RECONSTRUCTION OF CRITICAL IZED OVINE MANDIBULAR …Reconstruction of Critical-Sized Ovine Mandibular Defects – A Pilot Study iii Results revealed an organised fibrous tissue network

RECONSTRUCTION OF CRITICAL-SIZED

OVINE MANDIBULAR DEFECTS – A PILOT

STUDY

Dr Brendan John Jones

BSc (UQ), MBBS (UQ), DTMH (Liverpool)

Submitted in fulfilment of the requirements for the degree of

Master of Engineering (Research)

Institute of Health and Biomedical Innovation

Science and Engineering Faculty

Queensland University of Technology

Submitted 2014

Reconstruction of Critical-Sized Ovine Mandibular Defects – A Pilot Study i

Keywords

Bone defect, Critical sized defect, Framework, Inflammation, Large segmental

mandible sheep model, Ovine Mandible, Pilot study, Polycaprolactone (PCL),

Scaffold, Sheep mandible, Tissue engineered constructs, Titanium plates.

ii Reconstruction of Critical-Sized Ovine Mandibular Defects – A Pilot Study

Abstract

The current gold standard for treatment of mandibular segmental defects is the

use of autologous, cancellous bone grafts(Ekholm et al., 2006). These autografts

possess excellent osteoconductive and osteoinductive properties. However,

autografts have associated donor site morbidity including haemorrhage, infection,

insufficient transplant integration, insufficient graft revitalization, limited availability

and the need for a second operative site(Reichert, Epari, et al., 2010). The preclinical

and clinical evidence for the application of newly developed tissue engineered

constructs (TEC) in mandibular segmental defect reconstruction remains unclear and

has not yet undergone sufficient rigorous scientific investigation to warrant

widespread adoption(Bell & Gregoire, 2009; Carter, Brar, Tolas & Beirne, 2008b;

Glied & Kraut, 2010b).

An effective standardized and systematic approach in large animal studies

investigating novel approaches to healing critical-sized mandibular segmental defects

has yet to be established. We propose that sheep provide the most advantageous

large animal model for translational studies to assess safety and efficacy of novel

TEC in healing a mandibular segmental defect. To ascertain viability and assess the

nuances of a large segmental mandible sheep model (LSMSM), a pilot study was

performed creating a 22mm-25mm bicortical osteotomy involving the

parasymphyseal (diastema) region of the mandible in Merino Ovis Aries. Sheep were

randomly assigned into two groups receiving either medical grade polycaprolactone

(PCL) scaffold or an empty defect with no scaffold. To achieve stability across the

defect, clinically relevant 2.4mm titanium mandible reconstruction plates (Synthes

UniLock, Australia) were employed, primarily due to robustness and ubiquitous use

both in the literature and clinic for fixation of large mandibular segmental defects in

humans(Carter, Brar, Tolas & Beirne, 2008a; Clokie & S·ndor, 2008; Glied & Kraut,

2010a; Herford & Boyne, 2008a). Unfortunately premature fracturing of 9 out of the

12 fixation plates resulted in an early sacrifice timepoint. The defect site and

surrounding tissue was extracted from 5 scaffold and 2 empty defect groups and

assessed histologically for early signs of bone regeneration and an inflammatory

response.

Reconstruction of Critical-Sized Ovine Mandibular Defects – A Pilot Study iii

Results revealed an organised fibrous tissue network present from the initiation

of the inflammatory response phase of wound healing, even in an unstable fracture

environment and the scaffold appeared to provide the framework for an organised

cellular response from an early stage with increased osteoid production and bone

mineralisation.

Whilst the pilot study was concluded prematurely the framework for relevant,

reproducible data sets was successfully established. The study verified that a

standardized large segmental mandible sheep model (LSMSM) is effectual and offers

the suitable framework from which additional experimentation and evaluation of

novel TEC’s may be undertaken, compared and collated. This will provide the

necessary data sets required in the assessment of current and future novel approaches

to mandible segmental defect reconstruction that may be transferable to the human

condition and, ultimately, the operative table.

iv Reconstruction of Critical-Sized Ovine Mandibular Defects – A Pilot Study

Table of Contents

[The Table of Contents can be updated with the F9 key – refer to Thesis PAM.]

Keywords ................................................................................................................................................. i

Abstract ................................................................................................................................................... ii

List of Figures ........................................................................................................................................ vi

List of Tables ........................................................................................................................................ vii

List of Abbreviations .......................................................................................................................... viii

Statement of Original Authorship .......................................................................................................... ix

Acknowledgements ................................................................................................................................. x

CHAPTER 1: INTRODUCTION ....................................................................................................... 1

1.1 Background ................................................................................................................................... 3

1.2 Context .......................................................................................................................................... 4

1.3 Purposes ........................................................................................................................................ 4

1.4 Significance, Scope and Definitions .............................................................................................. 5

1.5 Thesis Outline ............................................................................................................................... 5

CHAPTER 2: LITERATURE REVIEW ........................................................................................... 9

2.1 Clinical context and historical background ................................................................................... 9

2.2 Animal Models ............................................................................................................................ 12 2.2.1 Small Animals ................................................................................................................ 13 2.2.2 Rabbit ............................................................................................................................. 13 2.2.3 Porcine ............................................................................................................................ 14 2.2.4 Canine ............................................................................................................................. 15 2.2.5 Nonhuman primates ........................................................................................................ 15

2.3 Sheep Model for Mandible .......................................................................................................... 16

2.4 Establishing a Model ................................................................................................................... 20

2.5 Mandibular Fracture Management in Sheep ............................................................................... 22 2.5.1 Post-explantation Biomechanical Testing ...................................................................... 23 2.5.2 HISTOLOGY AND COMPUTED TOMOGRAPHY .................................................... 26

2.6 Conclusion ................................................................................................................................... 26

CHAPTER 3: PILOT STUDY .......................................................................................................... 29

3.1 INTRODUCTION ....................................................................................................................... 29

3.2 Materials and mETHODS ........................................................................................................... 30 3.2.1 Scaffold fabrication and preparation............................................................................... 30 3.2.2 Surgical Procedure .......................................................................................................... 30 3.2.3 Post operative management ............................................................................................ 34 3.2.4 Post-operative Assessment ............................................................................................. 35 3.2.5 Explantation .................................................................................................................... 36

3.3 Results ......................................................................................................................................... 36 3.3.1 Radiographs .................................................................................................................... 36 3.3.2 Biomechanical Testing ................................................................................................... 38 3.3.3 Histology and Immunohistochemistry ............................................................................ 40

3.4 Discussion: .................................................................................................................................. 42

CHAPTER 4: HISTOLOGY: ........................................................................................................... 47

4.1 Abstract: ...................................................................................................................................... 48

Reconstruction of Critical-Sized Ovine Mandibular Defects – A Pilot Study v

4.2 Introduction: ................................................................................................................................ 49

4.3 Materials and Methods: ............................................................................................................... 53 4.3.1 Scaffold fabrication and preparation ............................................................................... 53 4.3.2 Surgical Procedure .......................................................................................................... 53 4.3.3 Post-operative management ............................................................................................ 56 4.3.4 Radiographic Analysis .................................................................................................... 56 4.3.5 Explantation .................................................................................................................... 57 4.3.6 Histology/Immunohistochemistry .................................................................................. 58 4.3.7 Microscopy ..................................................................................................................... 58 4.3.8 Statistical Analysis.......................................................................................................... 59

4.4 Results ......................................................................................................................................... 60 4.4.1 Plate fracture ................................................................................................................... 60 4.4.2 Radiographic Analysis .................................................................................................... 60 4.4.3 Histology/Immunohistochemistry .................................................................................. 62

4.5 Discussion ................................................................................................................................... 69

CHAPTER 5: CONCLUSIONS AND FUTURE WORK ............................................................... 74

BIBLIOGRAPHY ............................................................................................................................... 85

APPENDICES ................................................................................................................................... 101 Appendix A Title ..................................................................................................................... 101

vi Reconstruction of Critical-Sized Ovine Mandibular Defects – A Pilot Study

List of Figures

[The List of Figures can be created automatically and updated with the F9 key – refer to Thesis PAM.]

FIGURE 1: HUMAN MANDIBLE (PHOTOS COURTESY OF DR KATHERINE CUNNEEN) .................................................... 1 FIGURE 2: ROAD MAP TO ESTABLISHING A CRITICAL-SIZED BONE DEFECT STUDY IN A LARGE ANIMAL MODEL.

[ADAPTED FROM (REICHERT, EPARI, ET AL., 2010)] ......................................................................................... 21 FIGURE 3: BIOMECHANICAL TESTING USING CANTILEVER FORCE WITH INSTRON 35KN. ......................................... 25 FIGURE 4: OPERATIVE PROCEDURE. ................................................................................................................................... 32 FIGURE 5: INITIAL RADIOGRAPHS POST-SURGICAL INTERVENTION. .............................................................................. 35 FIGURE 6: RADIOGRAPHS DISPLAYING INTACT AND FRACTURED 2.4MM TITANIUM MANDIBLE RECONSTRUCTION

PLATES .......................................................................................................................................................................... 37 FIGURE 7: BIOMECHANICAL TESTING USING CANTILEVER FORCE WITH INSTRON 35KN. ......................................... 39 FIGURE 9: OVERVIEW OF STAINS FOR THE SCAFFOLD GROUP AND EMPTY DEFECT GROUP ....................................... 41 FIGURE 10: RADIOGRAPH OF PLATE FRACTURE ............................................................................................................... 43 FIGURE 11: OVERVIEW OF THE OPERATIVE PROCEDURE ............................................................................................... 55 FIGURE 12: INITIAL RADIOGRAPHS ..................................................................................................................................... 56 FIGURE 13: HISTOLOGY PREPARATION. ............................................................................................................................ 57 FIGURE 14: IMAGE J ANALYSIS. ............................................................................................................................................ 59 FIGURE 15: PLAIN RADIOGRAPHS ....................................................................................................................................... 61 FIGURE 16: GOLDNER’S TRICHROME STAIN OF SCAFFOLD AND NATIVE BONE ........................................................... 62 FIGURE 17: OVERVIEW OF STAINS FOR THE SCAFFOLD GROUP AND EMPTY DEFECT GROUP ..................................... 64 FIGURE 18: CELL NUMBERS AT THE PROXIMAL AND DISTAL DEFECT SITE. ............................................................... 65 FIGURE 19: CELL NUMBERS AT THE PROXIMAL END OF THE DEFECT SITE. .................................................................. 66 FIGURE 20: CELL NUMBERS AT THE DISTAL END OF THE DEFECT SITE. ........................................................................ 66 FIGURE 21: WHOLE RESIN SECTIONS ................................................................................................................................. 67 FIGURE 22: BONE MINERALIZATION SEEN IN VON KOSSA STAINING. ........................................................................... 68 FIGURE 23: CUSTOMISED STAINLESS STEEL PLATES THAT WILL BE BIOMECHANICALLY TESTED ............................ 79

Reconstruction of Critical-Sized Ovine Mandibular Defects – A Pilot Study vii

List of Tables

[The List of Tables can be created automatically and updated with the F9 key – refer to Thesis PAM.]

TABLE 1: CRITICAL-SIZED MANDIBULAR DEFECTS IN ANIMAL MODELS ................................................................... 16 TABLE 2: SHEEP MANDIBLE STUDIES ................................................................................................................................ 19 TABLE 3: SHEEP OPERATIONS PERFORMED FOR THE LARGE SEGMENTAL MANDIBLE SHEEP MODEL ..................... 34

viii Reconstruction of Critical-Sized Ovine Mandibular Defects – A Pilot Study

List of Abbreviations

If appropriate, list any abbreviations used in the thesis.

BMP-2 BONE MORPHOGENETIC PROTEIN – 2

CaP CALCIUM PHOSPHATE

CT COMPUTED TOMOGRAPHY

μCT MICRO COMPUTED TOMOGRAPHY

EtOH ETHANOL

FDM FUSED DEPOSITION MODELLING

LSMSM LARGE SEGMENTAL MANDIBLE SHEEP MODEL

NaOH SODIUM HYDROXIDE

PBS PHOSPHATE-BUFFERED SALINE

PCL Polycaprolactone

PRP PLASMA RICH PROTEIN

rhBMP-2 RECOMBINANT HUMAN BONE MORPHOGENETIC

PROTEIN -2

rhBMP-7 RECOMBINANT HUMAN BONE MORPHOGENETIC

PROTEIN -7

rhOP-1 RECOMBINANT HUMAN OSTEOPONTIN-1

TCP Tricalcium Phosphate

TEC TISSUE ENGINEERING CONSTRUCT

QUT Verified Signature

x Reconstruction of Critical-Sized Ovine Mandibular Defects – A Pilot Study

Acknowledgements

Profosser Dietmar Hutmacher and Dr Anthony Lynham for their support and

overview of the project.

Special thanks to Dr Arne Berner for his help, guidance and friendship over the

duration of the research program. His sustained enthusiasm and encouragement was

essential and much appreciated, without which the project would have failed to get

off the ground.

Dr Siamak Siadezafah for his extensive experience and essential support with animal

handling, operative assistance and most importantly, theatre playlist selection.

Associate Professor Maria Woodruff for her friendship and support throughout the

project, for her expertise in organisation and histology.

Dr Mostyn Yong for his assistance and friendship over the duration of the research

program.

Dr Lance Wilson and Dr Roland Schleck for their humour, support and expertise in

biomechanical testing.

Dr Keith Blackwood for his technical know-how and assistance with histology.

Ms Kendra Haughland- for her indispensible assistance in the histology.

Edward Ren and Dr Giles Kirby for their help with preparations of histological

specimens.

Dr Gary Brierly for his assistance and energy with the review article and the final

stages of histology with preparation to further the research.

Toby Brown, Dr Verena Reichert for their support, guidance and encouragement

throughout the project.

To all the staff at MERF who were involved at one point or another in the project,

your patience and guidance was much appreciated.

To fellow students and staff at IHBI for their dedication, friendship and repartee that

helped keep me sane.

Chapter 1: Introduction 1

Chapter 1: Introduction

The human mandible is the largest and strongest bone of the face occupying a

position of prominence and vulnerability, supported by surrounding musculature on a

hinge joint(Gray, 2000). As a consequence, it is the second most commonly fractured

bone of the maxillofacial skeleton(Singh, Mohapatra & Kumar, 2010). The mandible

is unique, being the only movable load-bearing bone of the skull that is required to

withstand the forces transmitted during function(Wong, Tideman, Kin & Merkx,

2010). It plays an integral role in mastication, speech and defines facial structure

whilst providing the supportive framework for soft tissues of the anterior

laryngopharynx and assisting airway patency.

Figure 1: Human Mandible (Photos courtesy of Dr Katherine Cunneen)

Mandible bone loss can occur following fractures, infections, tumour

resections or congenital abnormalities. This can lead to a critical-sized defect of the

mandible, is a segmental bone defect that cannot bridge spontaneously or shows less

than 10 percent bony regeneration and requires surgical intervention (Hollinger &

Kleinschmidt, 1990). These bone defects are destructive, causing significant

impediment to normal function and aesthetics as well as being technically difficult to

reconstruct(Hollinger & Kleinschmidt, 1990; Schmitz & Hollinger, 1986). Further

development and optimization of the surgical management of mandibular bone

2 Chapter 1: Introduction

defects is crucial to improving outcomes and decrease patient morbidity. Tissue

engineering continues to pioneer novel and exciting approaches to restoring,

replacing and regenerating injured or diseased biological tissue(Chan & Leong,

2008).

In the field of craniomaxillofacial surgery several biosynthetic bone

supplements are already approved for human clinical use to treat mandibular bone

defects. Whilst these biosynthetic supplements have provided additional therapeutic

options, the current gold standard for treatment of bony defects remains autologous,

cancellous bone grafts(Ekholm et al., 2006). These grafts possess excellent

osteoconductive and osteoinductive properties, although they are hindered by donor

site morbidity including haemorrhage, infection, insufficient transplant integration,

insufficient graft revitalisation, limited tissue availability and the need for a second

operative site(Reichert, Epari, et al., 2010). In addition, the failure rate of autologous

bone graft is up to 30%(Gautschi, Frey & Zellweger, 2007). These complications are

avoidable and provide the rationale for research into viable therapeutic alternatives

that eliminate the need for a second operative site and provide effective management

at the initial operative intervention to improve patient outcome, limit patient

morbidity and avoid the need for re-intervention(Gautschi et al., 2007). The pursuit

of a viable alternate in the management of mandibular critical-sized bone defects is

thus of significant clinical implication.

The myriad of approaches employed in investigating mandibular defect bone

regeneration has resulted in many animal models and case-reports but often with a

lack of depth and reproducibility in scientific data sets. Currently, preclinical and

clinical evidence for more widespread indications and applications of newly

developed tissue engineering techniques is unclear and has not yet undergone

sufficient rigorous scientific investigation to warrant widespread adoption(Bell &

Gregoire, 2009; Carter et al., 2008b; Glied & Kraut, 2010b). For novel tissue

engineering approaches in mandible segmental defect regeneration to be translated

from the bench top to the bedside requires vigorous in vivo interrogation via a well-

designed and validated preclinical animal model.

Establishing an animal model is a necessary step in the study of in vivo

mandibular bone healing to provide clarification and scientific acumen for the most


efficient and effective therapeutic option to treat bone defects(Nunamaker, 1998). A

standardised systematic approach, employing a reproducible preclinical animal

model with consistent post-explantation analysis, investigating novel approaches to

healing critical-sized mandibular defects, has yet to be established. A reproducible

animal model is the necessary step in the study of in vivo mandibular bone

regeneration constructs to provide clarification and scientific acumen for the most

efficient and effective therapeutic option. In addition, establishing an effective

model will generate a framework for further studies into the wide-ranging

implementation of tissue engineered construct’s (TEC). The ability to construct

polycaprolactone (PCL) with fusion deposition modelling may result in customized

scaffolds prepared to the defect specifications. This provides a myriad of options for

therapeutic application from larger defects, including hemi- or full mandible

reconstructions to embedding with growth hormones and stem cells to assist

regeneration in difficult clinical scenarios including post radiotherapy, poorly

vascularised tissue. These extraordinary but achievable goals will provide the

inspiration to further research in innovative techniques for healing critical-sized

mandibular defects.

1.1 BACKGROUND

A variety of animal models have been employed in the investigation of

mandibular defect regeneration, ranging from small animal models to larger animals

including dog, pig, sheep and non-human primates. Whilst these studies provide an

increasing volume of data there has yet to be consistency in developing a

standardized systematic approach to evaluating mandibular defect regeneration.

What is lacking is a standardised large animal model that more effectively mimics

the human condition and wound healing environment and employs consistent

standardisation of in vivo application and post-explantation analysis to investigate

tissue engineering approaches for reconstructing mandibular critical-sized

defects(Muschler et al., 2010). Establishing this model is essential so that the

innovative approach to mandibular bone regeneration, when applied to clinical

practice, is both reliably efficacious and maintains appropriate patient safety.


1.2 CONTEXT

To present a viable therapeutic alternative for mandibular defect regeneration

via a tissue engineered construct (TEC) requires building a reliable and reproducible

body of research data. The data sets can then be compared and collated to further

develop the TEC so that it may be transferable to the human condition and,

ultimately, the operative table. To translate research from the bench-top to the

bedside necessitates the foundation of a large animal model with a standardised

evaluation process prior to implementation in the clinical setting(Berner et al., 2012;

Muschler et al., 2010). Whilst this can be expensive and time consuming, it is

imperative that novel constructs undergo rigorous scientific evaluation prior to

implementation. The current literature on translational research of mandibular defect

regeneration includes a variety of animal models that have been employed to

investigate novel approaches to mandibular defect regeneration. Whilst these studies

provide an increasing volume of data there has yet to be neither a reproducible model

nor a standardized systematic approach to evaluating mandibular bone regeneration.

Ideally, the preclinical animal model should emulate clinical surgical

techniques, provide appropriate tissue size and handling that would be encountered

clinically, be versatile in a controlled environment and allow quantitative assessment

of results(Cheng et al., 2005). In addition animals with comparable bone architecture

and remodelling rates to humans will provide greater accuracy in extrapolating data

and making predictions about the potential clinical success or failure of novel tissue

engineering approaches(Muschler et al., 2010; Reichert, Epari, et al., 2010; Reichert

et al., 2009a). Through a review of the current literature and an established

experience with sheep tibial defect studies we propose that the sheep model will

provide the necessary framework for collating and comparing data sets for novel

tissue engineering approaches.

1.3 PURPOSES

The purpose of this study is to review the current animal model research in

bone regeneration techniques for mandibular bone defects and provide a line of

reasoning for employing sheep as the preclinical translational model. In addition, a


pilot study was performed to provide guidelines for the large segmental mandible

sheep model (LSMSM) in vivo investigation and post-explantation analysis of novel

approaches to mandibular bone defects.

The research problem is that there has yet to be a consistent systematic

approach to animal studies of the in vivo application of mandibular defect

regeneration. The safety evidence for more widespread indications and applications

of newly developed tissue engineering techniques is unclear and has not yet

undergone sufficient rigorous scientific investigation to warrant widespread

adoption(Bell & Gregoire, 2009; Carter et al., 2008b; Glied & Kraut, 2010b). It is

believed that sheep will provide the best model for translational research; however,

stable fixation of the bony defect site has provided an impediment that must be

overcome prior to progression of further research

.

1.4 SIGNIFICANCE, SCOPE AND DEFINITIONS

Ultimately the objective is to provide and implement a viable, efficacious

alternative to bone regenerative fixation of a critical-sized mandibular defect that will

provide both therapeutic advantages and improve patient outcome whilst decreasing

patient morbidity. Establishing an effective model will generate the framework for

further research comparing novel approaches to large segmental mandible defects

and solving complicated therapeutic problems ranging from even larger segmental

mandible defects to bone regeneration in a difficult clinical setting such as a tumour

model with post-irradiated, poorly vascularised tissue.

1.5 THESIS OUTLINE

This Masters thesis will review the current literature on animal models for

novel regeneration of mandibular bone defects and endeavour to provide a line of

reasoning in favour of establishing a preclinical sheep model in the application and

analysis of novel tissue engineered constructs (TEC). It will determine whether or

not the sheep model provides the most suitable large animal model from which

additional experimentation and evaluation of TEC’s for healing mandibular defects

may be undertaken, compared and collated. It is hypothesized that the consistent


application of a large segmental mandible sheep model (LSMSM) will provide a

valuable framework in the assessment of a variety of novel TEC’s so that they may

be transferable to the human condition and, ultimately from bench to bedside.

Based on this background a pilot study was performed to ascertain the viability

and nuances of the sheep mandibular segmental defect model consisting of a 22mm-

25mm bicortical defect within the parasymphyseal diastema region of the mandible

in Merino Ovis Aries. In the absence of an established critical-sized defect for

Merino Ovis Aries, the defect size was determined intra-operatively as the greatest

distance available involving the diastema region without the prior removal of a

premolar tooth. Two sheep groups were randomly assigned to either the control

group, with an empty defect, or to the scaffold group with polycaprolactone (PCL)

scaffold. Premature fracturing of the mandibular reconstruction fixation plates

unexpectedly occurred. This presented an opportunity to uniquely analyse the early

stages of the host response to a polycaprolactone (PCL) construct in vivo, which, to

our knowledge, has not been previously performed. Histology and

immunohistochemistry findings reinforced the biocompatible nature of the scaffold.

They further highlight the importance of the scaffold system in providing a platform

for cells promoting a regenerative pathway, such as osteoblasts and fibroblasts, to

result in organised fibrous tissue deposition, a decreased immune response and

enhanced bone mineralization potential compared to that of the empty defect group.

Future prospects for this study involve the resolution of the plate fixation

system in repair of a bicortical bone defect in sheep mandible model. This is

imperative for further evaluation of regenerative repair. Whether the solution is a

thicker fixation plate or an innovative plate fixation design, it will require additional

scrutiny prior to implementation, including biomechanical testing currently being

undertaken in our group.

Establishing an effective model will generate the framework for further

research comparing novel approaches to large segmental mandible defects and

solving complicated therapeutic problems ranging from even larger segmental

mandible defects to bone regeneration in a difficult clinical setting such as a tumour

model with post-irradiated, poorly vascularised tissue.


Chapter 2: Literature Review 9

Chapter 2: Literature Review

2.1 CLINICAL CONTEXT AND HISTORICAL BACKGROUND

The human mandible is the largest and strongest bone of the face occupying a

position of prominence and vulnerability, supported by surrounding musculature on a

hinge joint(Gray, 2000). As a consequence, it is the second most commonly fractured

bone of the maxillofacial skeleton(Singh et al., 2010). It plays an integral role in

mastication, speech and defining facial structure whilst providing the supportive

framework for soft tissues of the anterior laryngopharynx and airway patency. The

mandible is unique, being the only movable load-bearing bone of the skull that is

required to withstand the forces transmitted during function(Wong et al., 2010).

Bone loss following fractures, infections, tumour resections or congenital defects can

lead to critical-sized defects of the mandible.

Critical-sized defects are defined as “the smallest size intraosseous wound in a

particular bone and species of animal that will not heal spontaneously during the

lifetime of the animal”(Schmitz & Hollinger) or as a defect which shows less than 10

percent bony regeneration during the lifetime of the animal(Hollinger &

Kleinschmidt, 1990). These defects are destructive, causing significant impediment

to normal function and aesthetics as well as being technically difficult to

reconstruct(Hollinger & Kleinschmidt, 1990). Thus, enhancement and optimization

of the clinical management of mandibular defects is essential. In the field of

craniomaxillofacial surgery several biosynthetic bone supplements are approved for

the use of sinus augmentation and also alveolar ridge augmentation associated with

extraction sockets(Davies & Ochs, 2010; Lo, Ulery, Ashe & Laurencin, 2012).

The current gold standard for treatment of continuity mandibular defects is the

use of autologous, cancellous bone grafts(Ekholm et al., 2006). These autografts

possess excellent osteoconductive and osteoinductive properties. However,

autografts have associated disadvantages such as donor site morbidity including

hemorrhage, infection, insufficient transplant integration, insufficient graft

revitalization, limited availability and the need for a second operative site(Reichert,

Epari, et al., 2010). In addition, the failure rate of autologous bone grafts is up to

30% and thus it is imperative any defect should be treated correctly at the outset to

10 Chapter 2: Literature Review

avoid the need for re-intervention(Gautschi et al., 2007). Clinically, the limitations of

autologous bone grafting include; inadequate quantity and quality of bone, potential

graft failure and the morbidity of a second operative site(Boyne, 1997; Schmidmaier,

Capanna, Wildemann, Beque & Lowenberg, 2009).

This has led to a search for alternative treatment modalities and substitutes that

restore facial form, speech, function and occlusion thus returning the patient to

normal oral function and providing a better quality of life(Bak, Jacobsen, Buchbinder

& Urken, 2010; Schrag, Chang, Tsai & Wei, 2006). These substitutes include the

successful use of autologous vascularized fibula, scapula, iliac crest, and rib

transplant(Cheng, Brey, Ulusal & Wei, 2006; Clokie & S·ndor, 2008; Deschler &

Hayden, 2000; Disa & Cordeiro, 2000; Ferretti & Ripamonti, 2002; Forriol et al.,

2009; Herford & Boyne, 2008a; Kahairi, Ahmad, Wan Islah & Norra, 2008; Kelley,

Klebuc & Hollier, 2003; Moghadam, Urist, Sandor & Clokie, 2001; Peled, El-Naaj,

Lipin & Ardekian, 2005; Terheyden et al., 2001; Warnke et al., 2004; Werle, Tsue,

Toby & Girod, 2000). Despite the success rates of these vascularized free flaps they

still possess disadvantages to the patient and clinician with long operative times and

donor site morbidity. (Bak et al., 2010; Bodde, de Visser & Duysens, 2003; Boyne,

1997; Daniels, Thomas, Bell & Neligan, 2005; Schmidmaier et al., 2009) Hence, a

growing interest in the application of tissue engineering to reconstruct segmental

mandibular defects has arisen to negate the disadvantages associated with current

techniques.

Warnke et al employed a novel approach to avoid a secondary bone defect with

the successful creation of a custom vascularised bone graft within the patient’s

latissimus dorsi muscle. Transplantation was carried out 7 weeks after the initial

surgery, harvesting the newly formed bone, latissimus dorsi muscle and the

associated vasculature of the bone-muscle flap. This bone-muscle flap was then

transplanted into the same patient’s mandibular defect utilizing a extraoral

approach(Warnke et al., 2004). This obviated the use of a second operative site with

the associated risks. In this case the patient developed a number of complications

several weeks and months after the surgery. In addition to vascularised free tissue

transfer, the use of guided bone regeneration with distraction osteogenesis has also

been employed as an alternative to bone augmentation(Kilic et al., 2011).


Bioimplants and distraction osteogenesis has shown promise in both animal trials and

in selected patients with benign disease and short segmental defects.(Chao, Donovan,

Sotelo & Carstens, 2006; Chin, Ng, William & Carstens, 2005; Clokie & S·ndor,

2008; Ferretti & Ripamonti, 2002; Herford & Boyne, 2008b; Herford, Boyne,

Rawson & Williams, 2007; Schuckert, Jopp & Teoh, 2009) Whilst distraction

osteogenesis avoids the complications associated with a second surgical site, the

technique requires tremendous patient compliance and is prone to infections

(Schroeder & Mosheiff, 2011). Attempts to overcome the limitations of a second

operative site and it’s associated morbidity lead to several case reports involving

reconstruction of mandibular continuity defects. These case reports were carried out

in the clinical setting with varying results using tissue constructs with or without

Bone Morphogenetic Protein-2.(Abukawa et al., 2004; Cheng et al., 2006; Clokie &

S·ndor, 2008; Ferretti & Ripamonti, 2002; Gautschi et al., 2007; Herford & Boyne,

2008a; Kahairi et al., 2008; Moghadam et al., 2001; Reddi, 2001) However, the

authors were inconsistent in their approach to patient care. Currently, the safety

evidence for more widespread indications and applications of newly developed tissue

engineering techniques is unclear and has not yet undergone sufficient rigorous

scientific investigation to warrant widespread adoption(Bell & Gregoire, 2009;

Carter et al., 2008b; Glied & Kraut, 2010b).

The myriad of approaches to mandibular defect bone regeneration and the lack

of scientific data highlight the need for a well-designed and validated preclinical

animal model. This model will provide the framework for reproducible scientific

acumen involving an appropriate tissue engineered construct (TEC) as well as

applicable angioinductive and osteoinductive factors, for extrapolation and clinical

application of tissue engineering techniques. A variety of animal models have been

employed in the investigation of mandibular defect regeneration, ranging from small

animal models to larger animals including dog, pig, sheep and non-human primates.

Whilst these studies provide an increasing volume of data sets there has yet to be

consistency in developing a standardized systematic approach to evaluating

mandibular defect regeneration. What is lacking is a standardised large animal model

with consistent in vivo application and post-explantation analysis to investigate tissue

engineering approaches for reconstructing critical-sized mandible defects.


Establishing this model will provide scientific rigor and confidence in a viable

alternative to current clinical practice that is both reliably efficacious and maintains

appropriate patient safety.

2.2 ANIMAL MODELS

Scaffolds designed for bone reconstruction have been progressively developed

through the interdisciplinary collaboration of medicine, science and engineering and

continue to be refined. The use of a preclinical animal model is a necessary step in

the study of in vivo bone healing. It provides scientific acumen for the most efficient

and effective way to treat clinical conditions(Nunamaker, 1998). The model plays a

crucial role along the developmental spectrum of regenerative medicine from

establishing the foundations of bone healing of TECs, to assessing their feasibility

and bioactivity for clinical implementation(Muschler et al., 2010). Thus, animal

models provide the necessary link between in vitro investigations to clinical

implementation, from the bench top to the bedside. Animal models range from small

animals such as rodents and rabbits, to larger animals including dogs, sheep and

nonhuman primates.

The choice of animal model is important. Each model possesses beneficial

attributes as well as unfavorable features. Animal selection should therefore be based

on a number of criteria. Foremost, the model should be able to fulfill the

requirements of the research question and be suitable for operative intervention,

employing a similar technique to that of the human clinical setting. In addition,

practical considerations including cost, animal availability, ethical acceptability,

tolerance to captivity, and ease of housing need to be addressed(Pearce, Richards,

Milz, Schneider & Pearce, 2007). It should be noted that the results obtained from

the use of animals must be interpreted within the context of inter-species disparity

prior to human application as animals of a lower phylogenetic order have, on

average, a higher potential for spontaneous bone regeneration(Salmon & Duncan,

1997). This advocates the use of a large animal model for in vivo investigation with

employment of an appropriate control arm. Though costly, large animal experiments

are necessary to replicate the limited vascular supply and slower bone healing


observed in humans and higher mammals compared with that of small

animals(Runyan & Taylor, 2010).

2.2.1 Small Animals

The advantages small animal models provide for investigating tissue

engineering constructs is that they are easy and inexpensive to house, easily

maintained and possess fast bone turnover rates(Mooney & Siegel, 2005; T. et al.,

2011). Furthermore, small animal breeding cycles are far shorter than large animals,

providing larger study sizes to assess healing of bony defects. Due to these

advantages, small animal studies have been used by several authors to investigate

TECs in healing mandibular defects.(Kahnberg, 1979; Mooney & Siegel, 2005;

Schliephake et al., 2009) Unfortunately, study designs have been inconsistent in

employing appropriate control arms for the critical-sized mandibular defects in these

small animal models(Nunamaker, 1998; Schliephake et al., 2009). Anatomically,

small rodents are disadvantageous, as they not only possess a more primitive bone

structure without a haversian system, but are technically difficult to perform

operative intervention due to the diminutive size of the mandible and intricacy of

operative access.(Hollinger & Kleinschmidt, 1990) Also, their biology differs to

humans in key aspects of bone healing. That is, they continue to model their skeleton

throughout their lives and their growth plates remain permanently open(Muschler et

al., 2010). However, the advantages appear to outweigh the disadvantages in small

animal models with a continuous rise in rodent fracture healing studies in Medline-

listed publications since the 1960s.(T. et al., 2011) Thus, small animal models can

provide a high-powered baseline proof-of-concept study prior to large animal studies

but do not serve well to provide a model to extrapolate to the human clinical scenario

due to their disparity in bone anatomy and healing mechanism.

2.2.2 Rabbit

The rabbit model has a similar profile to the rodent model being relatively

inexpensive whilst possessing a high bone turnover rate allowing for moderate sized

groups(Mooney & Siegel, 2005; Nunamaker, 1998). Akin to the rodent model, they

are beneficial for initial testing of TEC’s for bone healing in mandibular

defects(Kahnberg, 1979; Ren et al., 2007). Unfortunately, they possess fatty marrow


that is distinctly different in physical properties to human marrow making assessment

of bone healing difficult to extrapolate(Muschler et al., 2010). Also, there has been

no standardization for the critical-sized defect with lack of an adequate control arm

in most studies(Kahnberg, 1979; Ren et al., 2007). Again they provide data sets for

high-powered baseline proof-of-concept studies.

2.2.3 Porcine

Miniature pigs have been widely used to investigate orthopaedic and dental

research. They possess a plexiform bone pattern which is closer in relation to human

bone microarchitecture, physiology and biomechanical properties than

rodents.(Reichert et al., 2009b) Like humans, they possess succedaneous dentition

but differ with continually erupting incisors(Mooney & Siegel, 2005; Reichert et al.,

2009b). They have been successfully applied in ‘proof-of-principle’ studies to

highlight the success of various methodologies in reconstructing mandibular defects

such as in vitro cultured mesenchymal stem cells,(Abukawa et al., 2004) the

application of biomatricies without osteoblasts,(Henkel, Gerber, Dorfling, Gundlach

& Bienengraber, 2005) fabrication of vascularized bone flaps using rhBMP-2 and

adipose-derived stem cells,(Runyan et al., 2010) and rhBMP-7 with xenogenic

bone.(Terheyden et al., 2004) These ‘proof-of-principle’ studies provide the

foundation required for application of scaffolds, mesenchymal stem cells and

vascularized flaps generated by rhBMP in large animal models in the future. Despite

the previous mandibular reconstruction studies conducted using a porcine model,

animal models investigating critical sized mandibular defects in pigs may face

difficulties due to recent dispute in the literature regarding critical defect size.

(Ruehe, Niehues, Heberer & Nelson, 2009) Henkel et al established that critical sized

mandibular defects in miniature pigs were defects greater than 5cm3.(Henkel et al.,

2005) That figure was superseded by Ma et al who demonstrated that critical sized

mandibular defects in minipigs are 6cm with periosteum and 2cm without

periosteum.(Ma, Pan, Tan & Cui, 2009) However, in a study conducted by Ruehe et

al they found that large defects of 10cm3

showed 75.5% newly formed bone within

the defect. Due to the small sample size of n=3 further investigation may be

warranted.(Ruehe et al., 2009) Overall, handling difficulties due to their demanding

disposition coupled with high healing rates has kept pigs from being a useful model

in investigating fracture healing (Nunamaker, 1998; Reichert et al., 2009b).


2.2.4 Canine

Canine models have been used to assess mandibular bone healing in multiple

mandibular defect models. Numerous studies have had success at regeneration of

segmental bone defects utilizing bone marrow stromal cells and tricalcium

phosphate, (He et al., 2007) rhBMP-2 in a collagen sponge carrier,(Hussein et al.,

2013) bone marrow stromal cells with porous β-tricalcium phosphate(Yuan et al.,

2010) and microporous polylactide membrane combined with iliac crest bone

graft(Sverzut et al., 2008). Defects sizes ranged from 30mm to 35mm except in the

study by Sverzut et al. 2006, where the defects were only 10mm.(Sverzut et al.,

2008). Consensus of the critical-sized mandibular defect for the canine model is

difficult as there is inter-breed variation in bone healing. A well-designed study by

Huh et al. 2005, found the critical-sized mandibular defect for mongrel dogs to be

15mm without periosteum and 50mm with periosteum(Huh et al., 2005). However

reproducibility is difficult due to the heterogenous nature of mongrel

breeding(Muschler et al., 2010). The canine model provides advantages for in vivo

investigation of mandibular defects but carries disadvantages such as low non-union

rate and a higher rate of solid bony fusion compared to humans.(Reichert et al.,

2009b) In addition, the use of canine models is beset with ethical concerns given the

nature of their high societal regard and difficulty in handling.

2.2.5 Nonhuman primates

Nonhuman primates represent the closest phylogeny to humans, and have been

employed for mandibular defect studies often with impressive regenerative

results(Boyne, 1996, 2001; Boyne, Salina, Nakamura, Audia & Shabahang, 2006;

Marukawa et al., 2002; Seto, Asahina, Oda & Enomoto, 2001; Seto, Marukawa &

Asahina, 2006; Zhou et al., 2010). Despite the advantage of phylogenetic proximity,

nonhuman primates have several drawbacks. These include the marked variation in

response between species and the practical issues such as cost and

availability(Muschler et al., 2010). Similar to canines there are ethical

considerations associated with their phylogenetic proximity to humans and societal

regard(Muschler et al., 2010). They also provide obstruction due to their disposition

and handling difficulties. These obstacles have served to hinder the application of the

primate model in bone tissue engineering. However, as Muschler et al 2010 noted,

despite the notion that they provide a closer affiliation to human semblance there is


no direct evidence that nonhuman primate models are systematically or consistently

superior to other large animal models(Muschler et al., 2010).

Model Defect Size Control Arm

Mouse 4mm round Inconsistent

Rat 4mm segmental No control arm

Rabbit 5mm segmental Lack of adequate evidence, only 1

paper with a control arm

Dog 15-50mm segmental Mongrel dog with well designed

control arm

Pig 20-60mm segmental ?critical-sized as growth >10%

control. Critical size in dispute

Nonhuman Primates 15mm No control arm

Table 1: Overview of Critical-Sized Mandibular Defects in Animal Models

2.3 SHEEP MODEL FOR MANDIBLE

Sheep are a suitable large animal model for research in bone healing as they

respond well to surgical procedures and are closer in the phylogenetic order to

humans than most other animal models (Salmon & Duncan, 1997). Sheep are not

beset with the ethical concerns of dogs and nonhuman primates. In addition, they

provide a mild-mannered disposition, being easy to handle pre-and post-operatively,

when conducting operative intervention.(Reichert et al., 2009b) The mandible

operative site is readily accessible for the creation of a defect and practical for testing

regenerative procedures and therapy concepts, thus providing a suitable model for

surgery with uneventful post-operative healing(Salmon & Duncan, 1997). For

mandible defect studies a sheep model is most advantageous as it is a large mammal

with a mandible size similar to that of humans possessing a similar bone formation

rate(den Boer et al., 1999; Nunamaker, 1998; Wittenberg, Mukherjee, Smith &

Kruse, 1997). As a model for investigating tissue engineering constructs (TEC) in


segmental mandibular defect healing they have been widely employed and continue

to provide a valuable tool for ongoing research in this field(Abu-Serriah et al., 2006;

Abu-Serriah et al., 2005; Alkan, Celebi, Ozden, Bas & Inal, 2007; Ekholm et al.,

2006; Forriol et al., 2009; Kontaxis, Abu-Serriah, Ayoub & Barbenel, 2004; Martola,

Lindqvist, Hanninen & Al-Sukhun, 2007; Nolff et al., 2010; Schliephake, Knebel,

Aufderheide & Tauscher, 2001). However, despite their advantages sheep are

ruminants and possess a dissimilar pattern of mastication to humans(Kontaxis et al.,

2004). This influences optimal post-operative care, as it is important that the sheep’s

diet not remain too soft for too long a time period. Another disparity involves ovine

dentition, which, although similar to humans by being succedaneous, differs by

maintaining continually erupting incisors(Mooney & Siegel, 2005). Sheep also

possess long, narrow heads and enlarged attachment sites for the masseter

muscle(Mooney & Siegel, 2005). While the anatomy differs to that of the human

mandible, this difference is actually of benefit as the edentulous span at the

parasymphyseal region permits ease of operation without the need for exodontia. On

the whole, sheep provide a valuable model in the assessment of tissue engineering

constructs (TEC) for healing of mandibular defects, which would be transferable to

the human condition. Whilst sheep provide a useful model for translational studies to

investigate novel TECs for mandibular defects, consistent in vivo application and

post-explantation analysis has not been standardised across studies(Abu-Serriah et

al., 2006; Abu-Serriah et al., 2005; Alkan et al., 2007; Ekholm et al., 2006; Forriol et

al., 2009; Kontaxis et al., 2004; Martola et al., 2007; Nolff et al., 2010; Schliephake

et al., 2001).


Study No. Defect Size Healing

Period

Treatment Outcome Biomechanical

Testing

(Abu-Serriah

et al., 2005)

Adult

Scottish

Grey

Face

(n=6)

35mm

osteoperiosteal

segmental

defect

3months -rhOP-1 +

type-1 collagen

-No control

arm

Inferior

mechanical

properties of

regenerated

bone

compared to

contralateral

mandible.

Cantilever

(Alkan,

Celebi,

Ozden, Bas

& Inal, 2007)

Sheep

(n=20)

Plating

techniques

fractured

angle

mandible?

Plating

technique

article not

segmental

defect article

Nil 4 different

rigid fixation

on

hemimandibles

of sacrificed

sheep

Assessment

of plating

technique

3-point bend

test- Suggest

removal of

article

(Martola,

Lindqvist,

Hanninen &

Al-Sukhun,

2007)

4-5yrs

Adult

Finnish

Sheep

(n=6)

Angle

Mandible

~2cm upper

border, 5-6cm

lower border?

Plating article

not segmental

defect

2months Assess

bridging plate

fractures

Nil implant

Assessment

of plates

Nil- Suggest

removal of

article

(Ekholm et

al., 2006)

Adult

Landrace

Sheep

(n=12)

23mm x

11mm

unicortical-

unicortical

study suggest

removal of

article

9, 14,

24 and

52wks

Right side –

P(-CL/DL-

LA)-TCP

Left side – no

material

Bone

formation

L>R at all

time points.

Inflammation

greater R

side.

Nil

(Salmon &

Duncan,

1997)

Sheep

(n=3)

9mm

unicortical

bilaterally-

unicortical

suggest

removal of

study

6, 8 and

12wks

Mucoperiosteal

flap

Unicortical

CSD >8mm

Nil

(Abu-Serriah

et al., 2006)

Adult

Scottish

Grey

Face

Sheep

(n=6)

35mm

osteoperiosteal

segmental

defect

3months -rhOP-1 +

type-1 collagen

-No other arm

Excessive

bone

formation

and

unsatisfactory

restoration of

bone contour

Nil

(Schliephake,

Knebel,

Aufderheide

& Tauscher,

2001)

Adult

Sheep

(n=8)

35mm

segmental

defect

5months -Scaffolds of

pyrolized

bovine bone +

OP cells (n=4)

-scaffold only

(n=4)

Seeded

scaffold

enhanced

bone growth

but scaffold

only had

>10% bone

regeneration

Nil


(Kontaxis,

Abu-Serriah,

Ayoub &

Barbenel,

2004)

Adult

Scottish

Grey

Face

Sheep

(n=12)

35mm

osteoperiosteal

segmental

defect

3months -Collagen

alone (n=6)

-rhBMP-7 +

collagen (n=6)

Collagen

only group

failed show

any bone

bridging

within the

defect.

Regeneration

of defect in

the rhBMP-7

group had

variable

quality of

bone.

Cantilever

(Nolff et al.,

2010)

Adult

German

Black

Head

Sheep

(n=10)

25mm

segmental

defect

3

months --TCP (n=5)

--TCP +

autologous

bone marrow

(n=5)

-no control arm

Conventional

CT is NOT

suitable to

objectively

evaluating

ossification

and

degradation

of a -TCP

graft in vivo

Nil

(Forriol et al

2009)

Adult

Ovis

Aries

(n=15)

60mm

segmental

defect

2

months

Group I:

Control, empty

defect

Group II:

Platelet Rich

Plasma (n=3)

Group

III:rhOP-1 in

the form of

3.5mg

eptotermin

alpha in 1g of

bovine

collagen type I

matrix

Group IV:

Frozen rib

allograft

Group V:

Frozen rib

allograft and

rhOP-1

Control

group and

PRP group

did not show

any bone

formation.

rhOP-1 group

showed

endochondral

ossification.

Greatest bone

density was

been in the

allograft and

rhOP-1

group.

However, no

direct union

between

newly formed

bone and

graft was

seen.

Nil

Table 2: Overview of Sheep Mandible Studies


2.4 ESTABLISHING A MODEL

Cheng et al surmised that, for an animal model to be of most benefit, it must

replicate clinical surgical techniques, provide appropriate tissue size and handling

that would be encountered clinically, be versatile in a controlled environment and

allow quantitative assessment of results(Cheng et al., 2005). Due to the diversity of

disease conditions and the variation in animal constitution there can be no perfect

model that will provide the exact clinical environment to mimic the human condition.

This is the inherent limitation of any animal model, however, it still remains

imperative that translational studies investigating the applicability and safety profile

of any intervention be thoroughly examined and validated prior to human

implementation.


Figure 2: Road map to establishing a critical-sized bone defect study in a large animal

model. [Adapted from (Reichert, Epari, et al., 2010)]

One of the key components to evaluating TEC’s for bone healing in an animal

model is to establish the specific critical-sized defect for that model. The

experimental critical-sized defect for a sheep mandible depends on many factors

related to both the sheep and the nature of the defect created. These range from age,

breed, defect shape, defect position (inferior or superior), degree of penetration

(unicortical or bicortical), plate and screw size, length of healing time and method of

analysis(Salmon & Duncan, 1997). It is important that these variables be addressed

Research Question

Previous clinical

experience implant

selection

Selection of animal

model

Literature based

selection of defect size

Defect non-critical

Revise Surgical

Protocol

Increase defect

size

Animal Study

Pilot Surgery

Biomechanical

Testing

FE Modeling

Implant Failure

Mock Surgery

Defect Critical Implant Suitable

Revision of

implant choice


when establishing a model with a critical-sized defect so that results are comparable

and may be reproducible. Unfortunately, studies performed on sheep have, to date,

all differed in the size of their critical defect and the employment of a control arm. In

a study by Abu-Serriah et al, a bicortical osteoperiosteal mandible continuity defect

was performed on adult Scottish grey face sheep where it was found that a 3.5cm

defect was critical-sized(Abu-Serriah et al., 2006; Abu-Serriah et al., 2005; Kontaxis

et al., 2004). While in a study of adult female black headed sheep, a 2.5cm bicortical

defect was employed for the critical size but with an insufficient control arm(Nolff et

al., 2010). A further study by Forriol et al 2009, employed a 6cm bony defect in the

mandible of 15 sheep, Ovis aries, aged 8 years where reconstruction of the defect

was carried out with allograft, frozen rib, rhOP-1, PRP, and a combination of frozen

rib and rhOP-1(Forriol et al., 2009). This study employed a successful empty defect

control arm at 6cm.

To sum up the current literature it can be stated that the defect size employed

for the critical-sized mandibular defect in adult sheep has ranged from 2.5cm to 6cm,

and an appropriate control arm has not been employed for any defects less than

3.5cm. The critical-sized mandibular defect has proved difficult to establish and will

vary between sheep breed and age. An appropriate control arm is required to discern

what is the minimal sized defect that is critical in bone healing for that particular

sheep breed at a certain age. Following establishment of the critical-sized defect it is

important that the surgical technique be reasonable and relevant. In addition the post-

explantation analysis must include histology, biomechanical testing and computed

tomography scanning.

The review of the current literature highlights the need to employ an

appropriate reproducible control arm when investigating critical-sized mandibular

defects as well a consistency of in vivo application and post explantation analysis to

assist interpretation and correlation of results between studies.

2.5 MANDIBULAR FRACTURE MANAGEMENT IN SHEEP

The management of a mandibular bone defect in animal models is fraught with

difficulties given the importance of the mandible in mastication and the inherent


nature of veterinary medicine. Management will be highly influenced by the

temperament, compliance and ease of animal handling. However, difficulties and

complications in managing animals post-operatively must be anticipated. Clinically,

mandibular fracture management in humans covers the spectrum of conservative

management to operative intervention. This includes antibiotics, analgesia, oral

hygiene and a soft diet with immobilization to aid healing of the fracture site. Whilst

most of these aspects of fracture management are transferrable to the sheep model,

there remain noncompliant facets that impart obstacles to optimal management.

Open reduction and internal fixation with plate and screws has proven to be the

most effective method of rigid fixation in humans, associated with minimal

morbidity and early return to function(Ajmal, Khan, Jadoon & Malik, 2007). Lack of

adequate stabilisation leads to chronic inflammation, which impairs normal healing

and may result in delayed union, non-union, or infection(Futran, 2008). Whilst

dietary advice, oral hygiene, relative immobilisation and patient input regarding pain

are cornerstones of clinical care in humans, they provide obstacles in post-operative

management of mandibular defect repair in sheep. This is unavoidable and requires

that appropriate intervention including altered diet and regular analgesia be

instituted.

2.5.1 Post-explantation Biomechanical Testing

The biomechanical properties of the mandible has been studied and described

with better understanding of the forces the mandible withstands during function and

also the forces the reconstructed mandibles must tolerate(Wong et al., 2010). Whilst

reconstruction of critical-sized mandibular defects has been investigated in a variety

of animal models, there are limited studies assessing the biomechanical properties of

the regenerated bone. The mandible is the only movable load-bearing bone of the

skull that is required to withstand the forces transmitted during function(Wong et al.,

2010). Therefore, biomechanical assessment of the healed mandible post intervention

is essential. The five ways to biomechanically assess or load a bone involve tension,

compression, bending, shear and torsion forces. The small number of studies that

engaged in biomechanical testing of sheep mandible, following reconstruction of a

defect, differed in their approach. They either employed a 3-point bending test(Alkan


et al., 2007; Yuan et al., 2010) or cantilever forces(Abu-Serriah et al., 2005; Kontaxis

et al., 2004).

The shape of the mandible and its muscular and ligamentous attachments

results in complex dynamics of the masticatory system. It involves the interplay of

the temporomandibular joint and various ligaments attaching to the mandible, hyoid

and styloid process. However, the dominant determinants of jaw movements revolve

around the muscles(Koolstra, 2002). The system is mechanically redundant, with an

infinite number of muscle contraction patterns that can cause the same movement. As

such, the mechanical strength and the forces at play in a reconstructed mandible are

complex and not fully understood(Wong et al., 2010). Therefore, it is important that

an appropriate, reproducible protocol for biomechanical testing be instituted to assess

load-displacement, stiffness and maximum applied moment for both the operated and

non-operated side of a post-operative mandible. In establishing a model, we propose

to biomechanically test the mandible with cantilever test in all four planes as

displayed in figure 2.


Figure 3: Biomechanical testing using cantilever force with Instron 35kN.

The hemimandible is embedded in PMA and a distance of 11cm measured

and marked on the hemimandible (A) and (B). The hemimandible is held

firmly in place during loading force application and tested in the four planes


of craniocaudal (C), mediolateral (D), caudocranial (E), and lateromedial

(F).

2.5.2 HISTOLOGY AND COMPUTED TOMOGRAPHY

Post-explantation histological examination will also be established in the

analysis of bone growth within the critical sized defect of the operated mandibles.

Histological analysis will involve paraffin and resin embedding techniques whilst

utilizing Von Kossa and Goldner’s trichrome staining to determine calcification and

bone growth. Immunohistochemistry will be carried out to determine the

inflammatory reaction to the polycaprolactone scaffold as well as μCT to visualize

mineralized matrix volume and mineral density as described in previous sheep tibial

defect studies performed in our group(Abu-Serriah et al., 2006; Abu-Serriah et al.,

2005; Berner et al., 2013; Berner et al., 2012; Cheng et al., 2005; Hutmacher et al.,

2001; Reichert, Epari, et al., 2010; Reichert et al., 2009a; Reichert, Woodruff, et al.,

2010).

2.6 CONCLUSION

There exists a recognized need in the literature to provide further data sets on

the efficacy and safety profile for novel tissue engineered constructs (TEC) in

healing mandibular segmental defects prior to their translation to clinical

implementation. The establishment of a standardized large animal model is

imperative in translational research of TEC’s. The standardized animal model must

be consistent in the animal’s age and breed, to the operative technique and post-

operative care, and finally the post-explantation analysis. This will provide consistent

in vivo application and post-explantation analysis for investigating and comparing

TEC’s in reconstructing mandibular critical-sized defects. It will afford rigorous

assessment of alternate techniques to current clinical practice so that they are reliably

efficacious whilst maintaining appropriate patient safety.

Whilst there have been a variety of animal models employed in the

investigation of mandibular defect regeneration there has yet to be consistency in

developing a standardized systematic approach.(Carter et al., 2008a; Clokie &

S·ndor, 2008; Glied & Kraut, 2010a; Herford & Boyne, 2008a) The establishment of


a standardized sheep model provides the most suitable framework from which

additional experimentation and evaluation of TECs for healing mandibular defects

may be undertaken, compared and collated. It will provide a valuable tool in the

assessment of a variety of mandibular segmental defect TECs that may be

transferable to the human condition and, ultimately, the operative table.

Chapter 3: PILOT STUDY 29

Chapter 3: PILOT STUDY

3.1 INTRODUCTION

A standardized and systematic approach to large animal studies investigating

novel approaches to healing critical-sized mandibular segmental defects has yet to be

established. A preclinical animal model is the necessary step for investigating in vivo

mandibular segmental defect regeneration TEC’s to provide clarification and

scientific acumen assessing both the efficacy and safety profile of novel TEC’s.

Once established, the model will generate the framework for future research to

investigate alternative approaches to solve ever more complicated therapeutic

problems ranging from large segmental mandible defects to bone regeneration in post

radiotherapy tissues.

When designing a preclinical animal model it is important to encompass the

scope of animal research from the operative technique and post-operative

management to the post-explantation histological analysis, biomechanical testing and

radiographic assessment of bone regeneration. From a review of the current literature

and enlisting prior practical experience with sheep tibial segmental defect projects,

we propose that sheep provide the most advantageous large animal model for

translational studies to assess the safety and efficacy of a mandibular TEC(Abu-

Serriah et al., 2006; Abu-Serriah et al., 2005; Berner et al., 2013; Berner et al., 2012;

Cheng et al., 2005; Hutmacher et al., 2001; Reichert, Epari, et al., 2010; Reichert et

al., 2009a; Reichert, Woodruff, et al., 2010). A pilot study was performed to

ascertain the viability and nuances of the sheep mandibular segmental defect model

consisting of a 21mm-25mm bicortical defect within the parasymphyseal diastema

region of the mandible in Merino Ovis Aries. The defect size was dependent on the

length of the sheep diastema and was determined intra-operatively. To achieve

stability across the defect our research group employed a clinically relevant 2.4mm

titanium mandible reconstruction plate (Synthes UniLock, Australia), primarily due

to its robustness and ubiquitous use in the literature, to fixate large mandibular

segmental defects in humans(Carter et al., 2008a; Clokie & S·ndor, 2008; Glied &

Kraut, 2010a; Herford & Boyne, 2008a).

30 Chapter 3: PILOT STUDY

Sheep were randomly assigned into two groups with six sheep receiving

medical grade polycaprolactone (PCL) scaffolds and six sheep receiving no scaffold,

consequently having an empty defect. It was hypothesized that this pilot study would

highlight any practical difficulties with the sheep model in providing the framework

for establishing the large segmental mandible sheep model (LSMSM) for future

research endeavours. The establishment of the tenets of operative technique, post-

operative management and post-explantation analysis were successful, however,

premature fixation plate fracture occurred in 9 out of 12 sheep. The plate fixation

system will require further investigation so as to provide stable fracture fixation prior

to future implementation. This chapter will describe the operative and immediate

post-operative management employed for our chosen model.

3.2 MATERIALS AND METHODS

3.2.1 Scaffold fabrication and preparation

Biodegradable scaffolds comprising medical grade polycarprolactone (80 kDa)

and β-Tricalcium Phosphate (20 kDa.) (outer diameter 10mm, height 30mm, inner

diameter 5mm) were obtained from Osteopore (Osteopore International, Singapore).

PCL was electropsun using an in-house device(Detta et al., 2010). All scaffolds were

treated for 6 hours with 1M NaOH and washed five times with phosphate-buffered

saline (PBS) prior to their operative implementation. Sterilization of the scaffold was

subsequently performed with the scaffold placed in 70% EtOH for 5 minutes and

under UV irradiation for a minimum of 30 minutes.

3.2.2 Surgical Procedure

Large Animal Model

Merino sheep (weight 44-52kg, age 6-7yrs) underwent operative intervention

as approved by the University Animal Ethics Committee at the Queensland

Universirty of Technology, Brisbane, Australia (Ethics number 1000001192). The

two experimental groups consisted of an empty defect and a scaffold only group.


Induction and Preparation

The sheep were housed overnight in a communal pen. They were assessed by

the vetinary surgeon and weighed the morning of the procedure. A 7French venous

central line (Arrow, Teleflex®) was inserted into the jugular vein and the sheep was

anaesthetized with intravenous induction of propofol (4 mg/kg, IV). An endotracheal

tube was inserted and anaesthesia maintained with 50% oxygen in air,

and isoflurane (1%), using a mechanical ventilator.

Operative Instruments

Standard operating equipment for mandibular fixation including: Oscillating

saw (Stryker) for creating bicortical osteotomies; Titanium mandible reconstruction

plate (2.4mm, 10 holes, Synthes UniLock); Bicortical Titanium screws (12mm-

20mm, Synthes); Periosteal elevator; Depth gauge; and screwdriver.

Operative Intervention and Timeline

Sheep were positioned supine, following induction of anaesthesia and the

procedure was performed under sterile conditions. The right hemimandible surgical

site was prepared with iodine povacrylex. Sterile drapes were employed and a sterile

operating field created.

An 8-10cm longitudinal incision was made along the inferolateral border of the

mid to distal mandible in the parasymphyseal diastema region. The subcutaneous

tissue was reflected laterally and held with a retractor. The periosteum of the

mandible was stripped circumferentially from the mental foramen to the first

premolar, above the diastema, with a periosteal elevator.

A titanium mandible reconstruction plate (2.4mm, 10 holes, Synthes UniLock)

was placed on the inferolateral border of the mandible, torque neutral. All proximal

and distal screw holes were drilled and depth measured for screw length. The plate

was temporarily fixed either side of the proposed defect site with bicortical locking

screws at the diastema of the mandible. The proposed defect site was marked with a

sterile permanent marker.


The fixation plate was then removed and saline soaked gauze inserted between

the bone and soft tissue to avoid damage to the tissue whilst performing an

osteotomy. The neurovascular bundle exiting the mental foramen was ligated with 3-

0 monocryl.

The bicortical bone defect was created via parallel osteotomies, perpendicular

to the long axis of the bone, with an oscillating saw (Stryker) under constant

irrigation with saline solution. Gauze, bone wax and Surgicel were applied to the

bone marrow to achieve haemostasis. In all procedures, haemostasis was achieved.

Figure 4: Operative Procedure.

Incision of inferolateral right hemimandible performed under sterile

technique (A); the periosteum is removed (B); the premolar tooth (arrow)

and the neurovascular bundle (*) are identified (C). The site of the bony

defect is marked (curved arrow) and the plate applied, torque neutral with

drill holes performed through plate and fixed with screw (D); Plate removed

(E), then an osteotomy performed and the plate reapplied (F); Scaffold cut to

size and placed in defect (G) & (H); and finally, wound closure (G).


In the experimental group, the PCL scaffold was adjusted to fit the defect site

by trimming with a scalpel to match the length of the bone defect. It was then gently,

but firmly, placed in the defect site and the locking plate fixed. The wound was

closed in two layers with a subcutaneous continuous suture (3-0 Monocryl) and

interrupted skin sutures (4-0 Novafil). The skin incision was sprayed with

oxytetracycline (Terramycin, Pfizer).

Participants

The 12 operations were performed two cases per day over six separate

operating days with the involvement and cooperation of four surgeons. Two

specialist maxillofacial surgeons, Dr Lynham and Dr Warnke, performed 6 of the

operations. The remainder of the procedures were performed by either an

experienced trauma surgeon or surgical trainee.

The surgical period from induction to extubation and recovery was

approximately 3-4 hours with operative times of 45-60 minutes. The mandibular

segmental defect size ranged from 22mm up to 25mm, with an average of 23mm.

The defect size was selected intra-operatively and based on the length of the sheep

mandible diastema and after placement of fixation plate.


Plate Group Animal

ID Age

Weight (kg)

Date of Surgery

Defect Size (mm)

Plate #

2.4mm AO Plate Empty 1089 6 47.9kg 27.09.2011 24mm

2.4mm AO Plate Empty 1048 ~6 47kg 27.09.2011 24mm



2.4mm AO Plate Empty 1169 6 45.5kg 01.02.2012 23.5mm

2.4mm AO Plate Empty 1176 6 51kg 01.02.2012 22.5mm

2.4mm AO Plate

Scaffold 1178 6 48kg 02.02.2012 22mm

2.4mm AO Plate

Scaffold 1177 6 46kg 02.02.2012 22mm

2.4mm AO Plate

Scaffold 1172 6 48kg 03.02.2012 23mm

2.4mm AO Plate

Scaffold 1180 6 47kg 03.02.2012 24mm

2.4mm AO Plate

Scaffold 1183 6 43.5kg 09.02.2012 25mm

2.4mm AO Plate

Scaffold 1182 6 43kg 09.02.2012 22mm

Table 3: Sheep operations performed for the large segmental mandible sheep model

3.2.3 Post operative management

Antibiotics and Analgesia

Antibiotics and analgesia were administered for the first 48 hours after the

procedure. The sheep were administered buprenorphine (Temgesic®, 0.3mg/ml)

(0.005mg/kg, IV) and ketorolac (Toradol®, 30mg/ml)(0.5mg/kg, SC) for preventative

and bi-modal pain management. Antibiotic prophylaxis was administered to all sheep

post-operatively via the parental antibiotic regimen ciprofloxacin

(200mg/100ml)(5mg/kg, IV); cefazolin (Kefzol®1gr)(20mg/kg, IV); gentamicin

(80mg/2ml)(5mg/kg, IV).


Housing and Diet

Operated sheep were housed in a separate pen to the remaining flock but with

the other sheep that had undergone the same procedure. These sheep were placed on

a soft diet of water-soaked pellets and Lucerne-oaten chaff for 48 hours before

returning to the flock and a normal diet.

3.2.4 Post-operative Assessment

Sheep were imaged immediately following the surgical procedure and then

monitored daily for the first 3 days post-operatively to assess feeding and activity by

both the operating surgeon, a veterinary surgeon and animal handlers. Antibiotics

and analgesia were administered at this time. Further lateral and craniocaudal

radiographs of the operated mandibles were taken two to three weeks after the

surgical procedure.

Figure 5: Initial radiographs post-surgical intervention.

All radiographs demonstrate an empty defect (*) and the plate fixation

(arrow).


3.2.5 Explantation

The scaffolds from the sacrificed sheep were explanted for

immunohistochemical analysis. The soft tissue surrounding the mandible was

removed and the bony mandible explanted. The mandible was divided with an

oscillating saw along the symphysis between the central incisors creating two

hemimandibles.

3.3 RESULTS

3.3.1 Radiographs

Lateral and craniocaudal radiographs of the operated mandibles were taken two

to three weeks after the surgical procedure. In 9 out of the 12 operated cases a

fracture of the fixation plate was identified from the radiographs and confirmed

clinically. The animals were euthanized by intravenous injection of sodium

pentobarbital.


Figure 6: Radiographs displaying intact and fractured 2.4mm titanium mandible

reconstruction plates

The 2.4mm titanium mandible reconstruction plates (Synthes UniLock) were

inadequate fixation devices for the LSMSM with fracturing occurring at the

proximal junction and intact titanium plates. (A) is a lateral view of the

sheep mandible demonstrating the proximal fixation of the plate (i) and the

segmental bony defect at the diastema of the sheep mandible (*). (B) is a

craniocaudal view and demonstrates the segmental bony defect (*) and the

distal fixation of the plate at the parasymphyseal region of the mandible (ii).

(C) is a lateral view of the sheep mandible demonstration fracturing of the

fixation plate (iii) at the junction of the proximal fixation and the empty

segmental defect with inferior displacement of the distal segment. (D) is a

craniocaudal view of the same fractured mandible plate (iii) with medial

displacement of the distal segment.


3.3.2 Biomechanical Testing

Biomechanical testing was unable to be performed due to premature plate

fracturing resulting in fracture instability and the short timeframe meant the inability

of bone bridging to occur across the critical-sized defect. However, a framework for

biomechanical testing was established on non-operated hemi-mandible and will

involve cantilever forces in all four planes. The unoperated hemi-mandible was

explanted following sheep sacrifice. They were embedded in polymethyl

methacrylate (Paladur, Heraeus Kulzer, Germany) by holding the hemi-mandible in a

custom designed embedding block. A distance of 11cm was measured along the body

of the mandible distal from the edge of the embedded ramus and marked. The

hemimandible was held firmly in place by the vice of the Instron 35kN (Instron,

Norwood, USA). Loading force application and cyclical bending stress was applied

and tested in the four planes of craniocaudal, mediolateral, caudocranial, and

lateromedial (data not shown).

Formal biomechanical testing of non-operated hemi-mandibles is currently

being performed to assess fatigue and finite element modelling.


Figure 7: Biomechanical testing using cantilever force with Instron 35kN.

The hemimandible is embedded in PMA and a distance of 11cm measured

and marked on the hemimandible (A) and (B). The hemimandible is held

firmly in place during loading force application and tested in the four planes

of craniocaudal (C), mediolateral (D), caudocranial (E), and lateromedial

(F).


3.3.3 Histology and Immunohistochemistry

Histology and immunohistochemistry were performed to investigate early

immune response to the scaffold as described in the next chapter.

Figure 8: Von Kossa Stain Overview

(A) Von Kossa staining of a scaffold group demonstrating the proximal and

distal native bone; (B) comparison with the intraoperative appearance of the

scaffold in situ and; (C) Von Kossa staining of an empty defect group with

fibrous tissue linked only to the distal native bone.


Figure 9: Overview of stains for the scaffold group and empty defect group

(A) Hematoxylin and Eosin stain for scaffold group, s (scaffold), Ds (Distal end),

Px (Proximal end), Md (medial side), Lt (lateral side). Stains B,D,F and H are from

the proximal defect site. Stains C, E, G and I are from the scaffold strut located

towards the distal end. J. Hematoxylin and Eosin stain for empty defect group. Stains

K, M, O and R are taken from the proximal defect site and stains L, N, Q and S are

from the distal defect site. (B) Positive stain for TRAP. (C) Negative stain for TRAP.

(D) Positive stain for CD68. (E) Negative stain for CD68. (F) Some positive stain for

ON. (G) Positive stain for ON along the scaffold strut. (H) Positive stain for OPN. (I)

Negative stain for OPN. (K) Large positive stain for TRAP. (L) Light positive stain

for TRAP. (M) Large positive stain for CD68. (N) Light positive stain for CD68. (O)

Positive stain for ON. (Q) Negative stain for ON. (R) Positive stain for OPN. (S)

Negative stain for OPN.


3.4 DISCUSSION:

The surgical management of fractures of the mandible body is principally via

open reduction and internal rigid fixation (ORIF). This allows a more rapid osseous

repair, with return of occlusion and masticatory function as well as maintenance of

periodontal tissue.(Korkmaz, 2007; Olate et al., 2013) An ORIF approach to human

mandible body fractures is primarily achieved with 2.0mm and 2.4mm mandibular

reconstruction locking or standard plates(Collins, Pirinjian-Leonard, Tolas &

Alcalde, 2004; Korkmaz, 2007; Olate et al., 2013; Scolozzi & Richter, 2003; Shaik,

Raju, Rao & Reddy, 2012). Evidence suggests that mandible fractures treated with

2.0mm locking plates and 2.0mm standard plates present similar short-term

complication rates(Collins et al., 2004; Shaik et al., 2012). Further studies of 2.4mm

titanium reconstruction plates used to treat severe mandible fractures demonstrated a

low rate of major complications (3%) and a high success rate(Scolozzi & Richter,

2003).

Thus to mimic the clinical environment and in light of the Sheep ruminant

masticatory pattern it was decided that the more robust 2.4mm mandibular

reconstruction plates would be employed for fixation device in a large segmental

mandible sheep model (LSMSM). It must be noted that in the human clinical

condition, ORIF is often employed in conjunction with maxillomandibular fixation

(MMF) which is not feasible in the sheep model(Collins et al., 2004; Korkmaz, 2007;

Olate et al., 2013; Shaik et al., 2012).

The mandible provides a unique challenge in the operative and post-operative

management of a LSMSM given the crucial role it plays in mastication. From the

results of our pilot study it is evident the 2.4mm titanium mandible reconstruction

plates were unable to tolerate the considerable forces generated during the

masticatory cycle of ruminants(Martola et al., 2007). The mechanics of sheep

mastication are such that they possess large masseter muscles and generate

considerable degree of torque during mastication. As such, the 2.4mm titanium

mandible reconstruction plate used in human surgery is an inadequate fixation device

for the sheep mandible. The understandable difficulty associated with the post-

operative care of the sheep model is the inability to limit the movement of the

mandible due to its pivotal role in mastication. The fixation plate complication was

not anticipated from reviewing the literature, with only one author citing difficulty


with plate fracture and instability of a bicortical defect in the mandibular

parasympheal region of sheep(Schliephake et al., 2001).

Fracture of titanium fixation plates has occurred in patients who have

undergone mandibular reconstructions and are more common among patients with a

segmental defect that does not cross the midline and in whom no bone grafting is

performed(Katakura, Shibahara, Noma & Yoshinari, 2004; Shibahara, Noma, Furuya

& Takaki, 2002).

Figure 10: Radiograph of plate fracture

Case that was referred to Dr Lynham. Fracture occurred in the angle of the

mandibular reconstruction plate (arrow) at the proximal end of the defect site

(*), a similar location to that encountered in our pilot study.

The cause of the fixation plate fracturing is thought to be the result of stress

concentration, the location of which varies depending on the form of the plate, with

frequent repeated application of stress or fatigue(Katakura et al., 2004). Evaluation

of clinical and experimental plate fracture among cases in which primary

reconstruction after mandibular resection employed titanium reconstruction plates

revealed that plate fracturing was commonly in L-type defect cases in which angle-

type plates were used, and the fracture mainly occurred in the anterior region of the

mandibular angle(Katakura et al., 2004). Often the instability at the plate fracture site


in a mandibular segmental defect resulted in bone graft failure(Futran, Urken,

Buchbinder, Moscoso & Biller, 1995; Hidalgo, 1989; Mooren, Merkx, Kessler,

Jansen & Stoelinga, 2010; Yerit et al., 2002). In addition, excessive instability at the

defect site is not conducive to bone formation and bridging of the defect(Giannoudis,

Einhorn & Marsh, 2007).

The plate fracturing in the LSMSM occurred due to masticatory forces and led

to instability of the defect site. This instability is not conducive to bone formation

and bridging of the defect(Giannoudis et al., 2007). We hypothesize that thicker

reconstruction plates coupled with longer screw lengths at the mandibular symphysis

engaging the contralateral mandible may be able to withstand the masticatory forces

applied to the defect, and result in greater fracture stability allowing accurate

assessment of bone formation and bridging of the segmental defect. With the results

of the pilot study, biomechanical testing is currently being performed on unoperated

sheep mandibles and those with an empty defect fixated with 2.4mm titanium

reconstruction plates, 2.8mm titanium reconstruction plates and custom stainless

steel plates respectively. This will further evaluate the disparity in strength between

plate systems and provide confidence in the selected plate fixation system employed

for future surgeries.

Once stability at the fracture site is accomplished, the critical sized defect can

be established in the LSMSM and long term implantation studies pursued. The post-

operative management will involve regular clinical and radiographic review. The

tenets of the post-explantation analysis have been well established with the sheep

tibial model and this framework will be emulated in the mandible model(Abu-Serriah

et al., 2006; Abu-Serriah et al., 2005; Berner et al., 2013; Berner et al., 2012; Cheng

et al., 2005; Hutmacher et al., 2001; Reichert, Epari, et al., 2010; Reichert et al.,

2009a; Reichert, Woodruff, et al., 2010). It will involve biomechanical testing, as

well as histological, radiographic and computed tomography analysis.

Whilst reconstruction of critical-sized mandibular defects has been

investigated in a variety of animal models, there are limited studies assessing the


biomechanical properties of the regenerated bone. Studies that engaged in

biomechanical testing of sheep mandible following reconstruction of a defect

differed in their approach. They either employed a 3-point bending test(Alkan et al.,

2007; Yuan et al., 2010) or cantilever forces(Abu-Serriah et al., 2005; Kontaxis et al.,

2004). The dominant determinants of sheep mandible movements revolve around the

muscles(Koolstra, 2002). The system is mechanically redundant, with an infinite

number of muscle contraction patterns that can produce the same movement. As

such, the mechanical strength and the forces functioning in a reconstructed mandible

are complex and not fully understood(Wong et al., 2010). Therefore, it is important

that an appropriate, reproducible protocol for biomechanical testing be instituted to

assess load-displacement, stiffness and maximum applied moment for both the

operated and non-operated side of a post-operative mandible. We propose to

biomechanically test the mandible with cantilever force in all four planes, and this

protocol was established on non-operated hemimandibles.

Post-explantation histological analysis of bone growth within the critical sized

defect of operated mandibles will employ paraffin and resin embedding techniques

utilizing Von Kossa and Goldner’s trichrome staining to determine calcification and

bone growth. Immunohistochemistry will be carried out to evaluate the

osteoinductive and osteoconductive properties of the polycaprolactone scaffold as

described previously in the sheep tibia models performed in our group (Berner et al.,

2013; Berner et al., 2012; Cheng et al., 2005; Hutmacher et al., 2001; Reichert,

Epari, et al., 2010; Reichert et al., 2009b; Reichert, Woodruff, et al., 2010).

Plain radiographs of in vivo scaffold will assess the stability of plate fixation

and early callous formation and be performed at the two week, one month, three

month and six month review or earlier if clinically a plate fracture is suspected.

Micro-Computed Tomography (μCT) will be used to assess and visualize

mineralized matrix volume and mineral density of the explanted segmental defect

site as established at our institution (Berner et al., 2013).


In the current pilot study performed at our institution, the failure of the 2.4mm

titanium reconstruction plates proved an obstacle to establishing the critical-sized

defect and assessing the efficacy of our scaffold in bone regeneration of a segmental

mandibular defect. Overcoming fixation plate fracturing must be remedied prior to

further development of the LSMSM. It is maintained that sheep provide an excellent

preclinical model for both operative intervention and post-operative management and

investigation of mandibular scaffold efficacy. The brief post-operative management

and subsequent analysis has been appropriately designed to provide results that are

reproducible and relevant. This includes radiographic assessment, histological

analysis and biomechanical testing. These three tenets, along with computed

tomography, will provide the necessary data sets required to evaluate the safety and

efficacy profile of a tissue engineered construct (TEC) prior to clinical

implementation.

Once established, the large segmental mandible sheep model (LSMSM) will

provide the basis for further studies into the wide-ranging implementation of TEC’s.

The ability to construct PCL with fusion deposition modelling may result in

customized scaffolds prepared to the defect specifications. This provides myriad

options for therapeutic application from larger defects, including hemi- or full

mandible reconstructions to embedding with growth hormones and stem cells to

assist regeneration in difficult clinical scenarios including post irradiated, poorly

vascularised tissue. Through the use of a preclinical LMSM, these extraordinary but

achievable goals will provide the inspiration to further research in innovative

techniques for healing critical-sized mandibular defects.

Chapter 4: Histology: 47

Chapter 4: Histology:

Short Term Mandibular Implantation of Polycaprolactone

Scaffold In Ovine Aries

Brendan Jones, Kendra Von Troschel, Gary Brierly, Flavia Savi, Kristofor Bogoevski, Anthony Lynham, Dietmar. W. Hutmacher, Maria Ann Woodruff.

Biomaterials and Tissue Morphology Group, Institute of Health and Biomedical

Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove

4059, Brisbane, Australia.

Key Words: Biocompatible, Bone-regeneration, Immune response, Jaw,

Macrophages, Mandible, Polycaprolactone (PCL), Ovine, Scaffold, Segmental

defect, Sheep.

48 Chapter 4: Histology:

4.1 ABSTRACT:

The literature possesses a growing body of knowledge surrounding

polycaprolactone (PCL) as a tissue engineering construct (TEC) for bone

regeneration with much long-term implantation data available (i.e. 1 month- 5 years

in vivo). However, the early tissue response to an in vivo PCL scaffold application

has not yet, to our knowledge, been investigated(Ai-Aql et al., 2008; Akintoye et al.,

2008; Anderson & McNally, 2011; Athanasou & Quinn, 1990; Berner et al., 2013;

Boyne, 1996; Carstens et al., 2005; Cipitria et al., 2012; Collin et al., 1992; Ekholm

et al., 2006; Forriol et al., 2009; Fritz et al., 2000; Glied & Kraut, 2010a; Herford &

Boyne, 2008a; Kahnberg, 1979; Kilic et al., 2011; Lemperle et al., 1998; Meyer et

al., 1999; Okafuji et al., 2006; Ren et al., 2007; Shigeno et al., 2002; Tatsuyama et

al., 2009; Wei et al., 2006; Yuan et al., 2010; Zheng et al., 2009). The initial response

is important to understand as it influences surrounding tissue as well as the time

taken for bone regeneration. To investigate this, 8 Merino Ovis aries (sheep) were

operated over a two week operative interval with 6 being implanted with a 22mm-

25mm PCL scaffold in the parasymphyseal region of the mandible and the remaining

two with an empty defect. An unexpected adverse event of premature fixation plate

fracturing in 7 of the 8 operated sheep resulted in early sacrifice three weeks post

implantation. The defect site and surrounding tissue was extracted from 5 scaffold

and 2 empty defect groups and assessed histologically for early signs of bone

regeneration and inflammatory responses using immunohistochemistry and

conventional techniques assessing tissue morphology. Results reinforced the

biocompatible nature of PCL based TEC and also indicated that an organised fibrous

tissue network is present from the early tissue response phase of wound healing, even

in an unstable fracture environment. It would suggest that the scaffold has provided a

degree of mechanical stability and the framework for an organised cellular response

from an early stage with a combination of both decreased inflammatory markers and

increased osteoid production and bone mineralisation.


4.2 INTRODUCTION:

Tissue engineering continues to pioneer novel and exciting approaches to

restore, replace and regenerate injured or diseased biological tissue(Chan & Leong,

2008). A critical-sized defect of the mandible is a segmental bone defect that cannot

bridge spontaneously and requires surgical intervention(Hollinger & Kleinschmidt,

1990; Schmitz & Hollinger, 1986). The current gold standard for therapeutic

intervention of critical-sized defects is the use of autologous cancellous bone grafts,

with the foremost advantage being a minimal host immune response as the graft is of

the same tissue origin(Ekholm et al., 2006). Autologous bone grafts have associated

disadvantages of donor site morbidity including haemorrhage, infection, insufficient

transplant integration, insufficient graft revitalisation, limited availability and the

need for a second operative site from the same patient(Reichert, Epari, et al., 2010).

In addition, the failure rate of autografts is up to 30%(Gautschi et al., 2007).

Autologous bone graft must be transported from an alternate site, usually the iliac

crest, and for mandibular segmental reconstruction the need for a second operative

site and limited tissue availability provide the two greatest obstacles to a successful

outcome. In addition to autologous bone grafting for segmental defect regeneration,

less invasive management options include allografts, using donated tissue from bone

banks and metallic implants. In most cases these techniques are effective, however,

they risk infection, haemorrhage, and, in the case of metal implants and allografts, an

immune reaction. Thus, a clinical alternative that reduces these risks and offers a

ready to use “off the shelf” product would be indispensable. Tissue engineering aims

to provide constructs that are viable therapeutic alternatives, limiting these risks and

providing effective management at the initial operative intervention to improve

patient outcome, limit patient morbidity and avoid the need for re-

intervention(Gautschi et al., 2007). The key components to tissue engineering are

scaffolds, growth hormones and cells. Scaffolds act as the extracellular matrix with

the aim to provide the framework and structural support in facilitating the

localisation of cell migration and organised tissue healing(Chan & Leong, 2008;

Kim, Baez & Atala, 2000). Effective tissue engineered scaffolds for healing critical-

sized bone defects necessitate biocompatibility, mechanical strength, porosity to

enable nutrient exchange and vascular ingrowth as well as the ability to be rapidly

and reliably reproduced(Dalton, Woodfield & Hutmacher, 2009). Synthetic

biomaterials, such as polycaprolactone (PCL), are advantageous as a tissue


engineered construct (TEC) for bone regeneration due to their bioinert nature and

design flexibility with the ability to manipulate their physical and mechanical

properties and tailor the design towards bone specific regeneration(Abbah, Lam,

Hutmacher, Goh & Wong, 2009; Chan & Leong, 2008).

Mandible fracture healing is the result of a vast and intricate interplay of

molecules and cells influenced by intrinsic and extrinsic patient dynamics,

coordinated by molecular and intercellular interactions(Hollinger & Schmitz, 1987).

The majority of mandible bone defects and fractures heal spontaneously, coordinated

by a sequentially organized cell population and microenvironment determinants. If

these coordinated events are compromised the potential for regeneration is limited,

leading to poor wound healing with resultant patient morbidity(Anderson &

McNally, 2011; Nguyen, Orgill & Murphy, 2009; Reichert, Woodruff, et al., 2010;

Reichert et al., 2011). The three phases of wound healing can be roughly divided

into the initial inflammatory response, followed by a proliferative phase and then

maturation. Thus bone healing commences with an inflammatory reaction in order to

initiate the regenerative process eventually resulting in the reconstitution of

bone(Schmidt-Bleek et al., 2012). An excessive host immune response during this

early phase of bone healing may disrupt the normal regenerative process and delay

healing, endangering a potentially successful outcome(Schmidt-Bleek et al., 2012).

The nature of the initial host immune response is dependent upon the host’s

physiology and the nature of the insult, for example, trauma or infection. Whilst

aiding in initial bridging of a bone defect site, an excessive host immune response is

detrimental to wound healing and will impede bone regeneration(Ai-Aql et al.,

2008). The failure of bone bridging will lead to non-union or malunion of mandible

bone fragments causing significant patient morbidity.

The application of TEC’s for regeneration of a critical-sized bone defect

requires that the scientific data sets provide the clinician and patient with confidence

in a viable alternative to current clinical practice by being both reliably efficacious

and maintaining appropriate patient safety. Understanding the host immune response

to a scaffold, from both a morphology and materials point of view, is vital in

developing an efficacious and biocompatible therapeutic option for bone


regeneration of mandibular critical-sized segmental defects. It is important that an

implant such as a scaffold invoke a minimal host inflammatory response to optimize

healing of a bony defect(Ai-Aql et al., 2008).

A prospective alternative to the current surgical management of a bicortical

mandibular segmental bone defect involves employing a bioinert and biodegradable

scaffold made from polycaprolactone (PCL). PCL can be implanted in the bone

defect site, providing mechanical support whilst facilitating tissue regeneration. Over

time, as tissue healing occurs, the construct is biodegraded by the body resulting in

bridging of the defect with only regenerated host tissue remaining(Woodruff &

Hutmacher, 2010). Bone healing following a segmental defect commences with an

inflammatory reaction which may play a role in initiating the regenerative process

that eventually results in the reconstitution of bone(Schmidt-Bleek et al., 2012). An

excessive immune response during the early phase of bone healing disrupts normal

regenerative process and delays healing, negatively impacting on a potential

successful outcome(Schmidt-Bleek et al., 2012). Thus, in establishing an effective

alternative to autologous bone grafting, it is important that the PCL based scaffold

invoke a minimal host inflammatory response to optimize healing of the critical-

sized defect.

Traditionally animal models employing a tissue construct for the regeneration

of bone have not been sacrificed until 3-6months post implantation to assess bony

regeneration(Abukawa et al., 2004; Berner et al., 2013; Berner et al., 2012; Cheng et

al., 2005; Forriol et al., 2009; Herford & Boyne, 2008a; Reichert, Epari, et al., 2010;

Reichert et al., 2009a; Ren et al., 2007). Long-term implantation allows significant

time to pass before assessing scaffold integration and bone regeneration as well as

being ethically acceptable. These studies have demonstrated the biocompatible

nature of PCL based scaffolds, which make it a valuable synthetic biomaterial for

bone regeneration and, along with its biodegradable properties of being resorbed

over the course of 3-4 years, allows an adequate period for bone healing(Holland &

Tighe, 1992; Middleton & Tipton, 2000) . Polycaprolactone has been FDA approved

and when combined with its biocompatibility, slow resorption and mechanical


strength makes it an ideal material in the design of medical scaffolds for bone tissue

engineering.

The acute phase tissue response to in vivo polycaprolactone scaffolds employed

for bone healing in a sheep model has, to the best of our knowledge, never been

investigated. A greater understanding of the initial response is important to establish

the biocompatibility and the influence on surrounding tissue as a guide for future

mineralisation(Cipitria et al., 2012).


4.3 MATERIALS AND METHODS:

4.3.1 Scaffold fabrication and preparation

Biodegradable scaffolds comprising medical grade polycarprolactone (80 kDa)

(outer diameter 10mm, height 30mm, inner diameter 5mm) was obtained from

Osteopore (Osteopore International, Singapore). PCL was electropsun using an in-

house device. All scaffolds were treated for 6 hours with 1M NaOH and washed five

times with phosphate-buffered saline (PBS). Sterilization of the scaffold was

subsequently performed with the scaffold placed in 70% ethanol (EtOH) for 5

minutes and under UV irradiation for a minimum of 30 minutes prior to their

operative implementation.

4.3.2 Surgical Procedure

Merino sheep (weight 44-52kg, age 6-7yrs) underwent operative intervention

as approved by the University Animal Ethics Committee at the Queensland

Universirty of Technology, Brisbane, Australia (Ethics number 1000001192). The

two experimental groups consisted of an empty defect and a scaffold only group.

Sheep were anaesthetized with an intravenous induction of propofol (4 mg/kg,

IV) and maintained with 50% oxygen in air, and isoflurane (1%), using a mechanical

ventilator.

The sheep were placed supine, following induction of anaesthesia. The

procedure was performed under sterile conditions. The right hemimandible surgical

site prepped with iodine povacrylex. A longitudinal incision was made along the

inferolateral border of the mid to distal mandible approximately 6-8cm in length.

The subcutaneous tissue was reflected laterally and held with a retractor. The

periosteum of the mandible was stripped circumferentially from the mental foramen

to the first premolar, above the diastema, with a periosteal elevator.


A mandible reconstruction plate (2.4mm, 10 holes, Synthes UniLock) was

placed on the inferolateral border of the mandible, torque neutral. All proximal and

distal screw holes were drilled and the plate temporarily fixed either side of the

proposed defect site at the diastema of the mandible.

The fixation plate was then removed and saline soaked gauze inserted between

the bone and soft tissue to avoid damage to the tissue whilst performing an

osteotomy. The neurovascular bundle exiting the mental foramen was ligated with 3-

0 monocryl.

The bicortical bone defect was created via parallel osteotomies, perpendicular

to the long axis of the bone, with an oscillating saw (Stryker) under constant

irrigation with saline solution. Gauze and Surgicel were applied to the bone marrow

to achieve haemostasis.


Figure 11: Overview of the Operative Procedure

(A) An Incision of inferolateral right hemimandible; (B) Periosteum

removed; (C) Diastema exposed with the first premolar (straight arrow) and

mental nerve (*) identified. (D) Defect marked (curved arrow) and plate

applied, torque neutral. Drill holes performed; (E) Osteotomy site marked

and performed once plate removed; (F) Plate reapplied following osteotomy;

(G) & (H) Scaffold cut to the length of the osteotomy and placed in defect;

(I) wound closure

In the experimental group, the 30mm PCL length was adjusted to fit the defect

site by trimming it with a scalpel to match the length of the bone defect. It was then

gently, but firmly, placed in the defect site and the locking plate fixed. The wound

was closed in two layers with a subcutaneous continuous suture (3-0 monocryl) and

interrupted skin sutures (4-0 Novafil).


4.3.3 Post-operative management

Antibiotics and analgesia were administered for the first 48hrs post procedure.

The sheep were administered buprenorphine (Temgesic®, 0.3 mg/ml) (0.005

mg/kg, IV) and ketorolac (Toradol®, 30 mg/ml) (0.5 mg/kg, SC) for preventative and

post-operative bi-modal pain management. All sheep received a prophylactic

postoperative parenteral antibiotic regimen [ciprofloxacin (200mg/100ml) (5 mg/kg,

IV); cefazolin (Kefzol® 1 gr) (20 mg/kg, IV); gentamicin (80 mg/2ml) (5 mg/kg, IV).

All animals underwent daily clinical evaluation by a veterinary surgeon. They were

placed in a pen with the other post-operative sheep and given a soft diet, including

water-soaked pellets and Lucerne-oaten chaff, for 48 hours before returning to the

paddock and normal diet.

4.3.4 Radiographic Analysis

A lateral and craniocaudal radiograph of the sheep’s mandible was performed

immediately following the procedure and then two weeks following operative

intervention. In almost all cases (5 of 6), a fracture of the fixation plate was

immediately identified on radiograph analysis. The animals were subsequently

euthanized by intravenous injection of sodium pentobarbital.

Figure 12: Initial radiographs

Initial post-operative radiographs. All radiographs demonstrate an empty

defect (*) and the plate fixation (arrow).


4.3.5 Explantation

The scaffolds and surrounding tissue from the sacrificed sheep were explanted

for immunohistochemical analysis. The soft tissue surrounding the mandible was

removed and the bony mandible explanted. The mandible was divided with an

oscillating saw along the symphysis between the central incisors creating two

hemimandibles.

In two of the five samples from the experimental scaffold group the implanted

tissue construct was insecurely attached to native bone and unable to be removed

with adjacent bone tissue, thus losing the in vivo orientation of the scaffold. In the

three remaining samples, a tenuous attachment to the distal bone at the site of the

defect was observed. The two empty defect samples were employed as a control arm.

After the mandible section containing the scaffold or empty defect was

extracted it was placed in 10% neutral buffered formalin (NBF) for 2 days for tissue

fixation then transferred to 70% ethanol.

Figure 13: Histology Preparation.

Explantation of scaffold and distal body of the hemimandible (A). The

scaffold (straight lines) were tenuously linked to the native mandible (curved

lines) (B). The explanted scaffold was sectioned along the longitudinal axis

and prepared for resin samples (C) and sectioned for paraffin (D).


4.3.6 Histology/Immunohistochemistry

Following explantation specimens were placed in 10% neutral buffered

formalin then transferred to 70% ethanol. The specimen was sectioned in the axial

plane to create superior and inferior samples, assigned either to paraffin or resin

embedding.

Scaffold samples for paraffin embedding were decalcified in 15% EDTA for 6-

8 weeks at 4oC. They were subsequently sectioned at 5µm using a microtome (Leica

RM 2265). The slides were deparaffinised with xylene and rehydrated before

staining with haematoxylin and eosin (Sigma Aldrich) and mounting with Eukitt

mounting media (Fluka Biochemical, Milwaukee, WI).

The remaining samples were embedded in methylmethacrylate resin (MMA,

Technovit 9100 NEU, Heraeus Kulzer, Wehrheim, Germany). Resin samples were

sectioned at 6µm and stained with Von Kossa/Van Gieson to assess new

mineralisation and Goldner’s Trichrome to assess cellular details.

Immunohistochemistry was performed on the paraffin sections. They were

deparaffinised with Xylene and subsequently rehydrated with increasing

concentrations of ethanol. Immunohistochemical staining employed primary

antibodies specific to the osteogenic markers osteonectin, osteocalcin, osteopontin

and type I collagen (Abcam Cambridge, UK), the macrophage markers CD 68

(Abcam, Cambridge, UK), and an osteoclast marker TRAP (Abcam, Cambride, UK).

4.3.7 Microscopy

The stained slides were observed under a Zeiss Axio Observer motorized

microscope (Zeiss, Germany) for cellular details and photographs taken for further

analysis. Photos were taken at x20 magnification.


4.3.8 Statistical Analysis

Analysis of the stained slides was performed through the Image J (Schneider,

C.A., Rasband, W.S., Eliceiri, K.W. "NIH Image to ImageJ: 25 years of image

analysis". Nature Methods 9, 671-675, 2012).

Figure 14: Image J analysis.

(A)-(D). An example of how Image J can be used to quantify histology

slides, in particular bone mineralization. (A). Native slide without

modifications. (B) Slide is converted into a grey scale image. (C) The whole

slide (stained area) is thresholded (the entire tissue area is made red). (D)

Only the black (positive black staining for bone mineralization) is then

thresholded (made red). A percentage of the bone mineralization can then be

calculated. (E)-(G). Shows an example of Image J being used to count the

number of positively stained cells. (E) The stained image. (F). Image J

converting to grey scale. (G) Image J then counts the number of positively

stained cells.


4.4 RESULTS

4.4.1 Plate fracture

The unexpected fracturing of the 2.4mm mandible reconstruction plates

(Synthes, UniLock) was discovered at radiographic assessment two weeks post

procedure in 7 out of 8 sheep. As such, the 2.4mm mandible reconstruction plate

used in human surgery is an inadequate fixation device for the sheep mandible. The

unforeseen plate fracturing resulted in the premature cessation of any further

procedures with sacrifice of the 2 sheep from the empty defect arm, and the 5 sheep

from the scaffold arm. It is evident that 2.4mm mandible reconstruction plates are

unable to tolerate the considerable forces generated during the masticatory cycle of

ruminants(Martola et al., 2007). The fixation plate complication was not anticipated

and warrants further investigation prior to establishing the ovine mandibular model,

currently being undertaken at our institution. All plates fractured between the plate-

hole fixated immediately proximal to the defect site and the adjacent empty plate-

hole within the defect site. This consistency would indicate that the maximal force

moment is passed through the plate at this point. Whilst the premature fracturing of

the fixation plates was unfortunate, it presented an opportunity to study early healing

response to PCL scaffold.

4.4.2 Radiographic Analysis

Plain radiographs of the sheep mandible were performed at two weeks post

implantation and demonstrated fracturing of the fixation plate, in 7 out of the 8

operations, with inferior and ventral displacement of the distal mandibular segment

of bone.


Figure 15: Plain Radiographs

Radiographs displaying fracturing of the 2.4mm titanium mandible

reconstruction plate (Synthes UniLock) at the proximal junction and intact

titanium plates. (A) is a lateral view of the sheep mandible demonstrating the

proximal fixation of the plate (i) and the segmental bony defect at the

diastema of the sheep mandible (ii). (B) is a craniocaudal view and

demonstrates the segmental bony defect (ii) and the distal fixation of the

plate at the parasymphyseal region of the mandible (iii). (C) is a lateral view

of the sheep mandible demonstration fracturing of the fixation plate (iv) at

the junction of the proximal fixation and the empty segmental defect with

inferior displacement of the distal segment. (D) is a craniocaudal view of the

same fractured mandible plate (iv) with medial displacement of the distal

segment.


4.4.3 Histology/Immunohistochemistry

The macroscopic overview of the implanted scaffolds revealed the evident

failure of host bone integration. No resorption of any scaffold had taken place and

they remained entirely intact in the defect site. Given the proximal fixation plate

fracturing and short time frame between implantation and explantation, two of the

scaffold samples failed to integrate with host tissue at all whilst the remaining three

samples were tenuously linked at the distal margin to the host bone via fibrous tissue.

The two samples without fixation to host tissue were unable to be analysed due to

there unknown orientation in the host tissue. Thus, only three samples from the

scaffold arm could be accurately assessed.

Goldner’s trichrome stain revealed osteoid tissue within the defect site,

arranged along the struts of the scaffold. In addition, multi-nucleated cells were seen

clustered together along the PCL struts and within the soft tissue at the proximal end

of the defect

Figure 16: Goldner’s Trichrome stain of Scaffold and native bone

The native bone proximal and distal to the scaffold with evidence of osteoid

(red) and mineralisation (blue) within the struts of the scaffold.


The three scaffolds connected with host tissue were decalcified for histological

examination. Hematoxylin and eosin stain for both the scaffold group and the empty

defect group revealed connective tissue comprising collagen fibres and fibroblasts

throughout the defect site. Blood clot was observed consistently on the medial sides

of the defect, with the lateral sides showing dense fibrous tissue formation.

Positive TRAP, CD68 and OP staining for osteoclasts and macrophages were

observed in both the proximal and distal areas in the empty defect group, with greater

staining in the proximal region. Whilst in the scaffold group osteoclasts and

macrophages were shown in the proximal area only, with no positive staining along

the struts of the PCL.

Osteonectin (ON) staining for active osteoblasts was quite dense along the PCL

struts of the scaffold group with some positive stain scattered through the fibrous

tissue located in the proximal region of the defect. ON positive staining was seen in

the proximal region only of the empty defect group with no positive staining at the

distal region.

Type-I collagen, an early non-specific maker for osteoblastic differentiation

during mineralisation, and Osteocalcin (OC), a late osteogenic marker, were

demonstrated through the entire scaffold with Osteocalcin showing a predilection

around the scaffold struts.


Figure 17: Overview of stains for the scaffold group and empty defect group

(A) Hematoxylin and Eosin stain for scaffold group, s (scaffold), Ds (Distal end),

Px (Proximal end), Md (medial side), Lt (lateral side). Stains B,D,F and H are from

the proximal defect site. Stains C, E, G and I are from the scaffold strut located

towards the distal end. J. Hematoxylin and Eosin stain for empty defect group. Stains

K, M, O and R are taken from the proximal defect site and stains L, N, Q and S are

from the distal defect site. (B) Positive stain for TRAP. (C) Negative stain for TRAP.

(D) Positive stain for CD68. (E) Negative stain for CD68. (F) Some positive stain for

ON. (G) Positive stain for ON along the scaffold strut. (H) Positive stain for OPN. (I)

Negative stain for OPN. (K) Large positive stain for TRAP. (L) Light positive stain

for TRAP. (M) Large positive stain for CD68. (N) Light positive stain for CD68. (O)

Positive stain for ON. (Q) Negative stain for ON. (R) Positive stain for OPN. (S)

Negative stain for OPN.


Figure 18: Cell numbers at the proximal and distal defect site.

(A) Von Kossa staining of a scaffold group demonstrating the proximal and

distal native bone; (B) comparison with the intraoperative appearance of the

scaffold in situ and; (C) Von Kossa staining of an empty defect group with

fibrous tissue linked only to the distal native bone. (D) Cell numbers at the

proximal end of the defect site. (E) Cell numbers at the distal end of the

defect site.


Figure 19: Cell numbers at the proximal end of the defect site.

The positive stains were quantified using Image J and results were

interpreted in graphical format. OP Empty defect group (EDG) have a m=

301 cells, whilst scaffold group (SG) has a m = 234 cells with positive stain.

ON the EDG show m= 219 cells, with the SG having m=121 cells. CD68,

the EDG show m=342 cells and the SG show 163 cells. TRAP, EDG – m=

12 cells and SG- m=7 cells.

Figure 20: Cell numbers at the distal end of the defect site.

The positive stains were quantified using Image J and results represented in

graphical format. OP: EDG- m=25 cells, SG- m= 0 cells. ON: EDG- m=8

cells, SG- m= 105 cells. CD68: EDG – m= 220 cells, SG- m= 0 cells. TRAP:

EDG – m=3 cells, SG- m= 0 cells.


Undecalcified sections were resin embedded (MMA) and stained with Von

Kossa / Van Gieson to get an entire overview of the defect site. This enabled host

bone plus the defect/scaffold to be visualised in one histological slice with resin able

to embed very large bone samples. In addition, it allowed observation and

assessment of bone mineralisation. Both groups showed mineral deposition

throughout the centre of the defect site. Despite the limited implantation time, the

scaffold group (n=3) showed an average of 3.4 % mineralization across the whole

defect site and the empty defect (n=2) showed mineralization of 0.5% across the

whole defect site.

Figure 21: Whole resin sections

A. Schematic of the tissue sections indicating the host bone (b) and the

location of the scaffold (s) within the sections. B. Von Kossa / Van Gieson

stain to show bone mineralization (Black stain). Black lines indicate host

bone (which was not included when calculating bone mineralization). C.

Goldner’s Trichrome stain used to identify osteoblasts (red with Golgi

complex pint), osteoclasts (light red), osteoid (orange-red), cartilage

(purple), nuclei (blue-grey) and mineralised bone (Dark green). D. Positive

osteoclast staining (light red, multi-nucleated cells) around the struts at the

proximal end of the defect site.


Figure 22: Bone mineralization seen in Von Kossa staining.

The percentage of bone mineralization calculated from Image J presented in

a graph. The scaffold group (n=3) show a significant number bone

mineralization (m=3.4%) (p= compare to the empty defect group (n=3) (m=

0.5%) .


4.5 DISCUSSION

Bone healing following bicortical disruption commences with an inflammatory

reaction which initiates the regenerative process that may eventually result in the

reconstitution of bone(Schmidt-Bleek et al., 2012). Bone is unique in connective

tissue healing because it heals entirely by cellular regeneration and the production of

a mineral matrix rather than just collagen deposition, known as scar(Marx, 2007). An

excessive immune response during the early phase of bone healing disrupts the

normal regenerative process and may delay healing, negatively impacting on a

potentially successful outcome(Schmidt-Bleek et al., 2012). Therefore, a greater

understanding of the acute host response is pivotal when designing and optimizing a

tissue engineered scaffold system in bone healing. In our pilot study of a large

segmental mandible sheep model (LSMSM), the premature fracturing of fixation

plates was an unexpected adverse event. Nonetheless, it provided an opportunity to

examine the early stages of the host response to the polycaprolactone (PCL) scaffold

construct.

Plain radiographic assessment revealed the unexpected adverse event of plate

fracturing in most procedures (7 out of 8). Early callous formation could not be

discerned radiographically at 2 weeks post implantation, in keeping with an unstable

fracture and the early time point of the investigation. The distal fracture fragment

displayed inferior and ventral displacement as expected with attachment of the

prominent masseter muscle to the body and ramus of the mandible. This large muscle

attachment would be expected to produce dorsal and superior displacement of the

proximal segment. We also undertook micro-CT analysis but could not detect any

mineralisation at only three weeks implantation (data not shown). Titanium plate

fracturing occurred at the proximal position of the defect site between the final

screw-hole of proximal fixation and the first empty screw-hole of the defect site,

indicating that the maximal load and moment forces of mastication are occurring at

this point of the plate. It is evident that 2.4mm mandible reconstruction plates

(Synthes UniLock), used in human surgery, is an inadequate fixation device for the

sheep mandible as they are unable to tolerate the considerable forces generated

during the masticatory cycle of ruminants(Martola et al., 2007). The biomechanics of


the sheep jaw are unique and warrant the adoption of alternate fixation devices for

future research endeavours. The fixation plate complication was not anticipated and

warrants further investigation prior to establishing the large segmental defect

mandible sheep model. Whatever plate fixation system is employed, it will require

rigorous biomechanical testing prior to implementation, a process being undertaken

in our group at present.

Histology and immunohistochemistry results have reinforced the

biocompatible nature of the PCL based scaffold and highlight the capacity of the

scaffold system in providing a platform for cells promoting a regenerative pathway,

such as osteoblasts and fibroblasts, to result in organised fibrous tissue deposition, a

decreased immune response and enhanced bone mineralization potential compared to

that of the empty defect group. The markers of the acute phase inflammatory

response are observed in both empty defect (ED) and scaffold (S) groups

predominantly at the proximal defect, site of the fixation plate fracturing, with

minimal staining along the PCL struts.

Macrophages are a heterogeneous subset of the mononuclear cell population,

activated in response to tissue damage and infection, involved in the host response to

implanted materials such as polycaprolactone (Brown, Valentin, Stewart-Akers,

McCabe & Badylak, 2009). There is an association between the early macrophage

response to implanted materials and the outcome of tissue remodelling with the

tissue healing outcome ranging from scarring to healthy functional tissue formation

suggesting a central, and perhaps determinant, role for macrophages in tissue

remodelling(Badylak, Valentin, Ravindra, McCabe & Stewart-Akers, 2008; Brown

et al., 2009). In this study the macrophage lineage cells are observed mostly at the

site of fixation plate fracture, consistently being the proximal end of the defect

(m=163 cells/field for scaffold group and m=342 cells/field for empty defect group).

The macrophage population contained numerous multi-nucleated cells with the

correlating TRAP stain much lower (7-12 cells per field) in both groups, indicating

the remaining multi-nucleated cells are likely foreign-body giant cells, created by the

fusion of macrophages and a likely response to the presence of a foreign body such

as the fractured titanium plate. The presence of large numbers of tissue macrophages


and their multinucleate giant-cell counterpart is one of the hallmarks of chronic

inflammation(Badylak et al., 2008). The macrophage and multinucleate giant-cell

population in the proximal defect site of the scaffold group was almost half that of

the empty defect group providing a lower probability of initiating a chronic

inflammatory reaction. At the distal end of the defect site a lower number of

macrophage cells are observed in both groups (ED m = 220 cells /field and SG m=0

cells/field). Firstly and foremost, this demonstrates the biocompatible nature of the

PCL based scaffold, which has not stimulated a proinflammatory response. Secondly,

with the macrophage cells predominantly at the proximal defect site in both groups it

is likely that the fractured titanium plate has produced a foreign body reaction,

influencing the recruitment of cells of the macrophage lineage in the acute immune

response.

The scaffold group did not demonstrate macrophages along the scaffold struts

or in the distal defect with TRAP, CD68 and OP staining demonstrated only at the

proximal site of the scaffold group. The minimal inflammatory reaction

demonstrated within the scaffold group, with an absence of osteoclasts and

macrophages, highlights the bioinert nature of the PCL based scaffold but may also

be a result of the provision of mechanical stability provided by the scaffold across

the defect site, potentially limiting the inflammatory response. Mechanical stability

at a wound site is critical in wound healing as it may alter mesenchymal stem cell

differentiation by influencing the degree of angiogenesis at the wound site(Le,

Miclau, Hu & Helms, 2001; Marsh & Li, 1999). Von Kossa staining revealed

evidence of blood vessel formation along the PCL struts implying a provision for

angiogenesis. This improves bone healing potential as it has been shown that

compromised angiogenesis at unstable fracture sites may induce osteoblasts to

produce fibrous tissue instead of bone(Remedios, 1999). Thus, by providing a degree

of mechanical stability in the microenvironment and a framework for cellular

regeneration the scaffold may organize the tissue response during healing, restraining

the immune response, encouraging angiogenesis and in turn assisting bone

regeneration.


Osteoid and fibrous tissue aligned along the struts of the scaffold with the

marker for activated osteoblasts, osteocalcin (OC), also exhibiting a predilection for

the scaffold struts. The appearance is of the scaffold providing a pathway for

organised fibrous tissue and subsequent cellular regenerative response. Studies

employing a longer implantation period have previously demonstrated that a scaffold

design can guide fibrous tissue formation in a controlled manner(Cipitria et al.,

2012). The scaffold group demonstrated not only organised fibrous tissue deposition

but also an overall increase in the markers of early and late osteoblastic

differentiation, namely osteonectin (ON), osteocalcin (OC) and Collagen-I. The

expression rate of osteocalcin and osteonectin has been shown to directly correspond

to crystal formation and mineralisation implying the scaffold group possesses greater

bone mineralisation potential than the empty defect group (Collin et al., 1992;

Hauschka, 1986; Meyer et al., 1999). These markers for early and late osteoblastic

differentiation were predominantly along the PCL scaffold struts, indicating the

important role the scaffold performs in organization the cellular response. The

provision of a framework guiding tissue healing was demonstrated recently by

Cipitria et al, who surmised the scaffold acts as supporting substrate for organised

fibrous tissue deposition prior to mineralisation(Cipitria et al., 2012). Whilst both

groups demonstrated mineralisation within the defect site, the average mineralisation

of the scaffold group (3.4%) was greater than the empty defect group (0.5%) even

after only three weeks implantation. Thus, findings are congruous with previous

studies indicating the PCL scaffold provided the framework for an organised cellular

response from an early stage with a combination of decreased inflammatory markers,

organised osteoid and greater osteoblastic differentiation markers. It illustrates that

the scaffold system may influence bone regeneration from the acute phase by

limiting the initial inflammatory phase of healing whilst also demonstrating a

significant increase in bone mineralization potential compared to the empty defect

group.

Resolution of the plate fixation system for a large segmental mandible sheep

model is imperative for future studies evaluating tissue engineered constructs used in

regenerative repair. Whether the solution is a thicker fixation plate or an innovative

plate fixation design, it will require further scrutiny including biomechanical testing


prior to implementation and is currently being undertaken in our group. Whilst the

plate fracturing was an unexpected adverse event it opened a unique opportunity to

investigate the early acute phase tissue response to an in vivo polycaprolactone

scaffold employed for bone healing in a sheep model, which has, to the best of our

knowledge, never been investigated. Our early time point results indicate that the

PCL based scaffold’s ability to provide a framework for an organised fibrous tissue

network may occur from the initiation of the inflammatory response phase of wound

healing, even in an unstable fracture environment. The macrophage response in both

groups was principally at the site of plate fracturing and likely influenced by the

“foreign body” nature of the plate fracture with a distinctly reduced response in the

scaffold group. Results suggest that after only three weeks following osteotomy, the

scaffold struts have provided a platform for cells promoting a regenerative pathway,

such as osteoblasts and fibroblasts. The fibrous tissue network is vital in guiding

future mineralization, and has been shown to influence the microstructure of newly

formed bone(Cipitria et al., 2012). The PCL scaffold displayed greater bone

mineralisation as compared to the empty defect group after only three weeks in vivo.

The results provide further evidence that a PCL construct invokes a minimal host

inflammatory response and by providing mechanical stability and a framework for

cellular regeneration may promote organized tissue healing with a restrained immune

response, encouraging angiogenesis and increasing bone mineralisation potential that

should ultimately improve the likelihood of structured bone regeneration.

74 Chapter 5: Conclusions and Future Work

Chapter 5: Conclusions and Future Work

The human mandible is a unique bone occupying a position of prominence and

vulnerability(Gray, 2000). Segmental bone defects of the mandible are destructive

and cause significant impediment to normal function and aesthetics as well as being

technically difficult to reconstruct(Hollinger & Kleinschmidt, 1990). The current

clinical gold standard for reconstruction of segmental bone defects is the application

of autologous bone grafting, as they possess excellent osteoconductive and

osteoinductive properties(Ekholm et al., 2006). However, the limitations of

autologous bone grafts range from inadequate quantity and quality of bone, to the

morbidity of a second operative site(Boyne, 1997; Schmidmaier et al., 2009). In

addition, the failure rate of autologous bone graft is up to 30%(Gautschi et al., 2007).

These complications are avoidable with the development of an “off the shelf”, ready

to use construct possessing superior osteoinductive and osteoconductive properties,

alleviating the requirement of a second operative site. By applying tissue engineered

constructs (TECs) of synthetic or natural biomaterials that promote the migration,

proliferation, and differentiation of bone regenerative cells, surgeons can overcome

the problems posed by autologous bone grafting (Petite et al., 2000). Returning the

patient to normal oral function whilst limiting patient morbidity and avoiding the

need for re-intervention will provide the patient with a better quality of life(Bak et

al., 2010; Schrag et al., 2006). This provides the rationale for translational research

into viable therapeutic alternatives that are easy to handle, restore facial form,

speech, function and occlusion, providing effective management at the initial

operative intervention (Bak et al., 2010; Gautschi et al., 2007; Schrag et al., 2006). A

prospective alternative to the current surgical management of a bicortical mandibular

segmental bone defect involves employing a bioinert and biodegradable scaffold

made from polycaprolactone (PCL). PCL can be implanted in the bone defect site,

providing mechanical support whilst facilitating tissue regeneration. Over time, as

tissue healing occurs, the construct is biodegraded by the body resulting in bridging

of the defect with only regenerated host tissue remaining(Woodruff & Hutmacher,

2010). The application of TEC’s for regeneration of a critical-sized bone defect

Chapter 6: Conclusions and Future Work 75

requires that the scientific data sets provide the clinician and patient with confidence

in a viable alternative to current clinical practice by being both reliably efficacious

and maintaining appropriate patient safety. The realisation of a safe and viable

alternative in the surgical management of segmental mandible defects is of

significant clinical implication.

A review of the literature highlighted the need for greater data on the efficacy

and safety profile for novel approaches to healing mandible continuity defects prior

to their translation to the human condition and clinical implementation. The disparate

approaches in investigating mandible defect bone regeneration has resulted in many

animal models and case-reports but often a lack of depth and reproducibility in

scientific data sets. Currently, preclinical and clinical evidence for more widespread

indications and applications of newly developed tissue engineering techniques is

unclear and has not yet undergone sufficient rigorous scientific investigation to

warrant widespread adoption(Bell & Gregoire, 2009; Carter et al., 2008b; Glied &

Kraut, 2010b). For novel approaches in mandible bone defect regeneration to be

translated from the bench top to the bedside requires vigorous in vivo interrogation

through a well-designed and validated preclinical animal model. The collation of a

reliable and reproducible body of research in preclinical animal model requires the

establishment of a standardized large animal model, an imperative step in

translational research of a tissue engineered construct (TEC). The standardized

animal model must be consistent in the animal’s age and breed, the operative

technique, post-operative care, and finally the post-explantation analysis. The model

will provide reproducible and relevant data sets required for comprehensive

investigation of novel tissue engineering approaches in reconstructing mandible

segmental defects. It will permit rigorous assessment of alternate techniques to

current clinical practice so that they can be reliably efficacious when applied in the

operating theatre whilst maintaining appropriate patient safety.

The literature demonstrates that a variety of animal models have been

employed in the investigation of mandible defect regeneration but there has yet to be

consistency in developing a standardized systematic approach. Whilst there can be

no perfect animal model, on review of the current literature and invoking prior


practical experience with sheep tibial segmental defect projects, we propose that

sheep provide the most advantageous large animal model for translational studies to

assess the safety and efficacy of a mandibular tissue engineering construct

(TEC)(Abu-Serriah et al., 2006; Abu-Serriah et al., 2005; Berner et al., 2013; Berner

et al., 2012; Cheng et al., 2005; Hutmacher et al., 2001; Reichert, Epari, et al., 2010;

Reichert et al., 2009a; Reichert, Woodruff, et al., 2010). Our line of reasoning

follows that sheep are closer to humans in the phylogenetic tree, easy to handle,

possess a readily accessible mandibular site that is a similar size and has similar bone

formation to humans and respond well to surgical procedures(den Boer et al., 1999;

Pearce et al., 2007; Reichert et al., 2009a; Salmon & Duncan, 1997; Wittenberg et

al., 1997). Through a pilot study to assess practicality, we verified that a standardized

large segmental mandible sheep model (LSMSM) is effectual and offers the suitable

framework from which additional experimentation and evaluation of novel TEC’s

may be undertaken, compared and collated. It will provide the necessary data sets

required in the assessment of current and future novel approaches to mandible

segmental defect reconstruction that may be transferable to the human condition and,

ultimately, the operative table.

The pilot study we conducted to establish a large segmental mandible sheep

model (LSMSM) provided the framework for generating relevant, reproducible data

sets in assessing bone healing. It is important that relevant data sets be generated,

correlated and applied to future research to provide a growing body of knowledge of

novel TECs that will permit their translation and implementation to clinical practice.

The LSMSM framework involves;

i) Establishing the critical-sized defect;

ii) Standard operative technique with appropriate construct application

and fixation prostheses;

iii) Post-operative management involving diet, antibiotics and analgesia;

iv) Regular clinical and radiographic review;

v) Scaffold implantation timeframe and explantation; and

vi) Post-explantation analysis


a. Biomechanical testing,

b. Immunohistochemistry, and

c. µ (micro)-CT analysis.

The tenets of this process have been well established at our institution with the

sheep tibial defect model to provide relevant data sets in bone healing and the pilot

study we performed established that the process is equally relevant and applicable to

the sheep mandible defect model(Berner et al., 2013; Berner et al., 2012; Hutmacher,

2001; Reichert, Epari, et al., 2010; Reichert et al., 2009a; Reichert, Woodruff, et al.,

2010; Reichert et al., 2011).

The primary stumbling block of our current research was the unexpected

fracturing of the titanium fixation plates resulting in is the instability across the

defect site. The surgical management of mandibular body fractures in the clinic is

principally open reduction and internal rigid fixation (ORIF). This allows rapid

osseous repair, with return of occlusion and masticatory function and maintenance of

periodontal tissue(Korkmaz, 2007; Olate et al., 2013). ORIF approach to human

mandible body fractures is primarily achieved with 2.0mm and 2.4mm titanium

mandible reconstruction locking or standard plates(Collins et al., 2004; Korkmaz,

2007; Olate et al., 2013; Scolozzi & Richter, 2003; Shaik et al., 2012) The 2.4mm

titanium reconstruction plates used to treat severe mandibular fractures demonstrate

successful stabilisation with a low rate of major complications (3%)(Scolozzi &

Richter, 2003). To mimic the clinical environment and in light of the Sheep ruminant

masticatory pattern it was decided that the robust 2.4mm titanium mandible

reconstruction plates (Synthes UniLock) would be used for fixation in our pilot

LSMSM. It must be noted that in the human clinical condition ORIF is often

employed in conjunction with maxillomandibular fixation, which is not feasible in an

animal model(Collins et al., 2004; Korkmaz, 2007; Olate et al., 2013; Shaik et al.,

2012). In addition, the sheep mandible provides a unique challenge in the operative

and post-operative management of a segmental defect given the crucial role it plays

in mastication and the ruminant digestive system. This resulted in fracturing of 9 out

of 12 of the 2.4mm titanium mandible reconstruction plates (Synthes UniLock)


employed in the pilot study. Failure of the titanium locking plates proved to be a

significant impediment to assessing the efficacy of our polycaprolactone (PCL)

scaffold in bone regeneration of a segmental mandibular defect. Plate fracturing is

encountered in the human clinical setting, though rarely published(Collins et al.,

2004; Doty et al., 2004; Esen, Ataoglu & Gemi, 2008; Katakura et al., 2004). The

cause of plate fracture is thought to be the result of stress concentration. The location

of a plate fracture will vary and depend on the type of fixation plate and the position

of fixation, requiring frequent repeated application of stress or fatigue at a particular

site(Katakura et al., 2004). Evaluation of clinical and experimental plate fracture

among cases in which primary reconstruction after mandibular resection employed

titanium plates revealed that plate fracturing was commonly in L-type defect cases in

which angle-type plates were used, and the fracture mainly occurred in the anterior

region of the mandibular angle(Katakura et al., 2004). Instability at a defect site is

not conducive to bone formation and bridging of the defect(Giannoudis et al., 2007).

Thus in the clinical application of a bone graft in a mandibular segemental defect,

instability at the plate fracture site often resulted in bone graft failure(Futran et al.,

1995; Hidalgo, 1989; Mooren et al., 2010; Yerit et al., 2002). To correctly assess

bone healing following the application of a novel TEC will require adequate

stabilisation of the mandible segmental defect site.

The obvious difficulty associated with the post-operative care of the LSMSM

is the inability to limit post-operative mobility of the mandible. As ruminants, it is

important that sheep return to a normal diet as soon as possible. It was noted during

the pilot study that sheep grind their teeth immediately following extubation and

during recovery following anaesthesia. In addition, the mechanics of sheep

mastication are such that they possess large masseter muscles and generate large

amounts of torque during mastication. As such, the 2.4mm titanium mandible

reconstruction plates (Synthes UniLock) used in human mandible surgery have been

shown to be inadequate fixation devices for the sheep mandible and this

complication must be overcome prior to proceeding with future research and

implantation of a TEC. It will require a fixation device that affords strength and

stability immediately upon application to manage early force from the immediate

recovery phase of anaesthesia. Further investigation and design into providing a


viable fixation plate substitute for the sheep model is currently being performed. We

hypothesized that thicker mandible reconstruction plates with longer screw lengths at

the symphysis that engage the contralateral mandible or a purpose-designed plate

may provide the fixation able to withstand the masticatory forces achieved in the

sheep mandible and alleviate this predicament, allowing the sheep to initiate

immediate mastication post-operatively.

Figure 23: Customised stainless steel plates that will be biomechanically tested

Whilst mimicking the clinical setting completely is ideal in translational

research, no animal model is perfect. In this study, it has been established that the

application of 2.4mm titanium reconstruction plates are inadequate in the sheep

mandible model despite the ubiquitous use in both the literature and clinic for

fixation of large mandibular segmental defects(Carter et al., 2008a; Clokie & S·ndor,

2008; Glied & Kraut, 2010a; Herford & Boyne, 2008a). Biomechanical testing is

currently being performed on the unoperated hemimandibles (i.e. the left

hemimandible) to establish the force required for stable fixation of a segmental

mandible defect. Testing of alternate fixation plates, ranging from 2.4mm titanium

mandible reconstruction plate, 2.8mm titanium mandible reconstruction plate and

custom made stainless steel fixation plate will establish the most appropriate fixation

system that can be implemented into the LSMSM. Early results suggest that the

titanium mandible reconstruction plates, whilst satisfactory for clinical application,

will not provide adequate stabilisation of a segmental mandible defect in the sheep

model (data not shown). Successful stabilisation across the defect site is likely to


require the application of a customised, stainless steel fixation plate as shown in

Figure 20. Stability and reliability of the fixation plate system is crucial and the

operative technique required to apply the custom plate will be analogous to that

experienced in the clinical setting. It does not require an additional skills subset and

is easy to handle intra-operatively, similar in application to the titanium fixation plate

system. Importantly, once plate stability is achieved, the critical-sized defect can be

established for our model and data sets for long-term implantation of novel TEC’s

can be attained, rigorously assessed and compared.

The premature fracturing of the fixation plates was unexpected but provided a

unique opportunity to examine the early stages of the host response to a

polycaprolactone (PCL) scaffold construct. Histology and immunohistochemistry

results have reinforced the biocompatible nature of the PCL scaffold providing

further evidence that a PCL-based construct invokes a minimal host inflammatory

response. The PCL scaffold displayed a more organised and reduced immune

response with significantly greater bone mineralisation as compared to the empty

defect group after only three weeks in vivo. This result is congruent with recent

studies suggesting the scaffold provides a framework for the initial fibrous tissue

deposition prior to mineralisation(Cipitria et al., 2012). The scaffold has provided the

framework for organised fibrous tissue and cellular response from an early stage with

a combination of decreased inflammatory markers, structured fibrous deposition and

increased osteoid production with early bone mineralisation. By providing

mechanical stability and a framework for cellular regeneration, scaffolds have been

shown to promote organized tissue healing, restraining the immune response and

encouraging structured bone regeneration(Cipitria et al., 2012). Our unique early

time point study indicated that the importance of the scaffold system in providing a

platform for cells promoting a regenerative pathway, such as osteoblasts and

fibroblasts, occurs in the initial phases of wound healing. This structured response

may lead to organised fibrous tissue deposition, a decreased immune response and

enhanced bone mineralization potential that should result in enhanced wound

healing. Future studies in the LSMSM will employ a longer implantation time and

the subsequent histological analysis will provide data assessing later stages of wound

healing including the adequacy of bone remodeling.


The post-operative management employed in the pilot study was practicable

and suitable. Sheep, being ruminants, must be returned to a full diet as soon as

possible. As stated earlier, it was noted in the pilot study that sheep grind their teeth

immediately following extubation, during recovery following anaesthesia. The

provision of a robust fixation plate system must allow the sheep to commence

mastication immediately post-operatively. Regular analgesia and antibiotics were

appropriate and it is noted that the sheep did not loose any weight between the

operative dates till the time of sacrifice. However, it is difficult to ascertain exactly

when fixation plate fracturing occurred as no radiographic evidence was available till

three weeks post-operative. In addition, no polymorphonuclear leukocytes were

observed in histology, nor clinical signs of infection evident in any sheep, suggesting

appropriate sterilisation and operative technique and adequate antibiotic therapy was

employed.

Post-explantation analysis has been appropriately designed to provide data sets

that are both reproducible and relevant. Whilst the pilot study was concluded

prematurely with only a short time frame of scaffold implantation due to fixation

plate fracture, the framework for relevant, reproducible data sets was successfully

established. This includes the regular radiographic assessment of live sheep and

micro-CT analysis of explanted defects, both of which were performed during this

study. Unfortunately, due to the short time frame of implantation, almost no

mineralisation was observed in the defect site with absence of bone bridging, thus the

micro-CT did not produce any data. The slide preparation and subsequent

histological analysis was relevant and significant with further analysis of bone

mineralisation and remodelling to be performed in long term implantation studies.

The embedding technique for biomechanical testing and loading analysis of

explanted hemi-mandibles has been established, currently producing relevant data to

assess and compare alternate plate fixation systems for use in future studies. Future

studies will employ all of the above tenets of the post explantation analysis to

provide the relevant preclinical data required to evaluate the safety and efficacy of

novel TECs prior to clinical implementation.


The LSMSM provides an excellent model for both operative intervention and

post-operative management and investigation of TECs once the limitation of the

premature fixation plate fracturing is overcome. Whilst the premature fracturing of

fixation plate was an unforeseen setback, the pilot study reinforced the advantages of

standardized sheep model for translational research on mandible segmental defect

regeneration. It offers the most suitable framework from which additional

experimentation and evaluation of novel TEC’s may be undertaken, compared and

collated to provide the necessary data sets required in the assessment of current and

future novel approaches to mandible segmental defect reconstruction that may be

transferable to the human condition and, ultimately, the operative table. The ability to

compose PCL-based rapid prototype TECs with fused deposition modelling may

result in customized scaffolds made to defect specifications. This ability provides

many options for therapeutic application from larger defects, including hemi- or full

mandible reconstructions to embedding with osteoinductive and osteoconductive

factors and stem cells to assist regeneration. Establishing an effective model will

generate the framework for further research comparing novel approaches to large

segmental mandible defects and solving complicated therapeutic problems ranging

from even larger segmental mandible defects to bone regeneration in a difficult

clinical setting such as a tumour model with post-irradiated, poorly vascularised

tissue. These extraordinary but achievable goals will provide the inspiration to

further the research.


[Extra page inserted to ensure correct even-page footer for this section. Delete

this when chapter is at least 2 pages long.]

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Appendices

Appendix A

Title

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