium). The sections were rinsed with distilled
water and phosphate-buffered saline (PBS). Endogenous peroxidase
was quenched by incubating the sections in a 3% hydrogen peroxide
solution in methanol. This was followed by rinsing in distilled water
and PBS. The sections were preincubated in bovine serum albumin
and rinsed with PBS. Consequently, immunohistochemical staining
was performed using the primary antibody. The sections were rinsed
with PBS and incubated with biotinylated goat anti-mouse for the
monoclonal primary antibodies and biotinylated goat anti-rabbit for the
polyclonal antibodies. Afterward, the sections were rinsed and
incubated with avidin-biotin complex-horseradish peroxidase (Dako).
The samples were rinsed with PBS and incubated in 3,3′-
diaminobenzedine (Sigma, Bornem, Belgium) and finally rinsed with
distilled water. Counterstaining was done by immersing the sections in
hematoxylin, tap water, and distilled water. Finally, the sections were
dehydrated and covered. The described protocol was used for all
immunostainings except for CD3, where the PBS was changed for
tris-buffered saline (Vertenten et al., 2008a).
Relationships between the age of the goats, and the volume of BM

and retrieved cells after culturing were studied by the Pearson's
correlation coefficient.
The radiographic scores for defect radiographic density, periosteal

reaction, and soft tissue reaction were compared between the three
composites and the control defect by the Friedman test with tibia and
time as block factor at the 5% global significance level. The three
composites and the control defect were pair-wise compared by the
stratified Wilcoxon rank sum test using Bonferroni's multiple
comparisons adjustment technique. The digital radiographic density
86
assessments were analyzed by a mixed model with tibia as random

effect and composite, time, position, and the interaction between
composite and time as categorical fixed effects at the 5% global
significance level.
Histological bone healing assessments were analyzed by a mixed

model with tibia as random effect and composite, time, and the
interaction between composite and time as categorical fixed effects at
the 5% global significance level.
Pairwise comparisons in the mixed model were based on

Bonferroni's multiple comparisons adjustment technique.
87
RESULTS
All of the graft materials were easily implanted into the tibial
defects and were considered to be stable before wound closure. None
of the goats expressed signs of pain or lameness during the study
period. Except for transient, discrete subcutaneous fluid accumulation
at the surgical incision in the left tibia of three animals 2–4 weeks after
surgery, no other clinically visible adverse reactions were observed.
These minor seromas were successfully treated in all cases by sterile
puncture and pressure bandages for several days.
Isolation and culture of BM-derived cells
The quantitative values of each BM sample and their

characteristics after culturing and seeding on microcarriers are
represented in Table 1. A mean volume of 11.5 ± 5.1 mL BM could be
6
aspirated. A mean amount of 4.07 ± 2.4 × 10 BM-derived
mesenchymal stem cells (BMSCs) was retrieved after a culture period
6
of 14 days. On average 1.70 ± 0.3 × 10 cells were loaded on 0.09 g
microcarriers for a mean period of 20.0 ± 3.7 days before implantation.
The result of the BM punctures differed largely between the goats.
There was no significant correlation between the age of the goats and
the volume of retrieved BM (Pearson's correlation coefficient, 0.198; P
= 0.639). No significant correlation could be demonstrated between
the volume of BM and the number of cells cultivated after 14 days
(Pearson's correlation coefficient, −0.093; P = 0.827) or between the
age of the goats and the amount of cells after 14 days of culture
(Pearson's correlation coefficient, −0.099; P = 0.816).
88
PART 3.2: Results
Table 1. Quantitative values of the bone marrow harvested from the iliac
arch in eight goats.
Goat Goat Goat Goat Goat Goat Goat Goat
1 2 3 4 5 6 7 8
Age (months) 34 91 32 35 35 21 60 62
Vol BM (mL) 6 11 8 9 8 17 21 12
# Cells 14 d 8.28 3.66 2.00 × 2.75 1.48 6.00 2.36 × 6.00
6 6 6 6 6 6 6 6
culture × 10 × 10 10 × 10 × 10 × 10 10 × 10
# Cells on 6.00 3.66 2.00 × 2.75 1.48 6.00 2.36 × 6.00
6 6 6 6 6 6 6 6
microcarriers × 10 × 10 10 × 10 × 10 × 10 10 × 10
# Carriers (g) 0.27 0.18 0.126 0.18 0.09 0.27 0.144 0.27
# Cells/0.09 g 2.00 1.83 1.42 × 1.38 1.48 2.00 1.48 × 2.00
6 6 6 6 6 6 6 6
carrier × 10 × 10 10 × 10 × 10 × 10 10 × 10
Period on 17 19 25 25 22 16 16 20
carriers (days)
Age (months): age of each goat in months at the day of bone marrow
puncture. Vol BM (in mL): volume bone marrow after removal of potential
blood clots. # Cells 14 d culture: total amount of cells after 14 days of
culture. # Cells on microcarriers: quantity of cells used for seeding on
microcarriers. # Carriers (in g): quantity of carriers. # Cells/0.09 g carrier:
quantity of cells/0.09 g carrier. Period on carriers (in days): period that
cells were seeded on carriers in osteogenic differentiation medium before
surgical implantation.
Radiographic follow-up
A significant difference between the composite-treated and the

control defects was found for the distribution of the periosteal reaction
and callus formation over time (P = 0.0218), without significant pair-
wise comparisons (Fig. 1). The size of the periosteal reaction and
callus formation around each defect differed significantly between the
composites (P = 0.0215), with significant pair-wise comparisons
between composite nos. 1 and 2 (P = 0.004). Composite no. 2 scored
significantly higher than composite no. 1 concerning the size of the
periosteal reaction and callus formation around each defect (Fig. 1).
No significant differences were found with respect to the radiographic
density of each defect over time and the soft tissue reaction (Fig. 1).
This was in accordance with digitally measured gray-scale levels that
revealed no differences between the defects (P = 0.81) and between
89
the time periods (P = 0.39). The mean calculated gray-scale values for
the complete study period of the control defect and the defects
containing composite nos. 1, 2, and 3 were 73.8 ± 2.3, 75.8 ± 2.1,
74.4 ± 2.2, and 77.7 ± 2.2, respectively.
Figure 1. Box plots representing the differences in assessments for

radiographic density (dens), distribution of the periosteal reaction and callus
formation (dis), size of the periosteal reaction and callus formation (peri), and
the soft tissue reaction (soft) between the several defects on the
radiographies. X-axis: differences between defects with 0 indicating control
defect, and 1, 2, and 3 indicating composite nos. 1, 2, and 3, respectively. Y-
axis: result of differences.
New bone formation was mostly pronounced in the control defects,

originating at the intact cortex surrounding the defects and causing
complete healing of the defect from 8 weeks after surgery (Fig. 2A).
Control defects were filled with mesenchymal tissue at the center until
complete bridging by bone tissue was achieved. Complete healing
was not observed in the defects filled with the different composites
during the study period. At 8 and 12 weeks postsurgery, composite-
90
PART 3.2: Results
filled defects were characterized by the presence of fibrous tissue

surrounding the centrally located polymer. New bone formation was
present in the periphery of the defects. Polymer degradation was
limited over time. Defects filled with composite nos. 2 and 3 were seen
to contain several cellular tissue islands (Fig. 2C, D) consisting of
microcarriers, connective tissue, and viable cells (Fig. 3 and 4).
However, the volume of tissue surrounding the carriers in composite
no. 3 was considered smaller than in polymer no. 2. Further, several
nuclei incorporated into composite no. 3 appeared pyknotic. This
composite contained also diffusely spread granular material that
stained von Kossa positive and was subsequently identified as α-TCP
(Fig. 4 and 5). In 8 out of 64 tibial defects, clusters of high
concentrations of cells were visible around the composite material.
91
Figure 2. Photomicrograph of tibial defects 8 weeks post surgery in a goat

(magnification 2 x, Von Kossa staining). (A) The control defect is completely
filled with bone. (B) Defect filled with composite no. 1. (C) Defect filled with
composite no. 2: the composite is surrounded by a capsule of fibrous tissue.
New bone formation is present around the fibrous capsule; several centres of
tissue are present in the composite. (D) Defect filled with composite no. 3:
several centres of tissue are present in the composite; the composite is
surrounded by a capsule of fibrous tissue.
92
PART 3.2: Results
Figure 3. Photomicrograph of a tibial defect treated with composite no. 2, 8

weeks after surgery (magnification 20×, H&E staining). Several centers of
tissue consisting of microcarriers (1), colonized and overgrowth by
extracellular matrix, and viable cells (2) are present in the polymer (3).

weeks after surgery (magnification 20×, H&E staining). Several centers of
tissue consisting of microcarriers (1) colonized and overgrowth by
extracellular matrix and viable cells (2) are present in the polymer. The
polymer (3) contains diffusely spread granular material (4).
93

weeks after surgery (magnification 2×, von Kossa staining). Several centers of
tissue (1) are present in the composite. The composite is surrounded by a
capsule of fibrous tissue. Several dots of von Kossa–positive tissue are
present in the composite material (2), whereby newly formed cancellous bone
(3) and a mature tibial cortex (4) can be noticed.
Histomorphometry
The von Kossa–positive surface area was significantly influenced

by the type of material (P < 0.0001), time (P < 0.0001), and the
material–time interaction (P = 0.0208). Control defects contained
significantly more von Kossa–positive material (mean 26.1 ± 6.0%)
than composite nos. 1 (P = 0.0025), 2 (P = 0.0001), and 3 (P =
0.0015) (mean values of 10.9 ± 3.4%, 7.3 ± 2.2%, and 10.2 ± 2.4%,
respectively) (Fig. 6). No differences were found for this parameter
94
PART 3.2: Results
between the three composites. The percentage of von Kossa–positive

staining increased significantly over time (Fig. 7).
80
70
60
Mean percentage
50 Control
Composite 1
40
Composite 2
30 Composite 3
20
10
0
Von Kossa Von Kossa Polymer and
positive negative empty space
Figure 6. Mean percentage (+standard error of the mean) of each tibial

defect for von Kossa–positive, von Kossa–negative, and polymer and/or
empty space.
The percentages of von Kossa–negative surface areas were

significantly influenced by the type of material (P = 0.0093), time (P =
0.0031), and their interaction (P = 0.0116). Control defects (41.8 ±
7.0%) contained significantly (P = 0.035) more von Kossa–negative
material than defects filled with composite no. 1 (24.1 ± 4.5%). The
other composites did not differ significantly (mean of composite nos. 2
and 3: 29.9 ± 5.1 and 36.1 ± 4.5%, respectively) (Fig. 6). The
percentage of von Kossa–negative staining decreased significantly
over time (Fig. 8).
95
Figure 7. Mean percentage (±standard error of the mean) of von Kossa–

positive material of the tibial defects filled with different composites in eight
goats at 2, 4, 8, and 12 weeks postsurgery.
Figure 8. Mean percentage (±standard error of the mean) of von Kossa–

negative material of the tibial defects filled with different composites in eight
goats at 2, 4, 8, and 12 weeks postsurgery.
96
PART 3.2: Results
The percentages of surface area containing colourless material

were significantly (P < 0.0001) influenced by the type of material, but
not by time (P = 0.1479) and their interaction (P = 0.4501). Control
defects contained significantly less colourless material (mean 32.1 ±
5.2%) than composite nos. 1 (P = 0.0001), 2 (P = 0.0004), and 3 (P =
0.0146) (mean values of 64.9 ± 6.0%, 62.4 ± 5.7%, and 53.6 ± 5.1%,
respectively). No differences were found between the three
composites (Fig. 9).
Figure 9. Mean percentage (±standard error of the mean) of colourless

material of the tibial defects filled with different composites in eight goats at 2,
4, 8, and 12 weeks postsurgery.
97
Immunohistochemistry
The CD3 activity examined by immunohistochemical analysis was

negative in all samples except in three samples from one single goat
with a few CD3-positive staining cells diffusely spread around the
implanted composite. Bone tissue and granular material in defects
filled with composite no. 3 stained metachromatically.
Immunohistochemical staining for MAC387 (macrophages)

revealed no positive cells or tissue in the defects at any time. Bone
tissue and the granular material in composite no. 3 stained
metachromatically.
Intensive positive staining for vimentin (mesenchymal tissue) was

observed in the tissue concentrations in composite nos. 2 and 3, the
connective tissue surrounding the composites, and the fibrous tissue
inside the cortical bone tissue (Fig. 10 and 11). Bone tissue and the
granular material in composite no. 3 stained metachromatically.

which stained immunohistochemically for vimentin 12 weeks after surgery.
The capsule of tissue around the polymer and the tissue centers in the
polymer are positively stained (magnification ×10). 1, newly formed bone
(metachromatic staining); 2, capsule of tissue around composite (vimentin
positive); 3, microcarrier; 4, tissue and cells around the microcarrier (vimentin
positive); 5, polymer.
98
PART 3.2: Results

which stained immunohistochemically for vimentin 8 weeks after surgery. The
tissue between the newly formed metachromatic staining bone, the capsule of
tissue around the polymer, and the tissue centers in the polymer are positively
stained (magnification ×10). 1, microcarrier; 2, tissue and cells around the
microcarrier (vimentin positive); 3, capsule of tissue around composite
(vimentin positive); 4, newly formed bone (metachromatic staining); 5,
polymer; 6, granular material in polymer (metachromatic staining).
Immunohistochemical staining for osteocalcin revealed a slightly

positive staining of tissues in and around the microcarriers in
composite nos. 2 and 3, the connective tissue surrounding the
different composites and the tissue inside the newly formed cancellous
bone (Fig. 12 and 13).
99
Figure 12. Photomicrograph of a centre of tissue in tibial defect treated with

composite no. 2, which stained immunohistochemically for osteocalcin 12
weeks after surgery. There is a slightly positive staining of the tissue and the
nuclei are staining negative (magnification x 40). 1, microcarrier; 2, tissue and
cells around the microcarrier (slightly positive); 3: polymer.

which stained immunohistochemically for osteocalcin 8 weeks after surgery.
There is a slightly positive staining of the tissue around the microcarriers, the
capsule around the polymer, and the tissue between the newly formed
metachromatically stained cancellous bone. The nuclei are staining negative
(magnification ×20). 1, microcarrier; 2, tissue and cells around the microcarrier
(slightly osteocalcin positive); 3, polymer; 4, granular material in polymer
(metachromatic staining); 5, capsule of tissue around composite (slightly
osteocalcin positive); 6, newly formed bone (metachromatic staining).
100
DISCUSSION
In the present study, the biological behavior of porous bone

scaffolds prepared from methacrylate-endcapped poly(D,L-lactide-co--
caprolactone) mixed with BMSCs was studied in a tibial cortical defect
goat model. Critical sized defects were not used for this study to
facilitate the surgical procedures during these initial basic steps in the
study. They should, however, be a logical sequel for further
experiments. Although BMSCs survived and showed signs of new
bone formation as stated in the first part of the research hypothesis,
bone healing was still in its initial stages in the polymer composites at
the end of the study period compared to control defects where
complete healing was present.
Bone tissue engineering has been shown to be an effective

approach for bone regeneration, and BMSCs have proved to be a
major cell source during the bone engineering process. The
osteogenic potential of BMSCs has been demonstrated extensively
both in vitro and in vivo, and many studies have succeeded in
repairing bone defects using BMSCs in different animal models
(Arinzeh et al., 2003, Bruder et al., 1998, Dai et al., 2005, Kon et al.,
2000, Maniatopoulos et al., 1988, Muraglia et al., 1998, Petite et al.,
2000, Shang et al., 2001, Yuan et al., 2007, Zhu et al., 2006). Not only
can osteogenically induced BMSCs provide a source for new bone
formation, but the transformed cells can also secrete important growth
factors regulating further bone healing.
The present study found no age-related influence on the number of

cultured BMSCs derived from BM punctures in goats. Kotobuki et al.
demonstrated that BMSCs could even proliferate from aged marrow
cells (viability greater than 90%) (Kotobuki et al., 2004). This might
suggest that not only young but also geriatric patients might be treated
with autologous BMSCs or osteoblasts derived from BM. Bone
101
reconstruction procedures are frequently performed in the older

individuals, for example, in oral surgery, to allow stable placement of
dental implants in an augmented bone area (Wang & Boyapati, 2006).
Also bone reconstruction after tumor resection is challenging in the
elderly patient (Marx, 2004).
Photopolymerizing materials used for bone reconstruction have

promising characteristics: these products can be injected into a bone
defect in moldable form and solidified in situ by exposure to a specific
light source, maintaining the form and shape of the implant (Burdick et
al., 2003, Burdick et al., 2002, Grijpma et al., 2005, Ifkovits & Burdick,
2007, Sharma et al., 2007, Storey et al., 1993, Temenoff & Mikos,
2000). In the present study, photopolymerizable scaffolds based on
D,L-lactide and ε-caprolactone to encapsulate BMSC-loaded
microcarriers were used in an experimental model for bone
regeneration. Declercq et al. (2005) demonstrated in a previous in
vitro study that the encapsulated cells did not show pyknotic nuclei,
pointing out that the photopolymerization and handling of the viscous
polymer/microcarrier paste were not detrimental for the survival of the
cells. The present study confirmed these results in an in vivo setting
and demonstrated the implanted cells' ability to proliferate and
produce a calcified matrix (osteocalcin positive) inside the polymer as
cell proliferation and matrix production were absent in the polymer
composite without BMSCs.
Although radiographic evaluation did not reveal major differences

between the control defects and the three different composites, the
control defects had the tendency to become more dense 6 weeks
after surgery than defects filled with composites. However, the lowest
overall mean gray-scale values were observed in the control defect
followed by composite nos. 2, 1, and 3, respectively (not significantly
different). This finding is in contrast with those of a previous study
(Vertenten et al., 2008b) in which the controls showed the highest
radiographic density. Bone healing was evaluated radiographically
102
using a fixed time interval. Small but significant differences were found
only for the occurrence of the periosteal reactions and callus
formation, but not for radiographic density and soft tissue reactions.
This was also in contrast with the previous study, where more
radiographic findings were significantly present (Vertenten et al.,
2008b). A possible explanation for the differences between each study
might be that in the present study, the radiographic evaluation was
done over a 12-week period after surgery compared to a 32-week
follow-up in the previous study. Major differences between composites
were seen from 8-week postsurgery in both studies. Not surprisingly,
standard radiographic techniques had a low sensitivity to detect
significant changes in the early phases of bone healing.
Von Kossa–positive material is indicative for the presence of

phosphate and carbonate, the anions that bind calcium in tissues. On
the bone sections in the present study, the von Kossa–positive
material corresponded with the presence of either the mineralized
bone or the α-TCP particles that were included in composite no. 3
(Fig. 5). On the other hand, von Kossa–negative staining mainly refers
to the presence of pure fibrous tissue, whereas colourless zones are
indicative for the presence of the pure polymer or possible artefacts.
Overall, the empty control defects healed very fast. The results of
the present study demonstrated insufficient osteoconductive
properties of the used composites that were characterized by the
presence of fibrous tissue surrounding the implant material. This was
in contrast with the results of the previous study (Vertenten et al.,
2008b), where bone ingrowth at the periphery of the composite
material was seen even at early stages. A possible explanation for this
lack of bone ingrowth in the present study might be the lower porosity
of the polymer compared to the previous study because no porogen
was used in the present study as the addition of gelatine as a porogen
seemed to have a negative influence on in vitro cell viability (Tielens
et al., 2007). The present study identified several tissue clusters in the
103
composites originating from the seeded BMSCs as tissue clusters

were absent in the polymer composite without BMSCs. This
underlines the cells' potential to proliferate and further differentiate in
an osteogenic direction. Survival and proliferation of the cells are only
possible when the polymer has an adequate porosity. No extra
porogen was included in the composition of the scaffolds. Preceding
experiments showed that porosity could be induced by the leaching of
the plastizer and as a result of the degradation of the polyester and
the CultiSpher microcarrier particles. Further, during the mixing of the
viscous crosslinkable polyester and the cell-seeded microcarriers, air
bubbles were trapped and contributed as macroporous pockets.
Preceding in vitro experiments with cells grown on microcarriers and
immobilized in the crosslinkable polyester proved to maintain an
acceptable viability. Further, triacetin has a very low molecular weight
and is washed out fast, generating some more micropores. The
observed degree of proliferation of the BMSCs was considered rather
limited. A higher porosity of the polymers would most likely have
allowed a more pronounced proliferation activity. It was surprising to
observe that α-TCP–supplemented polymers demonstrated less
BMSC proliferation characterized by less amounts of tissue and cells
found around the microcarriers. Although α-TCP is most likely not
toxic for the cells, this calcium phosphate has been reported to be
inferior with regard to seeding efficacy, increase in osteogenic marker
genes, and three-dimensional cell alignment compared to other bone
grafts like mineralized collagen (Niemeyer et al., 2004). The addition
of α-TCP might also decrease the overall porosity of the polymer, thus
possibly interfering with proliferation potential.
The presence of clusters of cells around the composites in a single

goat might be indicative for the occurrence of an inflammatory
response because a small number of these cells were identified as T-
lymphocytes. This might have been induced by the presence of the
slowly degrading polymers, although no adverse reactions were
recorded in previous studies (Tielens et al. , 2007, Vertenten et al. ,
104
2008b) and no clinical signs of inflammatory reactions (lameness;

swelling, redness, and hyperthermia at the operated legs) were
observed during the study period in the other goats. A more obvious
explanation might be that these findings represented a reaction
against a possible contamination during surgery.
Histomorphometrical evaluation confirmed a slower bone healing

and remodeling in bone defects containing composites in comparison
with control defects, although no differences were found between the
three composites. The absence of ingrowth of new bone into the
polymer composites reflects the limited osteoconductive properties of
the polymers.
Similar to the previous study in goats without the addition of

BMSCs (Vertenten et al., 2008b), polymer degradation failed to
become visible during the first 12 weeks postsurgery. The present
study accentuates the need for further physicochemical adaptations of
the polymers used to improve their osteoconductive properties.
Recently, hydrogels seeded with bone-forming cells have been
reported as promising alternative scaffolds for bone regeneration
purposes (Arinzeh et al., 2003, Bianco & Robey, 2000, Bianco &
Robey, 2001, Endres et al., 2003, Srouji & Livne, 2005, Srouji et al.,
2005, Trojani et al., 2006, Tsuchida et al., 2003, Vehof et al., 2002).
These substances can also be crosslinked by photopolymerization
(Buxton et al., 2007, Li et al., 2006).
The BMSCs in the present study have been cultivated in an

osteogenic medium to stimulate their differentiation to bone-forming
cells. A clearly bone-forming potential was encountered in the
composite-seeded cells (Fig. 12), proving the osteogenic capacity of
the BMSCs in the used polymer.
In a previous study (Vertenten et al., 2008a), metachromatic

staining was also observed, which was suggestive for the presence of
mature bone. Most likely, all calcified tissues will have a
metachromatic staining after immunohistochemical staining with the
105
techniques of the present protocol. The metachromatic granular

material in composite no. 3 was identified as α-TCP particles using the
standard light microscopic techniques.
The second part of our research hypothesis, in which we

anticipated an improvement in bone healing characteristics of the
treated cortical defects compared to earlier reported results (Vertenten
et al., 2008b), is not true due to a too low resorption and porosity of
the polymers impeding BMSCs' proliferation and bone ingrowth.
106
PART 3.2: Conclusion
CONCLUSION
The use of osteogenic differentiated BMSCs is promising in the

process of bone tissue engineering and has been reported in different
animal models (Arinzeh et al., 2003, Bruder et al., 1998, Dai et al.,
2005, Kon et al., 2000, Maniatopoulos et al., 1988, Muraglia et al.,
1998, Petite et al., 2000, Shang et al., 2001, Yuan et al., 2007, Zhu et
al., 2006). Despite their excellent biocompatibility, the osteoconductive
characteristics of the in situ crosslinkable methacrylate-endcapped
poly-(D,L-lactide-co-ε-caprolactone) combined with BMSCs were
limited in the first weeks after implantation. The addition of α-TCP
seemed to have a less positive effect on expansion and growth of the
BMSCs than other polymer composites. A faster resorption and higher
porosity of the polymer seems imperative to promote BMSCs'
proliferation and encourage bone ingrowth from the surrounding
tissues. Further biochemical adaptation of the present polymer is
mandatory to improve its bone grafting properties. Their in situ
crosslinkable characteristic in combination with osteogenic
differentiated BMSCs offers potentials for the restoration of complex
orthopaedic situations.
107
ACKNOWLEDGEMENTS
The authors thank Cindy De Baere, Sarah Loomans, Bart De

Pauw, and Leen Pieters for their excellent technical assistance.
108
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112
3.3
Evaluation of bone regeneration with an
injectable, in situ polymerizable
Pluronic® F127 hydrogel derivative
combined with autologous
mesenchymal stem cells in a goat tibia
defect model
Adapted from: E. Lippens*, G. Vertenten*, J. Gironès, H. Declercq, J. Saunders, J.

Luyten, L. Duchateau, E. Schacht, L. Vlaminck, F. Gasthuys, M.
Cornelissen. (2009). Evaluation of bone regeneration with an

injectable, in situ polymerizable Pluronic® F127 hydrogel derivative
combined with autologous mesenchymal stem cells in a goat tibia
defect model. Tissue Engineering Part A: doi:

10.1089/ten.TEA.2009.0418
*: equally contributed
PART 3.3: Summary
SUMMARY
In situ forming bone substitute materials are very attractive to

fill irregularly shaped defects. In this study a chemically modified
®
form of the Pluronic F127 hydrogel was used. Similar to the
parent form, this derivative underwent a sol-gel transition in the
body while additional radical curing resulted in a stable 3D
network gel with a controllable degradation rate. An extra cell
source of autologous bone marrow derived mesenchymal stem
cells was mixed with the hydrogel to increase the ossification
process, when implanted in non-critical size unicortical tibia
defects. These cells were cultured and predifferentiated on 2
®
types of cell carrier systems i.e. gelatin CultiSpher-S
microcarriers and hydroxyapatite tubular carriers. Radiographic
and histological evaluation revealed that bone regeneration was
comparable in the defects with the bone substitute compositions
and the untreated control defects at 2 and 4 weeks post-
implantation and that newly formed bone originated from the
®
cells on the CultiSpher-S carriers. This resulted, 6 and 8 weeks
post-implantation, in faster bone repair in the defects filled with
®
the hydrogel plus CultiSpher-S carriers in comparison to the
control defects. Surprisingly, there was no formation of new
bone originating from the hydroxyapatite carriers. The hydrogel
by itself seemed to stimulate the natural repair process.
115
Evaluation of a hydrogel based scaffold with stem cells
INTRODUCTION
Bone tissue has an excellent ability of self-regeneration in healthy

individuals. However, when the defect exceeds a critical size,
impaired bone formation can occur, whereby surgical intervention is
mandatory. To date, the most used repair strategy for bone defects is
the use of natural bone grafts whereby surgeons can choose either
patients-own bone grafts (autografts), grafts obtained from other
individuals (allografts) or from other species (xenografts). Up to now,
the standard treatment includes the implantation of autografts which
can induce new bone formation due to the presence of osteogenic
cells (De Long et al., 2007, Finkemeier, 2002). Autograft harvesting,
however, is associated with increased patient pain and high risk of
donor side morbidity, while the amount of harvested bone is limited
(Laurie et al., 1984). On the other hand, a larger quantity of grafting
material can be implanted when using allo- or xenografts. The
downfall with these non patient-owned grafts is that the native cells
and proteins have to be destroyed to prevent harmful immunological
reactions in the patient (Finkemeier, 2002, Wheeler & Enneking,
2005). By doing so, the graft properties are altered, including a
reduction of the osteoinductive potentials of the grafts (Wheeler &
Enneking, 2005). Furthermore, the risk of disease transfer remains a
realistic complication.
An alternative approach for bone repair is bone tissue engineering.

Tissue engineering has as its primary purpose, the repair,
regeneration and reconstruction of damaged, lost or degenerative
tissue (Sopyan et al., 2007). For bone repair procedures an
engineered bone substitute that mimics the architecture of the native
bone can be incorporated into the defect (Langer & Vacanti, 1993,
Tabata, 2009). One of the main points for success is adequate contact
between the implant and the native bone. However, it is generally
116
difficult to fit in preformed scaffolds when the defect has an irregular

shape, which often occurs in clinical situations. The use of in situ
forming scaffolds that perfectly match with the defect is therefore
justified. In previous studies by our group, an in situ hardening,
biodegradable, methacrylate-endcapped poly(D,L-lactide-co-ε-
caprolactone) polymer was tested to fill standardized unicortical tibia
defects in a goat model (Vertenten et al., 2008, Vertenten et al.,
2009). To increase the osteoinductive properties of the implant, an
extra source of bone forming cells, namely bone marrow derived
mesenchymal stem cells (BMSC), was additionally mixed with the
polymer (Cancedda et al., 2007, Yang et al., 2007). BMSC, which
have a proven in vitro and in vivo osteogenic potential, are anchorage
dependent i.e. need a substrate for optimal cell activity. For this
reason, autologous BMSC were predifferentiated in vitro after seeding
on a cross-linked gelatin microcarrier. The cells cultured on the
carriers survived the mixing with and curing of the polymer.
Unfortunately, after implantation into the bone defect for a prolonged
period of time the cell survival was limited. A limited amount of newly
formed bone could be observed. One of the major problems of the
cross-linked methacrylate-endcapped poly(D,L-lactide-co-ε-capro-
lactone) polymer was the low porosity, restricting the essential oxygen
and nutrient flow needed for an acceptable bone repair of the defect.
Even more, the relative slow degradation of the polymer may also
explain the limited bone regeneration (Vertenten et al., 2009).
In the present study, the lactide-caprolactone polymer was

®
replaced by a chemically modified Pluronic F127 hydrogel. In
contrast to the first polymer, hydrogels were proven to be able to
retain large amounts of water. They are highly permeable for oxygen
®
and nutrients. Pluronic F127 is a thermoreversible hydrogel that
undergoes a sol-gel transition at body temperature above a critical
gelation concentration. The gel formation is also highly temperature
and concentration dependent (Dumortier et al., 2006, Escobar-Chavez
et al., 2006, Jeong et al., 2002, Klouda & Mikos, 2008). It has been
117
reported that the presence of body fluids reduces its concentration,

resulting in rather fast disintegration of the gel in vivo (Jeong et al.,
2002). By chemically converting the hydroxyl endgroups of this
polymer into a cross-linkable N-methacryloyl-depsipeptide unit, a
more stable gel was obtained. By adding a controlled amount of
photo-initiator, a 3D network with controllable degradation rate was
formed via radical polymerization using UV light. The depsipeptide
unit, alanine combined with L-lactic acid, determines the degradation
®
rate. Indeed, 50% of the gel fraction of a 30 wt% modified Pluronic
F127 (the so called Plu ALA-L) hydrogel was reported to hydrolyze
after approximately 38 days in phosphate buffered saline solution at
37°C (Swennen et al., 2006, Swennen, 2008). Additionally, in vitro
studies revealed good cell viability and proliferation of encapsulated
MC3T3-E1 pre-osteoblasts cultured on gelatin carriers in Plu ALA-L
hydrogel (Lippens et al., 2009).
The main objective of this study was to evaluate de novo bone

synthesis in non-critical size unicortical goat tibia defects, filled with a
cross-linkable hydrogel and pre-differentiated BMSC on a cell carrier
system. Two types of cell carrier systems were tested i.e. porous
®
spherical cross-linked gelatin CultiSpher-S (ø 130-380 µm) and
tubular sintered hydroxyapatite (HA) (ø 1 mm, length 2 mm). The first
aim of the present study was to provide evidence of bone formation
originating from the cells on the cell carrier systems, resulting in an
increased bone repair process when compared to the natural healing
capacity of bone. Secondly, the hypothesis that more newly formed
bone would be obtained starting from the hydroxyapatite carriers was
investigated, since clear osteoconductive properties have been
attributed to this material. Finally, the possible beneficial effects for
bone repair of the modified hydrogel without the addition of cells was
evaluated.
118
The animal experiments were performed with respect to all aspects

of the well being of the animals. The study was approved by the
Ethical Committee of the Faculty of Veterinary Medicine of the
University of Ghent (EC2008/028). Eight milk goats were housed in
groups and had ad libitum access to water and food. After starvation,
surgical procedures were performed under general anesthesia and all
procedures (surgical as well as bone substitute preparation) were
conducted under sterile conditions. The appropriate medical
treatments including antibiotics and antiphlogistic drugs were
administered after each procedure.
BMSC harvesting and cell construct preparation
Mesenchymal progenitor cells from goat bone marrow were

harvested from the iliac crest and culture expanded in vitro. Before
expansion, the bone marrow was thoroughly rinsed with culture
medium, i.e. αMEM medium (minimum essential medium, Life
Technologies, Merelbeke, Belgium) supplemented with 10 vol% FBS
(fetal bovine serum, Life Technologies), 1 vol% Fungizone
Amphotericin B (Life Technologies) and penicillin-streptomycin
(50U/ml-50µg/ml, Life Technologies). The cells were resuspended in
the same medium supplemented with 100 µM L-ascorbic acid 2-
phosphate (Sigma-Aldrich, Bornem, Belgium) and 10 nM
dexamethasone (Sigma-Aldrich) and seeded in 5 T75 culture flasks
(Greiner Bio-One, Frickenhausen, Germany). After 2 days incubation
at 37°C in a 5 vol% CO2 atmosphere, the stromal cells had adhered to
the bottom of the flask. By renewing the medium, the non-adherent
hematopoietic cell population was removed. After 2 weeks of culture,
119
the cells were collected, counted and seeded onto 2 different carrier
systems.
®
CultiSpher-S carriers:
®
Gelatin based CultiSpher-S cell carriers (Percell Biolytica, Åstorp,
Sweden), with an average diameter of 130 to 380 µm and pore size of
20 µm, were used. A standard amount of dry carriers (0.09 g) were
hydrated in phosphate buffered solution (PBS) and heat sterilized.
After rinsing in culture medium, the carriers were equally divided over
5
5 wells of a 12 well suspension plate (Greiner Bio-One) and 4.10
cells were added to each carrier containing well. The plate was
incubated for a further 48 hours under static conditions in a 5 vol%
CO2 incubator. Subsequently, the medium was renewed and the
constructs were collected and transferred to a shaker flask for
dynamic culturing (70 rpm). One week after seeding, 10 mM β-
glycerophosphate (Sigma-Aldrich) was added to the medium and the
constructs were cultured for an additional week. Two weeks after
seeding, the highest level of cell colonization was obtained, confirmed
qualitatively under the fluorescent microscope and quantitatively with
the cell proliferation assay MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, Promega,
Leiden, The Netherlands). An average MTS absorbance value of
1.212 was measured after 2 weeks in culture for 100µl constructs.
Hydroxyapatite carriers:
The cylindrical HA carriers were provided by VITO (Flemish

Institute for Technological Research, Mol, Belgium). The
hydroxyapatite powder (Merck, Darmstadt, Germany) was ball-milled
for 30 minutes in water and lyophilized. A 14 wt% solution of
polysulfone (PSF, Merck) in N-methyl-2-pyrrolidone (Merck) was
120
prepared to dissolve HA, resulting in a PSF/HA ratio of 1 out of 9

(w/w).
Tubular HA carriers were produced by a spinning technique based

on phase inversion. The suspension was extruded (0.1 ml/s) through
a nozzle with a hollow needle in the middle through which water was
pumped (0.6 ml/s) to keep the tube open and to induce phase
inversion. The internal diameters of the nozzle and the hollow needle
were 1.9 and 0.9 mm, respectively. An air gap of 10 mm was applied
before reaching the coagulation bath, filled with water to complete
phase inversion.
The tubes had an inner diameter of 700 µm and were cut 2 mm in

length. Afterwards, the smaller tubes were calcined at 600°C,
presintered for 1 hour at 1100°C and sintered for 3 hours at 1300°C in
air atmosphere. The carriers were sterilized with ethylene oxide gas at
37°C and ± 800 mbar for 5 hours and then thoroughly aerated for 2
days. Five carriers were placed upright into each well of a 96 well
5
suspension plate (Greiner Bio-One) and 10 cells were dropped in a
volume of ca. 30 µl onto the carriers. The carriers were pulled through
the cell suspension with tweezers and incubated for 30 minutes
without extra medium to allow maximal cell contact with the inner and
outer surface of the carrier. Subsequently, 200 µl medium was added
to each well and the plate was incubated in a humidified CO2
incubator. The constructs were transferred after 2 days to a 24 well
suspension plate (Greiner Bio-One) and further cultured under static
conditions for a total of 2 weeks. In the last week of culture, 10 mM β-
glycerophosphate was added. The carriers were fully colonized after 2
weeks of in vitro culture which was confirmed microscopically and by
the MTS assay. An average MTS absorbance value of 1.136 was
measured after 2 weeks in culture for 5 HA constructs.
121
Hydrogel preparation
The synthesis and characterization of the chemically modified

®
Pluronic F127 polymer into Plu ALA-L was worked out by Swennen
et al. (2006, 2008) and a scheme of the chemical modifications steps
is presented in Fig. 1. In summary, the modified polymer powder was
sterilized by ethylene oxide sterilization and a 30 (w/v)% solution of
sterile Plu ALA-L in physiological solution was prepared on a magnetic
®
stirrer at 4°C overnight. The photo-initiator Irgacure 2959 (Ciba,
Brussels, Belgium) was added to the mixture in a 1 mol%
concentration just before implantation. Until the moment of
implantation, the solution was stored in an ice bath protected from
light to prevent spontaneous gelation. After implantation, radical
polymerization of the vinyl side groups of the Plu ALA-L macromer
was achieved by UVA (λ=365nm) irradiation in the presence of the
photo-initiator.
Figure 1. The three steps reaction scheme of Plu ALA-L. a/ The hydroxyl
®
groups of the Pluronic F127 polymer were converted into a bromine ester
(STEP 1). b/ The syntheses of N-methacryloyl-L-alanine via the Schotten-
Baumann acetylation reaction (STEP 2). c/ After coupling of the N-
methacryloyl-alanine to the modified Pluronic (STEP 3), Plu ALA-L was
formed
122
Surgical procedure
The surgical procedure has been described in depth by Vertenten

et al. (2008). Briefly, 4 unicortical holes (ø 6mm) were drilled in the
medial diaphyseal cortex of the tibiae with a trephine burr (3I; Implant
Innovations, Copenhagen, Denmark). To reduce bleeding during the
surgical procedure, epinephrine soaked gauzes were inserted into the
defect holes prior to implantation of the bone substitutes. In each tibia,
each substitute was randomly assigned to one of the 4 holes. After
implantation, the surgical incision was closed secundum artem.
To be able to evaluate the bone healing of the different substitute

conditions, 2 goats were euthanized 2, 4, 6 and 8 weeks after
implantation and the tibia defects were harvested and fixated in 10%
buffered formaldehyde for further histological analysis.
Implantation conditions
In a first substitute condition, only the modified UV cross-linked

hydrogel was implanted. The cold Plu ALA-L solution was aspirated in
a syringe and carefully dropped in the defect. Due to the increase in
temperature, this thermosensitive hydrogel underwent a sol to gel
transition, and by subsequent irradiation for 2 minutes with UVA light
(I = 3000 mW/cm²) a stable 3D network gel was created. After
confirmation of the consistency of the gel with a spatula, the next
defect was filled.
The second substitute condition was a combination of the modified

UV cross-linkable hydrogel with predifferentiated BMSC cultured onto
®
CultiSpher-S microcarriers. The small sized cell loaded gelatin
carriers were easily mixed with the Plu ALA-L and aspirated in the
syringe. The deposition of the polymer and the irradiation step was
similar as for substitute condition 1.
In substitute condition 3, the BMSC were cultured onto the HA

tubes. As these carriers were too large to be injected in the defect,
123
approximately 20 carriers were manually inserted one by one into the

injected thermal gelated Plu ALA-L gel with tweezers. After UV
irradiation and confirmation of the consistency of the hydrogel, the
wound was closed.
The forth bone hole remained untreated and served as control.
A daily clinical evaluation of the surgical site for possible

complications related to the surgical intervention was performed.
Immediately after surgery and every 2 weeks, standardized

craniocaudal and mediolateral digital radiographs (Digivex 40 15kW
high-frequency generator, Medex Loncin S.A., Loncin, Belgium) of
each tibia were taken in the sedated goats. All radiographs were
evaluated blindly for defect density, periosteal reaction and soft tissue
reaction using the criteria of Dorea et al. (2005). Scores ranging from -
1 to 5 were used to evaluate the radiopacity (in comparison to the
scores of Dorea et al. (2005), a score of 5 was introduced as an
indication of fully radiopaque), while scores between 0 and 4 were
used for the estimation of the size of the periosteal reactions. For the
distribution of the periosteal reactions, the shape and location of the
reactions was taken into account by scores ranging between 0 and 4.
Additionally soft tissue reactions were evaluated (no reaction,
moderate or severe). Gray-scale densities at the defect region were
quantitatively evaluated using a standard imaging software packet
(Image J 1.41o; National Institutes of Health, Bethesda, USA).
Histological analysis
After euthanasia, the soft tissue around the tibiae was removed,
and the bones were split longitudinally. After removal of the bone
marrow, the defect sites were separated, fixed in 10% buffered
124
formaldehyde for 12 hours and embedded in the destabilized

®
Technovit 9100 New (Heraeus Kulzer, Wehrheim, Germany)
methacrylate. Five µm sections were cut with a heavy duty microtome
(SM2500; Leica Microsystems, Wetzlar, Germany). Sections were
stretched on a slide with 70% ethanol and dried for 12 hours at 60°C.
The extended description of the embedding protocol has been
described by Vertenten et al. (2008).
For reasons of standardization, only the sections of the central part

of the defect were analyzed microscopically (Jena Carl Zeiss
microscope, Zaventem, Belgium). These sections were stained with
hematoxylin & eosin (morphological evaluation) and von Kossa
(visualization of the present mineralization). The sections were
evaluated on the formation of de novo bone, the occurrence of
inflammatory reactions, tissue type and the presence of residual graft
material.
Histomorphometric analysis was performed on the von Kossa

M
stained sections using a software program (Cell , Imaging Software
for Life Science Microscopy; Olympus, Aartselaar, Belgium). The area
of the mineralization front which was stained black, was calculated by
the determination of von Kossa positive area and the percentages of
von Kossa positive material in the total defect area.
The radiographic scores were compared between the 3 substitute

conditions and control defects by the Friedman test with tibia and time
as block factors. Pairwise comparison between the 3 substitute
conditions and controls were based on the Wilcoxon rank sum test
using Bonferroni’s multiple comparisons adjustment technique at the
global 5% significance interval. The measured gray-scale levels in the
radiographs, and the percentages von Kossa positive material were
compared between the substitute conditions and controls by a mixed
125
model with tibia as random effect and substitute condition, time and
their interaction as categorical fixed effects. Pairwise comparisons
between the substitute conditions and controls were adjusted by
Tukey’s multiple comparisons technique at the global 5% significance
level.
126
PART 3.3: Results
RESULTS
Surgical procedure and clinical follow-up
Eight female goats with an average age of 47.3 ± 17.5 months and
an average body weight of 66 ± 12 kg were included in the study. The
two tibiae were surgical treated in each goat. Injection and
subsequent UV cross-linking of the hydrogel in the defect holes went
very smoothly. After UV irradiation of the hydrogel, the consistency of
the gel, which was checked with a spatula, was acceptable. One goat
was slightly lame for 5 days, which resolved without treatment.
Another goat developed a mild seroma 2 weeks after surgery, which
was successfully treated by sterile puncture and a pressure bandage
every 2 days for one week. No other signs of clinical pain, lameness
or increase in body temperature were observed during the entire study
period in the goats.
Radiographic follow-up
A significant difference was found between the bone substitute

conditions and control defects for the size of the periosteal reaction
and callus formation over time (P < 0.0001) with pairwise significance
(P = 0.0004) between the defects treated with Plu ALA-L and Plu ALA-
L plus HA carriers (Fig. 2). The periosteal reaction was significantly
higher when HA carriers were added to the Plu ALA-L in comparison
to the defects filled with the hydrogel alone (P = 0.0004). No
significant differences were found between the defects for the
distribution of the periosteal reaction and callus formation and for the
soft tissue reaction around the defects over time. The density of the
defects differed significantly between the substitute conditions and
control defects (P < 0.0001), whereby the density was significantly
higher for the HA carriers treated defects compared to the other
127
defects (P < 0.0001). It has to be mentioned that HA is a highly radio-

opaque material, which will increase the density scores of these
materials irrespective of the presence of newly formed bone.
Significant differences between the substitute conditions and controls
also occurred in the measured gray-scale levels in the radiographs (P
< 0.0001). The average gray-scale levels were 164.59 ± 2.69 for the
control defects, 164.45 ± 2.78 for the Plu ALA-L treated defects
without a cell delivery system, 168.28 ± 2.78 for defects filled with Plu
®
ALA-L with cell seeded CultiSpher-S carriers and 174.66 ± 2.78 for
Plu ALA-L with the HA carriers containing defects. The gray-scale
levels also changed significantly over time (P < 0.0001), the longer the
implantation period, the higher the measured density. An overview of
the digital radiographs taken at two weeks time intervals from a goat
with implant residence time of 8 weeks is presented in Fig. 3.
Figure 2. Boxplots representing the pairwise comparison for the differences in

scores between the substitute conditions and controls of the digital
radiographs evaluated for defect density (density), size of the periosteal
reaction and callus formation (size), distribution of the periosteal reaction
(distribution) and soft tissue reaction (soft tissue) averaged over time. In the
X-axis the 2 compared defects are presented, with 0: control defect, 1: Plu
®
ALA-L, 2: Plu ALA-L + CultiSpher-S carriers, 3: Plu ALA-L + Ha carriers. *
denotes a significant difference.
128
PART 3.3: Results
Figure 3. An overview of the radiographic pictures taken at a 2 weeks time

interval of the tibia of the same goat. The upper radiographies were taken
from the mediolateral axis, while the radiographies underneath are taken from
the cranio-caudal axis. a/ just after implantation, b/ 2 weeks after implantation,
c/ 4 weeks after implantation, d/ 6 weeks after implantation, e/ 8 weeks after
implantation. The 4 defects are treated respectively from the top defect to the
bottom with Plu ALA-L plus BMSC loaded CultiSpher-S carriers, Plu ALA-L,
Plu ALA-L plus BMSC loaded HA carriers and untreated control defect.
Histological follow-up
2 weeks: The defects were mainly filled with connective tissue and
blood clots whereby a limited bone formation, originating from the
intact bone surrounding the defects and bone marrow cavity, was
observed in all defects. The defects filled with Plu ALA-L plus
®
CultiSpher-S carriers showed additional bone formation starting from
®
the cells on the carriers (Fig. 4a,b). As for the CultiSpher-S carriers,
the HA carriers could also be well distinguished in the defects. The
defects filled with Plu ALA-L plus HA carriers failed to reveal any
additional newly formed bone originating from the carriers since the
carriers were surrounded by fibrotic tissue and a lot of red blood cells.
Blood clots could be visualized in and around the tubular HA carriers.
There were no signs of cell proliferation nor matrix production.
129
®
Figure 4. Overview CultiSpher-S (arrow head) treated defects in the early
bone regeneration process: a/ Matrix production (arrow) originating from the
BMSC on the carrier, 2 weeks post-implantation (H&E), b/ Mineralization of
the bone matrix, same carrier as picture a (von Kossa staining), c/ Eosinophile
®
matrix production around the CultiSpher-S carrier 4 weeks post-implantation,
this matrix was not yet mineralized. d/ Cuboidal osteoblast-like cells in the
centre of the defect 4 weeks post-implantation.
4 weeks: Newly formed bone originating from the defect margins at

the bone marrow cavity was present in all the defects after 4 weeks
(Fig. 5). At the bone marrow cavity side, most defects were completely
bridged by new bone trabeculae, especially in the Plu ALA-L defects
®
and the defects with CultiSpher-S carriers embedded in the hydrogel.
However, the majority of the defects was filled with connective tissue.
Within this connective tissue, additional newly formed bone was
®
observed in Plu ALA-L plus CultiSpher-S treated defects. CultiSpher-
®
S carriers were hardly visible but foci containing cuboidal osteoblast-
like cells, in some cases lining trabeculae of extracellular matrix, were
observed (Fig. 4c), occasionally not in the close proximity of a carrier
(Fig. 4d). Around the HA carriers, no matrix formation, mineralization
nor osteoblast-like cells could be observed. These carriers were
130
PART 3.3: Results
surrounded by connective tissue and red blood cells and most of them
were empty inside. Apparently, the HA carriers seemed to be pushed
out from the defect towards the periosteal site.
Figure 5. Von Kossa staining of the defects 4 weeks post implantation. a/

®
Control defect, b/ Plu ALA-L treated defect, c/ Plu ALA-L plus CultiSpher-S
carrier treated defect, d/ defect with implantation of Plu ALA-L with embedded
HA carriers.
6 weeks: In all the defects, the increase in matrix production was

rather limited while connective tissue was abundantly present.
Especially in some of the control defects, bone matrix production was
restricted. The highest level of newly formed bone was found in the
defects filled with Plu ALA-L with and without cell loaded CultiSpher-
® ®
S carriers. The CultiSpher-S carriers were not distinguishable
anymore, while the HA carriers resided in the connective tissue at the
periosteal side of the defects. No indications of newly formed bone
originating from the HA carriers were present after 6 weeks (Fig. 6).
131
Figure 6. Defect treated with Plu ALA-L with HA carriers, 6 weeks post
implantation. a/ Overview picture of the bone regeneration in the defect area
(HE), b/Detail of a HA carrier surrounded by fibrotic tissue and a blood cloth
with red blood cells.
8 weeks: Most defects were filled with new bone 8 weeks after the
surgical intervention (Fig. 7). The defects treated with Plu ALA-L with
®
encapsulated CultiSpher-S carriers showed the most bone formation,
and were all completely filled with new bone. In the control defects, an
ingrowth of connective tissue and fat cells could be visualized at the
bone marrow cavity and the periosteal sides of the defect. Some of
the defects treated with the Plu ALA-L plus HA carriers were
completely filled with new bone, while the carriers resided in the
connective tissue callus above the original defect. However, in some
other defects the HA carriers were still present in the defect at the
periosteal side, and de novo bone formation was hampered.
132
PART 3.3: Results
Figure 7. Defects 8 weeks post-implantation stained with von Kossa. a/ bone

regeneration in a control defect, b/ defect treated with Plu ALA-L, c/ complete
®
bone repair after implantation of Plu ALA-L with CultiSpher-S carriers, d/
defect treated with Plu ALA-L and HA carriers.
Histomorphometry
The percentage of newly formed bone was determined with a

software program as the percentage of von Kossa positive stained
material in the total defect area on a histological slide (Fig. 8).
A minimum of mineralization was present in the defects, two weeks

after implantation. The mean percentage of de novo formed bone
varied between 2.29 ± 1.32 % for the control defects and 3.08 ± 2.89
®
% for defects treated with Plu ALA-L plus cell loaded CultiSpher-S
133
carriers. The treated defects, especially, contained a lot of newly

formed bone after 4 weeks: 18.52 ± 4.29 % for the pure Plu ALA-L
defects, 17.63 ± 8.06 % for the Plu ALA-L plus HA defects and 13.92
®
± 4.04% for defects treated with Plu ALA-L plus CultiSpher-S . An
average percentage of newly formed bone of 13.35 ± 4.66 % was
calculated for the control defects. Six and 8 weeks post-implantation,
the percentage de novo formed bone was the highest in defects
®
treated with Plu ALA-L plus CultiSpher-S carriers (resp. 32.20 ± 8.61
% and 56.08 ± 5.57 %), followed by the defects filled only with the
hydrogel (resp. 32 ± 6.95 % and 51.09 ±15.83 %).
No significant difference between the 3 bone substitute conditions

and controls occurred (P = 0.3098). Only a significant change in time
(P < 0.0001) was observed in the percentage of newly formed bone,
i.e. there was more newly formed bone in all the defects at the end of
the study period.
Figure 8. Mean percentages and standard deviation of de novo formed bone

in the different defects at every observation time point as determined by von
Kossa positive stained area.
134
DISCUSSION
In the present study, de novo bone synthesis was evaluated in a

standardized goat model in the presence of 2 types of cell constructs
with an in situ cured hydrogel filling material. BMSC were cultured and
differentiated in the osteogenic lineage on gelatin and hydroxyapatite
cell carriers. All compositions were implanted in non-critical size tibia
defects whereby an empty bone defect served as control. The aim
was to evaluate if the addition of an extra cell source could enhance
bone healing in comparison to the untreated control defect. Secondly,
the role of the hydrogel on the regeneration process was investigated.
Based on the histological analysis, the following conclusions could

be made. The mineralization process of a newly synthesized bone
matrix was present and progressively increased in all the defects 2
and 4 weeks after the surgical implantation of the polymers. At those 2
moments in time, the amount of newly formed bone was limited and
the majority of the defects was filled with pure connective tissue. The
level of newly formed bone was more or less comparable between the
®
3 treatment conditions and the controls. However, in the CultiSpher-S
treated defects bone matrix synthesis also originated from the
implanted BMSC on the carriers. As a result, evaluation at the last two
time points after 6 and 8 weeks showed, although not significantly, a
®
better bone repair in the CultiSpher-S treated defects.
®
The use of cell seeded CultiSpher-S microcarriers as cell delivery
systems in in vivo applications is recent and up till now limited.
However, the available studies clearly indicate the potential of these
constructs, for instance in the repair of soft tissue (Gustafson et al.,
2007, Huss et al., 2007) or for nerve regeneration (Clavijo-Alvarez et
al., 2007). Similar to the results of these reports, the gelatin cross-
linked carriers did not elicit an inflammatory reaction and degraded
over time in the present bone tissue engineering study. The use of
135
®
BMSC seeded CultiSpher-S carriers for in vivo bone regeneration
was up to now used in one single study whereby the effect of these
cell constructs on the regeneration process was reported to be dual
(Yang et al., 2007). First of all, a direct production of matrix from the
osteogenically differentiated BMSC on the gelatin carriers was seen,
which was confirmed in the present study starting 2 weeks after
implantation (Fig. 4a-c). Secondly, the secretion of bioactive factors
was suggested to recruit progenitors in order to increase
osteogenesis. In the present study, this role of the implanted carriers
was confirmed 4 weeks post-implantation by the presence of cells with
an osteoblastic phenotype in the central part of the defect (Fig. 4d).
®
Since these cells were not in the vicinity of a CultiSpher-S carrier,
they may be considered as recruited endogenous progenitor cells.
Furthermore, in comparison to the control defects, there was a

general tendency in increased osteogenesis 6 and 8 weeks post-
®
implantation in the defects filled with Plu ALA-L plus CultiSpher-S
carriers.
Surprisingly, this dual role was not preserved for the BMSC loaded
on the small hydroxyapatite tubes. No newly formed bone originated
from the cells when using these carriers, nor were there any signs of
recruitment of osteoprogenitor cells. Although a significantly increased
radiopacity was present when compared to the other defects, this
phenomenon was most likely not induced by an increase in bone
formation rate, but rather by the specific composition of the used
carrier. These results were rather unexpected and surprising. This
ceramic has a chemical composition similar to the mineral phase of
bone and has biocompatible and osteoconductive characteristics and
is therefore one of the most used materials for bone tissue
engineering (Woodard et al., 2007). In addition, it provides a source of
calcium and phosphate, both essential elements for the production of
new bone (Matsumoto et al., 2007). In vitro tests showed a fast
colonization of these carriers with cells combined with high cell
136
PART 3.3 : Discussion
survival. Apparently, the transfer of these carriers into an in vivo

setting, as done in the present study, does not confirm the findings of
the in vitro tests. The reason for this contradiction remains unclear at
the moment. Perhaps, the influences of the cross-linked hydrogel on
the HA carriers in vivo, or even the physical characteristics of the used
HA carriers, for instance pore size and/or pore distribution may play a
major role (Fischer et al., 2003, Sopyan et al., 2007). The discrepancy
in results between in vitro and in vivo tests can be supported by the
findings that osteogenesis in vitro is favored by lower porosity and is
independent of pore size, while in contrast, in vivo osteogenesis is
favored by higher porosity and pore size (Baroli, 2009). Additional
tests are ongoing, to find a plausible explanation for these rather
disappointing in vivo results of the present study.
The use of in situ forming scaffolds is an attractive approach to fill

and repair irregular shaped defects, as this kind of scaffold will take
over the shape of the defect, allowing good integration in and contact
with the native bone. Moreover, only minimal invasive surgery is
needed for the implantation. In the present study, the main function of
the in situ formed material was to deliver the cell loaded carriers into
the defect and to keep them in place. However, the cell mixing with
and curing of the polymer may not interfere with the cell viability and is
of major importance to achieve reliable results. Once in place, the
scaffold should not only allow the smooth supply of nutrients and
oxygen to the cells, but also the migration of cells and vascular
sprouting. In a previous study (Vertenten et al., 2009), a
photopolymerizable L-lactide caprolactone methacrylate polymer was
implanted into unicortical tibia defects in a goat model whereby BMSC
®
seeded on the CultiSpher-S carriers survived the implantation
process. However, the cell proliferation and bone synthesis was
limited, most likely because of the low permeability of the polymer
restricting the essential neo-vascularization and/or cell infiltration. In
®
the present work, a chemical UV polymerizable Pluronic F127
derivative was used (Swennen et al., 2006) into the same
137
standardized goat model. Not only an acceptable cell survival on the

encapsulated CultiSpher-S® carriers, but also good cell proliferation
and bone formation, was observed. Smaller blood vessels could be
found in the defect area, assuring the essential nutrient and oxygen
flow. Consequently, cells could infiltrate the hydrogel whereby 2
weeks post-implantation a lose connective tissue network bridged the
defect. Implantation of the hydrogel in the defect without
encapsulation of a cell delivery system showed, from 4 weeks on,
slightly better bone regeneration than the control defects. This
hydrogel probably facilitated the migration of cells and bioactive
molecules through the defect site, and as such ameliorated the
healing process.
The strength of a bone implant is also of major importance in bone

tissue engineering strategies. The modified hydrogel polymer used in
the present study is not suitable for load bearing applications in critical
size defects where the mechanical stability is impaired since the
mechanical properties of this polymer are low. However, the
combination of this modified hydrogel with standard external fixation
techniques can circumvent this shortcoming in clinical situations (Lee
et al., 2005).
For most bone tissue engineering scaffolds, it is difficult to possess

mechanical properties similar to that of natural bone while maintaining
a certain porosity to allow cell infiltration and being able to biodegrade
in a controllable way (Cancedda et al., 2007). In the present study, the
mechanical properties of the hydrogel were improved by using a HA
carriers system to deliver the extra cell source in the defect. HA, and
especially the sintered form used in the present in vivo experiment,
showed higher mechanical stiffness. However, in vivo studies have
revealed that sintered HA remained undegraded for a long period
post-implantation (Matsumoto et al., 2007), while the ideal bone tissue
construct should degrade at the speed of new bone formation. In the
present study, it was noticed that the HA carriers were still present 8
138
PART 3.3 : Discussion
weeks post-implantation, while most defects were already completely

regenerated. In contrast, 4 weeks post-implantation, it was still difficult
®
to distinguish the cross-linked gelatin CultiSpher-S carriers in the
defects. Both carrier materials are naturally occurring components of
bone since HA is part of the mineral phase of bone (Mastrogiacomo et
al., 2006, Sopyan et al., 2007, Woodard et al., 2007), while gelatin is a
hydrolyzed form of collagen that accounts for 90% of the organic
matrix of bone (Baroli, 2009). Accordingly, both cell delivery systems
did not induce an unwanted foreign body reaction.
In the present study, a standardized goat model was preferred

because of the similarities in metabolic rate and bone remodeling rate
compared to humans. In larger body weight animals, such as goats,
larger implants or multiple implant conditions can be tested (Pearce et
al., 2007). In the present study, 4 non-critical size defects were
created in each tibia. This means that 3 different implantation
conditions were simultaneously evaluated in the same animal, serving
as its own control without animal to animal variability and as such also
reducing the number of animals. The bone healing capacity was also
compared with the natural bone healing regeneration capacity of the
control defects. This is an initial step prior to testing in critical size
defects were the size of the defects restricts complete spontaneous
osseous regeneration (Hollinger & Kleinschmidt, 1990). Since the
®
addition of BMSC loaded CultiSpher-S carriers mixed in the Plu ALA-
L hydrogel resulted overall in a better bone repair in comparison to the
natural bone healing, application in critical size defects could be very
promising. The role of the highly permeable hydrogel would be to
facilitate cell ingrowth and cell migration and also diffusion of nutrients
and oxygen to the cells on the gelatin carriers. These cells in turn
would guarantee bone matrix production even in the centre of the
defect and the release of osteoinductive factors. Therefore a follow-up
experiment, for instance in the iliac crest of goats in which the bone
regeneration capacity of the BMSC loaded CultiSpher-S carriers
encapsulated in the Plu ALA-L hydrogel could be tested in a critical
139
size defect in comparison to an autologous bone graft, would be a

necessary step towards its clinical usefulness. In the present study the
®
ratio CultiSpher-S carriers to hydrogel volume was fixed to 1 to 5.
The amount of cell loaded carriers could be increased to further
increase the de novo bone synthesis in this model.
140
PART 3.3 : Conclusion
CONCLUSION
The objective of this study was to evaluate de novo bone synthesis

in non-critical size uni-cortical goat tibiae defects. For this purpose, a
modified in situ cross-linkable and biodegradable hydrogel was used
whereby predifferentiated BMSC cultured on 2 different cell delivery
systems (gelatin and hydroxyapatite) were mixed in the gel. The
®
chemically modified Pluronic F127 hydrogel was an excellent in situ
forming gel keeping the cell delivery systems in place when implanted
into the bone defect. Most likely this polymer allowed the essential cell
and nutrient migrations because of its high permeability. However,
there was a clear difference between the 2 incorporated cell carriers.
The formation of new bone starting from the cells on the carrier
systems was present after 2 weeks implantation when the gelatin
carriers were used. In contrast and surprisingly, newly formed bone
was not observed around the cell loaded HA carriers since these
carriers were surrounded by connective tissue and remained in the
centre or on the periosteal side of the defect throughout the entire
experiment. Even more, these HA constructs seemed to hamper new
bone formation.
The present study showed that an increase in newly formed bone

®
can be achieved by injecting BMSC loaded CultiSpher-S carriers
mixed in the Plu ALA-L hydrogel in a goat tibial defect when compared
with the natural bone regeneration rate.
141
ACKNOWLEDGEMENTS
The authors would like to thank Leen Pieters, Cindy De Baere, Bart
De Pauw, Nelly François and Roger De Vos for their excellent
technical assistance.
142
PART 3.3 : References
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Caprolactone) Combined with Autologous Mesenchymal Stem Cells in
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145
CHAPTER 4
Adapted analytical methods
4.1
Immunohistochemical analysis of low-
temperature methylmethacrylate resin-
embedded goat tissues
Adapted from: G. Vertenten, L. Vlaminck, R. Ducatelle, E. Lippens, M.

Cornelissen, F. Gasthuys (2008). Immunohistochemical analysis of
low-temperature methylmethacrylate resin-embedded goat tissues.

Anatomia Histologia Embryologia: doi: 10.1111/j.1439-
0264.2008.00881.x
PART 4.1: Summary
SUMMARY
Goats are frequently used as a suitable animal model for

tissue engineering. Immunohistochemistry can be helpful in
improving the understanding and evaluation of the in vivo tissue
responses at a molecular level. Several commercially available
antibodies (anti-KI67, anti-vimentin, anti-CD31, anti-core-binding
factor alpha-1, anti-osteocalcin, anti-alkaline phosphatase, anti-
MAC387, anti-CD3, anti-CD20, anti-CD20cy, anti-CD79 and anti-
CD45) were evaluated on Technovit 9100 New® embedded goat
tissues. Only immunohistochemical staining for vimentin,
osteocalcin, MAC387 and CD3 were positive. These antibodies
can be routinely used to evaluate goat tissues at molecular level.
The use and development of alternative antibodies might further
supplement and complete the possibilities for
immunohistochemical analysis of goat tissue samples.
151
Immunohistochemical analysis of goat tissues
INTRODUCTION
The main goal for using bioactive graft material in bone

regeneration in men and animals is the activation of bone formation
and stimulation of the differentiation of osteoprogenitor cells into
osteoblasts at their surfaces. The evaluation of the specific
characteristics of the implant materials is primarily focussed not only
on examination of the induced immunological response but also on
their effect on osteoblastic differentiation. Immunohistochemistry is a
frequently used technique to evaluate the characteristics of tissue in
bone regeneration studies (Christgau et al., 2007, Morgan et al., 2007,
Schwarz et al., 2007). Paraffin is the standard embedding medium for
immunohistochemistry. The major disadvantage, however, of paraffin
embedding is that it requires bone decalcification, a time consuming
process that eliminates essential information about mineralization and
locations of recent bone formation.
Routine histology of resin-embedded undecalcified bone has been

practiced for over 40 years (Burkhardt, 1966, Schenk, 1965, Te Velde
& Haak, 1977). However, Knabe et al. (2006) stated that it can be
hard to visualize the expression of immunological and osteogenic
markers in undecalcified sections of bone. This handicap is partly
because of the highly exothermic polymerization procedure of classic
methylmethacrylate (MMA) destroying both enzyme activity and tissue
antigenicity. More recently, low temperature embedding resins
preserving antigenicity combined with improved embedding
techniques have been developed allowing immunohistochemical
analysis of resin-embedded undecalcified tissue samples (Johansson
et al., 1999, Roser et al., 2000). Yang et al. (2003) demonstrated good
histological and immunolabelling results with several bone matrix
markers for human, bovine and ovine undecalcified cancellous bone
embedded in Technovit 9100 New® (Heraeus Kulzer GmbH,
152
Wehrheim, Germany). Immunohistochemical evaluation of

undecalcified bone samples using this improved embedding technique
have not been documented in goats despite their growing popularity
as animal models in bone and cartilage regeneration research (An &
Friedman, 1999, Cancedda et al., 2007, Dai et al., 2005, Lamerigts et
al., 2000, Pearce et al., 2007). A major handicap is the lack of goat-
specific primary antibodies.
The purpose of this study was to evaluate several commercially

available antibodies on Technovit 9100 New® (Heraeus Kulzer
GmbH) embedded goat tissues. Such techniques having adequate
staining are certainly useful for analysis of bone regeneration
experiments using the goat as animal model.
153
Fixation and embedding
In a previous study by the same authors, three different polymer

bone replacement mixtures were tested in a unilateral cortical tibial
defect model in eight goats. The polymers were randomly inserted in
one of four 6.0-mm-diameter defects leaving a fourth defect unfilled as
control. Biocompatibility and bone-healing properties were evaluated
by serial radiographies, histology and histomorphometry (Vertenten et
al., 2008). A sample of the tibial cortex containing a 4-week-old
cortical control defect of a goat used in this study and different 'normal'
tissue samples from another goat not included in the bone
regeneration study including tibial cortex, liver, lung, muscle and
lymph node were harvested. All samples were fixed in formalin 10%
for 12 h. The samples were rinsed with tap water and dehydrated at
4°C using an ethanol gradient (48 h in 50, 75 and 96% and 72 h in
100% ethanol). Afterwards, the samples were defatted in xylene for 48
h at 4°C and embedded in destabilized Technovit 9100 New®
(Heraeus Kulzer GmbH) which then polymerized for 24 h at 0°C. Four
micrometre sections were cut with an osteomicrotome (SM2500®;
Leica Microsystems, Wetzlar, Germany), stretched with 70% ethanol
on a slide and dried for 12 h at 60°C.
154
Immunohistochemistry
Antibodies
Different antibodies for the immunohistologic evaluation of the

samples were tested in appropriate dilutions:
1. polyclonal rabbit antibodies against human T lymphocytes

CD3 (Dako A0452, Heverlee, Belgium) and B lymphocytes
CD20 (Neo Markers RB-9013-P, Duiven, the Netherlands),
and antibodies for identification of osteoblast differentation
using the core-binding factor alpha-1 (cbfa-1) (alpha
Diagnostic Int Inc Cat.#CBFA11-A, San Antonio, TX,
USA);
2. monoclonal mouse antibodies against:
• human mitotic cells KI67 (Bio SB BSB5709, Santa

Barbara, CA, USA), endothelium cells CD31 (Dako
M0823), macrophages MAC387 (Serotec MAC387,
Oxford, UK), B lymphocytes CD20cy clone L26 (Dako
M0755), B lymphocytes CD79 clone HM57 (Dako
M7051), osteocalcine (OC) (Biogenex RTU 386M, the
Hague, the Netherlands) and plasma cells CD45
(UCDavis CA21.4B3, Davis, CA, USA);
• bovine vimentin (Vim) for identification of

mesenchymal cells (Dako M7020);
• human, mouse and rat alkaline phosphatase (AP)

(R&D Systems MAB1448, Abingdon, UK).
155
Immunolabelling
Prior to immunohistochemical staining, sections were de-acrylized

by immersing the sections in xylene (2 × 20 min), 2-
methoxyethylacetate (1 × 20 min), acetone (2 × 5 min) and distilled
water (2 × 2 min). Initially epitope retrieval was achieved using the
microwave with pressure cooker protocol (Taylor et al., 1996).
Because this technique induced destruction of the samples, antigen
retrieval was performed using proteinase K (Dako) for 15 min at 21°C.
The sections were rinsed with distilled water (2 × 5 min) and
phosphate-buffered saline (PBS) (2 × 5 min). Endogenous peroxidase
was quenched by incubating the sections in a 3% H2O2 solution in
methanol for 5 min. This was followed by rinsing in distilled water (2 ×
5 min) and PBS (2 × 5 min). The sections were pre-incubated in
bovine serum albumin during 30 min at 25°C and rinsed with PBS (2 ×
5 min). Subsequently, immunohistochemical staining was performed
using the primary antibody for 2 h at 25°C. The sections were then
rinsed with PBS (2 × 5 min) and incubated with biotinylated goat anti-
mouse for the monoclonal primary antibodies and biotinylated goat
anti-rabbit for the polyclonal antibodies for 30 min at 21°C. The
sections were again rinsed and incubated with avidin-biotin complex-
horseradish peroxidase (ABC-HRP) (Dako) for 30 min at 21°C.
Afterwards, the samples were rinsed (2 × 5 min) with PBS and
incubated in 3,3'-diaminobenzidine (DAB) (Sigma, Bornen, Belgium)
for 5 min and finally with distilled water (2 × 5 min). Potential
counterstaining was done by immersing the sections in haematoxyline
for 10 s, tap water for 1 min and distilled water (20 times up and
down). Finally, the sections were dehydrated and covered.
The above described protocol was used for all immunostainings

except for CD3, KI67 and CD31 stainings. For CD3, staining the PBS
was changed by tris-buffered saline. Immunohistochemical staining for
KI67 was performed using the primary antibody for 12 h at 21°C.
156
The tibial cortex sample containing a 4-week-old defect was

stained using anti-cbfa-1 and anti-OC antibodies. Native tibial cortex
(without a defect) was stained using anti-OC and anti-AP. Liver
samples were stained with anti-CD31 and anti-MAC387 antibodies.
Lung and muscle tissue were used to perform immunostaining for
MAC387 and Vim, respectively. The lymphe node was stained for
KI67, CD3, CD20, CD20cy, CD79 and CD45.
All stained sections were individually analysed by standard light

microscopy. The tissues were examined for antibody attachment to
cellular and matrix components.
157
RESULTS
The results of the immunohistochemical stainings and the optimal

antibody dilution of the positive stainings are represented in Table 1.
Table 1. Results of immunostainings of several tissues of goats

stained with an appropriate dilution of the primary antibody.
tibial cortex normal liver lung muscle lymphe
with defect tibial cortex node
KI67 neg
Vim 1:200
CD31 neg
Cbfa-1 neg
OC 1:2 1:2
AP neg
MAC387 1:100 1:100
CD3 1:100
CD20 neg
CD20cy neg
CD79 neg
CD45 neg
Blank, not tested; neg, negative immunostaining; 1:x, the best

immunostaining obtained when the commercial available primary
antibody is x times diluted.
Negative immunohistochemical staining was observed for KI 67,

CD31, cbfa-1, AP, CD20, CD20cy, CD79 and CD45. Immunostaining
for Vim in muscle demonstrated positive staining (brown colouring) of
the wall of the capillaries between the muscle cells and fibres together
with some slight staining of fibrous tissue between the muscle fibres
158
(Fig. 1). Anti-OC produced a metachromatic staining of the tibial

cortex characterized by the presence of an intensive purple colour
(Fig. 2). Even more, the stainings on tibial cortex sections without use
of primary antibody and immunostaining for AP resulted in the same
metachromatic staining of cortical bone tissue. OC staining without
counterstaining demonstrated positive staining of the bone tissue as it
was generally light brown coloured (Fig. 3). OC staining of the tibial
cortex sample containing a 4-week-old control defect showed intense
positive (brown) staining of the cells and slight positive (brown)
staining of the matrix around the newly formed lamellar bone tissue,
which stained metachromatically (Fig. 4). MAC387 staining revealed
positive staining of several cells in the alveolar tissue (Fig. 5) and in
the cells around hepatic vessels (Kuppfer cells) (Fig. 6). Positive
staining of lymphatic nodules became clear with CD3-staining. The
greater part of the nuclei of the cells between the lymphatic nodules
was surrounded by a positive-stained border (Fig. 7).
159
Figure 1. Resin-embedded goat muscle stained immunohistochemically for

vimentin (magnification ×20). 1, artery; 2, transverse section of a muscle fibre;
3, fibrous tissue between muscle fibres; 4, nucleus of muscle cell.
Figure 2. Resin-embedded goat full thickness tibial cortex stained

immunohistochemically for osteocalcin (magnification ×60).
160
Figure 3. Resin-embedded goat full thickness tibial cortex stained

immunohistochemically for osteocalcin without counterstaining (magnification
×60).
Figure 4. Resin-embedded goat tibial cortex with a 4-week-old defect stained

immunohistochemically for osteocalcin (magnification ×20). 1, mesenchymal
stromal tissue; 2, newly formed bone (metachromatic staining); 3, osteoblasts.
161
Figure 5. Resin-embedded goat lung stained immunohistochemically for

MAC387 (magnification ×100). 1, alveolus; 2, pneumocytes; 3, positively
stained macrophages.
Figure 6. Resin-embedded goat liver stained immunohistochemically for

MAC387 (magnification ×60). 1, hepatocyt; 2, sinusoids; 3, central vein; 4,
positively stained Kuppfer cells (macrophages).
162
Figure 7. Resin-embedded goat lymph node stained immunohistochemically

for CD3 (magnification ×60). 1, paracortical area containing mostly positively
staining T cells; 2, lymphoid follicle.
163
DISCUSSION
The use of in vivo animal models in bone regeneration research is

often an essential step before proceeding with human trials (Pearce et
al., 2007). The goat has been reported as being a suitable animal
model, especially for the similarities in metabolic and bone
remodelling rate compared with men (Cancedda et al., 2007, Pearce
et al., 2007, Spaargaren, 1994). Dai et al. (2005) further supported the
use of goats for bone-healing studies because of their comparable
bone-healing capacity and tibial blood supply. Lamerigts et al. (2000)
also demonstrated that the mechanisms accompanying bone graft
incorporation during bone-healing is similar in humans and goats.
However, the rate at which a bone graft is revascularized and
converted into a vital cancellous structure was reported to be faster in
the goat (Pearce et al., 2007). There is little information comparing the
pros and cons of the use of goats versus sheep for implant-related
bone regeneration studies. Milk goats were preferred over sheep in
this study because of their easiness in handling during all procedures
needed before, during and after surgical procedures (Vertenten et al.,
2008).
The ability to characterize the in vivo tissue responses at a

molecular level can be helpful to improve understanding and
evaluation of graft integration in receptor tissues. The use of several
immunohistochemical stainings on undecalcified bone sections has
been reported in man, rat, bovine, sheep and dogs (Knabe et al.,
2006, Rammelt et al., 2007, Schwarz et al., 2007, Yang et al., 2003).
To the authors' knowledge, similar reports have not been published in
goats.
Various protocols have been described for antigen retrieval prior to

immunohistochemical staining including heat and proteolytic-induced
techniques. Many papers have compared different methods in search
164
of the gold standard (Elias et al., 1999, Fraenkel-Conrat et al., 1947,

Fraenkel-Conrat & Olcott, 1948, Hunt et al., 1996, Kahveci et al.,
2003, Pileri et al., 1997, Ramos-Vara & Beissenherz, 2000, Shi et al.,
1991, Taylor et al., 1996). To the authors' knowledge, these
techniques have not been described for use in goat bone tissue
samples embedded in low temperature methylmethacrylate. The 4-
µm-undecalcified sections used in this study were very fragile
requiring a more-than-gentle manipulation. The microwave with
pressure cooker protocol initially used for antigen retrieval was too
aggressive for the routine handling of the sections because fracturing
of the samples and detachment of the samples from the glasses
occurred frequently. A proteolytic protocol using the proteinase K
technique caused less damage to the tissue sections. Knabe et al.
(2006) advocated the use of an ethanol-based fixative in undecalcified
sheep mandibles to preserve the antigenicity of the tissues and avoid
the need for high-temperature recovery of epitopes. Although this
method was not further investigated in this study, the authors are
convinced that this technique also needs more explorative research in
goat tissues. Negative staining in this study might be caused by the
used antigen retrieval technique. Studies using other retrieval
techniques might clarify this hypothesis.
Several specific antigens have been used to assess new bone

formation and immune responses in and around bone grafts (Knabe et
al., 2006, Rammelt et al., 2007, Yang et al., 2003). The antibodies
used in this study were specifically selected for immunohistochemical
analysis of early bone tissue response to grafting of cultured
osteoprogenitor cells combined with a synthetic scaffold in a goat tibia
model. The antibodies allow evaluation of histocompatibility (anti-CD3,
anti-CD20, anti-CD79, anti-CD45 and anti-MAC387), identification and
characterization of cell proliferation and mesenchymal cells (anti-KI67,
anti-Vim), identification of blood vessel ingrowth in the bone graft
(anti-CD31) and new bone formation (anti-cbfa-1, anti-OC, anti-AP
and anti-MAC387).
165
Only a few studies have reported on the use of

immunohistochemistry in low temperature MMA-embedded
undecalcified bone sections (Knabe et al., 2006, Rammelt et al., 2007,
Schwarz et al., 2007, Yang et al., 2003). Just as it was the case with
this study, some researchers also encountered negative
immunostainings with other specific markers. Yang et al. (2003)
produced positive immunostaining for AP on human cancellous bone
using a specific commercial marker, but the same technique yielded
negative results on bovine and ovine bones. Rammelt et al. (2007)
reported positive staining for CD3 and keratin markers on human
bone but rat and ovine bone did not stain positive. Furthermore, they
were not able to produce reliable staining results for several specific
markers including bone sialoprotein, CD8, CD31, CD44 and TGFβ
with rat, sheep or human specimens. Yang et al. (2003) explained that
the fact that anti-human AP did not recognize bovine or ovine AP was
partly because of it being a monoclonal antibody. In this study using
goat tissues, positive stainings with anti-Vim, anti-OC, anti-MAC387
and anti-CD3 were demonstrated. The absence of positive results
using the other markers might partly be caused by a possible
incomplete epitope retrieval associated with the use of the proteinase
K technique. Second, different authors (Rammelt et al., 2007, Ramos-
Vara & Beissenherz, 2000) have postulated that positive
immunohistochemical staining results largely depend on the
characteristics of the antibody used, the species in which the antibody
is applied and finally the technical specifications of the laboratory
protocol. It has been demonstrated that the majority of commercially
available antibodies have no cross-reactivity between all species and
that different antibodies are available for detection of one specific
antigen (Ramos-Vara & Beissenherz, 2000). These considerations
stimulate the further evaluation of other commercially available
variants of specific markers which might yield other positive results in
goat tissue.
166
A surprising finding in this study was the detection of a

metachromatic staining of mature bone in every OC or AP staining
used on bone tissue. Although the authors have no direct explanation
for this finding, the counterstaining had to be directly related to this as
no metachromatic staining was found when counterstaining was
omitted from the protocol. Because of its persistent occurrence, the
presence of a metachromatic colouring might be used as an indicator
for the presence of mature bone.
167
CONCLUSION
This study resulted in the optimization of four staining techniques

(Vim, OC, MAC387 and CD3) which can be routinely used for
evaluation of goat tissues for the presence of mesenchymal tissue
(Vim), bone formation and remodelling (OC and MAC387), T-cells
(CD3) and macrophages (MAC387) in goat tissues using
methylmethacrylate-embedded samples. All these staining techniques
can be useful for the evaluation of the osteo-integration of bone
substitutes in goat undecalcified tissues. Even more, with these
antibodies a sequential evaluation of the bone regeneration process
can be performed, allowing monitoring of the proliferation of
osteogenic cells, the production of mesenchymal and bone tissue in
and around bone substitutes as well as the biocompatibility of the
bone substitutes in goats. Implementation of different antigen retrieval
protocols and the use of alternative commercially available antibodies
for the same markers might further supplement and complete the
possibilities for immunohistochemical analysis of goat tissue samples
in experimental bone regeneration research.
168
PART 4.1: Acknowledgements
ACKNOWLEDGEMENTS
The authors thank Cindy De Baere, Sarah Loomans, Bart De Pauw

and Leen Pieters for their excellent technical assistance.
169
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172
4.2
Agreement between micro-computed
tomography and histomorphometry for
evaluation of new bone formation in a
tissue-engineered cortical tibial goat
model
Submitted as: G. Vertenten, E. Lippens, M. Dierick, L. Van Hoorebeke, E.
Schacht, M. Cornelissen, F. Gasthuys, L. Vlaminck. Agreement

between micro-computed tomography and histomorphometry for
evaluation of new bone formation in a tissue-engineered cortical
tibial goat model.
to European Cells & Materials.

PART 4.2: Summary
SUMMARY
In recent years, micro-computed tomography (µCT) has

emerged as an evaluation technique for new bone formation in
tissue-engineering models. µCT protocols are currently quite
different depending on the used equipment and software. The
aim of this study is to validate a high resolution µCT scanner as a
tool for assessing the micro-architecture of regenerating cortical
bone by evaluating the correlation with results obtained by
histomorphometry. A non critical sized tibial defect model in
goats was used. Technovit 9100 New embedded tibial defect
samples were scanned (2D and 3D) and analyzed with an in-
house developed µCT scanner and then histomorphometrically
analyzed on 4µm sections. Bone volume density, bone surface
density, bone-specific surface, trabecular thickness and
trabecular separation were calculated. There were strong positive
correlations between histomorphometric analysis, 2D and 3D
µCT for bone volume density analysis. The difference plot
method reported by Bland and Altman revealed agreement
between the used methods. Our results show that µCT analysis
is a useful and adjunctive tool for imaging and nondestructively
quantifying new bone healing in bone tissue engineered models.
175
Agreement between µCT and histomorphometry
INTRODUCTION
For bone regeneration purposes, autografts are still considered the

“golden standard”, but inherent disadvantages include limited
availability of sufficient quantity and possible donor morbidity (Damien
& Parsons, 1991). Prosthetic materials overcome some of these
issues, but their effectiveness is limited by unpredictable graft
resorption, infection, structural failure, and/or unsatisfactory aesthetic
outcomes. The search for a reliable implantable material has spurred
a new line of research on biocompatible scaffolds.
In biomaterials and tissue engineering research, multiple research

methods such as histology, immunohistochemistry and
histomorphometry are combined to understand scaffold-to-tissue
relation and interaction (Cuijpers et al., 2009). Analysis of the bone
healing process is routinely done using two-dimensional (2D)
analyses techniques such as radiography and histomorphometry
based on Von Kossa and alizarin red positive distribution analysis
(Vertenten et al., 2009, Barros et al., 2003). More adapted
methodologies have been developed for research on bone healing,
including microradiographic analysis, mechanical testing,
densitometry and the development of new specific biological markers
for immunohistochemical analysis using light microscopy (Vertenten et
al., 2008a, Cornelis et al., 2008, Tyler et al., 2008, Saran & Hamdy,
2008).
Because bone formation is a 3D process, techniques that offer 3D

analysis of tissue samples would be able to provide more detailed
information on the complex changes that occur during bone healing
within tissues. In recent years, micro-computed tomography (µCT) has
gained more interest as a technique to analyze calcified structures in
bone tissue engineering experiments (Cuijpers et al., 2009). It allows
non-destructive and high-resolution imaging of different kinds of
176
objects. It produces 2D slices where contrast is generated by

differences in X-ray absorption that arise from a mixed combination of
density and compositional information within the object. 3D models
can be generated by reconstruction from 2D slices (Chappard et al.,
2005). The 3D data can be used as well for descriptive as for
objective analyses regarding bone regeneration. Several objective
parameters can be measured on bone samples using µCT (e.g. bone
volume, total volume, bone surface, trabecular thickness, trabecular
separation, trabecular number, interconnectivity index, number of
nodes, number of terminus, node-to-node strut count, node-to-
terminus strut count, terminus-to-terminus strut count, marrow space
star volume, Euler number, and fractal dimension) and can be
compared with the same parameters obtained using
histomorphometry as an analysis tool (Cortet et al., 2004).
Some studies have reported correlations between

histomorphometric and µCT analysis (Cortet et al., 2004, Muller et al.,
1998, Odgaard, 1997, Park et al., 2005, Uchiyama et al., 1997). Most
of these studies focused on metabolic diseases causing changes in
bone mineral density of cancellous bone (Chappard et al., 2005,
Cortet et al., 2004, Muller et al., 1998, Uchiyama et al., 1997). Others
have used the technique for the analysis of the degree of
mineralization in intramembranous bone (Buchman et al., 1998, Verna
et al., 2002, Dalstra et al., 2001) or the investigation of the correlation
between histomorphometry and µCT in intramembraneous bone
healing (Yeom et al., 2008). Few authors have published on µCT
analysis of cortical bone healing (Basillais et al., 2007, Raum et al.,
2006). Moreover, the correlation between histomorphometry and µCT
in cortical bone healing is still unclear. The aim of this study is to
validate a high resolution µCT scanner as a tool for assessing the
micro-architecture of regenerating cortical bone by evaluating the
correlation with results obtained by histomorphometry. As hypothesis
we assumed that 2D and 3D µCT analysis of cortical bone healing
provides comparable results to histomorphometric analysis (golden
177
standard). For this purpose a non critical sized tibial defect model in
goats was used (Vertenten et al., 2008b).
178
Bone biopsies
In a previous study by the same authors (Lippens et al., 2009),

three different polymer bone replacement (Pluronic ALA-L, Pluronic
ALA-L mixed with autologous bone marrow derived mesenchymal
stem cells (BMSC) loaded onto CultiSpher-S®, Pluronic ALA-L mixed
with autologous BMSC loaded on hydroxyapatite pipes) mixtures were
tested in a unilateral cortical tibial defect model in goats. The polymers
were randomly inserted in one of four 6.0-mm-diameter defects
leaving a fourth defect unfilled as control. Biocompatibility and bone-
healing properties were evaluated by serial radiographies, histology
and histomorphometry. Standardized samples of the tibial cortex
containing 4-week-old defects were harvested and embedded in
destabilized Technovit 9100 New (Heraeus Kulzer, Wehrheim,
Germany) as described earlier (Vertenten et al., 2008b).
µCT analysis
The samples were scanned at the in-house developed µCT

scanner of the Centre for X-ray Tomography at the Ghent University
(UGCT) (Masschaele et al., 2007). This modular CT scanner offers
resolutions down to 1 micrometer, can handle samples up to 20 cm
diameter and has a 20-160 kV tube voltage range. It has a dual head
X-ray source (a high resolution low power transmission tube head and
a lower resolution high power tube head). These can be combined
with a variety of detectors with different energy range, pixel size and
field-of-view. The samples were embedded in a 2 cm diameter
methacrylate resin. Based on the sample size and composition the
high-power directional tube head was used at a tube voltage of 100
keV and with a beam hardening filter of 550 µm Aluminum. The
179
chosen detector was a Varian Paxscan 2520 flat panel detector with
CsI scintillator. This has 1800x1496 pixels of 127 micron each. The
magnification was around 90, resulting in a voxel size of 10 µm and a
field of view of 15 mm diameter and 18 mm in height. A total of 1000
projections were recorded covering an angle of 360 degrees. These
were reconstructed using the in-house developed reconstruction
software Octopus (Vlassenbroeck et al., 2007). The resulting 3D
volume consisted of 1000 slices of 1496x1496 pixels each. The 3D
morphological analysis was performed using Morpho+ software which
was also developed within UGCT (Vlassenbroeck et al., 2007).
Morpho+ offers real 3D morphological analysis of volume data (as
opposed to a slice-per-slice approach). In order to analyze the newly
formed bone, the cylindrical shaped defect had to be selected as the
volume of interest (VOI). Morpho+ only allows the selection of a VOI
along one of the main axes of the reconstructed volume. Because the
defect could not be aligned exactly to the rotation axis of the µCT
scanner for practical reasons, the reconstructed data had to be
resliced prior to analysis in order to align the VOI with one of the main
axes. This was done in VGStudio Max 2. Once the defect is selected
the bone fraction inside this volume can be quantified by thresholding
the grayscale data based on a dual thresholding technique. This
technique uses two threshold levels. The strongest threshold selects
pixels that are certainly bone, but may not select the entire bone
fraction. The lighter threshold is set to select the entire bone fraction
but may also select pixels that do not belong to the bone fraction, due
to image noise for example. Both levels are then combined to retain
only those pixels that are selected by the lightest threshold and are
connected to pixels selected by the strongest threshold. This
thresholding technique is more accurate and less susceptible to noise
than using a single threshold.
Parameters common to 2D and 3D measurements were chosen to

investigate the relationship between methods. The following
parameters were determined: bone volume density (BVD = BV/TV,
180
-1
%), bone surface density (BSD = BS/TV, mm ), bone-specific surface
-1
(BSS = BS/BV, mm ), trabecular thickness (TT), and trabecular
separation (TS). The 2D measurements were done on slices that were
made perpendicular to the longitudinal axis of the tibia and situated in
the central part of each defect. This corresponds with the sections
used for histomorphometric analysis.
Histomorphometric analysis
After completion of the 2D and 3D image acquisition using µCT,

the samples were cut perpendicular to the longitudinal axis of the tibia
with a heavy duty microtome (SM2500®, Leica Microsystems,
Wetzlar, Germany) to obtain 4 µm sections which were stained with
von Kossa. Histomorphometric analysis was performed on the
M
sections using specific image software program (Cell , Imaging
Software for Life Science Microscopy; Olympus, Belgium). Only
sections from bone tissue located in the central part of each defect
were analyzed as these corresponded with the bone levels analyzed
using 2D µCT analysis. The same parameters as with µCT were
calculated (bone volume density, bone surface density, bone-specific
surface, trabecular thickness and trabecular separation).
Statistical methods
Data were used to evaluate the correlation between measuring

techniques. Mean measurements were summarized by the number of
observations. Pearson’s correlation coefficient was used to assess the
strength of the linear relationship between the histomorphometric
measurements and respective 2D and 3D µCT analyses. To assess
how well 2D and 3D measurements agree with histomorphometric
measurements, paired t-tests were calculated. The paired t-tests
assess whether there is a significant non-zero mean difference
between measurements from the samples. The difference plot method
181
reported by Bland and Altman (Bland & Altman, 1986) was also used
to assess the agreement between methods. Statistical processing was
done by SPSS 15 for Windows at the 5% global significance level.
182
PART 4.2: Results
RESULTS
µCT analysis could be performed on all 16 samples. All µCT

images were of good quality and all parameters could be easily
calculated. Newly formed bone entered the created gap from the
borders. 2D (Fig. 1B, C) and reconstructed 3D images (Fig. 1A) nicely
illustrated the regeneration process in the defects. A remarkable
finding was the rough border of the created cylinder which was clear
on the 2D images parallel to the axis of the tibia (Fig. 1B). Only 13
samples could be used for histomorphometrical analysis as technical
difficulties excluded 3 samples from further processing (Fig. 1D).
Figure 1. µCT and histomorphometric images of a unicortical cylindrical tibial

goat defect of 6mm diameter filled with Pluronic ALA-L 4 weeks post surgery.
A: reconstructed 3D µCT image; B: 2D µCT image parallel to the tibial axis; C:
2D µCT image perpendicular to the tibial axis; D: photomicrograph of a Von
Kossa stained undecalcified 4 µm section perpendicular to the tibial axis.
183
Table 1 presents correlations for the three different methods

calculating BVD. There were strong positive correlations between
histomorphometric analysis (BVDhisto), 2D (BVD2D) and 3D µCT
(BVD3D) measurements (Pearson’s correlation coefficient varying
between 0.75 and 0.87). However, mean BVD measured by the
histomorphometric analysis was more than double of the values
obtained with 2D and 3D µCT .
Table 1. Bone volume density between histomorphometric (BVDhisto) and two-

dimensional (BVD2D) or three-dimensional (BVD3D) micro-computed tomography
measurements.
Pearson's Correlation
Coefficient
n mean SD histo 2D 3D
BVDhisto (%) 13 27 14.606 1 0.75* 0.77*
BVD2D (%) 16 12.063 7.47 0.75* 1 0.87*
BVD3D (%) 16 12.875 11.057 0.77* 0.87* 1
*: statistically significant for P = 0.05.
The correlations for BSD and BSS measurements between the

three different analyzing methods were moderate and only
significantly correlated for BSD measured by histomorphometry
(BSDhisto) and 2D µCT (BSD2D) and BSS measured by 2D (BSS2D)
and 3D µCT (BSS3D) (Tables 2 and 3). Bland-Altman’s plots revealed
good agreement among the three different analyzing methods for BSD
and BSS (Fig. 2). However, absolute values for BSD and BSS were
higher by histomorphometry compared to µCT measurements (Table
2 and 3). Mean value for BSD obtained with histomorphometrical
analysis tripled mean values measured by µCT analysis (Table 2).
184
PART 4.2: Results
Table 2. Bone surface density between histomorphometric (BSDhisto) and two-

dimensional (BSD2D) or three-dimensional (BSD3D) micro-computed tomography
measurements.
Coefficient
BSDhisto (1/mm) 13 18.932 7.977 1 0.63* 0.46
BSD2D (1/mm) 16 6.677 2.734 0.63* 1 0.21
BSD3D (1/mm) 16 4.446 2.18 0.46 0.21 1
Table 3. Bone-specific surface between histomorphometric (BSShisto) and two-
dimensional (BSS2D) or three-dimensional (BSS3D) micro-computed tomography
measurements.
Coefficient
BSShisto (1/mm) 13 76.087 17.207 1 0.3 0.43
BSS2D (1/mm) 16 61.813 22.257 0.3 1 0.66*
BSS3D (1/mm) 16 46.659 13.145 0.43 0.66* 1
The correlations for TT and TS were moderate with significant

correlation between TT measurements for 2D (TT2D) and 3D µCT
(TT3D) and TS measurements for histomorphometry (TShisto) and 3D
µCT (TS3D) (Table 4 and 5). Also for TT and TS Bland-Altman’s plots
revealed acceptable agreement between the three different analyzing
methods (Fig. 2).
185
Table 4. Trabecular thickness between histomorphometric (TThisto) and two-

dimensional (TT2D) or three-dimensional (TT3D) micro-computed tomography
measurements.
Coefficient
TThisto (µm) 13 32.769 22.756 1 0.16 0.54
TT2D (µm) 16 35.563 11.105 0.16 1 0.56*
TT3D (µm) 16 36.125 12.988 0.54 0.56* 1
Table 5. Trabecular separation between histomorphometric (TShisto) and two-
dimensional (TS2D) or three-dimensional (TS3D) micro-computed tomography
measurements.
Coefficient
TShisto (µm) 13 97.539 61.218 1 0.42 0.79*
TS2D (µm) 16 285.063 174.244 0.42 1 0.41
TS3D (µm) 16 455.813 288.629 0.79* 0.41 1
Results of paired t-tests for the 5 parameters are shown in Table 6.

The mean of differences in measurements for the same samples were
represented by P values. Histomorphometric analysis resulted in
generally higher values (except for BSShisto and BSS2D) than
measurements obtained by µCT for BVD, BSD, BSS. Comparison
between 2D and 3D µCT were significantly different for BSD and BSS
(P = 0.012 and 0.003) but not for BVD (P = 0.589). There were no
significant differences between the three different methods for
measurement of the TT. On the other hand, the three different
methods revealed statistical significant different values for TS. TS
values obtained by histomorphometric analysis were significantly
lower compared to 2D and 3D measurements.
186
PART 4.2: Results
Figure 2. Relationships between three measurement methods for bone

-1
surface density (BSD, mm-1) bone specific surface (BSS, mm ), trabecular
thickness (TT, µm) and trabecular separation (TS, µm) by Bland-Altman’s
difference plots.
187
Table 6. Summary of paired comparisons

P
Difference n mean SD
Value
BVDhisto - BVD2D 13 14.538 10.096 < 0.001
BVDhisto - BVD3D 13 12.692 9.358 < 0.001
BVD3D - BVD2D 16 0.813 5.879 0.589
BSDhisto - BSD2D 13 11.968 6.544 < 0.001
BSDhisto - BSD3D 13 14.136 7.228 < 0.001
BSD3D - BSD2D 16 -2.231 3.11 0.012
BSShisto - BSS2D 13 11.994 25.108 0.111
BSShisto - BSS3D 13 28.381 17.048 < 0.001
BSS3D - BSS2D 16 -15.154 16.801 0.003
TThisto - TT2D 13 -2.077 16.297 0.654
TThisto - TT3D 13 -0.692 20.686 0.906
TT3D - TT2D 16 0.563 18.984 0.907
TShisto - TS2D 13 -175.385 173.936 0.003
TShisto - TS3D 13 -302.769 223.533 < 0.001
TS3D - TS2D 16 170.75 269.641 0.023
188
DISCUSSION
Several methods to evaluate bone healing/regeneration are

currently available. Descriptive methods such as histology,
histomorphometry and immunohistochemistry are often combined with
image analysis to obtain the maximal amount of information on the
bone healing process (Vertenten et al., 2009). However, at this
moment each method has inherent disadvantages. Histology,
histomorphometry and immunohistochemistry evaluate tissue
characteristics on 2D slices taken out of a 3D structure such as bone.
This may lead to different outcome results depending on the position
of the histological slice in the total 3D structure. Furthermore, it is not
always obvious to extrapolate results from two-dimensional analysis of
tissue samples to the three-dimensional in vivo situation. The
preparation of histological slides is very time consuming and as
paraffin is the standard embedding medium for histological analysis,
more time consuming preparation is needed to decalcify the tissue
samples. Essential information about the ongoing mineralization
process and the sensitivity to locate ‘hot spots’ of active bone
formation is lost when using decalcified bone histology (Yang et al.,
2003). Resin embedded tissue allows histological examination of
undecalcified bone but includes the major challenge of preparing
tissue sections thin enough to allow light microscopic evaluation
(Blokhuis et al., 2000, Kessler et al., 2001, Vertenten et al., 2009).
With the heavy-duty microtome used in the present study,
undecalcified sections of 4 µm could be made. However, 3 samples
were of too poor quality for histological analysis due to inadvertent
tearing of the sections. This complication is frequently encountered
during the processing of undecalcified tissue samples especially when
the tissues contain centers of different hardness. Moreover, the
embedding process is crucial, because the whole tissue should be
189
fully impregnated by the resin, and air bubbles should be removed.

Good impregnation and preventing air bubbles is possible by working
under constant vacuum during the embedding process.
Every research group is actually using its own protocol for

histomorphometrical analyses based on general principles but with its
particular differences. Different embedding protocols, microtomes,
slice thicknesses, staining protocols, stain concentrations, thresholds,
evaluation protocols and material, to identify the investigated tissue,
have been used in several studies (Barros et al., 2003, Vertenten et
al., 2009, Chappard et al., 2005, Cortet et al., 2004, Muller et al.,
1998, Park et al., 2005, Uchiyama et al., 1997, Yeom et al., 2008).
During the thresholding process, individual pixels in an image are
marked as “object” pixels if their value is greater than some threshold
value (assuming an object to be brighter than the background) and as
“background” pixels otherwise. This convention is known as threshold
above. Variants include threshold below, which is opposite of
threshold above; threshold inside, where a pixel is labeled "object" if
its value is between two thresholds; and threshold outside, which is
the opposite of threshold inside (Sezgin & Sankur, 2004). This makes
it very difficult to compare studies. Also immunohistochemical analysis
has some typical problems that preclude standardization of the
technique: various protocols have been described for antigen retrieval
prior to immunohistochemical staining, the majority of commercially
available antibodies have no cross-reactivity between all species,
different antibodies are available for the detection of one specific
antigen, and quantification of the technique is not straight forward
(Ramos-Vara & Beissenherz, 2000, Vertenten et al., 2008a). 2D
techniques are unable to quantitatively supply measurements of
certain useful 3D architectural properties of new formed bone (Kuhn et
al., 1990). The need for more accurate and more reproducible data in
the evaluation of bone healing mandates the development of
technologically advanced approaches for the analysis of newly formed
bone.
190
µCT is an example of just this type of technologically advanced

approach serving as a highly accurate and automated tool to precisely
measure changes in bone stereology, bone volume and projection,
and bony microarchitecture. µCT technology is capable of measuring
changes in the 3D architecture of bone providing the ability to directly
analyze the organization and ultra-structure of specimens in three
dimensions (Buchman et al., 1998). However, a big disadvantage of
µCT is the fact that tissues can only be distinguished when they have
a different density. An evaluation at cellular and molecular level is still
impossible with µCT but obvious with histology and
immunohistochemistry respectively. Moreover, µCT protocols are
currently quite different depending on the used equipment and
software. A standardization and user-friendly analysis method of µCT
is necessary.
Differences appeared in the morphometric results presented in our

study when comparing 2D and 3D µCT data with histomorphometric
analysis and significant linear correlations were only obtained
between the various techniques in less than 50% of the comparisons.
However, to evaluate the agreement between two or more techniques,
the estimation of the Pearson’s correlation coefficient is clearly
inadequate. In certain medical areas the comparison of a new method
with a reference method plays a major role in the statistical evaluation
of experiments. A large body of the chemical literature is concerned
with statistical analyses for method-comparison experiments: the use
of regression analysis tends to be discouraged and replaced with
other graphical presentations in the form of a difference/average plot
according to Bland-Altman (Hollis, 1996). The Bland-Altman plots are
useful to reveal a relationship between the differences and averages
(indicating a proportional error), to identify a systematic bias and
outliers. If there is no bias, the mean of differences is centered on 0; in
other words, the mean difference ± 1.96 standard deviation (SD) can
easily be plotted on the graph. As in this study the mean difference ±
1.96 SD can easily be plotted on the Bland-Altman graphs illustrating
191
the use of µCT as a valid tool for evaluating the bone micro-
architecture in bone regeneration models. We could obtain
quantitative measurements of bone morphology and stereology in
three dimensions on all specimens. We have shown that among the
parameters investigated in the present study BVD showed the
strongest agreement between the three evaluation methods (3
statistically significant Pearson’s correlation coefficients). BVD is the
most frequently used quantitative parameter to evaluate bone healing
in bone tissue engineering models (Bodde et al., 2008, Kruyt et al.,
2008, Vertenten et al., 2009). The correlation between
histomorphometry and µCT (2D and 3D) analysis in BSD, BSS, TT
and TS was moderate to weak. Mean values of BVD, BSD and BSS
measured by histomorphometry are generally significantly higher than
measured by µCT (2D and 3D) and mean values of TS measured by
histomorphometry are significantly lower than measured by µCT. This
means that in the present study more bone tissue is measured by
histomorphometry than by µCT. In other words, histomorphometrical
analysis significantly overestimates bone volume fraction in a given
tissue or µCT significantly underestimates bone volume fraction. This
was also clear when comparing the Von Kossa stained sections
(Figure 1D) with the comparable 2D µCT images (Figure 1C).
Although µCT images have a quasi-histological appearance, the
trabecular boundaries are less well defined than on histological
sections stained by Von Kossa. Besides it is clear that new bone will
be identified at an earlier time interval with histological evaluation
methods as new bone is less dense than mature bone and thus is
more difficult to highlight using x-ray techniques. On the other hand,
µCT images revealed extra information compared to
histomorphometric analysis. Histological evaluation of the defects
suggests that necrosis of the bone due to burring was not present.
However, a nicely bordered cylinder was not present on µCT images.
At some places cylindrical borders were roughly aligned suggesting
potential bone resorption or crumbling off of small bone pieces.
192
When we compare 2D evaluation methods (histomorphometry and

2D µCT) with the 3D µCT, we observed that most methods are
significantly different except for BVD between 2D and 3D µCT and for
TT. Although all methods are quite similar (Bland-Altman graphs), the
results of the 2D and 3D evaluation methods are in general different. It
is clear that a result calculated on a section at more or less the central
part of the defect can not be blindly extrapolated for the complete
defect.
Different correlations between histomorphometry and 2D and 3D

µCT analysis are described in literature. Muller et al. (1998) evaluated
human cancellous bone using iliac crest bone biopsy samples. A
correlation coefficient of 0.93 for BVD, 0.91 for BSD, and 0.84 for TT
was found when a fan-beam-type 3D µCT (ScancoMedical AG) was
compared with histomorphometric analysis. The correlations in the
present study were much lower (0.77, 0.46 and 0.54 for BVD, BSD
and TT respectively), but we evaluated newly formed bone which is
less dense than mature bone. Cortet et al. (2004) reported
correlations between 3D µCT and histomorphometry by analyzing
human calcaneus that are lower than in the present study. Correlation
coefficients were 0.69 and 0.24 for BVD and TT respectively.
Chappard et al. (2005) reported high correlations by evaluating human
iliac bone from various metabolic bone disease patients with an Elite
Plus µCT. Similar to the present study strongest agreement was
between histomorphometry and µCT (2D and 3D) when BVD was
compared. Chappard et al. (2005) stated that quantities expressed as
a percentage are more comparable between studies. Yeom et al.
(2008) analyzed the micro-architecture in intramembraneous bone
regeneration and found strong correlation between histomorphometry
and µCT (2D and 3D) in BVD but not for the other parameters, similar
to our study and the study by Cortet et al. (2004). However
comparisons between these studies may not be appropriate because
of differences between used protocols for histomorphometry
(standardized processing, staining and thresholding) and µCT
193
(standardized equipment, processing and thresholding).

Nevertheless, a uniform threshold is inappropriate for µCT
measurements because X-ray attenuations through the
nonhomogeneous material are not uniform and there may exist
trabeculae of varying densities throughout the samples (Feldkamp et
al., 1989, Kuhn et al., 1990). In contrast, Muller et al. (1998) used a
uniform threshold for analysis of one type of bone to be able to have
more comparable results within the site. They argued that a uniform
threshold is possible for µCT using a resolution of 14µm for the
evaluation and differentiation of normal and osteoporotic bone in
human iliac bone.
Micro-tomographic imaging is a nondestructive method to quantify

the structural properties of bone in an efficient and quick manner. A
long lasting embedding of the samples before scanning is not
necessary at all. The embedding of the samples was done in this
study to allow further histomorpometric analysis on undecalcified bone
tissue. µCT could evaluate bone healing in the animal model (under
anesthesia) at several time points in vivo. Thus far, successful in vivo
scanning of small animals such as rats and rabbits has been
described and the benefits of longitudinal studies are being
demonstrated (Voor et al., 2008, Boyd et al., 2006b, Boyd et al.,
2006a, Gasser et al., 2005, Kinney et al., 1995, Laib et al., 2001,
Waarsing et al., 2004a, Waarsing et al., 2004b). Scanning of larger
bone volumes from larger animals such as sheep and goats would be
helpful to take full advantage of the ability of µCT to analyze bone
graft incorporation, implant bone interfaces, and skeletal assessment.
194
PART 4.2: Conclussion
CONCLUSION
We can conclude that µCT imaging is a nondestructive and precise

procedure allowing the measurement of bone healing in unprocessed
biopsies as well as the determination of morphometric indices. Indices
from the µCT analysis are not always in agreement with the indices
assessed from conventional histomorphometry. However, µCT
analysis is a reliable technique to analyze bone healing and
regeneration. In the future, µCT may help to investigate the bone
healing in bone tissue engineering studies as a valuable adjunctive
analysis technique complementary to radiography, histology and
histomorphometry. However a further modification and standardization
of the technique is indispensable.
195
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199
GENERAL DISCUSSION
The engineering of functional bone constructs is a rapidly growing

branch in the field of tissue engineering. A considerable number of
international research groups as well as commercial entities work on
the development of new bone grafting materials, carriers, growth
factors and specifically tissue-engineered constructs for bone
regeneration. There is a strong interest in evaluating the concepts in
highly reproducible large segmental bony defects in preclinical and
large animal models. To allow comparison between different studies
and their outcomes, it is essential that animal models, surgical
procedures including fixation devices, and measurement methods are
well standardised in order to obtain reliable data pools and to create
firm foundations so the development of orthopaedic and tissue
engineering experiments with specifically translation into clinic
applications can be further guided (Reichert et al., 2009).
Development of an in vivo model
The first aim of this PhD study was to evaluate bone enhancing
products in an in vivo model. For that purpose, a new in vivo model
suitable to study bone healing and to screen biomaterials had to be
developed. The translation of tissue engineering concepts from bench
to bedside is a difficult, expensive and time consuming process. It
requires the confirmation of safety and efficacy of tissue-engineered
constructs under investigation.
The entire process can be defined as the “value chain of

regenerative therapies” and consists of: 1) in vitro essays; 2)
screening and/or proof of concept in small animal models; 3)
preclinical studies in large, immunocompetent animals; 4) clinical
201
GENERAL DISCUSSION
studies in a larger patient cohort; and 5) application for FDA approval

and/or CE Mark. We can suppose that the lack of end translation in
the field of bone engineering might be related to the fact that most
groups pursuing their projects no further than the second phase of the
value chain. However, to establish a tissue-engineering concept into a
clinical human setting, a rigorous demonstration of the level of

therapeutic benefit in clinically relevant animal models is a conditio
sine qua non. In literature most of the reported in vivo models focuses
on laboratory animals. Even more, these preclinical models are often
not well documented, defined and/or standardised. Based on a recent
literature review (Reichert et al., 2009), one can conclude that only a
small number of bone engineering groups have established and
validated a well described and standardised preclinical large animal
model.
Animal models in bone repair research include representations of

normal fracture healing, segmental bone defects, and non-union
fractures in which regular healing processes are compromised without
the presence of a critical sized defect site (Tseng et al., 2008). In
critical sized segmental defect models, bridging of the respective
defect does not occur despite an active biological micro-environment
mainly due to the removal of critical amounts of bone substance. In
contrast, biomechanical stimuli or cellular responses may prevent
healing of the defect more than the influence of the defect size. The
use of a critical sized defect model was considered too invasive in the
current stadium of this ongoing PhD research, mainly because of the
need of sophisticated support devices.
The use of uni-cortical cylindrical defects has been reported for

experimental work on bone substitutes and healing by many groups
(Dorea et al., 2005, Griffon et al., 1996, MacNeill et al., 1999, Meinig,
2002, Dalkyz et al., 2000). The medial diaphyseal cortex of the tibia is
preferred for creation of cortical defects because it can be easily
approached with minimal dissection. Furthermore, the ability of these
202
GENERAL DISCUSSION
defects to heal spontaneously is diminished because of the minimal

soft tissue coverage. Poor soft tissue coverage increases not only the
risk of bone loss but also complicates the treatment (DeCoster et al.,
2004).
Fracture models in osteotomized tibiae have also been well

documented in different large animal species. A number of papers
described tibial fracture models in dogs (Gilbert et al., 1989, Tiedeman
et al., 1990, Markel et al., 1990, Faria et al., 2007, Edwards et al.,
2004, Hupel et al., 2001, Jain et al., 1999, Nakamura et al., 1998),
sheep (Gugala & Gogolewski, 2002, Gugala & Gogolewski, 1999, den
Boer et al., 1999, Schemitsch et al., 1994, Tepic et al., 1997, Schell et
al., 2005, Epari et al., 2008, Claes et al., 2008, Gao et al., 1997,
Bloemers et al., 2003, Meinel et al., 2003, Mastrogiacomo et al., 2006,
Wolf et al., 1998, Schell et al., 2008), goats (Liu et al., 2008, Curtis et
al., 1995, den Boer et al., 2002, Xu et al., 2005) and pigs (Bail et al.,
2002, Pek et al., 2008, Raschke et al., 2001).
When selecting a specific animal species as a model system, a

number of factors need to be considered. In comparison to humans or
some domestic animal species including dogs, cats and horses, the
chosen animal model should clearly demonstrate both significant
physiological and pathophysiological similarities respecting the
scientific question that needs to be answered before the investigation.
Moreover, it must be manageable to operate and observe a
multiplicity of study objects post-surgery over a relatively short period
of time (Egermann et al., 2005, Liebschner, 2004). Further selection
criteria include costs for acquisition and care, animal availability,
acceptability to society, tolerance to captivity and ease of housing
(Pearce et al., 2007).
During the last decades, several groups used dogs as model for
human orthopaedic research. Although clear differences in bone
microstructure and remodeling do exist between men and dogs, dogs
have been considered as the animal the closest to humans with
203
GENERAL DISCUSSION
regard to bone weight and density and bone material constituents

such as hydroxyproline, extractable proteins, insulin-like growth factor
I, organic, inorganic and water fractions (Aerssens et al., 1998).
However, the use of dogs as experimental models has recently
decreased mainly due to ethical objections.
In some other study set ups, pigs were considered the animal of
choice because they are a highly representative model of human bone
regeneration processes with respect to anatomical and morphological
features, healing capacity and remodeling, bone mineral density and
concentration (Aerssens et al., 1998, Thorwarth et al., 2005).
However, pig models are often abandoned in favor of sheep and
goats because the handling of this species is rather intricate (Newman
et al., 1995). Furthermore, the length of the tibiae and femora in the
pig is relatively small, which requires the need for special implants, as
one cannot use implants designed for human use (Pearce et al.,
2007).
Mature sheep and goats possess a body weight comparable to

adult humans and long bone dimensions enabling the use of human
implants. Since no major differences in mineral composition exist
between small ruminants and men and both metabolic and bone
remodeling rates are analogous to humans, small ruminants are
considered as a valuable alternative model to study bone turnover and
remodeling activity (den Boer et al., 1999, Anderson et al., 1999). In
the period between 1990 and 2001, sheep as model were used in 9 to
12% of orthopaedic research, compared to only 5% between 1980
and 1989 (Martini et al., 2001). In the last decennium, the number of
studies including sheep and goats as models have even increased to
11 to 15% (O'Loughlin et al., 2008).
Preliminary work prior to this PhD study was originally performed in

sheep using a uni-cortical tibial defect model. Despite an acceptable
and easy surgical technique and postoperative care, 2 out of the 8
sheep involved in this preliminary experimental set-up developed a
204
GENERAL DISCUSSION
tibial fracture shortly after the surgical intervention. A potential

explanation for the occurrence of the fractures is the fleetly character
and herd instinct of sheep whereby sheep often showed abrupt
reactions, mainly when entering the housing facilities (Van
Keymeulen, 2008). Therefore, a non-critical size uni-cortical tibial
defect model was tested in goats. The milk goats used in the present
studies were quiet and easy to handle because they are accustomed
to men and manipulation. However, 2 of the 30 operated goats (of
which 24 included in the described studies) still fractured one leg
during this PhD research proving the importance of gentle
manipulation of the animals.
In the present goat model, relatively small defects of 6 mm

diameter were created which allowed the comparison of several
composites including a control defect in the same leg of one animal.
No major problems were encountered during the surgical interventions
in which standardised tibial defects were created using a standard 6
mm trephine burr. The burring was quite easy to perform and the
produced heat was counteracted by cooling with a saline solution.
Radiographically, the diameter of the defects did not increase at any
time compared to the diameter of the defects after surgery, suggesting
that possible thermal necrosis induced by burring was effectively
avoided. Even more, histological evaluation of the defects confirmed
that necrosis of the bone due to burring did not occur. However, a
nicely bordered cylinder was not observed on µCT images. Indeed, at
some places cylindrical borders were roughly aligned, suggesting
potential bone resorption or crumbling off of small bone pieces most
likely secondary to ‘trauma’, stressing the importance of cooling and
gentle drilling. Gentle handling of the drilling device is imperative to
avoid slipping of the burr but also to prevent the accidental deposit of
the burred cylindrical cortex into the bone marrow. This minor
complication occurred 6 times on a total of 240 burred holes
throughout the experiments.
205
GENERAL DISCUSSION
‘Immobilisation’ of the different bone substitutes in the cortical

defects was enhanced by suturing the periostal layer of the tibia over
the defect which additionally improved healing conditions. Indeed, the
periosteum consists of an outer fibrous layer with collagen fibers and
fibroblasts and an inner cambium layer composed of flattened cells,
the so-called osteoprogenitor cells, which have the capacity to divide
by mitosis and to differentiate into osteoblasts (Nather et al., 2005).
The recovery from general anesthesia of the first series of goats

was relatively long. Recovery times of 2 hours and more were
recorded. This phenomenon was most likely attributed to the
occurrence of post-operative hypothermia since recorded post
surgical rectal temperatures in these first series of goats ranged from
32 to 35.5°C. The low body temperatures associated longer recovery
times were effectively counteracted in the following series of
experiments by increasing the room temperature to 25°C and the use
of water heated pads during surgery to preserve normal body
temperatures.
Evaluation of experimental injectable bone enhancing

products
One disadvantage of the currently used orthopaedic materials for

bone filling purposes is the fixed and firm volume of the majority of the
commercially available bone substitutes, making them unsuitable for
fitting into an irregularly shaped bony defect. A promising alternative is
an injectable and in situ polymerizable material, which allows the filling
of bone defects of various sizes and shapes so less invasive surgical
methods can be applied.
The injectable materials have to fulfill the following requirements

(Temenoff & Mikos, 2000, Peter et al., 1998):
Biocompatibility – the material must not elicit an inflammatory

response nor demonstrate extreme immunogenicity or cytotoxicity.
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GENERAL DISCUSSION
Mechanical properties – these properties must be similar to those

of the tissue that needs to be regenerated.
Promotion of tissue formation – the material should encourage not

only tissue growth but also vascularisation.
Setting time – the material should polymerize in situ in a relatively

short period of time without destructive effects to the surrounding
tissue.
Viscosity, sterilizability and ease of handling.
Nowadays numerous medical applications benefit from in situ

formed biomaterials including the commercially available
photopolymerization of bismethacrylate monomers used in dentistry or
the polymethylmethacrylate based bone cement commonly applied in
orthopaedic surgery (Mano et al., 2004). Although recent advances in
polymer chemistry and processing have induced new methods for in
situ formation of biomaterials, only few polymers are available that can
be formed in situ and possess suitable properties for orthopaedic
applications. Creating scaffolds with the same properties as bone is
still an ultimate goal since hard biomaterials are often not
bioresorbable and even impede the ingrowth of new tissue.
One of the aims of the present PhD work was to evaluate several
experimental in situ crosslinkable and biodegradable composites in
the tibial goat model with the focus on the biocompatible properties
and ease of handling of these scaffolds. This study was performed in
collaboration with the Polymer Material Research Group of the Faculty
of Sciences of the Ghent University and the Department of Basic
Medical Sciences of the Faculty of Medicine and Health Sciences of
the Ghent University. The tested scaffolds were based on
methacrylate-endcapped poly(D,L-lactide-co-ε-caprolactone) in the
first 2 studies and hydrogels in the last one.
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GENERAL DISCUSSION
Table 1. Overview of the results of the experimental work.
Chapter Product Result
Poly-(D,L-lactide-co-ε- Biocompatible
3.1 caprolactone) +HEMA
Moderate osteoconductive
3.1 caprolactone)
More osteoconductive than pure
+HEMA +Bioglass polymer
3.1 caprolactone) +HEMA + α-TCP
More osteoconductive than
polymer+Bioglass
3.2 caprolactone)
Not osteoconductive
+ triacetin
3.2 caprolactone)
Not osteoconductive
+ triacetin +BMSCs
Survival and proliferation of BMSCs
3.2 caprolactone)
Not osteoconductive
+ triacetin +BMSCs
Less survival and proliferation of
+ α-TCP BMSCs than without α-TCP
Plu ALA-L Biocompatible
3.3
Similar to faster healing than empty
control defect
Plu ALA-L Biocompatible
3.3
+ BMSCs (Cultispher-S) Faster healing than empty control
defect
Plu ALA-L + BMSCs (HA) Biocompatible
3.3
BMSCs did not survive and
proliferate on HA.
HA hampered healing
First of all, a variety of in situ, photopolymerizable 3D scaffolds

were synthesized with variables in polymer type (lactide, glycolide, ε-
caprolactone, trimethylene carbonate...), copolymer ratio and initiator
systems. The degradation time, mechanical strength and
biocompatibility of these scaffolds were investigated in depth in
208
GENERAL DISCUSSION
standardised in vitro set ups (Declercq et al., 2006, Jackers, 2002,

Schacht et al., 2004, Gorski, 2006) before they were used in the in
vivo model.
A polymer scaffold should ideally degrade at a rate equal to the

rate of tissue ingrowth, whereby the scaffold structure and mechanical
support can be maintained during the early stages of tissue formation
(Hedberg et al., 2005). In chapter 3.1 of the present PhD work, an in
situ crosslinkable, biodegradable, methacrylate-endcapped bone
scaffold composed of D,L-lactide, ε-caprolactone and 1,6-hexanediol
(cf. Appendix), which was proven to be bioresorbable and
osteoconductive in different in vitro tests (Declercq et al., 2005), was
tested in the new goat model.
Since the scaffold needs to polymerize in the in vivo goat model,

several options had to be considered. Polymers can be triggered
using the so-called redox systems in which a base and catalyst
substance react when mixed together. This allows the polymerization
of bigger volumes at the same time in contrast to polymerization using
a light source where only a relatively small surface of the polymer can
be treated. On the other hand, redox systems often produce heat and
even toxic substances. Both can be disastrous for cell and tissue
survival. Further research focused on the potential toxic effects of
those redox systems is definitely justified (Pashkuleva et al., 2005).
Photopolymerization is the alternative option for the redox system.

The photopolymerization reaction is induced by chemicals that
produce free radicals when exposed to specific wavelengths of light.
As a photon from a light source excites the photo-initiator into a high-
energy radical state, this radical induces the polymerization of a
macromer solution. Photopolymerization has several advantages over
conventional polymerization techniques including spatial and temporal
control over polymerization, fast curing rates (less than a second to a
few minutes) at room temperature or physiological temperatures, and
minimal heat production (Nguyen & West, 2002). Furthermore,
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GENERAL DISCUSSION
polymers can be cross-linked in the presence of photo-initiators when

using visible or ultraviolet light. As ease of handling is crucial for
clinical use, the viscous properties of the polymer solution must
balance between the requirement of the material to remain at the site
of injection and the request of the surgeon for an easy manipulation
during implantation (Temenoff & Mikos, 2000). Because of the
advantages of the photopolymerization over the redox triggered
polymerization, the photopolymerization process was used in all
experimental set ups of the present PhD.
One of the initial perspectives of our group was to create a syringe

containing the so-called prepolymers which could set when exposed
to daylight after injection in the bone defect site. These materials
should also be able to fill efficiently irregular bone defects. The initially
used pre-polymers (chapter 3.1) were rather granular which reduced
their ease of manipulation resulting in the spillage of several granules
during the surgical procedures. However, the created defects could be
easily filled with the granules. Once the cross-linking process was
achieved by photo-initiators activated by a commercial dental lamp,
the polymer was very firm, hard and well fixed in the created defect.
This firm characteristic was additionally obtained by the addition of 2-
hydroxyethyl methacrylate (HEMA) (cf. Appendix) as the co-cross-
linker, which significantly reduced the viscosity of the initial
polymerization mixtures. An injectable material should be activated in
situ in several minutes, not only to minimize the length of the
procedure but also to allow surgeons enough time for placement
before hardening of the material.
An adequate porosity of the scaffold is required to allow cellular

infiltration and bone formation into the scaffold. Therefore, gelatin (cf.
Appendix) particles were mixed with the scaffold since the gelatin
melts at the normal body temperature producing a network within 4 to
7 hours inside the hardened scaffold. In previous in vitro tests it was
proven that cellular infiltration was possible in scaffolds with high
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GENERAL DISCUSSION
porosity (70%) obtained by gelatin (250–355 µm) leaching, which was

accepted as the correct porosity of the scaffold (Declercq et al., 2008).
For scaffolds with lower porosity (60%) and gelatin particle size 250–
355 µm, no ingrowth was observed due to a polymer top layer,
although cellular infiltration was possible when the gelatin particle size
increased to 355–500 µm. An appropriate amount of gelatin particles
was added to the scaffolds used in our study in order to have the
desired high porosity of 70%. In spite of the adaptation, rather
negative results in our study were obtained since the histological
results only revealed a slight ingrowth of bone at the periphery of the
composite materials. No signs of inflammatory or immunologic
reactions were present which was further illustrated by the negative
immunohistochemical staining for CD3, a marker for the presence of
lymphocytes (non-published results). As tissue infiltration into the
scaffold without histological signs of inflammation was present, and
the polymers showed no signs of rejections, this newly developed
polymer was considered as being highly biocompatible but had only
moderate osteoconductive and manipulation characteristics in an in
vivo setting.
Because of the relatively disappointing osteoinductive results

obtained with the initial scaffold, a modification of the procedure was
justified. Therefore, osteoinductive substances, such as autologous
BMSC, were inserted into the scaffold to promote new bone formation
inside the osteoconductive scaffold. This adaptation was tested in the
same tibial unicortical defect model in goats (chapter 3.2).
The incorporation of the bone marrow derived mesenchymal stem

cells into the scaffolds caused a lot of difficulties. First of all, several
modifications were needed to the bone marrow aspiration protocol to
achieve a sufficient amount and quality of bone marrow. Although a lot
of sites can be used to aspirate bone marrow, we performed the
aspiration from the dorsal iliac crest as it is readily accessible and
could be performed in sedated goats using an 11 gauge Jamshidi
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GENERAL DISCUSSION
needle. Some initial aspiration samples were unfortunately

contaminated during the cultivation process, most likely due to less
sterile conditions. Respecting a strict aseptic protocol for the
prelevation of bone marrow induced optimal results. In summary, after
the surgical preparation of the area, a skin incision was made, the
subcutaneous fat was dissected and the iliac crest was exposed.
Copiously flushing of the Jamshidi needle with a sterile ACD
anticoagulant during the procedure was found to be of major
importance to avoid unwanted clotting of the bone marrow.
A second problem of adding cells to the scaffold was the protection

of the BMSC, not only during the photopolymerization process but
also during handling of the viscous polymer paste containing the cells.
Therefore, the cells were loaded on microcarriers which can carry
large numbers of cells and can directly and controllably be transferred
into an injectable in situ forming scaffold. Our group reported
previously that CultiSpher-S® carriers (cf. Appendix) are an excellent
3D culture system since mesenchymal and embryonic stem cells not
only attached on and infiltrated into these carriers, but also
differentiated into the osteogenic lineage when cultured in appropriate
culture media (Declercq et al., 2005, Tielens et al., 2007).
The HEMA co-cross-linker was used as a solvent in the

experimental set ups (chapter 3.1) but seemed to be detrimental for
the bone marrow derived mesenchymal stem cells. When HEMA (with
or without gelatin) or triacetin (used as a plasticizer) (cf. Appendix)
with gelatin was tested in vitro to encapsulate the microcarriers
immediately after the cross-linking procedure, no viable cells could be
found in the carriers. However, promising results were obtained in
vitro using methacrylate-endcapped poly(D,L-lactide-co-ε-
caprolactone) with triacetin as solvent whereby a positive effect on the
viability of the encapsulated cells could be demonstrated.
Unfortunately, triacetin induces some negative effect such as
decreases in the viscosity of the polymer and final hardness of the
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GENERAL DISCUSSION
polymerized scaffold (Tielens et al., 2007). Although this lower

hardness was hardly justified for clinical use, it became no drawback
as both mixtures (with HEMA or triacetin) never reached the ultimate
hardness of bone and could not be used to provide support to the
defect site in this phase. Even more, ease of manipulation was, in this
stage of bone scaffold development, of greater importance than final
hardness. Additionally, the use of a polymer with lower viscosity
(polymer/triacetin) clearly had a positive influence on the cell viability
in comparison with polymer/HEMA. Nevertheless, this positive effect
of the reduced viscosity was lost when gelatin was added as a
porogen. Although gelatin is not toxic and is often used in vitro as well
as in vivo, addition of gelatin as porogen had a negative influence on
cell viability, most likely because the cells loaded on the microcarriers
probably suffered from shear stress when mixed with the
polymer/gelatin paste. Also, the gelatin started to liquify immediately
after cross-linking. Incubating the scaffolds in an in vitro culture
medium at 37°C, this liquification induced a very high concentration of
gelatin in the culture dish, which could also explain the observed lower
viability of the cells.
Because of the observed low cell viability, an additional experiment

was performed. Therefore, bone marrow derived mesenchymal stem
cells (BMSC) were seeded on 500 to 1200 µl of CultiSpher-S®
microcarriers and aseptically injected subcutaneously around the
sternum in each anesthetized goat. Only 6 of the 9 subcutaneous
injections could be retrieved after a time period of 2 to 8 weeks. Five
out of 6 contained a lot of cells and in 3 samples the CultiSpher-S®
microcarriers were clearly visible on histology. Some cells of the
subcutaneous injections stained positive for CD3, MAC387 and
vimentin but negative for osteocalcin. This latter finding was in
contrast with the staining observed in the tibial bone samples, where
the tissue around the seeded microcarriers and the polymer stained
positive for osteocalcin. The absence of a mineralized matrix of the
osteogenic differentiated subcutaneous injected bone marrow derived
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GENERAL DISCUSSION
mesenchymal stem cells confirmed the highly multipotent

characteristic of the cells and the need for an osteogenic environment
to continue differentiation into bone cells.
Despite the use of BMSC, our results showed a rather limited

proliferation of these cells and lack of ingrowth of new bone at the
borders of the scaffolds. This might be explained by the limited size of
pores in the composite as no porogen was used in this study. The
scaffolds were encapsulated by fibrous tissue which is generally
accepted as a classic response to a foreign material (Fournier et al.,
2003). However, the encapsulation in the present study was most
likely not a foreign body reaction as the former study using the porous
scaffold without BMSC clearly demonstrated osteoconductive and
biocompatible characteristics (chapter 3.1).
Due to the limited proliferation and survival of the BMSC,

probably induced by the slow degradation rate, and the difficulty to
enhance vascularisation into the methacrylate-endcapped poly(D,L-
lactide-co-ε-caprolactone) polymer, the original polymer was replaced
by a chemically modified hydrogel in a final study (chapter 3.3).
Hydrogels have the advantage that they can imbibe large amounts of
water and biological fluids, and combine the cohesive properties of
solids and the diffusive transport characteristics of liquids. They
exhibit a high permeability for oxygen, nutrients and other water-
soluble metabolites (Peppas et al., 2000). These hydrogels can cross-
link in situ in response to physical triggers including changes in
temperature, pH or an exchange in ions (Klouda & Mikos, 2008,
Gutowska et al., 2001). Hydrogels can also be seeded with bone-
forming cells forming promising alternative scaffolds for bone
regeneration purposes (Bianco & Robey, 2001, Arinzeh et al., 2003,
Bianco & Robey, 2000, Endres et al., 2003, Srouji et al., 2006, Srouji
& Livne, 2005, Srouji et al., 2005, Trojani et al., 2006, Tsuchida et al.,
2003, Vehof et al., 2002). These substances can be cross-linked
using photopolymerization (Buxton et al., 2007, Li et al., 2006).
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GENERAL DISCUSSION
Pluronic® F127 (cf. Appendix) and F68 hydrogels have been

approved by the Food and Drug Administration (FDA) as injectable
materials for use in the human body. Pluronic® F127, also referred to
as Poloxamer 407 or Lutrol® F127, is a water-soluble tri-block
copolymer of poly(ethylene oxide) – poly(propylene oxide) –
poly(ethylene oxide) and undergoes a sol to gel transition at a
concentration higher than 14 wt% and a temperature of 30°C (Khattak
et al., 2005). Consequently, Pluronic® F127 solutions can be injected
as a liquid into the body, where it will form a gel without toxic effects
(Escobar-Chavez et al., 2006, Ruel-Gariepy & Leroux, 2004). The
collaborating Polymer Material Research group of this PhD thesis
developed a method for preparing a cross-linked Pluronic® F127
hydrogel with biodegradable building blocks, whereby the hydroxyl
end-groups were chemically converted to N-methacryloyl-
depsipeptides creating a more stable gel, called Pluronic ALA-L
(Swennen et al., 2006, Swennen, 2008)(cf. Appendix). This gel is
characterised by an increased degradation rate since 50% of the gel
fraction of a 30% Pluronic ALA-L hydrogel in phosphate buffered
saline solution at 37°C was hydrolyzed only after 38 days (Swennen,
2008). The modified hydrogel is liquid at 0°C, allowing an efficient
mixing with seeded CultiSpher-S® microcarriers. Exposure to
moderate temperature (lukewarm) and light makes the hydrogel more
jelly, allowing aspiration in a syringe and proper placement in the
created defect. Finally, after UV irradiation, a 3D network with tailored
degradation rate can be formed. The final consistency of the polymer
was comparable with that of a bouncing ball.
Preliminary tests revealed no cytotoxicity induced by the initial

cross-linking of the Pluronic ALA-L hydrogel by irradiation with UV
light (I = 3000 mW/cm²) for 2 minutes. In contrast, the viability dropped
dramatically to around 20% after 5 days of culture in a thermal gelated
hydrogel. This was a significant decrease in cell viability in
comparison to the encapsulated cells in the UV cross-linked gels.
Using the modified Pluronic ALA-L hydrogel, higher viability
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GENERAL DISCUSSION
percentages of the encapsulated cells were found compared to the

results of Khattak et al. (2005) who investigated the encapsulation of
HepG2 liver cells in a parent 20wt% Pluronic® F127. Since the
modified hydrogel gave promising results in all in vitro tests, this gel
was also investigated in the goat model in which not only the
Cultispher-S® microcarriers loaded with cells but also a
hydroxyapatite carrier was incorporated into the trials (chapter 3.3).
Hydroxyapatite (cf. Appendix) was included in our studies because

its chemical composition resembles the mineral phase of bone. Even
more, this substance shows excellent biocompatibility to bone tissue
and meets the desired requirements for materials applicable in bone
tissue engineering and bone regeneration (Hench, 1998). In bone
tissue engineering, hydroxyapatite based materials have been used
as scaffolds as well as cell delivery systems. When applied as a
scaffold, not only osteoconductive but also osteoinductive properties
have been reported. Scaffolds of pure hydroxyapatite (Ripamonti,
1996, Yuan et al., 2001), but also of biphasic calcium phosphate
cements (Le Nihouannen et al., 2006, Le Nihouannen et al., 2008), or
of tricalcium phosphate ceramics (Cancedda et al., 2007,
Mastrogiacomo et al., 2005) were also reported to be able to induce
bone formation when implanted ectopically. Variability in results
seemed not only to depend on the composition of the material or of
the selected species model (Cancedda et al., 2007, Ripamonti, 1996),
but also on the geometry and pore structure of the material.
Although hydroxyapatite has been reported to be a good carrier

structure for osteogenic cells (Sopyan et al., 2007) and the in vitro
results of this material performed by our group were very promising
(Lippens et al., 2008), the cell-loaded hydroxyapatite based cylindrical
structures surprisingly failed to form bone in the in vivo goat model in
the present PhD work. In contrast, a marked bone formation was
observed when these cell-loaded CultiSpher-S® carriers were applied.
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GENERAL DISCUSSION
The 2 types of carriers used in our study differed in the

composition and geometry of the carrier material. Cells that were
seeded on the inner surface of the hydroxyapatite cylinders did not
result in any bone formation, nor was there an indication of
vascularization inside the carrier. Although hollow hydroxyapatite
cylinders with an inner diameter of about 700µm were used in the
present study, this did not seem to meet the requirements needed for
efficient nutrition of the cells. This was in contrast with in vitro
investigations, in which proliferation and cell viability were extremely
pronounced both on the inside as well as on the outside of the
cylinders (Lippens et al., 2008). It has to be mentioned that the
required nutrition of bone substitutes is clearly different between in
vitro and in vivo conditions whereby in vitro results can not blindly be
extrapolated to in vivo work. Other studies have shown that highly
porous hydroxyapatite cylinders (70-80% pore volume and average
pore sizes more than 150µm) loaded with BMSC can result in bone
formation when implanted in tibial defects in sheep (Kon et al., 2000).
A higher porosity of the microporous cylinder wall used in our study
may be needed to fulfill the requirements of a sufficient supply of
blood vessels since the pores in the wall of the used hydroxyapatite
carriers were only on average 2µm in diameter and the overall
porosity was only 24 volume %. Research is ongoing to find the
optimal pore-size of the hydroxyapatite cylinders to allow nutrition of
the seeded cells but also protect the cells during polymerization of the
materials.
The present study showed that an increased bone formation can

be achieved by injecting BMSC loaded CultiSpher-S® carriers mixed
in the Pluronic ALA-L hydrogel in a goat tibial defect when compared
with the natural bone regeneration rate.
217
GENERAL DISCUSSION
Optimalisation of the evaluation techniques for bone healing
Bone healing is routinely analysed using histological techniques to

assess the process of bone production and remodelling. However,
paraffin, which is the standard embedding medium for the evaluation
of bone enzymes in immunohistochemical analysis techniques, is only
suitable for use on demineralised tissue samples that are often
characterised by an insufficiently preserved cancellous structure (Yang
et al., 2003). At the present moment, few embedding resins are
available for both morphological and immunohistochemical analyses
on mineralised tissue. Problems with cutting the samples, morphology
and reliable staining protocols are often encountered in non-
demineralised resin embedded tissue including bone. Technovit 9100
New® is a polymerization system based on methyl methacrylate
(MMA) which hardens at low temperature. It has been developed for
embedding mineralized tissues and allows extensive possibilities of
staining for light microscopy purposes. Our research group found
similar results between routine and immunohistochemical staining of
non-demineralised bone embedded in Technovit 9100 New®
compared to demineralised bone embedded in paraffin. Occasionally,
some scattering and less clear osteocytes were encountered in the
Technovit 9100 New® sections (Vandemaele, 2005).
In two parts of this PhD work (chapter 4.1 and 4.2), less frequently
used evaluation methods of bone healing were investigated in depth,
namely immunohistochemistry and micro computed tomography.
Immunohistochemistry has become a standard tool for

investigating the microscopic localization of cells, structural proteins
and cellular products. As mentioned previously, Technovit 9100 New®
was used in all experiments as embedding medium for histological
follow up in non-decalcified tissues, allowing optimal
immunohistochemical analysis of the bone samples. However, in the
first series of goats (chapter 3.1), immunohistochemical results were
218
GENERAL DISCUSSION
not reported as the majority of the sections were ambiguously or

negatively stained. A further standardisation of the technique was
indispensable since the immunohistochemical evaluation of
undecalcified bone samples in Technovit 9100 New® has not been
documented in goats. Even more, negative staining using a non
optimal technique can be caused by inaccurate preserved antigens.
Therefore the technique was optimized in the present PhD study to

improve the immunohistochemical staining results. Until recently, an
acetone fixed cryostat tissue section was accepted as the “gold
standard” for immunostaining, although tissue morphology was rather
poor. Additionally, acetone fixation dissolves lipid-containing
membranes and coagulates proteins although this mild fixation
technique preserves antigenic structures. Therefore, acetone fixed
antigens generally are recognized by their corresponding antibodies.
This mild fixation contrasts with sections cut from a tissue block that
are fixed using neutral buffered formalin, processed through alcohols,
cleared in xylene and finally embedded. Formalin is known to
crosslink so many epitopes that may be concealed and become not
stained by their corresponding antibodies when sections of the
standard prepared tissue blocks are used. To overcome the effects of
fixation and processing, different methods have been proposed to
expose the antigens in the tissue section, making them suitable for
immunostaining. Several groups introduced heat-induced epitope
retrieval methods using different buffers whereby high temperatures
combined with pH and additives such as EDTA break down the
existing cross-links and allow antibodies to bind with their
corresponding antigens (Shi et al., 2001, Shi et al., 1991). Problems
inherent to cross-linking are theoretically absent when a non-cross-
linking fixative including methacarn composed of methanol, chloroform
and glacial acetic acid is used (van der Loos, 2007). In our studies of
this PhD work, all samples were fixed in formalin 10% for 12 hours.
Longer fixation times were avoided to preserve antigenicity. Generally,
prolonged as well as insufficient fixation times may affect the
219
GENERAL DISCUSSION
sensitivity of the immunohistochemical staining (James & Hauer-

Jensen, 1999, Werner et al., 2000). However, before the introduction
of the antigen retrieval technology, it was highly recommended to fix
the tissue for the minimum of time (Fox et al., 1985, Battifora &
Kopinski, 1986, Leong & Gilham, 1989). It is presently accepted that
heat-mediated antigen retrieval can reduce or even eliminate the
staining variations caused by a prolonged formalin fixation (Shi et al.,
1997). The staining patterns of some antigens, such as the
proliferation cell nuclear antigen, are relatively independent of fixation
time (6 hours to one week). Munakata and Hendrickx (1993) stated
that for unknown fixation times, longer heating times were preferred to
shorter times, since long microwave treatments did not show negative
effects under the applied experimental conditions. However, extended
retrieval time can be potentially harmful to the tissue and cause
damage “over retrieval”. A study on the effects of prolonged formalin
fixation on the immunohistochemical reactivity of breast tissue (Arber,
2002), showed that formalin fixation did not significantly reduce
immunoreactivity for different important markers including Ki67, p27,
or vimentin, even in tissue fixed for 154 days. In conclusion, a
complete and rapid fixation minimizes false localizations and antigen
diffusion artifacts.
The most important factor to improve antigen retrieval efficiency is

heat (Shi et al., 1991, Stirling & Graff, 1995). It was demonstrated that
optimal and efficient results are correlated with the production of
temperature and irradiation (Shi et al., 1998, Koopal et al., 1998).
Most protocols recommend the use of 10 to 20 minutes of irradiation
at about 100 °C. However, many authors recommend higher
temperatures and irradiation times longer than 20 minutes
(Vonwasielewski et al., 1994, Brorson, 2004, Evers & Uylings, 1997).
During the initial trials with undecalcified bone sections in the present
PhD work, the microwave pressure cooker protocol for antigen
retrieval was found to be too aggressive for the samples since
fracturing of the samples and even detachment of the samples from
220
GENERAL DISCUSSION
the glasses frequently occurred. Although one report stated that the
bone sections attached better to the microscope slides compared to
paraffin (Vandemaele, 2005), sample sections easily came loose
using the pressure cooker protocol for antigen retrieval in our study. In
order to avoid this complication, the proteinase K technique to retrieve
the antigens was applied since this technique gave a better
preservation of the tissue sample embedded in the specific MMA
resin. Introduced in the 1970s and still used for certain antigens,
proteolytic induced epitope retrieval consists of the controlled
treatment of tissue section with proteolytic enzymes (Huang et al.,
1976, Huang, 1975, Curran & Gregory, 1977, Mepham et al., 1979,
Battifora & Kopinski, 1986, Ordonez et al., 1988). Overall, the use of
enzymatic digestion is a more aggressive approach in comparison to
the heating methods. Indeed, it can damage some epitopes as well as
the tissue morphology when used for extensive time (Pileri et al.,
1997). However, the use of proteinase K represents an efficient
approach for the retrieval of some antigens (Rojo et al., 2006, Jessie
et al., 2004). Other proteases, such as trypsin, chymotrypsin, pepsin
and pronase can also be used for uncovering antigen sites (Ward et
al., 2006).
Initially, relatively disappointing immunostaining results were

obtained in the present PhD work. This phenomenon can partly be
explained by the lack of goat-specific primary antibodies since most
antibodies are only indicated for application on human and lab animal
tissues. It has also been demonstrated that the majority of
commercially available antibodies have little to no cross-reactivity
between all species (Ramos-Vara & Beissenherz, 2000). Therefore,
several commercially available antibodies were tested at different
concentration on positive and negative control goat tissues in the
present PhD work (chapter 4.1). Using the results of this research,
only 4 out of 12 antibodies stained positively and could be used for
evaluation of goat tissues embedded in Technovit 9100 New®. Those
4 staining techniques can be useful for the future evaluation of the
221
GENERAL DISCUSSION
osteo-integration of bone substitutes. However, complementary

antibodies (e.g. anti-cbfa-1, anti-alkaline phosphatase, anti-collagen I,
anti-bone sialoprotein, anti-osteopontin) may allow a more complete
evaluation of potential bone regeneration, so more research of their
applicability is justified.
Histological analysis of samples remains the standard method for

evaluation of host tissue reaction to biomaterials and extracellular
matrix formation within the interconnected pores of scaffolds. The
combination of exceptional in-plane resolution and cellular detail
provided by histology is unparalleled. Immunohistochemistry can be a
complementary analysis technique to quantify cell numbers and tissue
ingrowth but also to identify the recruitment of macrophages and other
cells associated with an inflammatory response to implanted
biomaterials. Histological sections can further more be stained with
appropriate polychromatic dyes to identify multiple tissue types.
Finally, these sections can be analysed using a polarized microscopy
to assess extracellular matrix organization. In the present PhD work,
histology was mainly used to evaluate and identify the different tissues
(bone, fibrous tissue and polymer) and their interfaces. This allowed
proper evaluation of tissue development on a cellular level (e.g.
presence of endothelial cells, cellular immunity, and survival of cells).
Most evaluations were performed using the standard hematoxylin-
eosin (H&E) and Von Kossa stained sections.
The assessment of biomaterial-tissue interfaces, which is of major

importance in the evaluation of bone tissue engineering models, is
often difficult due to physical and chemical mismatches that result in
distortion artifacts or tissue separation during sectioning. Histological
processing is overall time consuming and the obtained 2D sections
provide not only an incomplete but also a potentially misleading
representation of 3D tissue formation within porous scaffolds.
Micro-CT imaging can offer a solution. Indeed, the recently

developed µCT in vivo scanners give the opportunity for longitudinal
222
GENERAL DISCUSSION
monitoring of bone formation within biomaterial scaffolds after

implantation. Through the use of selected contrast agents, the
boundaries of quantitative µCT analysis can also be expanded beyond
mineralized tissues to include soft tissues such as cartilage and blood
vessels. In addition to detailed 3D morphology, local voxel attenuation
can provide additional information regarding the type or density of
locally synthesized tissue. Several groups have used µCT scanners to
assess mineralised matrix formation within a variety of biomaterial
scaffolds using in vitro and in vivo setups (Oliveira et al., 2007, Porter
et al., 2007, Jones et al., 2004). In chapter 4.2 of the present PhD
work the validation of µCT was investigated using an in-house
developed µCT scanner of the Centre for X-ray Tomography at the
Ghent University. The goal was to assess the micro-architecture of
the regenerated cortical bone by evaluating the correlation between
the images obtained by µCT and those obtained by
histomorphometry. Several problems were encountered using the µCT
technique in our studies. First of all, image-based quantification of
mineralised matrix volume requires accurate segmentation of
mineralised tissue from other materials or tissues within the scanned
region (Jones et al., 2007). Selection of one or more global thresholds
for X-ray attenuation is the simplest and most efficient method of
segmentation. This approach works well for detection of mineralized
extracellular matrix formed within low attenuating materials such as
polymeric scaffolds. However, more sophisticated segmentation
algorithms may be needed to separate multiple materials with
overlapping density distribution (Hilldore et al., 2007). For these
reasons, it is difficult to isolate small volumes of bone formed on the
surface of hydroxyapatite or other bioceramic material scaffolds. In
our studies, no new bone was formed on the hydroxyapatite carriers
which have the same density as the newly formed bone.
Consequently, the hydroxyapatite had to be removed manually on 2D
image slices. Another important issue was the density of newly formed
mineralized matrix which is substantially lower and more variable than
223
GENERAL DISCUSSION
mature bone. Quantification of mineralized matrix volume can be

highly sensitive to the chosen threshold value. The selection of an
appropriate threshold value is often a compromise between not over-
estimating the volume of relatively larger or strongly mineralized
structures and not missing smaller or less mineralized regions. A
common approach to address this issue is to compare 2D grayscale
and threshold regions visually side by side and then maintain a
consistent selected threshold value for all groups within a given
experiment. As we did in the present PhD work, it is advisable to
evaluate the sensitivity of the results to threshold value and compare
threshold image slices to registered histological sections. Compared
to µCT, histomorphometrical analysis significantly overestimated bone
volume fraction in the analysed tissue of our studies. However, µCT
may be an appropriate method to analyse bone healing in our model
since results have similar tendency compared to histomorphometry.
For applications involving highly variable attenuation levels or very
thin structures, it may be informative to report data at more than one
threshold value (Guldberg et al., 2004).
For implantation studies, µCT can be applied either to analyse

samples or to scan defect regions in vivo using new generation live
animal scanners (Jones et al., 2004, Liu et al., 2008, Byers et al.,
2006, Gauthier et al., 2005, Hu et al., 2007, Lin et al., 2006, Rai et al.,
2007). Because µCT imaging is nondestructive, samples are still
available for histological evaluation or biomechanical testing. The
volume of bone ingrowth into polymeric scaffolds implanted into bone
defects correlates well with functional integration strength as
measured by postmortem biomechanical testing (Gauthier et al.,
2005). Noninvasive in vivo scanning allows the collection of data over
the time course and rate of mineralisation data (Oest et al., 2007).
However, nonmetallic fixation devices must be used in models using
fractures of segmental bone defects to avoid artifacts in the
reconstructed 3D µCT images. Although repeated exposure to
ionizing radiation can be a concern, scanning at lower resolution in a
224
GENERAL DISCUSSION
limited region and at a reduced frequency over the course of time can
minimize this potentially confounding factor (Ford et al., 2003). In our
experiment, the surface of the Technovit 9100 New® was slightly
damaged. This caused a less fluent cutting by the microtome through
the borders of the cubes, but had no negative influence on the final
quality of the sections. Similar harmful effects of µCT scanning on
tissues have never described in literature so a suitable explanation for
this observation was not available.
225
GENERAL DISCUSSION
FUTURE PERSPECTIVES
Critical sized defects were not used in the different parts of the
present PhD work but remain a logical sequel for further experiments.
As some results are promising, several tested scaffold combinations
could be used in clinical trials for different applications (sinus lifting,
palatoschisis, …) not only in goats but also in other animal species
(dogs, cats, horses). However, the development of a suitable pre-
clinical critical sized animal model to analyse these scaffolds is not
straightforward. In the area of tissue engineering and related animal
studies, stabilization of the defect with internal fixators offers a great
advantage since there is a minimal influence of the fixation device on
the created defect site not only on the space for scaffold implantation
but also on the biological factors. When compared to external fixators
or intramedullary nails, rates of infections (pin-track infection),
infections related complications and non-union rates (6-25%) are
lower. However, higher numbers of malalignment may be observed
(Beardi et al., 2008). Especially in large animal models, fixation and
immobilization of the bony defect with eccentrically placed devices
remains a challenge and has been used in only a few studies up to
now (Mastrogiacomo et al., 2006, Meinel et al., 2003, Wefer et al.,
2000). Overall, the establishment of a critical size segmental defect
model in an immunocompetent large animal is a challenging task. A
highly standardised and reproducible tibial, critical sized defect model
in a goat based on a plate fixation system may be the most suitable
model for further research.
A further modification of the polymers (hardness) and the carriers

(increase of the porosity of hydroxyapatite) is necessary before clinical
applications become a reality. In our research, we try to develop a
product that stimulates bone healing requiring additional stabilization
of the defect by osteosynthesis. In contrast, a product with a high
hardness that does not require extra stabilisation of the defect will
automatically have a lower porosity. Even more, this latter product will
226
GENERAL DISCUSSION
be a less attractive environment for cell viability. Although the used

mixtures have some promising results, none of them are ideal. The
addition of other bone enhancing products could stimulate adequate
new bone formation further more.
The applicability of photopolymerization used in these studies is

certainly a subject for improvement. Polymerization by a light source
can be easily performed on small defects but is hard to realize in
bigger critical sized irregular defects. Other methods of polymerization
(e.g. chemical) have to be investigated.
The unique population of multipotential cells has been isolated

from various sources, including bone marrow, adipose, umbilical cord
blood, periosteal and muscle tissues. Bone marrow stromal cells were
already used in the early days of tissue engineering. However, the
harvesting can be painful (Vanhelleputte et al., 2003) and might be
associated with increased morbidity. Therefore, alternative sources for
MSC’s need to be identified. Especially ‘waste material’ as adipose
tissue, femoral head, umbilical cord blood and placenta may be
interesting sources as they are easily accessible without large
negative issues (van Griensven, 2008, Jang et al., 2008).
The use of BMSC in our study revealed promising results, but

there is still a long way to go to improve the efficiency of those cells.
The results of using autologous MSCs have to be compared to those
of allogeneic mesenchymal stem cells and even frozen MSCs. If
results are comparable, stem cell banks can be set up to have a direct
access to the cells, avoiding the time consuming culturing process.
MSCs appeared to be immunologically privileged since mismatched
allogeneic stem cells were demonstrated in dogs to regenerate bone
without inciting an immunologic response (Kraus & Kirker-Head,
2006).
The use of direct gene delivery is also promising for in vivo bone
repair. Several viral and non-viral methods have been used to achieve
substantial bone tissue formation in various sites in animal models. To
227
GENERAL DISCUSSION
advance these platforms into clinical settings, it will be mandatory to

overcome specific hurdles, such as control over transgene
expression, viral vector toxicity, and prolonged culture periods of
therapeutic stem cells (Kimelman et al., 2007).
Growth factors will most likely be a major part of any successful

strategy to create synthetic bone. It is important to consider the
endless combinations of growth factors that might be used and the
limitless methods of delivery (e.g. direct delivery, gene therapy)
(Calvert et al., 2003).
In order to refine the evaluation methods of bone healing, further

research is required to improve the immunohistochemical analysis of
low-temperature methylmethacrylate resin-embedded goat bony
tissues. Different fixation and antigen retrieval protocols have to be
tested as well as alternative commercially available antibodies for the
same markers. The development of more goat specific antibodies
would be of great interest in the further research since antigens such
as cbfa-1 and collagen I can be used to detect early bone formation,
while alkaline phosphatase, osteopontin and bone sialoprotein can be
used to confirm bone mineralization, remodulation and formation,
respectively. Also a lot of hematologic, immunologic and tissue
markers can additionally be used to identify more precisely the cells
and formed tissues.
The in-house developed µCT scanner confirmed that the

measurement of new bone formation was in agreement with the
indices obtained by the conventional histomorphometry. The µCT
equipment and the analysis protocol can be further improved to be
able to evaluate more parameters relevant to the bone healing
process including 3D vascular ingrowth, cartilaginous tissue formation,
pore size of biomaterials etc. A µCT scanner in which live animals can
be scanned without loss of high-resolution data would allow in depth
longitudinal studies on bone regeneration with reduction of the
number of experimental animals.
228
GENERAL DISCUSSION: References
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239
GENERAL DISCUSSION
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240
APPENDIX
1. Filling Materials
1.1 D,L- Lactide-co-ε-caprolactone polyester
Step 1 : ring opening polymerization of the

lactone monomers
Step 2 : derivatization of the hydroxyl endgroups into

methacrylate esters (TEA = triethylamine)
Step 3 : in situ photopolymerization - formation of the 3-D

polymer network
241
APPENDIX
Katalyst : hydroxyethylmethacrylate (HEMA)
Plasticizer : triacetin
Porogen : gelatin
BioGlass : The key composition features of BioGlass is that it

contains less than 60 mol% SiO2, high Na2O and CaO
contents, high CaO/P2O5 ratio, which makes BioGlass highly
reactive to aqueous medium and bioactive.
TCP: tricalciumphosphate : Ca3(PO4)2
242
APPENDIX
1.2. Pluronic ALA-L
The three steps reaction scheme of Plu ALA-L. a/ The hydroxyl

®
groups of the Pluronic F127 polymer were converted into a bromine
ester (STEP 1). b/ The syntheses of N-methacryloyl-L-alanine via the
Schotten-Baumann acetylation reaction (STEP 2). c/ After coupling of
the N-methacryloyl-alanine to the modified Pluronic (STEP 3), Plu
ALA-L was formed
2. Carriers
CultiSpher-S : crosslinked gelatin
Hydroxyapatite : Ca5(PO4)3(OH)2
243
SUMMARY
The self-healing capacity of bone is widely used for the repair of

small fractures. However, if segments of bone are lost or damaged so
severely that they have to be removed, a bony union is not possible
and bone grafts are required to achieve an acceptable healing.
Chapter 1 describes the present possibilities in veterinary

orthopaedics to enhance bone healing and regeneration. The
currently used methods to restore bone defects are often not
satisfactory. Especially the healing of large, irregular defects results in
the formation of tissues with inferior qualities compared to the original
structures. For these reasons, several new approaches are currently
being explored to improve bone healing capacities in different
situations. The use of bone enhancing grafts can be indicated in
different surgical disciplines including surgery of the head, dentistry,
long bone and joint surgery. Its use in veterinary surgery is rather
limited but promising results in human studies might create similar and
new surgical techniques and opportunities for veterinary indications in
the near future. Autografts still represent the “gold standard” material
for enhanced bone regeneration. Allo- and xenografts are alternatives
but these latter two posses considerable less capacity for
osteoinduction and osteoconduction. Many synthetic materials are
available to the surgeon, including ceramics and/or ceramics-collagen
composites, natural corals, coralline hydroxyapatite, and resorbable
polymers. As natural substances bone marrow, stem cells and several
growth-promoting substances as bone morphogeneic protein-2 are
245
SUMMARY
used to enhance bone growth. “Bone tissue engineering” has become

a new approach to enhance bone regeneration. In this field, a
synthetic 3D porous template (scaffold) is combined with an
osteogenic potent cell population to finally develop bone tissue
equivalents that can induce total regeneration of a large affected area.
The application of enhanced bone regeneration in veterinary medicine
is relatively limited to experimental studies using animal models for
human purposes, apart from few case reports and a small number of
clinical trials.
The general aim of this PhD work (Chapter 2) is to evaluate

potential bone substitutes and bone enhancing products in an in vivo
tibial goat model.
In Chapter 3 several ‘home-made’ in situ crosslinkable and

biodegradable polymers with attractive characteristics to stimulate
healing of bone defects in standardized in vitro tests, are tested in an
in vivo uni-cortical tibial non critical sized defect model in goats. In
Part 3.1 of this PhD work an in situ crosslinkable, biodegradable,
methacrylate-endcapped porous bone scaffold composed of D,L-
lactide, -caprolactone and 1,6-hexanediol, in which crosslinkage is

achieved by photo-initiators, is evaluated. Three different polymer
mixtures (pure polymer and 30% bioactive glass or α-tricalcium
phosphate added) are tested. The polymers are randomly applicated
in one of four (6.0 mm diameter) defects leaving a fourth defect
unfilled. Biocompatibility and bone healing properties are evaluated by
serial radiographies, histology and histomorphometry. The pure
polymer clearly shows excellent biocompatibility and moderate
osteoconductive properties. The addition of α-tricalcium phosphate
increases the latter characteristics.
246
SUMMARY
In a next experiment (Part 3.2) different combinations of the

polymer with bone marrow-derived mesenchymal stem cells seeded
on gelatin CultiSpher-S® microcarriers and α-tricalcium phosphate are
tested in the same goat model. The results demonstrate cell survival
and proliferation in the polymer-substituted bone defects. The addition
of phosphate is associated with less expansion and growth of the
bone marrow-derived mesenchymal stem cells than other polymer
composites. A faster resorption and higher porosity of the polymer
seems imperative to promote bone marrow-derived mesenchymal
stem cells' proliferation and encourage bone ingrowth from the
surrounding tissues. Further biochemical adaptation of the polymer is
mandatory to improve its bone grafting properties. In a last experiment
(Part 3.3) a chemically modified form of the Pluronic® F127 hydrogel
is tested in the same goat model. An extra cell source of autologous
bone marrow derived mesenchymal stem cells is mixed with the
hydrogel to increase the ossification process. These cells are cultured
and predifferentiated on 2 types of cell carrier systems, i.e. gelatin
CultiSpher-S® microcarriers and hydroxyapatite tubular carriers.
Radiographic and histological evaluation reveal that bone
regeneration is comparable in the defects with the bone substitute
compositions and the untreated control defects at 2 and 4 weeks post-
implantation and that newly formed bone originates from the cells on
the CultiSpher-S® carriers. This results, 6 and 8 weeks post-
implantation, in faster bone repair in the defects filled with the
hydrogel plus CultiSpher-S® carriers in comparison to the control
defects. Surprisingly, there is no formation of new bone originating
from the hydroxyapatite carriers. The hydrogel by itself seems to
stimulate the natural repair process.
247
SUMMARY
During the experiments, several evaluation techniques are used. A

lot of problems have been encountered during the
immunohistochemical evaluation. Micro tomographical analysis offers
promising perspectives to evaluate bone healing. Chapter 4 describes
the optimalisation of both techniques for use on bony tissue
embedded in a low-temperature methacrylate resin.
Immunohistochemistry can be helpful in improving the understanding
and evaluation of the in vivo tissue responses at a molecular level. In
Part 4.1 several commercially available antibodies (anti-KI67, anti-
vimentin, anti-CD31, anti-core-binding factor alpha-1, anti-osteocalcin,
anti-alkaline phosphatase, anti-MAC387, anti-CD3, anti-CD20, anti-
CD20cy, anti-CD79 and anti-CD45) are evaluated on Technovit 9100
New® embedded goat tissues. Only anti-vimentin, anti-osteocalcin,
anti-MAC387 and anti-CD3 reveal positive staining. These antibodies
can be routinely used to evaluate goat tissues at molecular level. The
use and development of alternative antibodies might further
supplement and complete the possibilities for immunohistochemical
analysis of goat tissue samples. In recent years, micro-computed
tomography (µCT) has emerged as an evaluation technique for new
bone formation in tissue-engineering models. µCT protocols are
currently quite different depending on the used equipment and
software. Part 4.2 describes the validation of a high resolution µCT
scanner as a tool for assessing the micro-architecture of regenerating
cortical bone by evaluating the correlation with results obtained by
histomorphometry. Technovit 9100 New embedded tibial defect
samples are scanned (2D and 3D) and analyzed with an in-house
developed µCT scanner and then histomorphometrically analyzed on
4µm sections. Bone volume density, bone surface density, bone-
specific surface, trabecular thickness and trabecular separation are
calculated. There are strong positive correlations between
histomorphometric analysis, 2D and 3D µCT for bone volume density
248
SUMMARY
analysis. The difference plot method reported by Bland and Altman

reveals agreement between the used methods. Our results show that
µCT analysis is a useful and adjunctive tool for imaging and
nondestructively quantifying new bone healing in bone tissue
engineered models.
All relevant findings about the development of the goat model, the
experimental studies and the optimalisation of the analytical methods
are discussed in the General Discussion. In conclusion, the presented
PhD shows that the new goat model is suitable to perform preliminary
in vivo tests of potential bone enhancing composites. The tested in
situ crosslinkable and biodegradable polymers have promising
characteristics to be applied in orthopaedic purposes especially when
they are mixed with bone enhancing molecules and bone promoting
cells. A further modification of the polymers (hardness) and the
carriers (increase of the porosity of hydroxyapatite) is necessary
before clinical applications become a reality. Finally
immunohistochemistry and µCT are useful techniques in the
evaluation of bone healing processes. In order to refine the evaluation
methods of bone healing, further research is required to improve the
immunohistochemical analysis of low-temperature methylmethacrylate
resin-embedded goat bony tissues and further development and
standardization of µCT scanners and protocols will be necessary to
develop a reliable 3D evaluation technique.
249
SAMENVATTING
Bij kleine breuken wordt geregeld gebruik gemaakt van de sterke

regeneratiecapaciteit van bot. Toch is botheling niet mogelijk bij groot
botverlies of ernstige botschade die het verwijderen van bot tot gevolg
heeft. In deze gevallen zijn botgreffen noodzakelijk om een
aanvaardbare genezing te bekomen.
In Hoofdstuk 1 worden de huidige inzichten om botheling en -

regeneratie te stimuleren belicht in de diergeneeskunde. De huidig
gebruikte technieken om botdefecten te herstellen zijn vaak
onvoldoende. Vooral grote, onregelmatige defecten kunnen aanleiding
geven tot de vorming van minderwaardig weefsel. Hierdoor worden
tegenwoordig verschillende denkpistes onderzocht om de botheling te
promoten in verschillende gevallen. Het gebruik van botstimulerende
greffen kan een indicatie zijn bij meerdere heelkundige disciplines
zoals hoofdchirurgie, tandheelkunde, lange been- en
gewrichtschirurgie. In de diergeneeskunde is het gebruik van deze
greffen eerder beperkt maar belovende resultaten in humane studies
geven ook hoopvolle perspectieven voor de diergeneeskundige
orthopedie. Autogreffen zijn de “gouden standaard” om botheling te
stimuleren. Allo- en xenogreffen kunnen als alternatief aangewend
worden maar zijn veel minder osteoinductief en –conductief.
Synthetische materialen zoals keramische producten en/of keramiek-
collageen composieten, natuurlijke koralen, koraalhydroxyapatiet en
resorbeerbare polymeren worden ook in de botheling aangewend. Als
natuurlijke substanties worden beenmerg, stamcellen en verschillende
groeifactoren zoals BMP-2 aangewend. “Bone tissue engineering” is
een nieuwe benadering om botregeneratie te stimuleren. Hierbij wordt
een 3D poreuze matrix (scaffold) met mogelijk osteogene cellen
251
SAMENVATTING
gecombineerd om uiteindelijk botgelijkend weefsel te vormen dat

grote botletsels kan doen helen. De toepassing van versnelde
botregeneratie in de diergeneeskunde is relatief beperkt tot
experimentele studies met diermodellen voor humane toepassingen.
Wel bestaan er enkele diergeneeskundige case reports en klinische
proeven.
Het doel van dit doctoraatswerk (Hoofdstuk 2) is de beoordeling

van potentiële botstimulerende producten in een in vivo tibia
geitenmodel.
In Hoofdstuk 3 worden verschillende ‘huisgemaakte’ biologisch

afbreekbare polymeren, die crosslinken in situ en bij in vitro testen
interessante botstimulerende eigenschappen hadden, getest in
unicorticale, ‘non-critical sized’ tibiadefecten van een in vivo
geitenmodel. In Deel 3.1 van dit doctoraatswerk wordt een poreuze
methacrylaat-endcapped botmatrix bestaand uit D,L-lactide, -
caprolactone en 1,6-hexaandiol beoordeeld die in situ crosslinkt onder
invloed van licht en biologisch afbreekbaar is. Drie verschillende
polymeermengsels (zuiver polymeer waaraan 30% bioactief glas of α-
tricalciumfosfaat werd toegevoegd) worden getest. De drie polymeren
worden ad random aangebracht in één van vier (6,0 mm diameter)
gaten. Het vierde defect wordt leeg gelaten als controle.
Biocompatibiliteit en bothelingsmogelijkheden worden beoordeeld
door radiografie, histologie en histomorfometrie op verschillende
tijdstippen post operatief. Het zuivere polymeer is zeer biocompatibel
en beperkt osteoconductief. Het toevoegen van α-tricalciumfosfaat
verhoogt het osteoconductief vermogen. In een volgend experiment
(Deel 3.2) worden verschillende combinaties van het polymeer met uit
beenmerg ontwikkelde mesenchymale stamcellen (gezaaid op
gelatineuze CultiSpher-S® microdragers) en α-tricalciumfosfaat in
hetzelfde geitenmodel getest. De resultaten tonen celoverleving en –
252
SAMENVATTING
vermeerdering in de met polymeer gevulde defecten. De toevoeging

van α-tricalciumfosfaat verhindert de groei van de uit beenmerg
ontwikkelde mesenchymale samcellen in vergelijking met de andere
polymeermengsels. Een snellere resorptie en hogere porositeit van
het polymeer is onontbeerlijk om meer celproliferatie en botingroei
mogelijk te maken. In het laatste experiment (Deel 3.3) wordt een
chemisch gemodificeerde vorm van Pluronic® F127 hydrogel getest in
hetzelfde geitenmodel. Uit autoloog beenmerg worden mesenchymale
stamcellen ontwikkeld, die gemengd worden met de gemodificeerde
hydrogel om de botvorming te stimuleren. De cellen worden gekweekt
en gepredifferentieerd op 2 soorten celdragers: gelatineuze
CultiSpher-S® microdragers en tubulaire hydroxyapatietdragers.
Radiografische en histologische beoordeling toont aan dat de
botheling vergelijkbaar is in de vier gaten 2 en 4 weken na het
inbrengen. De cellen op de CultiSpher-S® dragers vormen nieuw bot.
Dit bot geeft 6 en 8 weken na de operatie een snellere botheling in de
gaten met hydrogel en CultiSpher-S® in vergelijking met het lege gat.
Een verrassende vaststelling is het uitblijven van botvorming vanuit de
hydroxyapatietdragers. De hydrogel zonder cellen blijkt ook het
natuurlijk helingsproces te bevorderen.
Tijdens de onderzoeken worden verschillende evaluatiemethoden

gebruikt. De immunohistochemische beoordeling verliep niet zonder
problemen. MicroCT analyse biedt interessante mogelijkheden om de
botheling te beoordelen.
Hoofdstuk 4 beschrijft het op punt stellen van beide methoden bij

de evaluatie van bot ingebed in methacrylaathars die polymeriseert bij
lage temperatuur (Technovit 9100 New®). Immunohistochemie kan de
inzichten en beoordeling van in vivo weefselreacties op moleculair
niveau verbeteren. In Deel 4.1 worden verschillende beschikbare
antilichamen (anti-KI67, anti-vimentine, anti-CD 31, anti-core-binding
factor alfa-1, anti-osteocalcine, anti-alkalische fosfatase, anti-
253
SAMENVATTING
MAC387, anti-CD3, anti-CD20, anti-CD20cy, anti-CD79 en anti-CD45)

getest op Technovit 9100 New® ingebed geitenweefsel. Enkel anti-
vimentine, anti-osteocalcine, anti-MAC387 en anti-CD3 kleuringen zijn
positief. Deze antilichamen kunnen routinematig gebruikt worden om
geitenweefsel op moleculair niveau te beoordelen. Het gebruik en de
ontwikkeling van andere antilichamen zou verder de mogelijkheden
van immunohistochemische analyse van geitenweefsel ten goede
komen. De laatste jaren kent microCT analyse een enorme opgang
als evaluatiemethode voor nieuwbeenvorming bij tissue engineering.
MicroCT protocols zijn tegenwoordig enorm verschillend afhankelijk
van het gebruikte materiaal en software. Deel 4.2 beschrijft de
validatie van een hoge resolutie microCT scanner als middel om de
micro-architectuur van regenererend corticaal bot te beoordelen door
de correlatie met histomorfometrie te bepalen. Stalen van
tibiadefecten uit de hydrogel geitenproef worden gescand (2D en 3D)
en geanalyseerd met een huisgemaakte microCT scanner en nadien
histomorfometrisch geanalyseerd op 4µm coupes.
Botvolumedichtheid, botoppervlaktedichtheid, botspecifieke
oppervlakte, trabeculaire dikte en trabeculaire scheiding worden
berekend. Er is een sterke positieve correlatie tussen de
histomorfometrische analyse, 2D en 3D microCT wat betreft de
botvolumedichtheid. De grafische weergave volgens Bland en Altman
toont de gelijkenis tussen de gebruikte technieken aan. MicroCT is
een handige en aanvullende manier om op een niet destrucieve
manier de nieuwbeenvorming in tissue engineering modellen te
kwantificeren.
Alle relevante bevindingen over de ontwikkeling van het

geitenmodel, de experimentele studies en de optimalisatie van de
analysemethodes worden besproken in de Algemene Discussie. De
resultaten van het huidig doctoraatswerk tonen aan dat het nieuw
ontwikkeld geitenmodel gepast is voor preliminair in vivo onderzoek
254
SAMENVATTING
van botgroeistimulerende composieten. De geteste biologisch

afbreekbare polymeren die in situ crosslinken hebben belovende
eigenschappen om ingezet te worden bij orthopedische chirurgie,
vooral wanneer ze gemengd worden met botstimulerende
bestanddelen en cellen. Een verdere aanpassing van de polymeren
(hardheid) en de dragers (hogere porositeit van hydroxyapatiet) is
noodzakelijk om ze effectief bij klinische toepassingen in te zetten.
Ten slotte zijn immunohistochemie en microCT handige technieken
om botheling te evalueren. Verder onderzoek is nodig om de
immunohistochemische analyse van geitenweefsel in
methacrylaatharsen te verbeteren en verdere ontwikkeling en
standaardisatie van microCT scanners en protocols zullen nodig zijn
om een betrouwbare 3D evaluatiemethode te ontwikkelen.
255
CURRICULUM VITAE
Geert Vertenten werd geboren op 3 januari 1978 te Gent. Na het

beëindigen van het secundair onderwijs, richting Wetenschappen-
Wiskunde, aan het Sint-Vincentiuscollege te Eeklo, is hij in 1996
gestart met de studies Diergeneeskunde aan de Universiteit Gent. Hij
behaalde in 2002 het diploma van Dierenarts met grote
onderscheiding.
In september 2002 ging hij werken in de ‘Clinique Vétérinaire La

Licorne’ in La Souterraine (Frankrijk), waar hij zich als
praktijkdierenarts vooral toelegde op de pathologie van het Limousin
rundvee. In juni 2004 trad hij in dienst bij de Vakgroep Heelkunde en
Anesthesie van de Huisdieren als voltijds assistent, dit onder de
leiding van dierenarts Lieven Vlaminck en de professoren Gasthuys,
Steenhaut, Martens en Desmet. Zijn voornaamste taak bestond erin
klinisch onderricht te geven aan de studenten van het laatste jaar en
hij was verantwoordelijk voor de chirurgie van de herkauwers. Hij
deed ook geregeld anesthesieën en kleine ingrepen bij paarden en
hielp mee aan de nacht- en weekenddiensten van de vakgroep. Naast
deze klinische activiteit deed hij ook wetenschappelijk onderzoek over
chirurgie en orthopedie bij landbouwhuisdieren en maakte hij een
doctoraat over potentiële botsubstituten in een geitenmodel wat
uiteindelijk tot deze thesis leidde. Sinds augustus 2009 is Geert
Vertenten werkzaam bij V.M.D. n.v. als Technical Service and Product
Manager.
Geert Vertenten is auteur of mede-auteur van 19 publicaties in

internationale en nationale tijdschriften, was spreker op meerdere
wetenschappelijk congressen en reviewer voor ‘Tissue Engineering’,
‘European Cells and Materials’ en ‘Current Nanoscience’.
257
BIBLIOGRAPHY
PUBLICATIONS
Steenhaut, M., A. Martens, L. Vlaminck & G. Vertenten, 2004:

Incarceration of the small intestine through the omental
(epiploic) foramen: a retrospective study of 100 horses. Vlaams
Diergeneeskundig Tijdschrift, 73, 17-30.
Vertenten, G., A. Martens, J. Declercq, S. Schauvliege, L. Weiland &
F. Gasthuys, 2006: Surgical repair of a tibial fracture in a
Belgian Landrace pig. Veterinary and Comparative
Vertenten, G., 2006: Claudicatieproblemen bij meststieren. Vlaams
Diergeneeskundig Tijdschrift, 75, 461-462.
Vertenten, G., 2006: L'évolution récente de la parésie spastique du
veau. L'Hebdo Vétérinaire, 176, 14-17.
Vertenten, G., 2006: Recente evolutie van spastische parese bij het
kalf. Het Dierenartsen Weekblad, 44, 14-17.
Vertenten, G., L. Vlaminck, T. Gorski, E. Schreurs, W. Van Den
Broeck, L. Duchateau, E. Schacht & F. Gasthuys, 2008:
Evaluation of an injectable, photopolymerizable three-
dimensional scaffold based on D,L-lactide and epsilon-
caprolactone in a tibial goat model. Journal of Materials
Science-Materials in Medicine, 19, 2761-2769.
Vertenten, G., L. Vlaminck, R. Ducatelle, E. Lippens, M. Cornelissen
& F. Gasthuys, 2008: Immunohistochemical Analysis of Low-
Temperature Methylmethacrylate Resin-Embedded Goat
Tissues. Anatomia Histologia Embryologia, 37, 452-457.
Vertenten, G., E. Lippens, J. Girones, T. Gorski, H. Declercq, J.
Saunders, W. Van den Broeck, K. Chiers, L. Duchateau, E.
Schacht, M. Cornelissen, F. Gasthuys & L. Vlaminck, 2009:
Evaluation of an Injectable, Photopolymerizable, and Three-
Dimensional Scaffold Based on Methacrylate-Endcapped
Poly(D,L-Lactide-co-epsilon-Caprolactone) Combined with
Autologous Mesenchymal Stem Cells in a Goat Tibial
259
BIBLIOGRAPHY
Unicortical Defect Model. Tissue Engineering Part A, 15, 1501-

1511.
Vertenten, G., 2009: Abord de la parésie spastique des veaux. Point
Veterinaire, 40, 39-42.
Vertenten, G., 2009: Quelles complications lors d’omentopexie? Point
Veterinaire, 40, 11.
Vertenten, G., J. Declercq, F. Gasthuys, L. Devisscher, S. Torfs, G.
van Loon & A. Martens, 2009: Abomasal end-to-end
anastomosis as treatment for abomasal fistulation and
herniation in a cow. Veterinary Record, 164, 785-786.
Lippens, E., G. Vertenten, J. Girones, H. Declercq, J. Saunders, J.
Luyten, L. Duchateau, E. Schacht, L. Vlaminck, F. Gasthuys &
M. Cornelissen, 2009: Evaluation of bone regeneration with an
injectable, in situ polymerizable Pluronic(R) F127 hydrogel
derivative combined with autologous mesenchymal stem cells
in a goat tibia defect model. Tissue Engineering Part A. In
press.
Pardon, B., G. Vertenten, I. Durie, J. Declercq, D. Everaert, P.
Simoens & P. Deprez, 2009: Four cases of omental herniation
in cattle. Veterinary Record, 165, 718-721.
Vertenten, G., F. Gasthuys, M. Cornelissen, E. Schacht & L.
Vlaminck, 2009: Enhancing bone healing/regeneration : present
and future perspectives in veterinary orthopaedics. Veterinary
and Comparative Orthopaedics and Traumatology. In press.
Lippens E., L. Vlaminck, H. Declercq, G. Vertenten, S. Mullens, J.
Luyten, E. Schacht, F. Gasthuys, M. Cornelissen, 2009: In vitro
and in vivo evaluation of gelatin and hydroxyapatite based cell
delivery systems for bone tissue engineering using a
subcutaneous implantation model in goats. Journal of Tissue
Engineering and Regenerative Medicine. Submitted.
Vertenten, G., E. Lippens, M. Dierick, L. Van Hoorebeke, E. Schacht,
M. Cornelissen, F. Gasthuys & L. Vlaminck, 2009: Agreement
between micro-computed tomography and histomorphometry
for evaluation of new bone formation in a tissue-engineered
cortical tibial goat model. European Cells & Materials.
Submitted.
Lippens, E., I. Swennen, J. Gironès, H. Declercq, G. Vertenten, L.
Vlaminck, F. Gasthuys, E. Schacht & M. Cornelissen, 2009:
Modified Pluronic® F127 in combination with cell-loaded
CultiSpher-S® for bone tissue engineering. Journal of Materials
Science-Materials in Medicine. Submitted.
260
BIBLIOGRAPHY
Pardon, B., G. Vertenten, S. Schauvliege, F. Gasthuys, G. van Loon

& P. Deprez, 2009: Left displacement of the abomasum in 2
cows during twin pregnancy. Journal of the American Veterinary
Association. Submitted.
Vertenten, G., 2009: Vaccinatie tegen paramyxovirus: een juiste
keuze geeft een juiste bescherming. Het Spoor der
Kampioenen, 8, 38-39.
261
BIBLIOGRAPHY
INTERNATIONAL ORAL PRESENTATIONS
Schacht E., P. Dubruel, T. Gorski, I. Swennen, S. Van Vlierberghe, M.

Cornelissen, F. Gasthuys, G. Vertenten, R. Unger & J.
Kirckpatrick, 2006: Tailor made matrices for tissue engineering
derived from crosslinkable polymeric precursors. Internat.
Conference on Advance in Biomaterials for Drug Delivery and
Regenerative Medicine, Capri, 61.
Lippens E., G. Vertenten, J. Gironès, J. Luyten, F. Gasthuys, E.
Schacht & M. Cornelissen, 2008: Gelatin and hydroxyapatite
cell delivery systems in bone tissue engineering. i-sup 2008,
Conference 1: Smart Materials for Sustainable Production.
Vertenten G., L. Vlaminck, S. Muylle & F. Gasthuys, 2008: Bovine
digit amputation with primary closure: surgical technique and
follow-up of 45 cases. 15th international symposium & 7th
conference on Lameness in Ruminants, Kuopio, 121-125.
Declercq J., G. Vertenten, L. Devisscher, V. Barberet, F. Gasthuys, P.
Verleyen & A. Martens, 2008: Bilateral Surgical Fracture Repair
in an Alpaca. XXV Jubilee World Buiatrics Congress, Budapest,
145-146.
Vertenten G., Declercq J., A. Martens, L. Devisscher, S. Torfs, G. van
Loon, F. Gasthuys, 2008. Abomasal end-to-end anasthomosis
as a treatment for an extensive abomasal hernia and fistula in a
cow. XXV Jubilee World Buiatrics Congress, Budapest, 257-
258.
Vertenten G., E. Lippens, J. Gironès, J. Saunders, W. Van den
Broeck, K. Chiers, L. Duchateau, E. Schacht, M. Cornelissen,
F. Gasthuys & L. Vlaminck, 2008: Evaluation of methacrylate-
encapped poly(D,L-lactide-co-epsilon-caprolactone) as
substrate for bone tissue engineering in a goat tibial unicortical
defect model. International Bone-Tissue-Engineering Congress,
Hannover, 71.
Schacht E., I. Swennen, J. Gironès, S. Van Vlierberghe, M.
Cornelissen, E. Lippens, F. Gasthuys & G. Vertenten, 2008:
Biodegradable scaffolds for tissue engineering prepared from
crosslinkable precursors. International Bone-Tissue-
Engineering Congress, Hannover, 127.
Vertenten G., E. Lippens, J. Gironès, J. Saunders, W. Van den
Broeck, K. Chiers, L. Duchateau, E. Schacht, M. Cornelissen,
F. Gasthuys & L. Vlaminck, 2008: Evaluation of an injectable,
photopolymerizable scaffold based on D,L-lactide and ε-
262
BIBLIOGRAPHY
caprolactone combined with autologous mesenchymal stem

cells in a goat model. 4th European Congress for Medical and
Biomadical Engineering, Antwerp, 277-278.
Lippens E., H. Declercq, G. Vertenten, F. Gasthuys, J. Luyten, E.
Schacht & M. Cornelissen, 2008: Cell delivery systems & in situ
cross-linkable filling materials in bone tissue engineering. 4th
European Congress for Medical and Biomadical Engineering,
Antwerp, 291.
263
DANKWOORD
Wat ik enkele jaren geleden als onmogelijk beschouwde is toch

gelukt. Geert Vertenten is gedoctoreerd in de diergeneeskunde over
een heel actueel en innoverend onderwerp dat naast
diergeneeskundige vooral humane perspectieven meebrengt. Ik heb
me gedompeld in een aanvankelijk voor mij onbekende wereld die ik
langzaam leerde appreciëren en leidde tot een echte obsessie. Een
heleboel mensen – het is een onbegonnen taak om deze allen bij
naam te noemen – hebben de voorbije jaren hun rechtstreekse of
onrechtstreekse bijdrage geleverd aan het tot stand komen van dit
proefschrift. Hierbij wil ik hen van harte danken. Tot een aantal
personen wil ik graag een persoonlijk woordje van dank richten omdat
ze één voor één, op hun eigen specifieke manier, voor mij heel wat
hebben betekend.
Mijn eerste woord van dank gaat uit naar mijn promotoren: Lieven
Vlaminck, Frank Gasthuys en Maria Cornelissen, zonder wie er
helemaal geen sprake zou zijn van het afgeleverde werk. Lieven
stond steeds klaar met een luisterend en begrijpend oor en wist
telkens de puntjes op de i te zetten als het op schrijven aan kwam.
Hekelpunten kon hij steeds op zijn eigen diplomatische wijze het
hoofd bieden. Prof. Gasthuys opende telkens opnieuw de juiste
deuren waardoor dit onderzoek zowel financieel als wetenschappelijk
de juiste richting is uitgegaan. Zijn immer kritische oog vormde een
continue stimulans om elke stap van dit proefschrift zorgvuldig te
plannen, uit te voeren en de resultaten te analyseren. Professor
Cornelissen, Ria, kwam als een geschenk uit de hemel. Dankzij haar
kreeg mijn onderzoek een enorme positieve stimulus en kwam het in
een stroomversnelling. Begripvol en vol toewijding hebben we de vele
resultaten overlopen op zoek naar een waardig vervolg van de
265
DANKWOORD
bekomen resultaten. Een persoon die ik nooit zal vergeten zowel op

intelectueel als emotioneel vlak. Ik vind het jammer dat onze
samenwerking na dit doctoraat geen vervolg kent. Het doet steeds
pijn iets wat je jarenlang met overgave opgebouwd hebt achter te
laten. Ria, hopelijk kruisen onze wegen zich nog meerdere keren.
Voor de eigenlijke planning en uitvoering van dit proefschrift is de

hulp en kennis van Professor Etienne Schacht van cruciaal belang
geweest. Zijn kennis en ontwikkeling van de gebruikte polymeren
vormen de basis van dit onderzoek. Zijn eenvoudige vraag om de
eigenschappen van de door zijn groep ontwikkelde polymeren te
testen leidde tot een constructieve samenwerking die hopelijk nog
jaren kan voortgezet worden. Interfacultair onderzoek verbreedt ieders
visie en brengt mensen met verschillende opleidingen samen
teneinde hetzelfde doel te bereiken.
De kunde en toewijding tot het ontwikkelen van polymeren met een

gewenste eigenschap is voor mij nog steeds iets magisch. Deze
magie werd ten volle beheerst door Dr. Tomasz Gorski. Tomek
ontwikkelde zijn eigen methacrylate-encapped polymeer bestaande uit
D,L-lactide, ε-caprolactone en 1,6-hexaandiol dat door ons
onderzoeksteam niet voor niets kortweg het polymeer van Tomek
genoemd werd. Jammer genoeg keerde je te vroeg terug naar je
moederland Polen. Je korte terugkeer naar Gent was een redding
voor dit onderzoek. Bedankt Tomek. Je was een echte dierenvriend
en de dierenwereld is je zeker dankbaar voor je hulp in de
behandeling van hun pathologieën.
Dr. Jordi Gironès was een waardige opvolger van Tomek. Stil en
onopvallend liep hij rond in de operatiezaal en draagde steeds
constructief zijn steentje bij. Elke vraag beantwoorde hij steeds to the
point. Jordi, ik wens je nog veel succes in je verdere ondernemingen.
Woorden schieten me tekort als ik Dr. Evi Lippens moet bedanken.

Waar zouden we staan zonder haar. De goede resultaten van dit
onderzoek zijn zeker te danken aan haar toewijding en perfectie. Haar
266
DANKWOORD
oog voor detail en vastberadenheid zijn ongeëvenaard. Stamcellen

voelen zich echt in hun sas als ze door Evi opgekweekt worden. Zo’n
vermenigvuldigingen werden zelfs niet in de bijbel beschreven. Evi,
bedankt dat je steeds bereid was om mijn vele vragen correct te
beantwoorden. Samen hebben we iets moois bereikt.
Naast Ria en Evi wil ik ook al de andere mensen van de vakgroep

Medische Basiswetenschappen bedanken. Het was een ware thuis
voor mij: aanvankelijk aan de stoffige Bijloke waar je aan de inkom
door de loslopende kippetjes verwelkomd werd, zelfs in tijden van
vogelgriep. De vernieuwde gebouwen aan het UZ waren een
reuzenstap vooruit. In deze rustige omgeving kan wetenschap
beoefend worden op ‘hoog’ niveau. Heidi, bedankt voor je in vitro
onderzoek van de cellen en polymeren. Leen, bedankt voor het
snijden van de coupes en het uitvoeren van de kleuringen. Nelly, je
stond steeds klaar om mijn beenmergpuncties te ontvangen. Eens ik
ze aan jou gegeven had, wist ik dat ze in goede handen waren.
Roger, als man van de vakgroep speel je een belangrijke rol.
Ook op de vakgroep Morfologie werd ik steeds met een

vriendelijke lach begroet. Dagen heb ik er doorgebracht om de
staaltjes in te bedden, te snijden en de coupes te beoordelen. Prof.
Wim Van den Broeck gaf een gepast antwoord op pertinente vragen
en vroeg geregeld of alles naar wens verliep. Hoe kon dit anders. Bart
is een toegewijde laborant die oneindig veel capaciteiten heeft en zeer
luisterbereid is. Ook Lobke, Liliana, Patrick en Eric hielpen me als ik
op zoek was naar product A, boek B of persoon C en hadden steeds
tijd voor een leuk babbeltje. Leuke en constructieve herinneringen heb
ik ook aan Prof. Paul Simoens, Anamaria, Pieter, Christophe, Ward,
Sofie M. en Sofie B.
Voor het radiografische luik van dit proefschrift ben ik veel dank
verschuldigd aan Prof. Jimmy Saunders. Bij Jimmy kon ik steeds
terecht voor alle praktische regelingen en de wetenschappelijk
voorbereiding en interpretatie van de radiografische evaluatie.
267
DANKWOORD
Hartelijk dank Jimmy voor de vlotte en aangename samenwerking!

Ook de medewerking van Elke Schreurs, Virginie Barberet en Yseult
Baeumlin aan dit proefschrift is van onschatbare waarde geweest.
Telkens opnieuw gestandaardiseerde opnamen maken bleek niet
altijd even makkelijk te zijn. Het is dankzij hun professionalisme en
perfectionisme dat ook dit deel tot een goed einde gebracht werd. Ik
hoop dat we de vriendschap die hieruit gegroeid is nog lang met ons
mogen meedragen.
Prof. Ducatelle, bedankt voor de professionele beoordeling van de

immunohistochemische preparaatjes van de geiten. Wat we onder de
microscoop zagen was niet altijd wat ik gehoopt had, maar dat hoort
nu eenmaal bij wetenschappelijk onderzoek. Bedankt ook voor het
filosoferen over bepaalde hypothesen. Sarah Loomans is een
laborante die voor dit doctoraat onmisbaar was. Ongelooflijk hoe ze
naast haar vele andere taken steeds mijn vragen met hetzelfde
enthousiasme en dezelfde glimlach beantwoordde. Het
immunomisterie leek aanvankelijk niet te doorgronden, maar dankzij
jouw werkijver ben ik er toch ingeslaagd een pad in het oerwoud te
leggen. Het was een leuke samenwerking. Vele groetjes aan Hans en
zus Liesbeth. Ook Prof. Koen Chiers wil ik bedanken voor het kritisch
nalezen van mijn studies.
Prof. Duchateau, de resultaten van een artikel kunnen pas goed

geïnterpreteerd worden als ze ook statistisch geanalyseerd worden.
Jij hebt me daarin op een voortreffelijke manier bijgestaan. Statistiek
is voor mij nog steeds een zoektocht naar de meest gepaste methode.
Maar ik moet toegeven dat het me echt boeit. Statistiek proeven
vraagt bij mij steeds om meer en meer. Jammer dat de totale brok zo
immens groot en zwaar verteerbaar is.
Ik heb nog nooit zo een dynamisch, enthousiast, talentvol en jong

team wetenschappers tegengekomen als dat van het Centre for X-
Ray Tomography (UGCT) zijnde Manuel Dierick, Bert Masschaele,
Denis Van Loo, Jelle Vlassenbroeck, Matthieu Boone en Yoni De
268
DANKWOORD
Witte. Supergedreven waren jullie steeds met jullie wetenschap bezig,

maar een prangende vraag van mijnentwege was nooit teveel.
Hopelijk komt er ooit een in vivo microCT scanner voor ons
geitenonderzoek.
Een woordje van dank ook aan de nog niet vernoemde leden van
de examencommissie voor jullie opbouwende commentaren: Prof. Dr.
Hubert De Brabander, Dr. Jan Luyten, Prof. Dr. Hilde De Rooster en
Prof. Dr. Evelyne Meyer.
Wie ik zeker niet mag vergeten zijn de mensen van onze vakgroep
die meegewerkt hebben aan dit doctoraatsonderzoek. Op de eerste
plaats komt Cindy De Baere. Bedankt voor het bijstaan van het maken
en kleuren van de ontelbare coupes. Ik was maar al te blij dat jij dit
snijden geregeld van mij overnam. Ik hoor de microtoom nog
knetteren als het blokje niet wilde meewerken. Vele groetjes aan
Kristof en Jeroen. Ook de talrijke anesthesisten die me bijstonden om
de ingrepen bij de geitjes zo pijnloos mogelijk te laten verlopen wil ik
bedanken: Stijn Schauvliege, Els Hermans, Véronique Martin-Bouyer,
Linda Weiland, Lindsey Devisscher, Miguel Gozalo Marcilla, Caroline
Gadeyne en Stefanie Segaert. En natuurlijk onze onmisbare mensen
die instonden voor de hospitalisatie: Annelies Van den Eede, Eva
Pint, de interns en last but not least de vele studenten.
Onvoorstelbaar hoe deze laatste groep zich dag in dag uit het lot van
mijn witte meisjes ten harte namen. Dit geldt zeker ook voor de
stalknechten Erwin, Hubert, Didier V., Nadine, Wilfried, Nico en Maité.
Een speciaal woord van dank ook voor den Guido. Je hebt me enorm
veel bijgebracht. De omgang met dieren werd dankzij jou een
aangenaam spel. Jammer dat je door je kritieke gezondheid niet
langer mijn copiloot kon zijn tijdens de operaties. Je wekelijks
donderdags bezoekje was steeds een aangenaam weerzien. Hou je
sterk Guido. Een speciaal woord van dank naar de gezellige
sterelisatiemeiden. Valérie, Caroline, Cindy en Nadine, we hebben
nogal een potje afgelachen. Mijn patiëntjes stonden steeds te blinken
269
DANKWOORD
na een beurtje van jullie. En vergeet niet: sluit steeds de deuren en

niet meer dan twee clippers per patiënt. Annemie, je bent een echte
hygiënisch specialiste, is het nu op de Ferrari, op den Porsch of aan je
radiokarretje. Nog eens sorry voor de vele keren dat we de net
gekuiste zaal direct weer in een smerig kot veranderden. Ook de
mensen van de vakgroep die niet rechtstreeks iets met dit onderzoek
te maken hadden maar die me wel dag in dag uit bijstonden in de
kliniek verdienen een plaatsje in dit dankwoord. Prof. Martens, Ann, je
bent een ongelooflijke vrouw. Zo bestaan er geen twee. Ik bewonder
je enthousiasme en vastberadenheid. Bedankt voor de vele kansen
die je me aanbood. Je zal steeds een voorbeeld voor me zijn. Jammer
dat ons ECVS avontuur zo abrupt afgebroken moest worden door
toedoen van anderen. Iets wat ik nooit begrepen heb en waarvan de
logica me volledig ontsnapt. Prof. Steenhaut, bedankt voor het met
raad en daad bijstaan van de runderoperaties. Relativeringsvermogen
is een element die de vakgroep door jouw vertrek is kwijtgespeeld.
Prof. Desmet, je vele verhalen over je runderervaringen wisten me
steeds te boeien. Je bracht me ook enkele essentiële knepen bij over
claudicatieproblemen bij herkauwers. Bedankt ook aan Jeroen
Declercq, ons runderscoopavontuur is maar een van de vele
projectjes waar we samen op de tandem zaten. Ook een woord van
dank voor de vele toffe momenten aan Stefaan, Michèle, Maarten,
Frederik, Tamara, Pieter, Kelly, Lies, Mireilla, Hanna, Eva V., Jan,
Johan, Didier R., Remi, Heidi en Veerle.
De rundercollega’s van de andere vakgroepen blijven in mijn

geheugen gelinkt aan vele aangename en succesvolle
samenwerkingen. Vier belangrijke vriendschappen zijn hierdoor
ontstaan die ik steeds zal koesteren: Dries Everaert, Bart Pardon,
Marcel Van Aert en Jef Laureyns.
Ook mijn ouders en schoonouders wil ik danken voor hun blijvende

belangstelling en steun tijdens mijn doctoraatsjaren. Mama en papa,
270
DANKWOORD
bedankt voor de vele kansen die jullie me gaven om te komen waar ik

nu sta.
Last but not least wens ik af te sluiten door twee heel belangrijke
personen in mijn leven op het ereschavotje te zetten zonder wie dit
hele proefschrift en alle moeite die ik ervoor gedaan heb geen enkel
betekenis zouden hebben. Valérie, het beste wat me in mijn leven
overkomen is, is dat ik jou mocht ontmoeten. We hebben samen al
veel doelen tot een magnifiek einde gebracht en maken dagelijks nog
nieuwe toekomstplannen. Door het beëindigen van deze thesis bieden
zich hopelijk nieuwe opportuniteiten aan die onze levenskracht elke
dag doen toenemen. Ons gezin is een echte thuis die ik levenslang
zal koesteren. Gustave, ongelooflijk hoe je dagelijks straalt. Je blik
geeft me telkens een enorme boost aan levenskracht.
Valérie, Gustave en … : veel liefs, gros bisous !!!
Geert
271

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Evaluation of potential bone

substitutes in a goat model

Proefschrift ter verkrijging van de graad van

Doctor in de Diergeneeskundige Wetenschappen (PhD)

aan de Faculteit Diergeneeskunde, Universiteit Gent

Evaluation of potential bone substitutes in a goat model

Cover Photo: Jozef Kusters, Schootseweg 50, 2381 Weelde

CHAPTER 2 AIMS OF THE STUDY 39

CHAPTER 3 EVALUATION OF BONE SUBSTITUTES 43

3.2 EVALUATION OF AN INJECTABLE,

3.3 EVALUATION OF BONE REGENERATION WITH

CHAPTER 4 ADAPTED ANALYTICAL METHODS 147

GENERAL DISCUSSION 201

CURRICULUM VITAE 257

ACD-A Anticoagulant Citrate Dextrose Solution Formula A

Prof. dr. Ulrich Libbrecht

The self-healing capacity of bone is widely used for the repair of

The clinical practice of bone grafts to repair, replace or supplement

Newer techniques involve the use of natural and synthetic bone

The increasing popularity of arthroscopic procedures in

In this PhD thesis, several potential ‘home made’ injectable bone

Adapted from: G. Vertenten, F. Gasthuys, M. Cornelissen, E. Schacht, L. Vlaminck

Comparative Orthopaedics and Traumatology.

Fracture healing typically results in restoration of the original

Intimate contact of the fracture fragments is required for secondary

generation of an adequate blood supply (angiogenesis) is mandatory

INDICATIONS FOR USE OF ENHANCED BONE

The use of bone enhancing grafts can be indicated in different

Surgery of the head and dentistry

Cleft palate deformity is a relatively common congenital

Dentistry related bone graft application in man focuses on repair of

whereby the thickness of the maxillary sinus floor is increased with a

Application of bone substitutes in veterinary dentistry has been

Long bone and joint surgery

Bone enhanced healing is an essential but not always obvious part

DIFFERENT SUBSTITUTES TO ENHANCE BONE

Bone grafts harvested from a donor site site can be transplanted to

Auto-, allo- and xenografts

Autografts still represent the “gold standard” material for enhanced

The use of cancellous and cortical bone autografts in veterinary

Lindner, 2008). On the other hand, both graft substances possess

Demineralized bone matrix (DBM) is a good option as an allograft

Deproteinized bovine bone is the most widely used xenograft

Alternative xenografts originate from the exoskeleton of

Synthetic and natural bone substitutes

Many synthetic and natural materials are available to the surgeon,

Bone marrow - stem cells

Bone marrow contains osteoprogenitor stem cells that are able to

Molecules enhancing bone healing

Several growth-promoting substances involved in local regulation

signalling molecules (generally referred to as growth factors) and

Growth factors exert multiple effects on cells at both local and

Urist (1965) noted that demineralized bone matrix (DBM) could

(Sciadini & Johnson, 2000). Interspecies amino acid sequence

Development of an optimal delivery system for BMP use is still of

A last delivery method consists of implanting BMP’s with a carrier

cell expression. Non-union, open tibial fractures, spinal fusions and

Allograft Osteoconductive Risk of disease transfer

Xenograft Osteoconductive Not osteoinductive (except

Calcium Osteoconductive filling Not osteoinductive (although

Calcium sulphate Bone filler Not osteoconductive

Bioactive glass Osteoconductive No structural support, although