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

Promotoren : Prof. Dr. L. Vlaminck1

Prof. Dr. F. Gasthuys1

Prof. Dr. M. Cornelissen2

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

Ad-BMP

α-TCP

AP

BM

BMP

BMSC

BS

BSD

BSS

BV

BVD

Cbfa-1

CD

cDNA

CT

DBM

ECM

EDTA

FBS

FDA

H&E

HA

HEMA

Histo

HRP

MEM

µCT

MMA

MSC

Anticoagulant Citrate Dextrose Solution Formula A

Adenoviral Bone Morphogenetic Protein

alfa TriCalcium Phosphate

Alkaline Phosphatase

Bone Marrow

Bone Morphogenetic Protein

Bone marrow-derived Mesenchymal Stem Cells

Bone Surface

Bone Surface Density

Bone-Specific Surface

Bone Volume

Bone Volume Density

Core binding factor alpha1

Cluster of Differentiation

complementary DNA

Computed Tomography

Demineralized Bone Matrix

ExtraCellular Matrix

EthyleneDiamineTetraacetic Acid

Fetal Bovine Serum

Food and Drug Administration

Hematoxylin and Eosin

HydroxyApatite

HydroxyEthylMethAcrylate

Histology

HorseRadish Peroxidase

Minimum Essential Medium

micro-Computed Tomography

Methyl MethAcrylate

Mesenchymal Stem Cell

List of abbreviations

MTS

no

OC

PBS

PLU

PSF

rhBMP

rpm

SD

TGFβ

TS

TT

TV

UGCT

UVA

Vim

VITO

VOI

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumsalt

number

Osteocalcin

Phosphate-Buffered Saline

Pluronic

Polysulfone

recombinant human Bone Morphogenetic Protein

revolutions per minute

Standard Deviation

Tumor Growth Factor Beta

Trabecular Separation

Trabecular Thickness

Total Volume

Centre for X-ray Tomography at the Ghent University

UltraViolet A

Vimentin

Flemish Institute for Technological Research

Volume of Interest

“De Weg is Wijzer dan de Wegwijzer”

Prof. dr. Ulrich Libbrecht

1

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.

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

4

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

CHAPTER 1

5

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.


6

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

CHAPTER 1: Indications

7

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).


8

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 &

CHAPTER 1: Different substitutes

9

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).


10

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


11

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


12

(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


13

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

Donor site morbidity Limited availability Longer operation time Additional operation site & scar

Osteoconductive Osteoinductive Residing cells No immune response No risk for disease transmission Minimal ethical concerns

Allograft

Osteoconductive Higher availabitlity Avoids donor site morbidity

Risk of disease transfer Immunogenicity of fresh allografts Variable quality Limited mechanical stability Faster resorption rate No to limited osteoinduction although debatable

Xenograft

Osteoconductive Graft extender Readily available Available in large quantities

Not osteoinductive (except DBM), although debatable Ethical/religious concerns Low mechanical support Risk for disease transfer Batch difference

Calcium phosphate

Osteoconductive filling material Not inflammatory, well tolerated

Not osteoinductive (although some reports claim the opposite) Brittle and poor tensile strength No mechanical strength

Calcium sulphate

Bone filler Not inflammatory

Not osteoconductive Not osteoinductive Fast resorption


14

Bioactive glass Osteoconductive Graft extender Able to bound to soft and bone tissue Actively aids the bone formation process

No structural support, although higher mechanical strength then calcium phosphates Slow resorption rate (12-16 months)

Bone marrow Osteoinductive

Low concentration of active component Inconsistent results No mechanical support

Stem cells

Osteoinductive Sufficient amount needed No mechanical support

Demineralized bone matrix

Osteoinductive Low concentration of active component No mechanical support

Bone morphogenetic protein

Different extraction methods possible Osteoinductive Commercially available for clinical applications

Best result if derived from same animal species Inconsistent results No mechanical support

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


15

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).


16

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

CHAPTER 1: Reports of veterinary clinical cases

17

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


18

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


19

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


20

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.,


21

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


22

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


23

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).


24

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

CHAPTER 1: Current research

25

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.


26

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CHAPTER 2

Aims of the study

CHAPTER 2: Scientific aims

41

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.

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

47

SUMMARY

An in situ crosslinkable, biodegradable, methacrylate-

endcapped porous bone scaffold composed of D,L-lactide, εεεε-

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.

Evaluation of an injectable scaffold

48

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

PART 3.1: Introduction

49

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


50

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.

PART 3.1: Materials and Methods

51

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,


52

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

and bottom of the defect and photopolymerized for 40 s (500 mW/cm2

blue light, 3M Unitek Visible Light Curing UnitTM). 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.


53

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


54

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.


55

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.


56

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).

PART 3.1: Results

57

0

0,5

1

1,5

2

2,5

3

3,5

4

0 4 8 12 18 24 32

Time post surgery (weeks)

Mea

n d

en

sit

y

Control

Composite1

Composite2

Composite3

0

0,5

1

1,5

2

2,5

3

3,5

0 4 8 12 18 24 32


Me

an

pe

rio

ste

al

reac

tio

n a

nd

ca

llu

s f

orm

ati

on

Control

Composite1

Composite2

Composite3

Figure 1. Evolution of the mean densities of unicortical tibial defects treated with different composites based on serial conventional radiographic evaluation in eight goats.

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.


58

0

0,5

1

1,5

2

2,5

3

0 4 8 12 18 24 32


Mean

dis

trib

uti

on

of

peri

oste

al

rea

cti

on

Control

Composite1Composite2

Composite3

0

0,1

0,2

0,3

0,4

0,5

0,6

0 4 8 12 18 24 32


Mea

n s

oft

tis

su

e r

eac

tio

n

Control

Composite1

Composite2

Composite3

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.

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.

PART 3.1: Results

59

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).


60

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).

Figure 7. Photomicrograph of a tibial defect treated with composite no. 2, 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).

PART 3.1: Results

61

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.

Figure 8. Photomicrograph of a tibial defect treated with composite no. 2, 32 weeks postoperatively. Almost no composite left anymore. (1: periost; 2: normal cortex surrounding the defect; 3: medulla) (Von Kossa).


62

0

10

20

30

40

50

60

70

Von Kossapositive

Von Kossanegative

Polymer andempty space

Me

an

pe

rcen

tag

e

Control

Composite 1

Composite 2

Composite 3

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.

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

PART 3.1: Results

63

(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).


64

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 &

PART 3.1: Discussion

65

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


66

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


67

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


68

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.

PART 3.1: Conclusion

69

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.


70

ACKNOWLEDGEMENTS

The authors thank Cindy De Baere, Bart De Pauw, Liliana

Standaert and Lobke De Bels for technical assistance.

PART 3.1: References

71

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Liljensten, E. L., A. G. Attaelmanan, C. Larsson, H. Ljusberg-Wahren, N. Danielsen, J. M. Hirsch & P. Thomsen, 2000: Hydroxyapatite granule/carrier composites promote new bone formation in cortical defects. Clinical Implant Dentistry and Related Research, 2, 50-59.

MacNeill, S. R., C. M. Cobb, J. W. Rapley, A. G. Glaros & P. Spencer, 1999: In vivo comparison of synthetic osseous graft materials - A preliminary study. Journal of Clinical Periodontology, 26, 239-245.

Malloy, K. M. & A. S. Hilibrand, 2002: Autograft versus allograft in degenerative cervical disease. Clinical Orthopaedics and Related Research, 27-38.

Meinig, R. P., 2002: Polylactide membranes in the treatment of segmental diaphyseal defects: Animal model experiments in the rabbit radius, sheep tibia, Yucatan minipig radius, and goat tibia. Injury-International Journal of the Care of the Injured, 33, 58-65.

Melo, L. G. N., M. J. H. Nagata, A. F. Bosco, L. L. G. Ribeiro & C. M. Leite, 2005: Bone healing in surgically created defects treated with either bioactive glass particles, a calcium sulfate barrier, or a combination of both materials - A histological and histometric study in rat tibias. Clinical Oral Implants Research, 16, 683-691.

Nguyen, K. T. & J. L. West, 2002: Photopolymerizable hydrogels for tissue engineering applications. Biomaterials, 23, 4307-4314.

Paprosky, W. G. & E. L. Martin, 2002: Structural acetabular allograft in revision total hip arthroplasty. American Journal of Orthopedics, 31, 481-484.

Rose, F. & R. O. C. Oreffo, 2002: Bone tissue engineering: Hope vs hype. Biochemical and Biophysical Research Communications, 292, 1-7.


74

Saadeh, P. B., R. K. Khosla, B. J. Mehrara, D. S. Steinbrech, S. A. McCormick, D. P. DeVore & M. T. Longaker, 2001: Repair of a critical size defect in the rat mandible using allogenic type I collagen. Journal of Craniofacial Surgery, 12, 573-579.

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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

77

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.

Evaluation of an injectable scaffold with stem cells

78

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


79

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


80

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).


81


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

groups (groups of four in boxes of 16m2) 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


82

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


83

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


84

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

photopolymerized for 40 s (500 mW/cm2 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


85

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


86

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


87

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.


88

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

aspirated. A mean amount of 4.07 ± 2.4 × 106 BM-derived

mesenchymal stem cells (BMSCs) was retrieved after a culture period

of 14 days. On average 1.70 ± 0.3 × 106 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).

PART 3.2: Results

89

Table 1. Quantitative values of the bone marrow harvested from the iliac arch in eight goats.

Goat

1 Goat

2 Goat

3 Goat

4 Goat

5 Goat

6 Goat

7 Goat

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 culture

8.28× 106

3.66× 106

2.00 ×106

2.75× 106

1.48× 106

6.00× 106

2.36 ×106

6.00× 106

# Cells on microcarriers

6.00× 106

3.66× 106

2.00 ×106

2.75× 106

1.48× 106

6.00× 106

2.36 ×106

6.00× 106

# Carriers (g) 0.27 0.18 0.126 0.18 0.09 0.27 0.144 0.27 # Cells/0.09 g carrier

2.00× 106

1.83× 106

1.42 ×106

1.38× 106

1.48× 106

2.00× 106

1.48 ×106

2.00× 106

Period on carriers (days)

17 19 25 25 22 16 16 20

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


90

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-

PART 3.2: Results

91

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.


92

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.

PART 3.2: Results

93

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).

Figure 4. Photomicrograph of a tibial defect treated with composite no. 3, 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. The polymer (3) contains diffusely spread granular material (4).


94

Figure 5. Photomicrograph of a tibial defect treated with composite no. 3, 12 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

PART 3.2: Results

95

0

10

20

30

40

50

60

70

80

Von Kossapositive

Von Kossanegative

Polymer andempty space

Me

an

pe

rce

nta

ge

ControlComposite 1Composite 2Composite 3

between the three composites. The percentage of von Kossa–positive

staining increased significantly over time (Fig. 7).

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).


96

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.

PART 3.2: Results

97

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.


98

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.

Figure 10. Photomicrograph of a tibial defect treated with composite no. 2, 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.

PART 3.2: Results

99

Figure 11. Photomicrograph of a tibial defect treated with composite no. 3, 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).


100

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.

Figure 13. Photomicrograph of a tibial defect treated with composite no. 3, 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).


101

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


102

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


103

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


104

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. ,


105

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


106

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.

PART 3.2: Conclusion

107

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.


108

ACKNOWLEDGEMENTS

The authors thank Cindy De Baere, Sarah Loomans, Bart De

Pauw, and Leen Pieters for their excellent technical assistance.


109

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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

115

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.

Evaluation of a hydrogel based scaffold with stem cells

116

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


117

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


118

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.


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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,


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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 wells of a 12 well suspension plate (Greiner Bio-One) and 4.105

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


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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

suspension plate (Greiner Bio-One) and 105 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.


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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


123

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,


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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


125

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

stained sections using a software program (CellM, 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


126

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.

PART 3.3: Results

127

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


128

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.

PART 3.3: Results

129

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.


130

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

PART 3.3: Results

131

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).


132

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.

PART 3.3: Results

133

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®


134

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.


135

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


136

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

PART 3.3 : Discussion

137

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


138

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

PART 3.3 : Discussion

139

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


140

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.

PART 3.3 : Conclusion

141

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.


142

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.

PART 3.3 : References

143

REFERENCES

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De Long, W. G., Jr., T. A. Einhorn, K. Koval, M. McKee, W. Smith, R. Sanders & T. Watson, 2007: Bone grafts and bone graft substitutes in orthopaedic trauma surgery. A critical analysis. Journal of Bone and Joint Surgery. American Volume, 89, 649-658.


Dumortier, G., J. L. Grossiord, F. Agnely & J. C. Chaumeil, 2006: A review of poloxamer 407 pharmaceutical and pharmacological characteristics. Pharmaceutical Research, 23, 2709-2728.

Escobar-Chavez, J. J., M. Lopez-Cervantes, A. Naik, Y. N. Kalia, D. Quintanar-Guerrero & A. Ganem-Quintanar, 2006: Applications of thermo-reversible pluronic F-127 gels in pharmaceutical formulations. Journal of Pharmacology and Pharmaceutical Sciences, 9, 339-358.

Finkemeier, C. G., 2002: Bone-grafting and bone-graft substitutes. Journal of Bone and Joint Surgery. American Volume, 84-A, 454-464.

Fischer, E. M., P. Layrolle, C. A. Van Blitterswijk & J. D. De Bruijn, 2003: Bone formation by mesenchymal progenitor cells cultured on dense and microporous hydroxyapatite particles. Tissue Engineering, 9, 1179-1188.

Gustafson, C. J., A. Birgisson, J. Junker, F. Huss, L. Salemark, H. Johnson & G. Kratz, 2007: Employing human keratinocytes cultured on macroporous gelatin spheres to treat full thickness-wounds: an in vivo study on athymic rats. Burns, 33, 726-735.

Hollinger, J. O. & J. C. Kleinschmidt, 1990: The critical size defect as an experimental model to test bone repair materials. Journal of Craniofacial Surgery, 1, 60-68.


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Huss, F. R., J. P. Junker, H. Johnson & G. Kratz, 2007: Macroporous gelatine spheres as culture substrate, transplantation vehicle, and biodegradable scaffold for guided regeneration of soft tissues. In vivo study in nude mice. Journal of Plastic, Reconstructive and Aesthetic Surgery, 60, 543-555.

Jeong, B., S. W. Kim & Y. H. Bae, 2002: Thermosensitive sol-gel reversible hydrogels. Advanced Drug Delivery Reviews, 54, 37-51.

Klouda, L. & A. G. Mikos, 2008: Thermoresponsive hydrogels in biomedical applications. European Journal of Pharmaceutics and Biopharmaceutics, 68, 34-45.

Langer, R. & J. P. Vacanti, 1993: Tissue engineering. Science, 260, 920-926.

Laurie, S. W., L. B. Kaban, J. B. Mulliken & J. E. Murray, 1984: Donor-site morbidity after harvesting rib and iliac bone. Plastic and Reconstructive Surgery, 73, 933-938.

Lee, O. K., M. J. Coathup, A. E. Goodship & G. W. Blunn, 2005: Use of mesenchymal stem cells to facilitate bone regeneration in normal and chemotherapy-treated rats. Tissue Engineering, 11, 1727-1735.

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 carriers for bone tissue engineering. Journal of Materials Science: Materials in Medicine.

Mastrogiacomo, M., S. Scaglione, R. Martinetti, L. Dolcini, F. Beltrame, R. Cancedda & R. Quarto, 2006: Role of scaffold internal structure on in vivo bone formation in macroporous calcium phosphate bioceramics. Biomaterials, 27, 3230-3237.

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Pearce, A. I., R. G. Richards, S. Milz, E. Schneider & S. G. Pearce, 2007: Animal models for implant biomaterial research in bone: a review. European Cells & Materials, 13, 1-10.

Sopyan, I., M. Mel, S. Ramesh & K. A. Khalid, 2007: Porous hydroxyapatite for artificial bone applications. Science and Technology of Advanced Materials, 8, 116-123.

Swennen, I., 2008: Thesis, Proefschrift voorgelegd tot het behalen van de graad: Doctor in de Wetenschappen, Groep Scheikunde. Promotor E. Schacht. Polymer Chemistry & Biomaterials Research Group, Ghent University, Belgium.

Swennen, I., V. Vermeersch, M. Hornof, E. Adriaens, J.-P. Remon, A. Urtti & E. Schacht, 2006: In-situ crosslinkable thermo-sensitive hydrogels for drug delivery. Journal of Controlled Release, 116, e21-e24.

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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-varepsilon-Caprolactone) Combined with Autologous Mesenchymal Stem Cells in a Goat Tibial Unicortical Defect Model. Tissue Engineering Part A, 15, 1501-1511.

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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

151

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.

Immunohistochemical analysis of goat tissues

152

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,


153

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.


154


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.


155

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).


156

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.


157

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.


158

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


159

(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).


160

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).


161

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.


162

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).


163

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.


164

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


165

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).


166

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.


167

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.


168

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.

PART 4.1: Acknowledgements

169

ACKNOWLEDGEMENTS

The authors thank Cindy De Baere, Sarah Loomans, Bart De Pauw

and Leen Pieters for their excellent technical assistance.


170

REFERENCES

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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

175

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.

Agreement between µCT and histomorphometry

176

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


177

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


178

standard). For this purpose a non critical sized tibial defect model in

goats was used (Vertenten et al., 2008b).


179


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


180

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,


181

%), bone surface density (BSD = BS/TV, mm-1), bone-specific surface

(BSS = BS/BV, mm-1), 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

sections using specific image software program (CellM, 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


182

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.

PART 4.2: Results

183

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.


184

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).

PART 4.2: Results

185

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).


186

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.

PART 4.2: Results

187

Figure 2. Relationships between three measurement methods for bone surface density (BSD, mm-1) bone specific surface (BSS, mm-1), trabecular thickness (TT, µm) and trabecular separation (TS, µm) by Bland-Altman’s difference plots.


188

Table 6. Summary of paired comparisons

Difference n mean SD P

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


189

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


190

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.


191

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


192

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.


193

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


194

(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.

PART 4.2: Conclussion

195

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.


196

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201

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

GENERAL DISCUSSION

202

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

GENERAL DISCUSSION

203

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

GENERAL DISCUSSION

204

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

GENERAL DISCUSSION

205

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.

GENERAL DISCUSSION

206

‘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.

GENERAL DISCUSSION

207

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.

GENERAL DISCUSSION

208

Table 1. Overview of the results of the experimental work.

Chapter Product Result

3.1 Poly-(D,L-lactide-co-ε-caprolactone) +HEMA

Biocompatible

Moderate osteoconductive

3.1 Poly-(D,L-lactide-co-ε-caprolactone)

+HEMA +Bioglass

Biocompatible

More osteoconductive than pure polymer

3.1 Poly-(D,L-lactide-co-ε-caprolactone) +HEMA + α-TCP

Biocompatible

More osteoconductive than polymer+Bioglass


+ triacetin

Biocompatible

Not osteoconductive


+ triacetin +BMSCs

Biocompatible

Not osteoconductive

Survival and proliferation of BMSCs


+ triacetin +BMSCs

+ α-TCP

Biocompatible

Not osteoconductive

Less survival and proliferation of BMSCs than without α-TCP

3.3 Plu ALA-L Biocompatible

Similar to faster healing than empty control defect

3.3 Plu ALA-L

+ BMSCs (Cultispher-S)

Biocompatible

Faster healing than empty control defect

3.3 Plu ALA-L + BMSCs (HA) Biocompatible

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

GENERAL DISCUSSION

209

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,

GENERAL DISCUSSION

210

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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211

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

GENERAL DISCUSSION

212

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

GENERAL DISCUSSION

213

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

GENERAL DISCUSSION

214

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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215

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

GENERAL DISCUSSION

216

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.

GENERAL DISCUSSION

217

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.

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218

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

GENERAL DISCUSSION

219

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

GENERAL DISCUSSION

220

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

GENERAL DISCUSSION

221

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

GENERAL DISCUSSION

222

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

GENERAL DISCUSSION

223

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

GENERAL DISCUSSION

224

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

GENERAL DISCUSSION

225

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.

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226

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

GENERAL DISCUSSION

227

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

GENERAL DISCUSSION

228

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.

GENERAL DISCUSSION: References

229

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241

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

APPENDIX

242

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

APPENDIX

243

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

245

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

SUMMARY

246

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, ε-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.

SUMMARY

247

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.

SUMMARY

248

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

SUMMARY

249

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.

251

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

SAMENVATTING

252

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, ε-

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 –

SAMENVATTING

253

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-

SAMENVATTING

254

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

SAMENVATTING

255

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.

257

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’.

259

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 Orthopaedics and Traumatology, 19, 180-183.

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

BIBLIOGRAPHY

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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.

BIBLIOGRAPHY

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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.

BIBLIOGRAPHY

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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-

BIBLIOGRAPHY

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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.

265

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

DANKWOORD

266

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

DANKWOORD

267

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.

DANKWOORD

268

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

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269

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

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270

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,

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271

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

phd geert vertenten definitieve versie

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