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
Chapter 1: Introduction 3
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.
4 Chapter 1: Introduction
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
Chapter 1: Introduction 5
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
6 Chapter 1: Introduction
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 1: Introduction 7
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).
Chapter 2: Literature Review 11
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.
12 Chapter 2: Literature Review
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
Chapter 2: Literature Review 13
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
14 Chapter 2: Literature Review
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).
Chapter 2: Literature Review 15
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
16 Chapter 2: Literature Review
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
Chapter 2: Literature Review 17
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).
18 Chapter 2: Literature Review
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
Chapter 2: Literature Review 19
(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
20 Chapter 2: Literature Review
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.
Chapter 2: Literature Review 21
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
22 Chapter 2: Literature Review
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
Chapter 2: Literature Review 23
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
24 Chapter 2: Literature Review
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.
Chapter 2: Literature Review 25
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
26 Chapter 2: Literature Review
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
Chapter 2: Literature Review 27
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.
Chapter 3: PILOT STUDY 31
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.
32 Chapter 3: PILOT STUDY
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).
Chapter 3: PILOT STUDY 33
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.
34 Chapter 3: PILOT STUDY
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 1046 ~8 43kg 01.11.2011 23mm
2.4mm AO Plate Empty 1123 ~8 38kg 01.11.2011 22mm
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).
Chapter 3: PILOT STUDY 35
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).
36 Chapter 3: PILOT STUDY
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.
Chapter 3: PILOT STUDY 37
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.
38 Chapter 3: PILOT STUDY
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.
Chapter 3: PILOT STUDY 39
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).
40 Chapter 3: PILOT STUDY
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.
Chapter 3: PILOT STUDY 41
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.
42 Chapter 3: PILOT STUDY
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
Chapter 3: PILOT STUDY 43
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
44 Chapter 3: PILOT STUDY
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
Chapter 3: PILOT STUDY 45
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).
46 Chapter 3: PILOT STUDY
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.
Chapter 6: Histology: 49
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
50 Chapter 4: Histology:
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
Chapter 6: Histology: 51
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
52 Chapter 4: Histology:
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).
Chapter 6: Histology: 53
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.
54 Chapter 4: Histology:
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.
Chapter 6: Histology: 55
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).
56 Chapter 4: Histology:
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).
Chapter 6: Histology: 57
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).
58 Chapter 4: Histology:
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.
Chapter 6: Histology: 59
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.
60 Chapter 4: Histology:
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.
Chapter 6: Histology: 61
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.
62 Chapter 4: Histology:
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.
Chapter 6: Histology: 63
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.
64 Chapter 4: Histology:
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.
Chapter 6: Histology: 65
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.
66 Chapter 4: Histology:
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.
Chapter 6: Histology: 67
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.
68 Chapter 4: Histology:
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%) .
Chapter 6: Histology: 69
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
70 Chapter 4: Histology:
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
Chapter 6: Histology: 71
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.
72 Chapter 4: Histology:
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
Chapter 6: Histology: 73
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
76 Chapter 5: Conclusions and Future Work
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
Chapter 6: Conclusions and Future Work 77
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)
78 Chapter 5: Conclusions and Future Work
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
Chapter 6: Conclusions and Future Work 79
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
80 Chapter 5: Conclusions and Future Work
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.
Chapter 6: Conclusions and Future Work 81
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.
82 Chapter 5: Conclusions and Future Work
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.
Chapter 6: Conclusions and Future Work 83
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this when chapter is at least 2 pages long.]
Bibliography 85
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Appendices 101
Appendices
Appendix A
Title
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referred to in the body of the thesis. That is, the first appendix referred to should be
Appendix A, the second appendix referred to should be Appendix B, and so on.
Appendix formatting can be different to the main document. Refer to Thesis PAM for
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