Characterisation of bone defect models in immunodeficient ...

Characterisation of bone defect models in immunodeficientanimals

Author:Gan, Jade Ho Yue

Publication Date:2005

DOI:https://doi.org/10.26190/unsworks/22500

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The University of New South Wales

Sydney, Australia

Characterisation of Bone Defect Models

in Immunodeficient Animals

Jade Ho Yue Gan

A dissertation submitted in fulfilment of the

requirements for the degree of

Doctor of Philosophy

Graduate School of Biomedical Engineering

Faculty of Engineering

April 2005

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Declaration of Originality

“I hereby declare that this submission is my own work and that to the best of my

knowledge and belief, it contains no material previously published or written by

another author, nor material which to substantial extent has been accepted for the

award of any other degree or diploma at UNSW or any other educational institution,

except where due acknowledgement is made in this thesis. Any contribution made to

the research by others, with whom I have worked at UNSW or elsewhere, is explicitly

acknowledged in this thesis.

I also declare that the intellectual content of this thesis is the product of my work,

expect to the extent that assistance from others in the project’s design and conception

or in style, presentation and linguistic expression is acknowledged.”

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Acknowledgement

My gratitude goes to A/Prof Bill Walsh for welcoming me into The Lab. His

unequivocal generosity and teachings is second to none; Dr. Yan Yu for her friendship

and technical advice and expertise; Dr. Mark Gillies for his support and unwavering

belief in me. A special mention goes to Gina O’Reilly for keeping the lab organised

and sane.

To my colleagues and laboratory staff members who have become an integral part of

my life: Rema Rajaratnam, Abe Lau, JB Chen, Adam Butler, Danè Dabirrahmani,

John Rawlinson, Alex Turner, Peter Smitham, Graham Matheson and all. Thank you

for providing joy and laughter to the lab. A special thank you to Susanne and Naomi

for their technical assistance and Ms. Sue Middleton for her advisory role in statistics.

A special note of appreciation to my friends, near and far, old and new, who have

provided pillars of support and guidance throughout my trials and tribulations.

This study would not be possible without the support from The Orthopaedic Research

Laboratories.

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Dedication

I would like to dedicate this dissertation to my parents and family who have provided

continuous love and support throughout the years. Thank you and may the blessings of

the triple jewels be with you.

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Abstract

Bone defects resulting from non-unions, fractures, significant revision joint

replacements, tumour resection and osteolysis present a clinical problem. While

autografts are considered the gold standard, ubiquitous use of this reparative technique

is limited by graft supply and site morbidity. Recent progresses in tissue engineering

using stem cells, bone enhancing molecules and gene therapy have provided more

hypotheses for bone defect treatment. In vivo assessment to test these hypotheses

requires animal models to mimic human conditions. Immunodeficient or nude animals

have the advantage of hosting materials from human and other xenographic origins

without immuno-intolerance or rejection. A thorough understanding of the biology in

nude animals is vital for the further advancement of connective tissue healing and

regeneration strategies. Nude mice are excellent xenographic hosts for in- vivo

characterisation and provide a reproducible animal source. The immune deficiencies

of nude compared to normal animals may however, influence bone healing and need to

be addressed.

This dissertation (a) investigated potential bone defect models in nude mice and nude

rats (b) incorporated the selected bone defect model to evaluate the effect of T cell

deficiency and age on bone defect healing in nude animals (c) determined the

feasibility of a critical size defect (CSD) in nude mice.

A distal-femur-condylar-defect (DFCD) model was successfully performed in nude

mice and rats. The model was found to have some advantages as a bone defect model:

(1) located at a weight-bearing skeletal site (2) no requirements for an internal or

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external fixator (3) does not obstruct or limit mobility (4) location is not in close

proximity to any major organs such as the brain (5) easy identification of surface

anatomy (6) defect size is standardised and reproducible (7) does not require lengthy

and complicated surgery and (8) cost effective.

This dissertation confirmed that bone healing in nude mice is similar to that of normal

immunocompetent mice. Absence of T lymphocytes did not delay or inhibit bone

repair. Use of older nude mice did not seem to affect the healing rate, in contrast to

older normal mice, which showed delay in bone healing in the initial phase.

Establishment of critical sized defects in mice at a weight-bearing location was not

feasible due to the robust healing of murine. This dissertation recommends that the

DFCD model could be utilized for the assessment of xenogenic materials at early time

point.

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Content

Declaration of Originality .............................................................................................. i Acknowledgement......................................................................................................... ii Dedication .................................................................................................................... iii Abstract ........................................................................................................................ iv Content ......................................................................................................................... vi List of Symbols and Abbreviations ............................................................................viii List of Figures ............................................................................................................... x List of Tables ..............................................................................................................xiii CHAPTER 1 Introduction ....................................................................................... 1

1.1. Bone trauma and treatment........................................................................ 1 1.2. Animal models .......................................................................................... 2

1.2.1. Bone defect models ....................................................................... 4 1.3. Scope of dissertation ................................................................................. 9

1.3.1. Objectives and Aims...................................................................... 9 CHAPTER 2 Background and Literature Review................................................. 11

2.1. Biology and histology of bone ................................................................ 11 2.1.1. Classification of bone.................................................................. 11 2.1.2. Macrostructure of bone ............................................................... 14 2.1.3. Microstructure of bone ................................................................ 17 2.1.4. Cellular and molecular level........................................................ 18 2.1.5. Related structure.......................................................................... 22 2.1.6. Mechanical properties of bone .................................................... 24

2.2. Bone damage and trauma ........................................................................ 26 2.2.1. Fracture........................................................................................ 26 2.2.2. Bone defect.................................................................................. 28

2.3. Bone healing............................................................................................ 30 2.3.1. Fracture healing........................................................................... 31 2.3.2. Bone defect healing ..................................................................... 35 2.3.3. Factors influencing bone healing ................................................ 36 2.3.4. Treatment of fracture and bone defects ....................................... 39

2.4. Animal models in orthopaedic research .................................................. 49 2.4.1. Nude and immunodeficient animals............................................ 50

CHAPTER 3 Methodology ................................................................................... 55 3.1. Criteria for study design and experimental model .................................. 55

3.1.1. Development and characterisation of bone defect model ........... 56 3.1.2. Selection of defect model ............................................................ 58 3.1.3. Influence of immune status ......................................................... 59 3.1.4. Influence of age ........................................................................... 60 3.1.5. Critical size defect (CSD)............................................................ 61

3.2. Surgery and anaesthesia procedure ......................................................... 62 3.2.1. Mouse models.............................................................................. 62 3.2.2. Rat models................................................................................... 66 3.2.3. Post-operative monitoring ........................................................... 69

3.3. End point analysis ................................................................................... 70 3.3.1. Radiography ................................................................................ 71

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3.3.2. Histology ..................................................................................... 72 3.3.3. Immunohistochemistry................................................................ 73 3.3.4. Microcomputed tomography (µCT) ............................................ 80 3.3.5. Quantitative analysis of histology ............................................... 81 3.3.6. Semi-quantitative analysis of immunohistochemistry ................ 83 3.3.7. Statistics....................................................................................... 83

CHAPTER 4 Results ............................................................................................. 84 4.1. Mouse models.......................................................................................... 84

4.1.1. Characteristics of defect models.................................................. 85 4.1.2. Immune status.............................................................................. 97 4.1.3. Age ............................................................................................ 104 4.1.4. Critical size defect (CSD)...........................................................111

4.2. Nude rat models..................................................................................... 113 4.2.1. Characteristics of defect models................................................ 114

CHAPTER 5 Discussion ..................................................................................... 121 5.1. Mouse defect models............................................................................. 121

5.1.1. Selection of defect model .......................................................... 122 5.1.2. Influence of immune system ..................................................... 124 5.1.3. Influence of age ......................................................................... 127 5.1.4. Critical size defect (CSD).......................................................... 130

5.2. Nude rat models..................................................................................... 132 5.3. Limitations............................................................................................. 132

CHAPTER 6 Conclusion..................................................................................... 135 6.1. Conclusion............................................................................................. 135 6.2. Future Direction .................................................................................... 136

Reference................................................................................................................... 139 Appendix ................................................................................................................... 164

Tissue Processing Protocol.................................................................................. 164 Haemotoxylin and Eosin (H&E) Protocol .......................................................... 165 Masson’s Trichrome Protocol ............................................................................. 166 Histology : Haemotoxylin and Eosin (H&E) ...................................................... 167 Histology : Masson’s Trichrome ......................................................................... 171 Immunohistochemistry........................................................................................ 176

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List of Symbols and Abbreviations

ANOVA Analysis of Variance

AR antigen retrieval

BMU bone multicellular unit

BMP bone morphogenetic protein

BRU bone remodelling unit

CBFa-1 core binding factor alpha-1

DAB diaminobenzamide

DBM demineralised bone matrix

DFCD distal-femur-condylar-defect

ECM extracellular matrix

FGF fibroblast growth factor

H&E Haematoxylin and eosin

HA hydroxyapatite

IgG Immunoglobulin G

IGF insulin-like growth factor

µCT microcomputed tomography

MSC mesenchymal stem cell

NCP non-collagenous protein

NK natural killer

PBS phosphate buffer saline

PDGF platelet-derived growth factor

PTH parathyroid hormone

SMAD Sma + Mad (mother against drosophila)

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Spf specific-pathogen-free

TGF-β transforming growth factor beta

TW tibial window

VEGF vascular endothelial growth factor

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List of Figures

FIGURE 2-1 RADIOGRAPHS OF LONG BONES IN DIFFERENT SPECIES (A) MOUSE (B) ESTROGEN DEFICIENT RAT (COURTESY OF REMA RAJARATNAM) (C) HUMAN .................................................................. 13

FIGURE 2-2 (A) HAVERSIAN SYSTEM IN HUMAN BONE. ADAPTED FROM [76]. (B) HISTOLOGIC SECTION OF OSTEONS OF CORTICAL BONE IN A MOUSE FEMUR (HAEMATOXYLIN AND EOSIN (H&E), 400X MAGNIFICATION).......................................................................................................................... 15

FIGURE 2-3 STRUCTURAL LAYERS OF A LONG BONE. ADAPTED FROM [77] ............................................ 16 FIGURE 2-4 HISTOLOGIC STAINING OF OSTEOBLASTS (SOLID ARROWS) AND OSTEOCYTES (OPEN ARROW)

IN MOUSE BONE (H&E, 400X MAGNIFICATION)............................................................................ 20 FIGURE 2-5 PRESENCE OF OSTEOCLASTS IN NUDE RAT BONE INDICATED BY ARROWS (H&E, 200X

MAGNIFICATION).......................................................................................................................... 21 FIGURE 2-6 STRUCTURE OF A BONE REMODELLING UNIT (BRU) IN CORTICAL BONE. ADAPTED FROM [82]

.................................................................................................................................................... 34 FIGURE 2-7 BONE FORMATION AND RESORPTION BY BRU UNITS IN TRABECULAR BONE. (A) RESORPTION

PROCESS IS COMPLETED BEFORE FORMATION BEGINS. (B) RESORPTION IS FOLLOWED CLOSELY BY FORMATION. ADAPTED FROM [82]................................................................................................ 35

FIGURE 3-1 A SCHEMATIC DIAGRAM OUTLINE OF EXPERIMENTAL DESIGN ............................................. 58 FIGURE 3-2 SURGERY OF DISTAL FEMUR CONDYLAR DEFECT MODEL IN NUDE MICE .............................. 66 FIGURE 3-3 SURGERY OF DFCD MODEL IN NUDE RATS. MAKING A SKIN INCISION USING A SCALPEL (TOP

LEFT) DRILLING A DEFECT USING A DENTAL BUR (TOP RIGHT) CHECKING AND MEASURING THE DEFECT SIZE BEFORE CLOSING THE WOUND (BOTTOM LEFT) A NEAT SUTURED SKIN CLOSURE (BOTTOM RIGHT).......................................................................................................................... 69

FIGURE 3-4 FLOW CHART OF POSTOPERATIVE PROCESSES ..................................................................... 70 FIGURE 3-5 FAXITRON MX-20. ADAPTED FROM [202] .......................................................................... 71 FIGURE 3-6 SCHEMATIC DIAGRAM SHOWING APPLICATION OF TWO-STEP INDIRECT METHOD WITH

STREP-BIOTIN COMPLEX. ADAPTED FROM [205]........................................................................... 75 FIGURE 3-7 POLYCLONAL AND MONOCLONAL ANTIBODIES. ADAPTED FROM [203] ............................... 75 FIGURE 3-8 (A) RECONSTRUCTION OF X-RAY SHADOWS INTO 3D IMAGES USING MICROCOMPUTED

TOMOGRAPHY (B) ACQUISITION OF MULTIPLE X-RAY SHADOW IMAGES OF AN OBJECT. ADAPTED FROM [207]. ................................................................................................................................. 81

FIGURE 3-9 SCREEN CAPTURE SHOWING THE USE OF BIOQUANT NOVA PRIME SOFTWARE FOR QUANTITATIVE BONE HISTOMORPHOMETRY. THE HIGHLIGHTED REGION IS THE AREA OCCUPIED BY NEW BONE.................................................................................................................................... 82

FIGURE 3-10 FLOW CHART OF QUANTITATIVE ANALYSIS PROCESS ......................................................... 82 FIGURE 4-1 TIBIAL WINDOW DEFECT AT (A) 3 WEEKS AND (B) 6 WEEKS POSTOPERATIVE (HAEMATOXYLIN

AND EOSIN (H&E), 40X MAGNIFICATION). AT 3 WEEKS, NEW WOVEN BONE HAD BRIDGED THE OPPOSITE ENDS OF THE TIBIAL WINDOW. AT 6 WEEKS, REGENERATION OF BONE MARROW AND CORTICAL BONE WAS EVIDENT...................................................................................................... 86

FIGURE 4-2 DIMENSIONS OF NUDE MICE LIMBS IN CENTIMETRES (CM) ................................................ 86 FIGURE 4-3 FAXITRON RADIOGRAPHS SHOWING PROGRESSIVE BONE HEALING OF A DFCD MODEL IN

NUDE MICE (A) DAY 3 (B) DAY 7 (C) DAY 10 (D) DAY 14 (E) DAY 21. FORMATION OF NEW BONE WAS DETECTED FROM AN INCREASE IN OPACITY DUE TO BONE MINERALIZATION FROM DAY 10 TO DAY 21. ........................................................................................................................................ 87

FIGURE 4-4 SEMI QUANTITATIVE ANALYSIS OF BONE HEALING FROM RADIOGRAPHS BASED ON SCORING SYSTEM IN TABLE 15. NO BONE FORMATION WAS DETECTED FROM THE RADIOGRAPHS IN NUDE MICE

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AT DAY 7. ERROR BARS REPRESENT STANDARD MEAN ERROR (SEM) OF EACH GROUP. ............... 88 FIGURE 4-5 A RECONSTRUCTION OF THE SLICES TO FORM A 2D MODEL ................................................. 89 FIGURE 4-6 A 3D RENDITION OF A MOUSE KNEE JOINT USING µCT........................................................ 89 FIGURE 4-7 CALCULATION OF BONE PROPERTIES USING CTAN AND CTVOL SOFTWARE ©.................... 90 FIGURE 4-8 HISTOLOGY OF BONE HEALING OF A DISTAL FEMORAL CONDYLE DEFECT IN NUDE MICE (H&E

STAINING, 100X MAGNIFICATION UNLESS INDICATED OTHERWISE) ............................................... 94 FIGURE 4-9 NOMENCLATURE FOR BONE GROWTH DIRECTION (H&E STAINING, 40X MAGNIFICATION)... 95 FIGURE 4-10 GRAPH INDICATING THE DIRECTION OF NEW BONE GROWTH IN THE DFCD MODEL IN NUDE

MICE. NOTE : DATA EXCLUDED SPECIMENS WITH NO BONE FORMATION OR UNDETERMINED DIRECTION OF BONE GROWTH. ..................................................................................................... 95

FIGURE 4-11 (A) (LEFT) EXPRESSION OF VEGF IN NUDE MICE AT DAY 21 (400X MAGNIFICATION) (B) (RIGHT) EXPRESSION OF FGF-2 IN NUDE MICE AT DAY 14 (400X MAGNIFICATION). ..................... 97

FIGURE 4-12 HISTOLOGY OF BONE DEFECT HEALING IN NUDE AND NORMAL MICE AT DAY 10 (H&E STAINING, 100X MAGNIFICATION). (A) CARTILAGE WAS OBSERVED IN NUDE MICE. (B) NO CARTILAGE WAS OBSERVED IN THIS SECTION OF NORMAL MICE DEFECT. ...................................... 98

FIGURE 4-13 GRAPH SHOWING PERCENTAGE BONE FORMED IN NORMAL AND NUDE MICE. PERCENTAGE NEW BONE FORMATION WAS NOT SIGNIFICANTLY DIFFERENT BETWEEN 12-WEEK-OLD NUDE AND NORMAL MICE AT ANY TIME POINT. ERROR BARS REPRESENT STANDARD DEVIATION (SD). .......... 99

FIGURE 4-14 GRAPH SHOWING PERCENTAGE CARTILAGE FORMED IN 12-WEEK-OLD NORMAL AND NUDE MICE. ERROR BARS REPRESENT STANDARD DEVIATION (SD). LARGE SD REPRESENTS LARGE VARIATION BETWEEN INDIVIDUALS IN THE GROUP...................................................................... 100

FIGURE 4-15 GRAPH COMPARING THE DIRECTION OF NEW BONE GROWTH IN NUDE AND NORMAL MICE. MAJORITY OF NEW BONE GREW FROM THE PROXIMAL AND POSTERIOR REGION WHERE VASCULAR TISSUES ARE ABUNDANT IN ADDITION TO SOFT TISSUE CONTRIBUTION....................................... 101

FIGURE 4-16 EXPRESSION OF SMAD 4 (LEFT) AND SMAD 5 (RIGHT) IN NORMAL MICE AT DAY 10 (400X MAGNIFICATION). THE ARROWS DEPICT STAINED CELLS............................................................. 102

FIGURE 4-17 GRAPH SHOWING VEGF EXPRESSION IN NUDE AND NORMAL MICE FROM DAY 3 TO DAY 21................................................................................................................................................... 103

FIGURE 4-18 GRAPH SHOWING SMAD -5 EXPRESSION IN NUDE AND NORMAL MICE FROM DAY 3 TO DAY 21................................................................................................................................................... 103

FIGURE 4-19 H&E SECTIONS OF TYPICAL BONE HEALING AT 14 DAYS (A) 12-WEEK-OLD NUDE MOUSE (B) 12-WEEK-OLD NORMAL MOUSE (C) 20-WEEK-OLD NUDE MOUSE (D) 20-WEEK-OLD NORMAL MOUSE (10X MAGNIFICATION) ............................................................................................................... 106

FIGURE 4-20 GRAPH SHOWING PERCENTAGE BONE FORMED IN 12-WEEK-OLD (YOUNG) AND 20-WEEK-OLD (ADULT) NORMAL AND NUDE MICE AT DAY 3, 7, 10, 14 AND 21. ANOVA WAS PERFORMED BETWEEN NUDE AND NORMAL MICE FOR THE SAME TIME POINT. PERCENTAGE NEW BONE FORMATION WAS SIGNIFICANTLY DIFFERENT AT DAY 7 BETWEEN ADULT NUDE AND NORMAL MICE (** P < 0.05). THERE WAS NO SIGNIFICANT DIFFERENCE BETWEEN YOUNG AND ADULT NUDE MICE OR YOUNG AND ADULT NORMAL MICE AT ALL TIME POINTS. ERROR BARS = SD.................. 107

FIGURE 4-21 GRAPH SHOWING CARTILAGE FORMATION IN NUDE AND NORMAL MICE AT 12 (YOUNG) AND 20 (ADULTS) WEEKS. ERROR BARS REPRESENT STANDARD DEVIATION (SD). THE LARGE SD DEPICTS LARGE VARIATION BETWEEN INDIVIDUALS AT EACH TIME POINT................................... 108

FIGURE 4-22 TRABECULAE THICKNESS DISTRIBUTION IN NUDE MICE .................................................. 110 FIGURE 4-23 TRABECULAE THICKNESS DISTRIBUTION IN NORMAL MICE ............................................. 110 FIGURE 4-24 TRABECULAE SEPARATION DISTRIBUTION IN MICE.......................................................... 111 FIGURE 4-25 CALCULATION OF RELATIVE DEFECT SIZE. DEFECT DIAMETER (SOLID LINE) TOTAL CONDYLE

DIAMETER (BROKEN LINE) ......................................................................................................... 112 FIGURE 4-26 GRAPH SHOWING PERCENTAGE BONE FORMED IN 1.0 MM AND 1.2 MM DEFECT SIZES IN 20

WEEK-OLD NUDE MICE. THERE WERE NO SIGNIFICANT DIFFERENCES BETWEEN THE TWO DEFECT

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SIZES AT ANY TIME POINTS. THE ERROR BAR = SD...................................................................... 113 FIGURE 4-27 OVERVIEW OF TW IN NUDE RATS ON A FAXITRON RADIOGRAPH (LEFT) AND POST OPERATIVE

(RIGHT) ...................................................................................................................................... 114 FIGURE 4-28 (A) A TYPICAL SECTION OF TIBIAL WINDOW IN NUDE RATS AT 6 WEEKS POSTSURGERY (H&E

STAINING, 100X MAGNIFICATION) AND (B) COLLAGEN TYPE I OF THE SAME SECTION UNDER POLARIZED LIGHT ...................................................................................................................... 115

FIGURE 4-29 RADIOGRAPHIC REPRESENTATION OF DFCD HEALING IN NUDE RATS. AT 1 WEEK, THE DEFECT WAS CLEARLY OUTLINED WITH LIMITED BONE FORMATION. AT 4 WEEKS, MORE NEW BONE FORMED IN THE DEFECT AREA. AT 6 WEEKS, THE DFCD OUTLINE WAS BLUR AND THE DEFECT AREA WAS FILLED WITH MINERALISED BONE. ...................................................................................... 116

FIGURE 4-30 TYPICAL REPRESENTATION OF BONE HEALING IN DFCD MODELS IN NUDE RAT (H&E, 40X MAGNIFICATION). AT 1 WEEK, THE DEFECT WAS FILLED WITH HEMATOMA AND FIBRIN. AT 4 WEEKS NEW BONE FORMATION PROGRESSED FROM THE DEFECT BORDER TO THE CENTER. AT 6 WEEKS, DEFECT WAS ALMOST HEALED WITH ~ 90% FILLED WITH NEW BONE AND BONE MARROW ......... 117

FIGURE 4-31 DIFFERENCE IN DFCD HEALING PATTERN IN (A) NUDE RATS (H&E, 40X MAGNIFICATION) AND (B) NUDE MICE (H&E, 100X MAGNIFICATION). (A) NEW BONE FORMED IN A CONVERGING MANNER TOWARDS THE CENTER OF THE DEFECT. (B) NEW BONE APPROACHED FROM A PROPAGATING FRONT FROM ONE END OF THE DEFECT. SOLID ARROW REPRESENTS THE DIRECTION OF HEALING................................................................................................................................ 117

FIGURE 4-32 CALCULATION OF BONE PROPERTIES IN NUDE RATS USING CTAN AND CTVOL SOFTWARE ©.................................................................................................................................................. 119

FIGURE 4-33 A SLICE RENDITION USING SKYSCAN (SKYSCAN, BE) IN COLOUR (LEFT) AND BINARY FORM (RIGHT) AT THE POSITION SHOWN IN FIGURE 4-32. ..................................................................... 119

FIGURE 4-34 ΜCT SLICES SHOWING THE CROSS-SECTIONAL AREA OF THE DEFECT IN NUDE RATS AT 1 WEEK, 4 WEEKS AND 6 WEEKS.................................................................................................... 120

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List of Tables

TABLE 1 DESCRIPTION OF DEFECT MODELS IN ANIMALS .......................................................................... 6 TABLE 2 MECHANICAL PROPERTIES OF BONE. ADAPTED FROM [77] ...................................................... 24 TABLE 3 CELLULAR CASCADE DURING FRACTURE HEALING. ADAPTED FROM [130] .............................. 33 TABLE 4 PROPERTIES AND FUNCTIONS OF GROWTH FACTORS IN BONE HEALING .................................... 47 TABLE 5 CHARACTERISTICS OF NUDE, SCID AND RAG1 MICE. ADAPTED FROM [175]............................. 50 TABLE 6 PROPERTIES OF NUDE MICE T CELLS. ADAPTED FROM [65] ...................................................... 53 TABLE 7 CELL PROPERTIES OF NUDE AND NORMAL MICE. ADAPTED FROM [171] ................................... 53 TABLE 8 OVERALL SURGERY DESIGN..................................................................................................... 57 TABLE 9 ANIMAL ALLOCATION FOR SELECTION OF DEFECT MODEL ....................................................... 59 TABLE 10 ANIMAL ALLOCATION TO INVESTIGATE INFLUENCE OF IMMUNE STATUS................................. 60 TABLE 11 ANIMAL ALLOCATION TO INVESTIGATE THE INFLUENCE OF AGE............................................. 61 TABLE 12 ANIMAL ALLOCATION TO DETERMINE THE FEASIBILITY OF A CSD ......................................... 62 TABLE 13 DECALCIFICATION PERIOD FOR DIFFERENT TISSUES............................................................... 71 TABLE 14 FAXITRON CONFIGURATION ................................................................................................... 72 TABLE 15 RADIOGRAPHIC SCORING SYSTEM. ADAPTED FROM LANE AND SANDHU [27]....................... 72 TABLE 16 LIST OF PRIMARY AND SECONDARY ANTIBODIES USED IN IMMUNOHISTOCHEMISTRY............. 78 TABLE 17 IMMUNOSTAINING PROTOCOLS .............................................................................................. 79 TABLE 18 GRADING SCALE OF IMMUNOHISTOCHEMICAL STAINING (ADAPTED FROM [208]). UNSTAINED

CELLS ARE GRADED AS NEGATIVE OR “-”...................................................................................... 83 TABLE 19 PARAMETERS OF THE TRABECULAR BONE STRUCTURE AS CALCULATED BY µCT. ADAPTED

FROM [83, 207, 209] .................................................................................................................... 91 TABLE 20 SUMMARY OF TYPICAL CELL AND TISSUE MORPHOLOGY IN THE DFCD MODEL.................... 93 TABLE 21 STATISTICAL RESULTS TO INVESTIGATE INFLUENCE OF DRILLING INTO GROWTH PLATE ON BONE

HEALING ...................................................................................................................................... 96 TABLE 22 IMMUNOSTAINING DATA FOR BMPS, VEGF, SMADS, FGF-2 AND CBFA-1 DETECTED IN NUDE

MICE ............................................................................................................................................ 97 TABLE 23 IMMUNOSTAINING DATA FOR BMPS, VEGF, SMADS, FGF-2 AND CBFA-1 DETECTED IN

NORMAL MICE ............................................................................................................................ 102 TABLE 24 µCT DATA OF THE TRABECULAR STRUCTURE IN BALB/C NUDE AND NORMAL MICE ............. 104 TABLE 25 ANALYSIS OF ΜCT SCANS USING CTAN AND CTVOL ® ...................................................... 109 TABLE 26 PROPORTION OF DEFECT SIZE IN COMPARISON WITH THE FEMORAL CONDYLE ..................... 112 TABLE 27 µCT DATA OF THE TRABECULAR STRUCTURE IN NUDE RATS WHICH WERE SACRIFICED AT 1 WEEK,

4 WEEKS AND 6 WEEKS............................................................................................................... 118

Introduction

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CHAPTER 1 Introduction

1.1. Bone trauma and treatment

Bone defects resulting from non-unions, fractures, trauma, revision joint replacement,

tumour resection and osteolysis are common, and account for a large proportion of

orthopaedic surgeries. Conventional clinical management for reparative treatment of

bone defects utilizes bone grafts harvested from the patient’s own tissue (autograft) or

donor tissue (allograft), and to a certain extent, xenografts and bone substitutes. The

most common bone graft materials used are autogenous iliac crest bone graft,

cancellous allograft chips and demineralised bone matrix. Autogenous bone graft

remains the gold standard for bone defect treatment [1, 2]. However, limited supply,

donor site morbidity, anatomical and structural problems, economical and

hospitalization period restrict the ubiquitous use of this treatment [3, 4]. Allografts are

widely used in response to autograft shortage, and are usually obtained from a bone

bank [5]. Since they are harvested from other individuals, bacterial infection,

immunogenicity and transmission of systemic diseases such as acquired immune

deficiency syndrome (AIDS) and hepatitis are the potential risks associated with using

allografts [6, 7]. Stringent sterilization methods using radiation, autoclaving, ethylene

oxide and antibiotic treatment [8] may reduce immunogenicity and decrease the risk of

infection [9] but they can damage the osteoinductive and structural properties [10, 11].

Xenografts harvested from other species such as bovine, porcine and ovine posed

similar problems. There have been a variety of bone graft substitutes, both natural and

synthetic based on calcium phosphate, calcium carbonate or calcium sulphate [2] but

none possess the regenerative attributes of autografts due to lack of inductive

molecules.

Introduction

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Clinical validation of safety and efficacy is crucial before bone graft substitutes are

applicable clinically. Since bone repair is a complex process of synergistic interactions

among internal and external factors, bone graft substitutes should ideally be assessed

in a biological setting i.e. an in vivo model. In vitro studies in controlled biological

samples such as cultured cells provide little insight on how living tissues respond in a

complete environment [12]. For some materials which have passed the safety

requirements and allowed to be used clinically, it is difficult to ascertain the dynamic

efficacy and mechanisms due to limited collection of biopsy tissues [13]. Human

clinical trials are also limited by high cost, ethical considerations and subject

suitability of a large population study [13]. Concerns over the sample site as well as

the number of compounding variables often make this choice of study difficult.

Measurements of bone formation using non-invasive methods such as radiography and

imaging methods are subjective and provide limited information [13]. Therefore,

animals are routinely used to evaluate proof of concept, feasibility, safety and efficacy

of experimental materials [14]. Animal models provide a living microenvironment for

comprehensive understanding of biological processes. Reproducible and quantifiable

data that is otherwise difficult to obtain from human subjects, cadaver studies and

computer simulations may be acquired from animal models [14].

1.2. Animal models

Animals are routinely used as preclinical screening tool to assess the feasibility of

surgical techniques or viability of implants before proceeding to human clinical trials.

In many fields, animal model exists that provide some degree of similarity to human

conditions and diseases. In orthopaedics, bone repair models are relevant to the extent

Introduction

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that the sequence of bone healing mimics that in humans. Long bone fractures [15-17],

calvarial defects [18], distraction osteogenesis [19] and mandibular distraction are

some of the commonly used animal models, which are not dissimilar to human

conditions. In the words of Perren, bone repair models “permits breakdown of

complex clinical problem into different physical entities, their interrelation and

biological reaction.”[20]

Animals, like humans, are influenced by their biological makeup and the environment.

As such, animals are expected to show multitude variations in gene expression due to

age, gender, physiological and genetic factors. They are however more practical and

clinically more relevant than in vitro study. Controlled biological samples such as

cultured cells have little sample-to-sample variation, but they provide little insight on

how living tissues respond in the complex in vivo environment [12].

Large animals that are commonly used in orthopaedic research range from rabbits,

sheep, goats, pigs, dogs and non-human primates. Non-human primates are closely

related to humans, with 98% genetic homogeneity and as a result, have been

established as the most valid test of preclinical efficacy [14], although the cost and

very limited supply restrict their use [21]. While larger animals are debated to

provide better presentation to human conditions due to their higher phylogeneticity

[22], small mammals have certain advantages such as large sample size, simple

housing and maintenance as well as being inexpensive [22, 23]. Rodents and murine

are well characterised and possess hardy and adaptable features suitable for research.

Gene manipulation capabilities in mice uphold its status as an invaluable animal model

[24].

Introduction

- 4 -

1.2.1. Bone defect models

Bone repair in animals has been investigated using ectopic bone and bone defect

models. Ectopic bone formation occurs at a non-skeletal site and involves implantation

of materials into muscles [25, 26] or other non-osseous i.e. subcutaneous sites. While

ectopic bone models reflect absolute osteoinductive capacity of the material under

study, the overall reparative response of test materials is better appreciated at an

osseous site, hence the need for bone defect models [27]. Defect models allow

assessment of both osteoconductive and osteoinductive potentials of bone graft

substitutes and other materials, proteins and tissue engineering based concepts.

Bone defect models can provide relevant clinical information for treatment of fracture

non-union, defects from bone disorders and iatrogenic origins. Non-union models in

mice and rats are used extensively, ranging from ribs fractures [28-31] to femora

[32-34] and tibiae [23, 34, 35]. Common defect models include calvarial defects

[36-38], segmental defects [39, 40] and window defects [41]. A comprehensive

description of bone defect models in animals is described in Table 1.

Segmental defects are usually achieved in long bone by removing a segment of bone

while still allowing the animal to maintain some kind of mobility. A defect larger than

one and half times or double the diaphyseal diameter produce a non-union in an adult

animal [21, 27]. Rib fractures are not uncommon and are performed in small mammals

[16, 28-31]. Rib fractures undergo similar morphological changes of a typical long

bone [16, 42] and do not require adjunctive form of immobilization [16].

Introduction

- 5 -

The calvarial defect model is widely used to assess implants and growth factors [38,

43]. The use of trephination assures standardization and reproducibility when

compared to other models such as a segmental defect. Critical sized defect (CSD) is

defined as a defect that would not heal throughout the lifetime of the animal [43, 44].

Critical-sized calvarial defects are attainable in mice [37, 38], rats [45, 46] and sheep

[43]. Nevertheless, the location and environment of the skull presents a number of

limitations for the assessment of bone defect. The local blood supply at the cranium is

naturally scarce and the skull is made up mainly of trabecular bone where load (weight

bearing) is minimal [47]. Therefore, the calvarial defect model falls short when it

comes to assessing the influence of angiogenesis and mechanical loading - two major

factors of bone healing. In mice, this model had some drawbacks due to the small size

and vulnerability of the dura mater [37].

Window defects can be performed with removal of the marrow such as in the tibia [48]

or ulna. The cortical layer and endosteum are removed while the underlying bone

marrow is left uncut. The result is a recess capable of supporting materials of

osteogenic potential.

Segmental defects, like non-union fracture models, usually require the use of a

stabilizer or fixator to assure immobilization and stability, especially when they are

located at weight bearing sites. Internal and external fixators, e.g. Kirschner (K) wires

may cause additional soft-tissue and vascular injury [49] or prevent normal movement

[50], thus complicating the healing process. A fixator-free defect model is favourable

to avoid this complication.

Introduction

- 6 -

Table 1 Description of defect models in animals

Author [reference]

Animal species

Defect type Dimensions Comments

Lee J.Y. et al. [38]

SCID mice Calvarial defect

5mm (diameter) Critical size defect. Close to the dura mater

Tamura et al. [45]

Sprague Dawley rats

Calvarial defect

4mm (diameter)

Oklund et al. [1]

Mongrel dogs Calvarial defect

18mm (diameter)

Critical size defect.

Prolo et; al. [11]

Mongrel dogs Calvarial defect

18mm and 20mm (diameters)

Tielinen et al. [51]

Wistar rats Defect at distal femur

2mm (diameter) 3mm (length)

Uusitalo et al.[52]

C57BL/DBA mice

Defect at femur

0.9mm (diameter)

Metaphyseal defect.

Campbell et al. [53]

129/Sv mice Defect at tibia

0.5mm (diameter)

Diaphyseal defect. Small defect size.

Many theories and hypotheses have been put forth to explain the factors contributing

to bone healing i.e. angiogenesis [54, 55], mechanical loading [56], age, soft tissue

trauma as well as contributions from the local environment such as the cortex, bone

marrow and periosteum [57]. The influence of the immune system (innate or otherwise)

has been highlighted as another factor to influence bone healing. More recent findings

showed that the immune system and bone repair are not two distinct responses, but are

in fact interrelated [58-60]. This is an area of little understanding and requires further

development.

The development of biological therapy from human origin necessitates the use of nude

and immunodeficient animal models. Tissue rejections have been linked to host

immune mechanisms, one of which is the cell-mediated response involving T

Introduction

- 7 -

lymphocytes [39, 61, 62]. Tissue rejection provided extra variables and unwanted

interference in the assessment of biological therapies. Immunodeficient animals are

seen to “eliminate” this problem since they lack a complete innate immune system,

which is conducive for studying the role of the immune system in different scenarios.

In oncology research, human tumours can develop in immunodeficient mice while

preserving their natural genetic and phenotypic characteristics [63]. Immunodeficient

animals were first discovered in 1960s with the appearance of nude mice [64], and

later nude rats in the 1970s, and severe combined immune deficiency (SCID) mice in

1983. Nude mice and rats (named for their lack of hair) suffer from thymic dysgenesis

(athymic) due to an autosomal recessive gene [65]. The thymus has been credited with

the development and maturation of thymus-derived (T) cells, and indirectly, bone

marrow-derived (B) cells. Although the role of T cells in the immune system is well

reported, the correlation between immune system (or the lack of it) and bone healing is

still unclear.

Immunodeficient animals are often used in bone research to investigate the viability of

bone graft substitutes such as demineralised bone [25], growth factors [66] and gene

therapy [67]. Bone formation has been reported to be enhanced and increased in

immunodeficient animals when similar investigations in immunocompetent animals

proved futile [68, 69]. Alden and colleagues injected BMP-2 into nude rats and

observed endochondral ossification whilst bone formation was inhibited in

immunocompetent rats [68]. Musgrave et al. observed less bone formation in the

muscles of immunocompetent mice when compared to SCID (immunodeficient) mice

[69]. Esses demonstrated that allogenic bone graft healed in Balb/c mice but healing

was impaired in immunocompetent C57BL/10 mice [70]. The few investigations of

Introduction

- 8 -

natural bone healing capacity in immunodeficient animals and their

immunocompetent counterparts provided contradicting findings [71-73]. Vignery et al.

suggested that immunodeficient mice have abnormal bone remodelling activity [71]

and McCauley et al. found differences in their bone turnover rate [72]. Kirkeby, on the

other hand, showed that athymic and normal rats have similar bone healing capacities

[73].

Animal selection is a critical aspect in conducting an in vivo experiment.

Understanding the advantages and limitations of using a certain animal model is an

integral part in the experimental design. A common mistake when selecting an animal

model is an attempt to study a particular human activity or function that does not occur

in animals. Bone formation and regeneration has been shown to be similar in rats,

sheep and human [47, 74]. Accurate representation of an animal model similar to that

of the human body is remains a challenge.

The significance of a well-planned experimental design is paramount as well as being

cost effective. Einhorn [44] outlined several important criteria for the selecting an

experimental model. Firstly, the model should mimic the clinical setting biologically

and physiologically close to human physiology and human clinical conditions [44].

Ultimately, experimental designs are to provide a better treatment and cure for

mankind. While in vitro models are less complex and provide a controlled

environment, the synergistic or antagonistic interactions between cells in a living

biological setting, i.e. in vivo are not reproducible in simple in vitro models [75].

Secondly, the experimental model should be planned such that its healing ability is

exclusive to the strategy under study. In orthopaedic research, for example, a bone

Introduction

- 9 -

defect or fracture non-union must fail to heal unless it is treated with therapeutic

intervention. The chosen model should be such that the methods used can show the full

therapeutic potential. However, having said this, even if the defect model heals, it is

useful to study the mechanism and rate of healing.

1.3. Scope of dissertation

Given the shortcomings of the current defect models in the study of human based

treatment strategies, the content of this dissertation addresses the concept of

establishing and characterizing a bone defect model that could be used in

immunodeficient and genetically manipulated (knockout and transgenic) mice.

1.3.1. Objectives and Aims

The objectives of this dissertation are two fold:

1. to establish a bone defect model that is viable, standardized and reproducible;

desirable model should be at a weight-bearing skeletal site without the need for a

stabilizing fixator;

2. to use this model to characterize the process of bone healing spatially and

temporally in immunodeficient animals.

The aims of the study are:

1. to establish a preliminary bone defect model in nude mice by evaluating potential

surgical sites;

2. to incorporate the selected bone defect model to characterise

a. the effect of T cell deficiency on bone defect healing in nude and normal

mice;

Introduction

- 10 -

b. the effect of age on bone defect healing in nude and normal mice;

c. the feasibility of a critical size defect (CSD) in nude mice;

3. to apply the selected bone defect model to a larger immunodeficient animal i.e.

nude rats.

Background and Literature Review

- 11 -

CHAPTER 2 Background and Literature Review

For the purpose of this dissertation, a brief outline of the basic structure, physiology

and biomechanics of bone and its auxiliary components is presented in Section 2.1.

For more in-depth reading, the author recommends Form and Function of Bone by

Bostrom et al. [76], Biomechanics of Bone and Fracture by Tencer [77] and The

Mechanical Adaptations of Bone by Currey [78].

2.1. Biology and histology of bone

Bone is a unique tissue that is capable of self-renewal, both in the physical and

biological sense. This specialized connective tissue is the foundation for mobilization

in vertebrates and non-vertebrates. Without bone and its supporting counterparts such

as the muscles, tendons, ligaments, movement would be impossible. Bone houses and

protects vital organs in the cranial and thoracic cavities from injuries. Apart from

providing support, protection and movement, the bone is a mineral reservoir for heavy

metal ions, calcium (Ca2+) and phosphorus (P) ions and acts as a site of hemopoiesis

[79].

2.1.1. Classification of bone

Bone is classified based on its physical features and composition. Bones are divided

into four classes: long, short, flat and irregular.

Long bones are found in the limbs, where they form the upper and lower extremities.

Examples of long bones include the femur and tibia. Most long bones are made up of a


- 12 -

long tubular shaft known as the diaphysis and two epiphyses found at the ends of the

diaphysis. An epiphyseal line can be found between the diaphysis and each epiphysis

of an adult bone. This line is the remains of the epiphyseal plate involved in the

lengthening of the bone during growth. An outer layer of cortical bone surrounds a

medullary or marrow cavity filled with fat or yellow marrow. Long bones act as a

system of levers to balance the weight of the trunk and for movement. The geometry of

the long bone shows that it is ideal for withstanding bending and torsional loads, with

the bony tissues distributed far from the neutral axis located at the center region of the

medullary canal [80]. Figure 2-1 illustrates examples of long bone in mice, rats and

human.

Short bones are named by virtue of their physical features: short and compressed.

Examples of these bones are the carpus and tarsus. Short bones are composed of

cancellous bone surrounded by a thin layer of cortical bones. The functions of short

bones are to provide strength and compactness, where movement is slight and limited.

Flat bones consist of thin cortical plates enclosing an interior cancellous bone. One

example of flat bones is the vitreous table located at the skull, composed of the

occipital, parietal, frontal, nasal, lachrymal, and vomer. Flat bones are also found at

the scapula, sternum and ribs. Flat bones are functionally relied upon to confer

protection. They also provide broad surfaces for muscular attachments such as the

bones of the skull and the scapulas.


- 13 -

Figure 2-1 Radiographs of long bones in different species (a) mouse (b) estrogen deficient rat (courtesy

of Rema Rajaratnam) (c) human


- 14 -

Bones that are not grouped under the preceding three categories are usually classified

as irregular or sesamoid bones, usually by virtue of their unusual form. The

composition of these bones is similar to other bones – a layer of dense, cortical surface

surrounding porous, cancellous tissues. Bones under this classification are the bones

forming the spine, some bones forming the skull, the hyoid, and the patella [79].

2.1.2. Macrostructure of bone

There are two forms of bone: cortical and cancellous bone. Each form is described in

detail in the following section.

2.1.2.1. Cortical bone

Cortical bone also known as compact bone makes up approximately 80% of the

skeletal system [81, 82]. The bone structural unit (BSU) of cortical bone is the

Haversian system, named after the 17th century physician, Clopton Havers. The

Haversian system or osteon consists of the lamellae, lacunae, canaliculi and the

Haversian canal. In an average human skeleton, there are a total of 21 x 106 Haversian

systems. On average, total cortical bone volume is approximately 1.4 x 106 mm3,

while the total internal surface area is approximately 3.5 x 106 mm2 [81, 82]. The

relative density of cortical bone is approximately 1.8g/cc [83] and the average

turnover is approximately 50%.

At the fundamental level of the Haversian system, millions of collagen fibrils are

embedded with hydroxyapatite (HA) crystals at the ends. Each fibril is approximately

200 – 400 Å in length. The collagen fibrils are flexible while HA confers the rigidity to

the bone structure. The collagen-HA fibrils are bundled together to form concentric


- 15 -

lamellae or sheets, approximately 3 – 7 µm [81]. The lamellae are then arranged

around a canal through the center of the Haversian system known as the Haversian

canal (Figure 2-2(a)). The Haversian canals contain blood vessels and lymphatic nodes,

and are attached to each other and to the outer periosteum by the transverse

Volkmann’s canals [84]. The blood vessels distributed through the Haversian canals

are closely linked, so that none of the cells are too isolated from any blood vessels [79,

85].

Figure 2-2 (a) Haversian system in human bone. Adapted from [76]. (b) Histologic section of osteons of

cortical bone in a mouse femur (Haematoxylin and Eosin (H&E), 400x magnification)

Canaliculi are minute canals in a network between lamellae and also between lamellae

and the Haversian canals. The canaliculi act as communication ports for osteocytes to

communicate with each other and with the Haversian canals. Through these channels,

substances such as calcium salts pass into the bone and vice versa, whereby calcium

from bone will be transported into the blood stream [79]. The structural outline of the

cortical bone is illustrated in Figure 2-3.


- 16 -

Figure 2-3 Structural layers of a long bone. Adapted from [77]

2.1.2.2. Cancellous bone

Cancellous bone is a maze of thin processes of bone or trabeculae with huge

marrow-filled spaces in between them. The trabecular structure is further divided into

series of rods and plates. The network contains irregularly-arranged lamellae and

osteocytes but no osteons.

Cancellous bone consists of a fifth of the skeletal system [82]. A total of 14 x 106

semilunar subunits exist in an average human skeleton. Similar to cortical bone, the

turnover rate of cancellous bone is approximately 50%. Although the total cancellous

bone volume is around a quarter of cortical bone volume (approximately 0.35 x 106

mm3), the total internal surface area is double (7.021 x 106 mm2) due to a large

surface area of the trabecular bone structure [81]. Trabecular bone has a relative


- 17 -

density of 0.05-0.7g/cc with a porosity value of 30% up to above 90% [83]. The

cancellous bone is nourished by nutrients that diffuse through the marrow spaces

between the trabeculae.

The trabeculae are arranged along the stress trajectories to enhance bone strength to

resist tension, compression and reduce bending. If the imposed load is equal,

trabecular bone tends to form approximately equally aligned cells while unequal load

results in a disproportional alignment to best support the load [83]. At low loading,

trabecular bone tends to form low density rod-like structures as opposed to perforated

plates at high loads [83].

2.1.3. Microstructure of bone

At the microscopic level, bone is further classified into woven and lamellar bone.

Woven bone is immature bone, characterized by a non-uniform collagen distribution

and random distribution of cells in a disorganized fashion [86, 87]. It is found during

embryonic development, in metaphyseal regions during growth, fracture repair or in

tumours and some metabolic bone diseases [88]. Lamellar bone or mature bone

replaces woven bone over time to provide structural integrity whereby the mineralized

collagen fibres are oriented in a rotated plywood-like structure [89].

Under tensile testing, the mechanical behaviour of woven bone is the same regardless

of orientation. This differs from lamellar bone which shows greatest resistance to load

in the longitudinal axis of the collagen fibres [80]. The mechanical properties of bone

are further discussed in Section 2.1.6.


- 18 -

Magnification of the bone exposes its minute composition of matrix, organic and

inorganic components. The extracellular matrix (ECM) consists primarily of Type I

collagen (90%) with non-collagenous protein (NCPs), and ground substance

(glycoproteins and proteoglycans) [84]. The matrix consists of an organic and an

inorganic component.

The organic component consists of cells, osteoblasts, osteoclasts, osteocytes and

osteoids. The osteoids include proteoglycans, glycoproteins, and collagen [90]. This

component forms one-third of total bone weight, and it provides stiffness and tensile

strength to the bone to resist tension and torsion. Immersing bone in dilute mineral

acid extracts the organic compound, after which the bone retains its original shape but

is brittle.

The inorganic part constitutes two-thirds of the total bone weight. Approximately 83%

of inorganic material is hydroxyapatite (HA) Ca10(PO4)6(OH)2 [76] or mineral salt, the

majority of which is calcium phosphate [84]. HA consists of plate-like crystal of 20–80

µm in length and 2-5 nm thick [87]. The remainder of the inorganic component is a

mixture of calcium carbonate, calcium fluoride, calcium chloride, magnesium

phosphate and traces of sodium chloride and sulphate. The inorganic component

confers hardness and rigidity to the bone. Removal of inorganic ingredient by

calcination leaves the bone intact in shape but extremely flexible.

2.1.4. Cellular and molecular level

Bone tissue consists of four major cell types: osteoprogenitor cells, osteoblasts,

osteocytes and osteoclasts.


- 19 -

Osteoprogenitor cells are the precursor to osteoblasts and subsequently osteocytes.

They are found on the periosteal and endosteal surfaces, and have the capacity to

differentiate and proliferate into osteoblasts, chondroblasts and fibroblasts. As they are

normally inactive cells, they appear as flattened cells with elongated or oval nuclei.

Similar osteoprogenitor-like cells found close to the bone matrix are known as bone

lining cells.

Osteoblasts are fully differentiated mesenchymal cells derived from stromal marrow

cells. Morphologically, active osteoblasts are plump and cuboidal in shape, typically

arranged in a row [87] with the nucleus at the furthest end from the bone surface as

shown in Figure 2-4. Osteoblasts are known as bone forming cells, as they are

responsible for the production and secretion of the constituent of the osteoids or

unmineralized bone matrix, mainly the proteoglycans, glycoproteins and collagen [90].

They also produce alkaline phosphatase (ALP), which initiates mineralization of the

matrix [90].

Osteocytes are osteoblasts that are embedded in the bone matrix during mineralization.

They are smaller than osteoblasts but have a higher nucleus to cytoplasm ratio [87].

Being the most abundant of bone cell types, their “position” as the terminal cells of the

osteoblastic differentiation line explains why they are metabolically active. Osteocytes

maintain both the mineral and matrix of bone. The interconnecting network between

osteoblasts, osteocytes and bone lining cells allows the bone to sense any deformation

and differential potentials occurring, and to coordinate the formation and resorption of

bone and the flow of mineral ions between the bone matrix and the extravascular fluid


- 20 -

spaces of the bone [91]. The sensitivity of this delicate intracellular network to

mechanical strains is reported to be minute (0.2-0.4%) change in the diameter of the

cellular membrane [92].

Figure 2-4 Histologic staining of osteoblasts (solid arrows) and osteocytes (open arrow) in mouse bone

(H&E, 400x magnification)

Osteoclasts are large, multi-nucleated giant cells (20-100µm) with numerous

mitochondria that are believed to originate from hematopoietic stem cells [91],

macrophages and monocytes [58]. They are found on the surface of endosteal,

periosteal and Haversian bone on occasion. An example of osteoclasts is shown in

Figure 2-5. Active osteoclasts are often found in shallow pits or cavities known as

Howship’s lacunae, where bone resorption occurs [79]. The role of osteoclasts is the


- 21 -

converse of osteoblasts, in that they are involved in bone resorption. This is a very

important role to allow space for blood cell formation and to prevent bones from

becoming too heavy. They secrete hydrogen ions, collagenase and acid phosphatase to

dissolve bone mineral and matrix [90].

Figure 2-5 Presence of osteoclasts in nude rat bone indicated by arrows (H&E, 200x magnification)

The delicate balance between bone formation by osteoblasts and bone resorption by

osteoclasts keep the bone mass constant. This intricate and dynamic system is known

as bone remodelling which important for the skeleton to adjust to changes in

mechanical loadings, to prevent accumulation of fatigue, to repair micro fractures [93],

to ensure the capability of the osteocytes and also for calcium homeostasis [94]. This

process responds to bone growth, bone repair or changes in mechanical loading in the

bone [95]. Further explanation on bone remodelling (and bone modelling) is given in

Section 2.3.


- 22 -

2.1.5. Related structure

2.1.5.1. Periosteum

Most of the bone structure is enclosed in a fibrous membrane called the periosteum;

except where it is coated with articular cartilage. The periosteum comprises of two

layers; an outer one of dense, connective tissue and an inner, osteogenic layer of

osteoblasts and osteoclasts [57]. Murakami and Emory, however, suggested that

periosteum is really a three-distinct-layer membrane; the third layer being a middle

layer of elastic fibres [96].

Innervated with nerve fibres, lymphatic vessels and blood vessels, the periosteum

provides an insertion or anchoring point for tendons and ligaments. Initial theory of the

periosteum consigned it as a mere inert, limiting membrane. This was proven

otherwise by Ollier in 1858 where the deepest layer was found to have the potential to

produce bone [97]. In vitro and in vivo experiments have shown that the periosteum

contains pluripotential mesenchymal cells capable of forming bone and cartilage [57].

Apart from the periosteum, there is a delicate tissue membrane lining the internal bone

surface called endosteum. The endosteum envelops the trabeculae of the cancellous

bone and the canals through the compact bone. It, too, contains osteoblasts and

osteoclasts.

2.1.5.2. Cartilage

In the skeletal system, cartilage is present in most long bones, articulating surfaces and


- 23 -

other specific locations in the body. Cartilage cells known as chondrocytes lie within

the lacunae in a fibrous matrix. Chondrocytes occupy only 5% of the cartilage ECM

and are isolated within the matrix [98]. Articular cartilage consists of water (68-85 %);

collagen (10-20 %), and proteoglycans (5-10 %) [99]. The water constituent partly

reflects the proteoglycan concentration [100] and the amount of dissolved ions in

solution [95].

Cartilage is intrinsically elastic and resilient, which makes it viable for weight bearing.

It is avascular and obtains nourishment from the surrounding perichondrium by

diffusion.

Cartilage is categorized into hyaline, fibrous and elastic. Hyaline cartilage is

characterized by a homogenous, amorphous matrix of predominantly Type II collagen

fibrils, ground substance (hyaluronic acid, chondroitin sulfate, keratan sulfate),

proteoglycans and water. In developing fetuses, hyaline cartilage is the precursor to

bone formation via a process known as endochondral ossification, which will be

elaborated in the next section. In the adult skeleton, hyaline cartilage is found at the

ends of long bone, joints, trachea, larynx, nose and ribs. Fibrocartilage has large

bundles of Type I collagen in its matrix. It is resistant to compression and shear forces,

hence its presence at the intervertebral discs, symphysis pubis, articular discs and

menisci. The distinguishable feature of elastic cartilage is the presence of elastin

fibres in the matrix. Elastic cartilage is normally found in the external ear, external

auditory canal, epiglottis and larynx.


- 24 -

2.1.6. Mechanical properties of bone

Bone is a complex material that is shaped and formed to fulfil its most vital role, i.e.

support and mobility. In fact, bone is the hardest of all connective tissues in the human

body. The mixture of organic and inorganic phase in the bone directly influences its

mechanical properties. One of the complexities of bone is that it is a viscoelastic or

time-dependant material, where the stress-strain characteristics and strength are

dependant on the applied strain rate. The stress-strain characteristics are also greatly

influenced by the orientation of the bone microstructure with respect to the direction of

loading, giving rise to its anisotropic behaviour. Bone is stronger and stiffer in the

longitudinal direction than in the transverse direction [101]. Therefore, it is not

surprising that cortical bone is strongest in compression, followed by tension and then

shear. In general, cortical bone is significantly much stronger than cancellous bone.

The mechanical properties of bone are given in Table 2.

Table 2 Mechanical properties of bone. Adapted from [77]

Bone type Load type Elastic Modulus Ultimate stress (109 N/m2) (106 N/m2 ) Cortical Tension 11.4-19.1 107-146 Compression 15.1-19.7 156-212 Shear 73-82 Cancellous Tension ~0.2-5 ~ 3-20 Compression 0.1-3 1.5-50 Shear 6.60 ± 1.66

The strength and mechanical properties of bone are greatly influenced by the dynamic

interaction between intrinsic and extrinsic factors. Intrinsic factors i.e. composition of

bone provide the architectural integrity. Mineral content in bone affects the stiffness

and rigidity of bone. Hypermineralized bone produces a stiffer but weaker impaction


- 25 -

tolerance in bone while decalcification decreases the ultimate strength and elastic

moduli in tension [102]. Effect of bone porosity is evident in osteoporotic patients

where loss in mineral content and thinning trabeculae and cortical bone weaken the

bone structure and its mechanical integrity, resulting in fractures. According to

Huiskes and Rietbergen, only a small variation of bone density from 0.1 to 1.0 g/cm3

is required to have a large influence on trabecular bone mechanical properties [83].

Extrinsic factors such as age, loading rate, use and disuse, and presence of defects

determine mechanical resilience. The effect of age on the mechanical properties on

bone can be observed in children where “greenstick” or ductile type fractures are more

common than brittle bone failures in adults [103]. The bending strength and elastic

moduli of bone increase from childhood to adulthood, and then begins to decrease with

aging [77]. Mineral content increases with age which leads to hypermineralization and

its effects. Bone becomes more brittle and less ductile with age, resulting in a

decreasing ability to absorb energy.

A revised version of Wolff’s Law has postulated that bone is a dynamic structure that

responds accordingly to its level of use [104]. Frost’s mechanostat hypothesis accounts

for the changes in biological mechanisms in relation to mechanical load [105]. The

concept of functional adaptation to its environment is clearly seen when bone loss

occurs due to endosteal surface resorption and thinning of the cortex as a result of

non-use. Another example is the formation of callus at a fracture site when primary

bone healing cannot occur. Callus provides a larger cross-sectional area to increase

resistance to bending.


- 26 -

The presence of defects in bone acts as a stress concentrator and subsequently

decreases the mechanical strength and integrity. McBroom et al. has shown that the

bending strength in long bones decreased as much as 80% of the normal strength when

a defect of diameter equal to 10% of the cross section is present [106]. Torsional

strength and energy absorbing properties decreased sharply when the defect size

surpasses 10% of the bone diameter [107].

2.2. Bone damage and trauma

The bone, unlike other organs, is capable of self-renewal into its original form when

damaged or injured. Bone damage is commonly due to fracture and presence of defect.

Bone repair is a cascade of events that involves a complex coordination of biological,

chemical and mechanical reactions.

2.2.1. Fracture

Fractures are varied and are classified according to pattern or etiology [103].

Disease-free bone may fracture due to sudden injury, either from direct or indirect

violence. Fatigue fractures caused by repeated stress normally occur at the tibia or

fibula shaft and the femoral neck. This may occur below ultimate stress limit due to

loading that exceeds bone’s ability to repair itself. Sports injuries usually belong to the

first two categories. Pathological fractures occur when bone, weakened by disease,

lesions and tumours yield spontaneously or from minimal impact [103]. Osteoporosis

is one of the major debilitating diseases that result in low energy fracture known as an

osteoporotic fracture. In Australia alone, 25% and 17% of its female and male

population respectively suffer from osteoporotic fracture [108].


- 27 -

2.2.1.1. Simple and complex fracture

Fracture can be classified as closed or simple fracture where a loss in bone continuity

occurs subcutaneously while the skin remains intact or open or compound fracture if

broken bones protrude through the skin [109]. Greenstick or incomplete fracture

occurs when the bone is partially broken with a segment still attached to the main

structure [103] as opposed to a complete fracture where the bone is completely broken

through both cortices [109]. This pattern is common in children whose bones are more

resilient to impact. Complete fragmentation of bone may result in transverse fracture,

oblique fracture, spiral fracture or comminuted fracture (with two or more fragments).

Bone loss occurs when a fractured portion is missing or lost.

2.2.1.2. Delayed and non-union fracture

Healing of a fracture occurs immediately after an injury or trauma and the end result is

the restoration of the structure and function similar to the original site. Unfortunately,

as with any biological system, not all fractures heal on time or completely. When

healing exceeds the expected healing period, a delayed union is said to occur [90].

Cessation of the periosteal response occurs before the fracture is successfully bridged

[110]. Healing is prolonged but will eventually produce a firm union. It is not arrested

unlike a non-union, which is defined as failure of union between fracture fragments

and complete cessation of bone repair [90]. In this case, the periosteal and endosteal

responses ceased without bridging the fracture [110]. In 1986, Food and Drug

Administration (FDA) defined non-union as “established when a minimum of 9

months has elapsed since injury and the fracture shows no visible progressive signs of

healing for 3 months” for the purpose of bone healing testing [111]. While this is a


- 28 -

general guideline, it cannot be applied to every fracture given that different sites have

different healing environment. A fracture that is healed artificially i.e. filled instead by

fibrous tissue or fibrocartilage with a synovial sac is a pseudarthrosis [53].

Causes of delayed unions or non-unions are due to systemic and local factors.

Systemic factors include general health of the patient, metabolic and nutrition status,

activity level and lifestyle. The most common local causes are inadequate fracture

fixation and loss of blood supply [90, 112]. Severe soft tissue trauma, infection,

interruption to fracture immobilization, separation of fracture fragments due to

distraction or loss of bone substance are other reasons of delayed union or non-union

[113].

2.2.2. Bone defect

Regions where bone is lacking or deficient are known as bony defects. Bone defects

are classified into two types. Contained defects have an intact rim of cortical bone

surrounding the defect while an uncontained defect lack a bony rim. Bone defects may

result from diseases and conditions such as bone disorders, arthritic angular deformity,

hypoplasia and avascular necrosis [114]. Other types of defects have iatrogenic origin

from surgery e.g. remnants of a joint replacement, drill holes or removal of large

pieces of bone to be transplanted in other parts of the body.

2.2.2.1. Bone disorders

Defects or cavities from local bone disorders are commonly due to infection, tumours

and bone cysts [114].


- 29 -

Acute and chronic osteomyelitis is caused by infection from pyogenic organisms such

as staphylococcus aureus (S.aureus) and salmonella [111]. Infection causes lesions to

form usually in the metaphysis of long bone at the tibia, femur and humerus. A special

form of chronic osteomyelitis is Brodie’s abscess where a localized abscess forms

within the bone near the metaphysis [111]. Tuberculosis and syphilitic infection are the

other notable bone infections [114].

Bone tumour is categorized as benign or malignant. Giant cell tumour, a benign

tumour, occurs commonly at the ends of long bone, at the epiphyseal region and often

extends to the joint surface. The tumour consists of a conglomeration of fused cells of

unknown origin that destroys bone substance resulting in pathological fracture.

Osteosarcoma, chondrosarcoma, Ewing’s tumour and multiple myeloma are examples

of malignant tumours [111, 114]. Primary carcinoma of the other organs such as lungs,

breasts and prostate may cause secondary (metastatic) tumour in bone. Radiographs

show erosion of bone usually at vertebral bodies, ribs, pelvis, proximal femur and

humerus that usually result in fractures.

Bone cysts are fluid-filled lesions lined with osteoclasts. Simple bone cysts occur in

children or young adolescents in the long bone. Enlargement of these cysts weaken the

bone, resulting in pathological fractures. Aneurysmal bone cysts have a “blow out”

characteristic where they can expand on one surface of the bone, and may extend to the

soft tissue [114].

Surgical removal of lesions, tumours, abscesses and cysts by curetting often lead to

large defects that do not heal adequately [114].


- 30 -

2.2.2.2. Revision of joint replacements

Revision surgeries account for 11% of TJR in Australia [115]. Loosening, joint

instability, misalignment and infection [111] are common causes for revision surgeries.

Removal and revision of the prosthesis may subsequently cause the formation of a

defect.

2.3. Bone healing

Characterisation of bone healing especially fracture repair in human and animals has

been presented from a biochemical approach [85, 97, 103, 116, 117] or in terms of

mechanical properties and biomechanics [118]. The fundamentals of fracture repair

and healing of bone defect are similar. While fracture repair involves the bridging of

two fracture fragments, the ultimate outcome of bone defect healing is the filling of the

void or missing bone. Details of the healing processes are presented in Sections 2.3.1

and 2.3.2.

The efficacy of bone healing is attributed to three biological processes: osteogenesis,

osteoinduction and osteoconduction [22, 119, 120] collectively named as The Triad of

Tissue Regeneration [121]. Osteogenesis is the initiation and stimulation of bone

formation from pre-committed osteoprogenitor cells [22, 27]. Osteoinduction refers to

formation of new bone by the active recruitment of host pluripotent cells that

differentiate into chondroblasts and osteoblasts to form new bone [120]. Enhanced

bone formation due to a favourable environment at the site of bone formation, usually

within a porous structure that acts as a temporary scaffold is known as

osteoconduction [110].


- 31 -

There are two known pathways by which bone heals, depending on the extent of injury

and the environment. Most fractures heal via a cartilaginous intermediate known as

endochondral ossification (endo = within, chondral = cartilage). A non-rigid fracture

with excessive interfragmentary movement and a fairly large fracture gap are some of

the milieu for endochondral ossification. When cells undergo endochondral

ossification, chondrocytes become hypertrophic and initiate calcification. The

calcified chondrocytes undergo apoptosis to allow vascular ingrowth and bone

formation [122]. Bone is then deposited by osteoblasts that reside in the inner layer

of the periosteum [123]. On the other hand, a rigid and stable fracture, e.g. a fracture

that is stabilized by an external fixator may undergo regeneration via

intramembranous ossification, which is the formation of bone directly from fibrous

membranes. Both processes occur during embryonic development and bone growth. A

third pathway was suggested by Yasui et al. [124] and Sato et al. [125] where bone

forms from cartilage but without proliferation of vascular tissues.

2.3.1. Fracture healing

Fracture healing is generally divided into three phases: inflammatory, repair and

remodelling. These phases are not distinct, in fact they are interrelated and may

overlap [90]. The inflammatory phase occurs immediately after an injury, and is

triggered by necrotic material from damaged local soft tissues, periosteum and bone

fragments. When a fracture occurs, damaged blood vessels cause the adjacent

osteocytes to be ischaemic and die. Blood seeps out of the torn vessels and forms a

hematoma. At this stage, macrophages, granulocytes, lymphocytes and mast cells

populate the fracture site, where they act to destroy dead cells, remove debris and also


- 32 -

release angiogenic and growth factors necessary for bone repair [90]. Osteoclasts are

found in the early stages of inflammation to resorb and remove dead bone. In vitro

studies have shown that osteoclasts require the presence of osteoblasts to function

optimally [126, 127].

Early repair phase shows the migration of osteoprogenitor cells from the periosteum,

endosteum and marrow into the fracture site. With the aid of fibroblasts and

macrophages, they form a periosteal callus that surrounds each fragment and grows

out towards the other fragment [90]. Vascularity to the fracture site at this stage is

provided by new blood vessels that exists as extraosseous, temporary blood supply

[128]. These blood vessels are different from the normal periosteal vascular tissues

and their contribution diminishes as bone repair progresses [90]. The periosteal callus

further differentiates into chondroblasts or osteoblasts. This stage is dependent upon

the local oxygen tension; when oxygen concentration is high, the cells form bone

while lower oxygen tension favours formation of chondroblasts [129]. Bassett

suggested that high oxygen partial pressure allowed cultured cell mass to contract and

form bone [129]. In situation where oxygen partial pressure is low, cartilage is formed

instead. Cartilage is gradually replaced by bone via endochondral ossification.

Osteoblasts lay down an intercellular matrix of collagen and polysaccharide which

soon becomes impregnated with calcium salts to form immature woven bone. The

primary fracture callus provides rigidity to the fracture, and gives the first radiological

indication that the fracture is uniting. A summary of the cellular cascade during

fracture healing is given in Table 3.


- 33 -

Table 3 Cellular cascade during fracture healing. Adapted from [130]

Timeframe post-injury Type of cells 0-20 minutes Trauma Platelet aggregation Blood clotting 0-1 day Neutrophils 1-2 days Macrophages 2-3 days Mesenchymal cells 3-4 days Endothelial cells 5-6 days Chondrocytes 9-10 days Osteoblasts

The remodelling process starts when the fracture fragments are bridged and continue

for months or even years. Woven bone is transformed into bone lamellar by the

combined activity of osteoclasts and osteoblasts. The osteoclasts remove woven bone

while the osteoblasts deposit lamellar bone. Gradually the bone is strengthened along

the lines of stress, and the surplus bone outside the lines of stress is slowly removed. In

children, remodelling is so perfect that the site of fracture becomes indistinguishable

on radiographs. In adult, there is usually an area of thickening or sclerosis left behind.

The healing process described previously is that of compact bone, and applies to

cancellous bone too. Cancellous bone has a much broader area of contact between

fragments and the open meshwork of trabeculae allows easier penetration by

bone-forming tissue.

Bone remodelling is not restricted to injury or clinical damage, but occurs

continuously throughout life. This is different from bone modelling which is an

adaptation by which the shape and size of the bone is “sculptured” during the growth

phase. During bone modelling, bone resorption does not necessary precede bone

deposition. Similar to other tissues, bone is subjected to wear and tear and thus, there is

a need to “rejuvenate” itself. In adults, more than 90% of the normal bone turnover


- 34 -

occurs during bone remodelling by a team of cells known as the bone multicellular unit

(BMU) [105]. The lamellar bone is replaced in minute quantities on the surfaces by a

“coupling” process of bone resorption and bone formation. In cortical bone, a

cylindrical cone-shaped top structure called the bone remodelling unit (BRU) bores a

tunnel through the hard and compact bone in a drill-like manner (Figure 2-6). The

front of the cutting cone is where osteoclasts resorb bone. Following closely are

osteoblastic progenitor cells, which initiate bone formation in the closing cone area.

The cyclic sequence of resorption and deposition is called the

activation-resorption-reversal-formation (ARRF) and takes about 3-6 months to

complete in human [87].

Figure 2-6 Structure of a bone remodelling unit (BRU) in cortical bone. Adapted from [82]


- 35 -

In cancellous bone, the BRU is a pancake-shaped structure. Bone remodelling is

greater in spongy or cancellous bone than in cortical or compact bone, generally about

5-10 times greater [94]. Parfitt presented two different theories on how trabecular

BRU carry out bone remodelling. One theory postulates that bone formation occurs

once resorption is completed while the second theory argues that the formation and

resorption of bone occurring simultaneous as in cortical bone [82]. It is quite possible

for both processes to occur depending on the circumstances. The theories are

illustrated in Figure 2-7.

Figure 2-7 Bone formation and resorption by BRU units in trabecular bone. (A) Resorption process is

completed before formation begins. (B) Resorption is followed closely by formation. Adapted from [82]

2.3.2. Bone defect healing

Healing of surgical defects is divided into three phases: inflammatory, osteoinduction

and osteogenesis, and remodelling. Immediately after surgery, the inflammatory phase


- 36 -

is characterised by the presence of hematoma. Often, very limited superficial cells

survive from the graft transplant through diffusive nourishment while most cellular

components undergo necrosis [131]. During this stage, granulation tissue, vascular and

mesenchymal cells are prominent. Osteoclasts are active at this stage where they

remove the dead bone to allow vascular ingrowth. Differentiation of mesenchymal

cells into osteogenic cells signals the next phase i.e. osteoinduction. New bone

formation is predominantly achieved by the differentiated mesenchymal cells.

Osteoblasts along the edges of necrotic bone produce new bone matrix, which

surrounds the resorbing necrotic osteoid. This process is termed as creeping

substitution [11, 85, 111]. The resorbing necrotic osteoid is indicated radiographically

by a gradual decrease in radiodensity and graft volume. Subsequent bone remodelling

delineates the sharp margins between graft and native bone and trabecular continuity is

eventually achieved.

2.3.3. Factors influencing bone healing

Successful fracture repair is dependent on a myriad of systemic and local factors from

the host and the surrounding tissues including the extent of the injury, injury site, age

and lifestyle of the patient. Given the extent of these factors, this study focused on the

factors that could be reproduced in laboratory animals.

2.3.3.1. Injury site

The sequence of bone healing is variable depending upon the location, type of bone

and their functional differences, i.e. a weight bearing tibial fracture vs. a flat,

non-weight bearing cranial fracture. Healing of flat bones is known to occur via

intramembranous ossification without an intermediate cartilaginous pathway.


- 37 -

2.3.3.2. Rigidity and stability

Rigidity and stability of a fracture site play a role in determining the healing pathway.

Intramembranous healing occurs at fracture site where fixation is rigid with minimal

motion or fracture gaps less than 1.0 mm. In this situation, lamellar bone is deposited

instead of cartilage. Endochondral ossification occurs when there is sufficient

micromotion present to trigger a fibrocartilage precursor prior to bone deposition.

Osteoprogenitor cells differentiate into hyaline cartilage to form a fibrocartilagious

callus, which subsequently calcifies and is replaced by lamellar bone. Perren

attempted to explain how a pathway is selected using an interfragmental strain concept

[20]. As a more rigid tissue, bone can only tolerate small changes in strain before

failing while cartilage is more flexible, hence cartilage has a higher failure strain.

Endochondral ossification may be preferred for several reasons. It is thought that

healing is accelerated when micromotion stimulates secretion of growth factors that

enhance healing. Furthermore, a rigid site may lead to stress shielding and

osteoporosis [90]. On the other hand, stable internal fixation and plating of a fracture

site commonly lead to primary bone healing without an endochondral pathway.

2.3.3.3. Age

Aging is associated with declining body functions and decreasing cell numbers. In

human, aging changes bone properties around 25-30 years of age, when peak bone

mass is achieved [78]. Bone mass drops steadily after this age, in cortical and

cancellous bone regardless of gender. In cortical bone, bone loss is caused by bone

removal via tunnelling or trabeculation of the endosteal cortical envelope [132].

Simultaneous expansion of the marrow cavity and outer cortical diameter is a


- 38 -

structural adaptation to compensate for decreasing bone strength. Usually, this is part

of bone remodelling where fatigued or damage bone is removed. However, unlike

bone remodelling, as more bone is progressively lost from the endosteal surfaces, a

positive feedback mechanism renders the bone loss irreversible, implying a

preferential recruitment of osteoclasts over osteoblasts or an impairment of

osteoblastic functions [133]. The reason for this is still yet unknown. There is a

decrease in fractional volume of trabecular bone (BV/TV, BV = bone volume, TV =

total volume) [134, 135] and mean thickness of trabecular bone and an increase in

inter-trabecular distance [136]. One theory to explain age-related decline of fracture

healing is a decrease in growth factor secretion [137].

2.3.3.4. Vascularity

Successful bone healing relies on the ingrowth of blood supply and angiogenesis [55].

At some stage in endochondral ossification, vascularisation signals impending bone

development as calcified cartilage is removed and replaced by bone. Oxygen tension

has been suggested as a regulator of angiogenesis. According to Bassett, when oxygen

concentration is high, the cells form bone while lower oxygen tension favours

formation of cartilage, which suggested that high oxygen partial pressure favours

vascularisation [129]. Contrary to that, Knighton et al. suggested that macrophages did

not produce angiogenic factors within the fracture callus under high oxygen tension

[112]. Differences in vascularity are reported to account for delayed and non unions

[90].

2.3.3.5. Inflammation and growth factors

Early ideas to instigate and recruit growth factors by stimulating an inflammatory


- 39 -

response was reported by Friedberg [138]. Interest in growth factors and cytokines to

aid bone healing escalated after an important breakthrough by Urist in 1965 [139]. The

balance between bone modelling and remodelling depends on the balanced action of

bone growth factors. The working mechanism of bone growth factors can be inhibitory

or stimulatory, depending on the presence of other growth factors and surrounding

environment. This is discussed in detail in Section 2.3.4.2.

2.3.3.6. Innate immune system

The primary role of B and T-cells is to defend the body against foreign body invasion

and infection. Recent findings however imply that these cells have further roles in

bone healing [58, 61, 140]. Takayanagi et al. highlighted a new role of Interferon-β, a

protein known mainly in cellular response to viruses, in osteoclast activation [58].

Horowitz and Friedlaender suggested that immunologic bone rejection caused

increased bone resorption due to increasing osteoclast activity and hypothesized that

responses from T-cells played a key role in this action [61]. This agrees with findings

by Weitzmann et al. that activated T-cells induce osteoclastogenesis and accelerate

bone resorption [140]. Alliston and Derynck, in a review of the work done by

Takayanagi et al. [58] hinted at a more interactive crossplay between the immune

system and bone remodelling than what was previously thought as mutually exclusive

[60].

2.3.4. Treatment of fracture and bone defects

Conventional clinical management for reparative treatment of bone defects and

fracture non-unions (when all other methods have failed) utilize bone grafts harvested

from patient’s own tissue (autograft), donor tissue (allograft) or bone substitutes


- 40 -

(xenograft). Bone grafts are also used in treatment of pseudarthrosis [131], spinal

fusion [141], arthrodesis of joints to promote extra-articular fusion [114] and

additional bone fillers. The most common bone graft materials used are autogenous

iliac crest bone graft, cancellous allograft chips and demineralised bone matrix (DBM)

[142].

2.3.4.1. Bone grafts and bone substitutes

Bone grafts originate from different anatomical sites, hence they come in different

structures and compositions namely cortical, cancellous and corticocancellous bone

[142]. Cortical bone is used primarily in long bones for discontinuity repair and as

onlays in the facial region for contour improvement [142]. Incorporation of cortical

grafts occur slowly involving an early resorption phase [143]. While this form of bone

graft is appropriate to withstand mechanical stress, revascularisation is complicated

and the bone may remain non viable for years after implantation while still providing

mechanical strength. The fibula is an example of cortical bone graft. Cancellous bone,

on the other hand, does not have the mechanical strength and therefore requires rigid

fixation to bridge the defect gap or discontinuity. Penetration of blood vessels is easily

achieved to produce a viable filler for healing purposes. Corticocancellous bone grafts

have the best response with good vascularisation and mechanical support [142].

Bone grafts are delivered in the form of bone strips, particulate, slurry and paste [142].

Particulate bone is composed of bone chips and used in discontinuity defects where

there is no requirement for mechanical strength. Bone blurry and bone paste or pâte are

ground cortical or corticocancellous bone used for quick revascularisation in

non-stress bearing area.


- 41 -

During an autograft transplant, surviving pre-osteoblasts, osteoblasts and growth

factors derived from the matrix of the bone graft result in synthesis of new bone by

osteogenesis [119, 120]. Growth factors and cytokines are osteoinductive agents; they

have been shown to enhance bone growth from differentiated mesenchymal cells.

Growth factors are discussed further in Section 2.3.4.2. Osteoconduction enhances

bone formation by providing a favourable environment to promote migration of

osteoprogenitor cells. During osteoconduction, the implanted material serves as an

inert scaffold, or trellis, for the growth of host bone, with ingrowth of vascular tissues

[120].

Bone regeneration and replacement has evolved from simple transplantation i.e.

autografts and allografts to sophisticated techniques involving electromagnetic field

and low-intensity ultrasound, growth factors, tissue engineering and the most recent

development - gene therapy. The ultimate goal is to replicate bone formation that will

restore full functionality.

2.3.4.2. Bone growth factors

Bone growth and repair involves a complex interaction among cytokines, growth

factors, hormones, and many other factors [84, 90, 117, 144-146]. Cytokine is a

generic name to describe protein chemical messengers that regulate immune responses.

Growth factors refer to proteins that affect cellular functions during growth and

development, injury and repair [147]. Though some factors are named specifically for

bone repair e.g. bone morphogenetic protein (BMP), their functions are not limited to

bone repair only. In this dissertation, we will limit our discussion to those related to


- 42 -

bone.

The notion that the body contains an inherent bone-inducing substance dated back to

Hippocrates, but Levander was the first modern author who described the

bone-inductive substance in 1938 [148]. In his experiment, Levander implanted bone

segments subcutaneously and intramuscularly and found newly-formed bone in

surrounding area. Lacroix named the hypothetical substance osteogenin [149]. In 1965,

Marshall Urist described his ground-breaking finding that demineralised bone matrix

from adult human bone could induce ectopic bone formation and named the

phenomenon of bone formation at non-skeletal site as osteoinduction. He coined the

term bone morphogenetic protein (BMP) for this substance [139].

The manner in which the bone proteins work is still an ongoing puzzle with new

theories and discoveries being put forward everyday. Questions on their origin source,

action point, functions and modus operandi are slowly being answered with the advent

of new and sophisticated tools and techniques. Generally, it is known that in the event

of trauma, injury or remodelling, bone growth factors act as chemoattractants for

progenitor cells and mitogens for mesenchymal cells as well as inducing

differentiation of chondrocytes, calcification of the cartilaginous matrix, and

angiogenesis for the invasion of vascular tissues [146, 147].

Growth factors act in one of three modes of actions: endocrine, autocrine and paracrine

[116]. Autocrine factors are synthesized and utilised by the same cell whilst paracrine

factors have different source of origin and target. Endocrine factors are delivered via

the blood system to their target location. Growth factors are also classified according


- 43 -

to their phase involvement. Competence factors are those that stimulate cells from its

resting cycle and are involved in the first few hours after trauma or injury. Progression

factors insert their influence in the latter stages [130].

Bone morphogenetic protein (BMP)

Bone morphogenetic proteins (BMP) are low molecular weight polypeptides family

with 12 different subtypes identified so far [22]. BMPs are multi-functional; they have

been showed to regulate growth, induce differentiation, chemotaxis, and apoptosis at

multiple sites and cause the differentiation of mesenchymal cells into osteoblasts [150].

It is the only growth factor known to stimulate differentiation of mesenchymal stem

cells into a chondroblastic and osteoblastic lineage [22]. Whether the whole family of

BMPs is required remains a debatable issue. During embryogenesis, BMPs are found

where early skeletal condensations are formed around developing cartilage and in the

periosteum. Numerous authors have demonstrated the presence of BMPs during

fracture healing in adult animals [29, 151, 152]. BMP-2 and BMP–4 stimulate

chondrogenic differentiation of mesenchymal cells (MSC) and are present during

endochondral and intramembranous ossification [151]. BMP-7 (also known as rhOP-1)

is found to stimulate cartilage maturation, growth of pre-osteoblastic cells [153] and

fibroblast differentiation into osteoblasts [154]. According to Urist, BMPs in bone

grafts are inactive unless the bone is demineralised [139].

Transforming growth factor beta (TGF-β)

Transforming growth factor beta (TGF-β) belongs to the superfamily of transforming

growth factor TGF that includes BMPs. Originated from platelets, MSC, osteoblasts

and chondrocytes, this group of proteins is involved in regulating cartilage and bone


- 44 -

formation in normal growth and also the remodelling phase. It is also found to be

expressed by osteoclasts, where it inhibits osteoclastogenesis and stimulates osteoblast

bone formation [155]. The complexity of the functionality of TGF-β extends to

regulation of the immune system, where it enhances the function of monocytes and

neutrophils, curbs lymphocytes functions and is involved in the differentiation of

several immune system lineages [156].

Fibroblast growth factor (FGF)

Fibroblast growth factor (FGF) family currently consists of 9 members (FGF1-9) [22]

and the ones that are of interest in the orthopaedic field are FGF-1 (also known as

acidic FGF or aFGF) and FGF-2 (basic FGF or bFGF). This growth factor is secreted

by fibroblasts, osteoblasts, chondrocytes and endothelial cells [90]. Although their

primary role is that of an embryonic inducers it is their role in angiogenesis and

revascularisation that have significant impact in bone formation and repair [157].

FGF-2 stimulates mitogenesis, chemotaxis [158], proliferation and differentiation of

osteoblast precursors as well as angiogenesis [157].

Insulin-like growth factor (IGF)

Insulin-like growth factors IGF-I and II are the most concentrated growth factors

found in bone. They are regulated by growth hormone and stimulate proliferation and

differentiation of osteoblasts [159]. They are expressed by the MSCs where they target

cells of mesenchymal origin. Their presence in bone matrix suggest that they are

regulators of bone metabolism [159].


- 45 -

Platelet-derived growth factor (PDGF)

Initially found to be expressed by platelets (hence the name), platelet-derived growth

factor (PDGF) is also secreted by monocytes, macrophages, fibroblasts and

endothelial cells [90, 160]. It is involved in fibroblast proliferation [90] as well as

chemotaxis and mitosis stages of bone induction [22].

Core binding factor (CBF)

CBFA-1 or CBFα-1 (core binding factor alpha) also known as Am13 or Runx2 is a

member of the Runt-domain family of transcription factors. CBFA-1 has two distinct

roles: differentiation of mesenchymal progenitors into osteoblasts and stimulation of

hypertrophic chondrocytes differentiation [161, 162]. Disruption to CBFA-1

expression in mice has shown to arrest the maturation of osteoblasts, and thus resulted

in incomplete bone formation.

CBFA-1 is an osteoblast-specific factor, and is expressed before osteoblastic

differentiation [163, 164]. It is also expressed in hypertrophic chondrocytes [161, 165]

and in prechondrogenic mesenchymal condensation, making it a unique factor to

govern both osteoblastic and chondrogenic differentiation.

Vascular Endothelial Growth Factor (VEGF)

Vascular Endothelial Growth Factor (VEGF) has similar roles in osteogenesis and

angiogenesis. VEGF has been shown to improve microcirculation by stimulating

angiogenesis at the injury site [166].


- 46 -

Smads

The term Smad is derived from human homologue of Drocophila Mad (Mad =

Mothers against decapentaplegic) and the gene Sma. Smads are a family of

cytoplasmic signalling molecules that act downstream of receptors for TGF-β and

BMPs [152]. Their role in bone healing is not fully comprehended. Smad 1, 5 and 8

are functional mediator of BMP-family signalling, working closely with co-Smad 4.

Smads 2 and 3 are transducers of TGF-β signalling while Smads 6 and 7 have the

opposite effect [167]. The expression patterns of Smads 1 and 5 closely resemble that

of BMPs-2 and 7 whereas the expression of Smads2 and 3 was consistent with that of

TGF-β [152].

A comprehensive list of growth factors related to bone healing and their functions is

summarised in Table 4.

- 47 -

Table 4 Properties and functions of growth factors in bone healing

Growth factor Origin Target Site Functions Phase Involvement

Other comments

Transforming Growth Factor Beta (TGF-β)

Platelets Mesenchymal Stem Cells (MSC) Osteoblasts Chondrocytes Osteoclasts[155]

Fracture hematoma Bone matrix

regulate cartilage & bone formation during injury and normal growth & remodelling suppress mineral formation in osteoid stimulate osteoid formation- inhibits markers of calcified bone matrix e.g. osteocalcin induce MSC to produce cartilage-specific proteoglycans and collagen II act on osteoblasts to proliferate and produce collagen induce matrix production and proliferation stimulate periosteal cells to undergo endochondral ossification [168] regulate immune system [156]

First 24 hours TGF-β secreted in latent form requires activation via enzyme cleavage, acidic pH or heat. The release of TGF-β in acidic microenvironment leads to osteoclasts formation

BMP-2 & BMP-4 (BMP-2b)

Primitive osteoblasts and chondroblasts [146] Undifferentiated osteoprogenitor cells [29]

osteoinductive factor stimulate chondrogenic differentiation of MSC induce endochondral ossification callus formation (BMP-4) [29]

Presence of one BMP enhances expression of other BMPs i.e. more potent

BMP-3 (osteogenin)

bone inductive factor induce rapid differentiation of extraskeletal tissue into bone

BMP-7 (osteogenic protein-1 OP-1)

Hypertrophic cartilage

stimulate cartilage maturation fibroblast differentiation into osteoblasts [154]

OP-1 + Collagen I produce effect comparable to autograft

Core binding factor alpha (CBFA-1 or CBFα-1)

Osteoblasts Hypertrophic chondrocytes

Osteoblasts Hypertrophic chondrocytes

stimulate osteoblasts differentiation [163, 164] stimulate hypertrophic chondrocyte differentiation [161, 165]

- 48 -

Growth factor Origin Target Site Functions Phase Involvement

Other comments

Platelet derived growth factor (PDGF)

Platelets Monocytes Macrophages Fibroblasts

Fibroblasts Smooth muscle cells

stimulate mesenchymal cell proliferation and initiate intramembranous bone formation [160] involved in mitogenesis [22]

Early phase of healing

Fibroblast Growth Factor 2 (FGF-2)

Fibroblasts, Osteoblasts, Chondrocytes Endothelial cells [90]

Fibroblasts Smooth muscle cells Vascular endothelial cells

stimulate mitogenesis

stimulates the proliferation and differentiation of osteoblast precursors stimulate angiogenesis [157]

Vascular Endothelial Growth Factor (VEGF)

Hypertrophic chondrocytes

Hypertrophic chondrocytes

stimulate angiogenesis [166] growth plate morphogenesis [169]

Insulin-like growth factor (IGF)

MSCs simulate proliferation and differentiation of osteoblasts [159] involved in mitogenesis [159]

Smad Smad 1, 5, 8

MSCs [152]

mediators of BMP family signalling [152]

Smad 2 MSCs transducer of TGF-β family signalling [167] positive regulator of ECM deposition

Smad 3 Mature

chondrocytes

transducer of TGF-β family signalling [170] negative modulator of keratinocyte proliferation

Smad 6,7 Mature

chondrocytes

inhibit TGF-β and BMP signalling [167]


- 49 -

2.4. Animal models in orthopaedic research

While rodents and murine are widely used in cancer research, immunology, toxicology,

metabolism, developmental biology, aging, diabetes and obesity [171], they also play a

vital role in orthopaedic research. There are many advantages in using small animals

as opposed to larger animals. Rats and mice are well characterized and understood

anatomically, physiologically and genetically. Apart from easy breeding, housing and

maintenance, their high reproductive potential, short generation time [172] and short

life span are favourable for research [24]. More importantly, the ability to produce

transgenic, knockout and inbred strains is highly appreciated, especially with the

advent of gene technology. On the other hand, the small sizes of mice confer a

technical challenge. However, Tay et al. [172] and Cheung et al. [49] have shown that

the minute size in mice is not a disadvantage by using custom-made external fixators.

Mechanical testing is particularly cumbersome, but not entirely impossible.

One of the greatest advantages of mice is their ability to withstand extensive

inbreeding. Inbreeding produces animals up to 99.9% similar genetic makeup [173]

when compared to outbred animals where each individual has a unique genetic

composition. Inbred strains eliminate variability considerably and make studies to

identify susceptibility genes easier. The phylogenetic level at which murine and

rodents reside in the hierarchy of species can be seen as a disadvantage, as opposed to

use of larger mammals and non-human primates. Sandhu and Khan commented on the

failure of bone graft materials in non-human primates although the same material was

successful in a lower species feasibility study [14]. Despite this, the advantages of

murine and rodents are substantial enough to warrant their use.


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2.4.1. Nude and immunodeficient animals

The discovery of immunodeficient animals has heralded a new era in research

especially in oncology and immunology research. These immunodeficient animals

possess physical and functional defects in their T cells, B cells or macrophages which

result in immunologic dysfunctions [174].

Nude, severe combined immunodeficient (scid) and Rag1 mice are some of the most

used mice models in research. While the former two are natural, spontaneous mutants,

Rag1 is the result of targeted mutation. Overall characteristics of these

immunodeficient mice are presented in Table 5.

Table 5 Characteristics of nude, scid and Rag1 mice. Adapted from [175]

Characteristics Nude Scid Rag1 Primary immune defects

T cell deficiency B and T cell deficiency

B and T cell deficiency

Secondary immune defect

Macrophages, NK cells



Advantages Well characterized Available on several different genetic background strains

More severe immunodeficient than nude

More severe immunodeficient than nude No B or T cell “leakiness”

Disadvantages Some extrathymic T cell functions due to functional T cell “leakiness” Significant B cell function

B and T cell “leakiness” High incidence of thymic lymphomas shortens lifespan

Not well characterised

The immune system may be artificially suppressed in animals through surgery (e.g.

thymectomy), irradiation [176], biological and genetic intervention or applying a


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suppressant (e.g. steroids, retinoic acid and cyclosporin A [177]). Prior to clinical

testing in human trials, materials that may be potentially used to induce, stimulate or

generate bone are usually assessed in mice, rats, rabbits or sheep. However,

inflammatory reaction and tissue rejection provide extra variables and unwanted

interference during assessment. Nude animals are seen to solve this problem since they

are inherently immunodeficient and therefore do not mount an immune response.

2.4.1.1. Nude mice

One of the most useful mutants is the “nude” mice which was named nu by Flanagan

[64]. Initial records showed that nude mice appeared in late 19th century but died out.

[65]. The reappearance of these mutants in 1962 gained immediate and vast

acceptance as an animal model for research particularly in immunology and oncology

[64, 178]. This mutation is inherited as an autosomal recessive gene on Chromosome

11 and thus appeared only in homozygotes [65].

Initially, nude mice were assumed to have a short-life span. Flanagan reported that

none survived past 25 weeks or 6-months period, although no specific cause of death

was given other than the evidence of extensive liver failure [64]. Then, nude mice bred

under standard conventional housing had an average survival time between 14-30 days.

From its initial discovery in the 1960s, the average lifespan of nude mice have

extended to 12-18 months. With proper care, however, these mutants may live as long

as the normal laboratory mice if kept in a germ-free, specific-pathogen-free (spf) or

defined flora environment [178, 179]. The average weaning age of homozygous nude

mice is 21 days and they are sexually and skeletally mature by 6-8 weeks [180].


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The most distinguishable physical feature of the nude mice is the lack of hair, hence

the name nude. The first coat of hair develops normally but further hair growth cease

after the first molt due to faulty keratinisation in hair follicle [64].

Homozygous nude mice suffer from thymic dysgenesis, i.e. lacking a functional

thymus [181], an organ which plays a central role in the development of mature T

lymphocytes. It is therefore not surprising that homozygous nude mice (nu/nu) have a

T-cell deficit, although not entirely devoid of T-cell lineage [182]. The numbers of

T-cell precursors are unaffected, which indicates that the T-cell deficit is due to a

defective thymus. The properties of T-cells in nude mice are shown in Table 6. Without

a complete immune system, the nude mouse is able to accept xenografts as well as

allografts. This discovery proved to be a boon for oncology and immunology research

since it provides a perfect model to investigate the role of the thymus and the effect of

thymus-processed lymphoid cells [181]. Before nude mice were discovered, human

tissues were grafted at immune-privileged sites such as the brain, cheek pouch and the

anterior chamber of the eye [178].

The level of macrophages and antigen presenting cells (APC) remained normal [183,

184] but level of natural killer (NK) cells were elevated [65]. Complement activity

also remained unaffected [175]. Serology data of in nude and normal Balb/c mice is

summarised in Table 7. While deficiency of T lymphocytes is a congenital defect,

grafting of a functional thymus or application of syngeneic splenic T lymphocytes may

reverse the effect and result in almost normal T lymphocyte count [65, 177].


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Table 6 Properties of nude mice T cells. Adapted from [65]

T helper cells Low numbers, delayed appearance T suppressor cells, Low numbers, delayed appearance cytotoxic cell precursor Functional T cells Low proportion among cells of the respective phenotype Cell size Cells larger than their normal counterparts

Table 7 Cell properties of nude and normal mice. Adapted from [171]

Normal Balb/c mice Nude Balb/c nu/nu mice Life span 2-3 years [166] 2+ years in an spf environment Red blood cells (106 cells/µl)

Male 10.397 ± 0.389 Female 10.35 ± 0.38

Male 9.63 ± 0.510 Female 9.04 ± 0.333

White blood cells (103 cells/µl)

Male 4.773 ± 1.494 Female 4.215 ± 1.907

Male 5.66 ± 1.57 Female 6.27 ± 1.21

Lymphocytes (103 cells/µl)

Male 3.494 ± 1.448 Female 2.917 ± 1.314

Male 4.437 ± 0.093 Female 5.085 ± 0.046

Monocytes (103 cells/µl)

Male 0.123 ± 0.070 Female 0.046 ± 0.051

Male 0.123 ± 0.006 Female 0.118 ± 0.0091

Platelets (103 cells/µl)

Male 1156 ± 93 Female 874 ± 106

Male 1159 ± 340 Female 1017 ± 338

The traits of nude mice resemble that of DiGeorge syndrome in human [65], although

parathyroidism and anomalies of great vessels (characteristics of DiGeorge syndrome)

are absent in the mutant.

2.4.1.2. Nude rats

Nude rats are similar to nude mice, albeit physically larger and heavier. Athymic nude

rats were discovered in 1975 and named rnu/rnu (Rowett nude) rat after the Rowett

Research Institute in Aberdeen. In 1979, another strain nzru/nzru appeared at Victoria

University, Wellington [185].

The athymic rats are similar to athymic nude mice in many ways. The physical

characteristics of the homozygous state include sparse, short, thin hair and a missing


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thymus, which rendered the animal severely immune deficient. Hairlessness and

thymus dysfunction are attributed to defect on Chromosome 10 [186]. The lifespan of

athymic rats is shorter than normal rats, but it is noted that the mutant may survive for

more than two years if kept under strict pathogen-free conditions. B-lymphocytes and

natural killer (NK) cells levels are normal, but T lymphocytes are severely deficient

[65, 187]. Athymic nude rats also lack CD4+ and CD8+ cells which render them

incapable of combating viral infections [188]. In the absence of mature T cells,

dendritic cells and natural killer (NK) cells are thought to contribute to specific innate

immune responses [189].

2.4.1.3. Current use of immunodeficient animals

Currently, there are about thirty immunodeficient strains, each with their advantages

and disadvantages [183]. Immunodeficient animals provided the first evidence of

major histocompatibility complex (MHC) and series of immunology studies [178].

Immunodeficiency and genetic manipulation are two important characteristics [24]

which make the mouse an attractive model for studies of bone healing. Nude mice are

used in extensively in immunology studies [190-192]. In orthopaedic research, nude

rats are widely used for studies involving bone grafts [39, 193], DBM [194] and

growth factors [66, 67, 195].

Methodology

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CHAPTER 3 Methodology

This chapter outlines the framework of this dissertation’s theoretical and experimental

design including the procedures, techniques and materials used. The following

variables were considered in the process of development and characterisation of a

suitable bone defect model: influences of surgical site, immune status, age and critical

size defect (CSD).

3.1. Criteria for study design and experimental model

Execution of a well-planned design is reflected in reliable and reproducible results and

outcomes. The significance of a well-planned experimental design is paramount as

well as being cost effective. The need to investigate the therapeutic potential of

xenographic substitutes from human origins necessitates the use of immunodeficient

animals to eliminate any interference from tissue rejection. Nude mice and nude rats

are wild-type animals with a T cell immuno deficient system [183], and therefore are

considered the most suitable animals for this thesis.

The problem presented is a lack of understanding of bone defect healing in

immunodeficient animals. Fracture healing commonly occurs via secondary healing

with the callus formation and requires a fixator for stability. Calvarial defects, on the

other hand, do not require a fixator for stability. Despite the ability to create a critical

size defect in the skull, the calvarial defect is not a feasible defect model because the

local blood supply is naturally scarce and the skull consists mainly of trabecular bone

with no load bearing capability [47]. Taking these points into consideration, the defect

Methodology

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model should possess the following criteria:

1. Located at a weight bearing site since most fracture non-union and defects occur

in the long bone;

2. Located at a skeletal site with cortical and cancellous bone to characterise healing

of both types of bone;

3. Requires a recess or crater-like space to hold biological materials for testing;

4. Does not require the use of a fixator or stabiliser for healing to occur;

5. Does not impede or hinder mobility;

6. Simple and reproducible;

7. Time and cost effective.

3.1.1. Development and characterisation of bone defect model

The thesis design was divided into three parts:

The first part was a preliminary study to determine the feasibility of a unilateral or

bilateral defect model without compromising mobility and integrity of the skeletal

structure. In section 3.1.2, the influence of surgical sites was studied based on the

understanding that different skeletal sites heal in different manners. While fracture

models are limited to unilateral models, bilateral defects are possible in bone defect

models.

The second part was to determine the end points of the experiment. Proper selection of

end points provides a sequential assessment of the healing process. Literature review

on murine models have emphasized that healing occurs by six weeks [52]. The relative

time points were chosen to reflect biological and chemical changes in fracture healing

Methodology

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process. For example, at one week postoperative, osteoblasts and chondrocytes were

shown to be proliferating and differentiating which resulted in significant biological

variation [12].

Thirdly, the thesis follows through to develop and characterize a viable bone defect

mouse model to host xenographic tissues. Several factors were considered: defect type

and surgery site; immune status of animals; age of animals; and critical-sized defects.

The mice models were then compared with similar models in nude rats.

The overall thesis design is summarised in Table 8. The outline of the experimental

design is illustrated in Figure 3-1.

Table 8 Overall surgery design

Animal strain Model Age Selection of defect model Nude mice TW 12 weeks DFCD 12 weeks Influence of immune system Nude mice DFCD 12 weeks Normal mice DFCD 12 weeks Influence of age Nude mice DFCD 12 weeks Nude mice DFCD 20 weeks Normal mice DFCD 12 weeks Normal mice DFCD 20 weeks Critical size defect Nude mice 1.0mm 20 weeks 1.2mm 20 weeks

Methodology

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Figure 3-1 A schematic diagram outline of experimental design

3.1.2. Selection of defect model

Part of this project looked at selecting a suitable defect model in a mouse that would be

utilised to assess implanted cells in future studies. Selection of a weight bearing

location was to fulfil practical clinical applications. Instead of a fracture model, a

defect model was chosen for several reasons, one of which was the fact that it is

non-fracture model and therefore does not impede mobility or require stability.

Methodology

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Initially, three defect models were considered: tibial window (TW),

distal-femur-condylar-defect (DFCD) and rib segmental defect. The rib segmental

defect was later excluded due to close proximity to the heart and lungs, and difficulty

in accessing the surgical site. In addition, rib bones have thin cortical layer that did not

contain adequate or well-developed Haversian system [97]. The other two models

were easily accessible, manipulated and standardised. The animals were allocated as

shown in Table 9.

Table 9 Animal allocation for selection of defect model

Age n Weight Selection of defect model Nude mice TW 12 weeks 9 20.75 ± 0.71 g DFCD 12 weeks 9 18.50 ± 1.20 g

3.1.3. Influence of immune status

Surgical intervention to heal bone defects using transplanted cells e.g. graft transplants

often fail due to rejection by cell-mediated response via phagocytosis and degradation

by lysosomal enzymes. While the role of T lymphocytes in wound healing is well

established [196], its role in bone healing is less known. Horowitz and Frienlaender

hypothesized that responses from T lymphocytes play a key role in the bone healing,

based on their findings on skin allografts [61]. An immunological manipulated model

as suggested by Heiple [197] presents a more suitable environment to study any

osteoinductive or conductive properties in a material. Nude mice are naturally

deficient in T lymphocytes and provide minimal interference from immune reaction

responsible for tissue rejection. The influence of immune status was examined using

Methodology

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normal and nude Balb/c as shown in Table 10.

Table 10 Animal allocation to investigate influence of immune status

Age n Weight Influence of immune system Nude mice 12 weeks 50 18.98 ± 0.94 g Normal mice 12 weeks 54 21.43 ± 1.45 g

3.1.4. Influence of age

This section is dedicated to investigate the influence of age factor while selecting a

particular animal model. Age is a complex phenomena generally associated with a

decline in body functions, including bone healing. Decline in trabecular bone mass

with increasing age regardless of gender has been shown, although there is also a

decrease in cortical bone mass in female [198]. In terms of systemic response, aging

had more profound effect on T cell functions compared to B-cells [199]. It has been

reported that functional T cells appear in lympoid tissues in nude mice at six months of

age [63, 200]. Nude rats develop T lymphocytes-like cells expressing mainly CD3,

CD4+ or CD8+ cells and T cell receptor (TCR) with increasing age, although they are

found to lack alloreactivity in vivo [186].

Two sets of skeletally matured mice (12-week and 20-week old) were compared to

determine the influence of T cell deficiency on animals of different age groups as

shown in Table 11.

Methodology

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Table 11 Animal allocation to investigate the influence of age

Age n Weight Influence of age

Nude mice 12 weeks 50 18.98 ± 0.94 g Nude mice 20 weeks 30 26.20 ± 1.78 g Normal mice 12 weeks 54 21.43 ± 1.45 g Normal mice 20 weeks 26 25.54 ± 2.07 g

3.1.5. Critical size defect (CSD)

A critical size defect is generally defined as a defect that would not heal throughout the

lifetime of the animal [43, 44], although Kleinschimidt and Hollinger went on further

to defined it as the smallest osseous size defect that will not heal over 10% of bone

growth [43]. CSDs in animal models would clearly justify that any healing response

is due to the properties of the implanted material. However, a critical size defect is not

a criterion for a model to be useful.

Failure to heal has been attributed to many factors including age and defect size. In this

thesis, a large defect of 1.2 mm was created in older adult animals in an attempt to

create a CSD. The 1.2 mm defect was compared to the standard 1.0 mm defect in nude

mice (Table 12).

Methodology

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Table 12 Animal allocation to determine the feasibility of a CSD

Age n Weight Critical size defect Nude mice 1.0 mm 20 weeks 30 26.20 ± 1.78 g 1.2 mm 20 weeks 28 25.43 ± 2.28 g

3.2. Surgery and anaesthesia procedure

Surgical procedures performed in a mouse (nude and normal) model were extrapolated

to nude rats. Postoperative analysis and end points were assessed using several

techniques: high-density radiography, microcomputed tomography (µCT), histology

and immunohistochemistry. Quantitative and semi-quantitative analyses were

performed on the stained sections.

3.2.1. Mouse models

Balb/c and Balb/c-nude mice were acquired from Biological Resource Centre,

University of New South Wales and Animal Resource Centre, Perth, Western Australia.

All studies were given ethical approval by the Animal Care and Ethics Committee of

the University of New South Wales (01/129).

The mice were operated on an electrical warm pad to prevent excessive heat loss. The

animals were anaesthetised using 4% Isoflurane (Forthane®, Abbott, Aust R 29656)

inhalation together with oxygen at a flow rate of 4 L/min via an anaesthetic machine.

Once the animal was unconscious, the anaesthetic was reduced to 2.5-3.0% and

monitored throughout the surgery. A skin incision was made after disinfecting with

10% povodine-iodine solution (Orion Laboratories, Australia) and 70% ethanol.

Methodology

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Normal immunocompetent Balb/c mice required more preparation prior to surgery.

The knee region was shaved off to expose the surgery site and disinfected with iodine

and 70% ethanol as mentioned above.

Post-operative analgesia buprenorphine (Temsegic®, Reckitt Benckiser, Aust R 15394)

was administered through intraperitoneal injection into the posterior quadrant of the

abdomen. Each animal was given a dosage of 0.03 mg /20 g body weight. The animals

were monitored closely until they recovered from anaesthesia. Food and water were

given ad libitum and unrestricted movement was allowed as tolerated postoperatively.

The animals were weighed on a scale after surgery and prior to sacrifice.

3.2.1.1. Tibial window (TW)

Unilateral tibial window (TW) model

The tibial window was made on one limb while its contralateral received sham

operation involved making an incision without creating a tibial window, and sealing

the skin incision with glue (Epiglue®, Meyer-Haake GmbH, Germany).

A 10-15 mm incision was made from the knee joint to one-third proximal tibial shaft

exposing the fascia and muscle on the anteromedial aspect. The tibial window defect

(4 x 2 mm) was created on the anterior aspect of the tibial crest with a 0.5 mm diameter

microburr attached to a dental handpiece (T1 Line, Sirona Dental Systems, GmbH,

Germany). The posterior cortex was left intact to necessitate support and movement.

The defect sites were cleaned with 0.9% sodium chloride (Baxter Healthcare,

Australia) to remove any bone and marrow debris to preclude any contribution from

Methodology

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external variables. All incisions were sealed with skin glue (Epiglue®, Meyer-Haake

GmbH, Germany). The animals were weighed on a scale after surgery and prior to

sacrifice.

At 7, 21 and 42 days postoperative, the animals were sacrificed via carbon dioxide

(CO2) asphyxiation. This defect model was compared with the unilateral DFCD (as

outlined below) as a preliminary study.

3.2.1.2. Distal femur condylar defect (DFCD)

Unilateral distal femur condylar defect model

A 10-15 mm incision was made from one-third distal femoral shaft to one-third

proximal tibial shaft to expose the knee joint. The distal femur condylar defect (DFCD)

was previously described in others [52, 201], whereby a 1.0 mm diameter defect was

drilled mediolaterally through the distal condyles until both cortices were exposed. A

1-mm diameter dental burr attached to a dental handpiece (T1 Line, Sirona Dental

Systems, GmbH, Germany) was used to drill the hole. In this manner, the defect size is

standardised.

The defects were created on one leg and the contralateral leg received sham operations

without any defect. The defect sites were cleaned with 0.9% sodium chloride (Baxter

Healthcare, Australia) to remove any bone and marrow debris to preclude any

contribution from external variables. All incisions were sealed with skin glue

(Epiglue®, Meyer-Haake GmbH, Germany). The sequence of the surgical procedure

is illustrated in Figure 3-2. After the surgery, the animals were weighed on a scale and

again prior to sacrifice.

Methodology

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Animals were sacrificed at day 7, 21 and 42 after surgery to allow a direct comparison

with the tibial window defect.

Bilateral distal femur condylar defect model

For the preliminary consideration of a suitable surgery site as described in Section

3.1.2, the unilateral model was carried out. Once a suitable defect model was selected

and mobility was not compromised, a bilateral model was carried out thereafter, with

ethical approval by the Animal Care and Ethics Committee of the University of New

South Wales. The bilateral distal femur condylar defect model used the procedure as

described in unilateral distal femur condylar defect model but on both limbs.

The animals were sacrificed at day 0, 1, 3, 7, 10, 14 and 21 days postoperative to study

the temporal and spatial pattern.

Methodology

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Figure 3-2 Surgery of distal femur condylar defect model in nude mice

3.2.2. Rat models

Following the results from the mouse models, similar procedures were carried out and

investigated in larger animals; a bilateral tibial window (TW) model and a bilateral

Methodology

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distal femur condylar defect model in nude rats.

CBH/rnu nude rats were acquired from Biological Resource Centre, University of

New South Wales. All studies were given ethical approval by the Animal Care and

Ethics Committee of the University of New South Wales.

3.2.2.1. Bilateral tibial window in nude rats

Four 13-week old CBH/rnu nude rats with an average weight of 300 g were used for

this study, as described in Yu et al. [10]. Prior to surgery, the rats were anaesthetised

using 4% Isoflurane (Forthane®, Abbott, Aust R 29656) at a flow rate at 4 L/min in

1500cc/min of oxygen. Once the animal was unconscious, the anaesthetic was reduced

and monitored throughout the surgery.

A bilateral tibial window (5 x 8 mm) was created on anteromedial aspect of the both

tibiae using a Hall Burr (S6-122, Midas Rex®, Medtronic), with the posterior cortex

left intact. The defect site was rinsed with 0.9% sodium chloride (Baxter Healthcare,

Australia) to remove any debris. Surrounding muscle and fascia were sutured with

DEXON 3.0 absorbable suture (Tyco Healthcare, MA, US) followed by skin closure

using similar suturing technique. Each animal was given a dosage of analgesia

buprenorphine (Temsegic®, Reckitt Benckiser, Aust R 15394) (0.1 mg/g of body

weight), which was injected intraperitoneally. The animals were monitored closely

until they regained consciousness. Food and water were given ad libitum and

unrestricted movement was allowed as tolerated postoperatively. The animals were

weighed on a scale after surgery and prior to sacrifice. The animals were sacrificed

three or six weeks postoperative via CO2 asphyxiation.

Methodology

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3.2.2.2. Bilateral distal femur condylar defect in nude rats

Three 13-week old CBH/rnu nude rats with an average weight of 300 g were used for

this study. The surgical and anaesthetic procedure was similar to that described in

Section 3.2.1.2 and Section 3.2.2.1 respectively. A defect of 2.1 mm in diameter and

4mm in length was drilled into the cancellous bone of the distal femur using a 2.1

mm-diameter dental burr. The end result is a plug or recess created similar to Tielinen

et al. [51]. After rinsing with 0.9% sodium chloride (Baxter Healthcare, Australia) to

remove residual bone, surrounding muscle, fascia and skin were sutured with

absorbable suture DEXON 3.0 (Tyco Healthcare, MA, US). Similar dosage of

analgesia buprenorphine (Temsegic®, Reckitt Benckiser, Aust R 15394) (0.1mg/g of

body weight) was injected intraperitoneally. After regaining consciousness, the

animals were returned to their respective cages. Surgical techniques are shown in

Figure 3-3. The animals were weighed on a scale after surgery and prior to sacrifice.

The animals were sacrificed one week, four weeks or six weeks postoperative via CO2

asphyxiation.

Methodology

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Figure 3-3 Surgery of DFCD model in nude rats. Making a skin incision using a scalpel (top left)

Drilling a defect using a dental bur (top right) Checking and measuring the defect size before closing the

wound (bottom left) A neat sutured skin closure (bottom right)

3.2.3. Post-operative monitoring

The animals were kept at ambient temperature (20 -22°C) in a 12 hour light and dark

cycle. Food and water were given ad libitum. Animal monitoring was carried out

everyday for the first week post operation and then on a weekly basis. The animals

were checked for wound healing, food and water intake and signs of distress and

immobility. Prior to sacrifice, the animals were weighed on a scale and the data

recorded on the monitoring sheet.

Methodology

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3.3. End point analysis

A flow chart of end point processes is shown in Figure 3-4. The limbs were harvested

and fixed in 10% phosphate buffered formalin prepared from formaldehyde for a

minimum of 48 hours. Fixation is essential to preserve tissue structure and prevent cell

autolysis by enzymes, cell breakdown, tissue degradation and protein degeneration.

Formaldehyde forms cross linkages between adjacent protein chains, and renders the

cells more amenable to staining. The tissues were decalcified in 10% formic

acid–formalin solution to remove inorganic components from the tissue and to

facilitate easy cutting of tissues for histology and immunohistochemistry.

Decalcification period for different tissues are recorded in

Table 13.

Figure 3-4 Flow chart of postoperative processes

Harvest tissue

Fix tissue in 10% phosphate buffered formalin

Decalcify tissue in 10% formic acid -formalin

Take radiographic images Scan tissue using micro-CT

Histology staining Immunohistochemical staining

Quantitative analysis

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Table 13 Decalcification period for different tissues

Species Decalcification period Mouse 24 hours Rat 6-7 days

3.3.1. Radiography

Clinically, radiology is often used to provide an estimation of the degree of

mineralization of bone as well as progression of healing. Radiographic images were

obtained prior to decalcification, which would otherwise cause the bone to be

demineralised and undetectable. Radiographs were taken in the medio-lateral aspects

using a high resolution Faxitron Xray Machine (MX20, Faxitron X-Ray Corporation,

Wheeling, IL, USA) on high-resolution mammography films (Eastman Kodak

Company, USA). An image of Faxitron MX-20 is shown in Figure 3-5. The

technical values and exposure times are given in Table 14.

Figure 3-5 Faxitron MX-20. Adapted from [202]

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Table 14 Faxitron configuration

Species Faxitron configuration Mouse 17 kV, 60 seconds Rat 20 kV, 60 seconds

Radiographic evaluation was undertaken using a radiographic scoring system adapted

from Lane and Sandhu [27] as illustrated in Table 15.

Table 15 Radiographic Scoring System. Adapted from Lane and Sandhu [27].

Score No bone formation 0 Bone formation occupying < 25% of defect 1 Bone formation occupying 25% - 50% of defect 2 Bone formation occupying 50% - 75% of defect 3 Bone formation occupying > 75% of defect 4

3.3.2. Histology

Fixed and decalcified tissues were processed using a standard laboratory protocol

(refer to Appendix). The tissues were dehydrated gradually from 70% to absolute

ethanol and cleared in xylene. The tissues were then embedded in paraffin and

sectioned on a Leica microtome RM2165 (Leica Instruments GmbH, Nussloch,

Germany). The slides were dried in a laboratory oven at 56 ºC overnight. The sections

were stained with Harris haematoxylin and eosin (H&E), Masson’s Trichrome and

selected immunohistochemical staining.

Haematoxylin and eosin (H&E) is a standard histological staining method used to

define cellular structures. Haematoxylin is a basic dye that stains the nucleus blue

while eosin, an acidic dye, stains positively charged structures such as cytoplasm, etc.

Methodology

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pink.

Masson’s Trichrome was utilised to distinguish certain tissue structures in defined

colours. A protocol using Weigert’s Haematoxylin, acid fuchsin, phosphomolybdic

acid and methyl blue or 2% Light Green was adopted for this thesis (refer to

Appendix). The nucleus was stained in purplish blue, collagen in light blue or light

green and marrow and blood vessels in pink.

3.3.3. Immunohistochemistry

Immunohistochemistry is used to detect protein expressions to validate histological

findings. Certain proteins are only expressed in certain cells, so the cellular expression

of the proteins may indirectly indicate the cellular function. Some proteins can be used

as a cell marker to identify cell type, location and distribution. For example, Cbfa-1 (or

Osf2) is expressed only in osteoblasts and osteoblasts-committed mesenchymal cells.

This protein can be used as a marker for osteoblasts lineage cells [163].

Immunohistochemical staining is based on the affinity between antigens and

antibodies [203]. Commercial reagents highlight the enzyme-substrate reactions in

colour for easy detection and visualization. Colour staining indicates the presence of

specific protein while the intensity correlates to the amount present.

Tissues fixed in formalin may result in a change in the formation of antigenic

determinants or epitomes of the proteins. Therefore, antigen retrieval (AR) is required

before staining to unmark antigen that may be masked by other molecules that obstruct

the access of the antibody molecules [203, 204]. There are two categories of AR

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procedures: heat-induced and non-heated-induced such as proteolytic digestion. The

former is considered better than the latter because there is less risk of digesting the

epitopes that are to be stained [203].

There are many staining methods available currently. The selection of the methods

depends on the cost, sensitivity and specificity. For this thesis, two methods were

adopted (1) a two-step indirect method with streptavidin-biotin complex method, and

(2) EnVision™ procedure. The schematic diagram is given in Figure 3-6.

Immunohistochemical staining requires the application of primary and secondary

antibodies (tertiary antibodies were not performed in this study). Primary antibodies

are produced by injecting purified quantities of a target protein into an experimental

animal (e.g. mouse, rabbit or goat). An immune reaction occurs whereby an antibody

is secreted, which is then isolated from the animal’s serum and collected. Secondary

antibodies are biotinylated fragments of antibodies raised against the same species as

the primary antibodies. Monoclonal antibodies have high affinity for specific epitopes

on the antigen due to their similar immunology. Polyclonal antibodies, usually

produced in rabbits, goats, sheep, are immunochemically dissimilar antibodies and

bind to different epitopes of the antigen. Polyclonal antibodies tend to produce

stronger staining intensity and are useful to detect antigens that are present in low level.

Figure 3-7 illustrates the differences between monoclonal and polyclonal antibodies.

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Figure 3-6 Schematic diagram showing application of two-step indirect method with strep-biotin

complex. Adapted from [205]

Figure 3-7 Polyclonal and monoclonal antibodies. Adapted from [203]

Staining Protocol A

A standard immunohistochemical staining protocol was used, as established by Yu et

al. [206]. After dewaxing the slides in xylene to remove the paraffin, the tissues were

rehydrated from absolute ethanol to deionised water. The tissues were treated in

Antigen Retrieval Citra HK086-09K (BioGenex, CA, USA) at 95 °C for 20 minutes

and allowed to cool down at room temperature (RT). The tissues were quenched in

freshly prepared 0.3% H2O2 in 50% methanol for 10 minutes to destroy any

endogenous peroxide, which would otherwise react with chromogen and produce

non-specific staining in the later steps. After quenching, the slides were brought back

to a neutral pH by rinsing with distilled water and then soaking in phosphate buffered

Methodology

- 76 -

saline-Tween 20 solution (PBS-T). The sections were then blocked in 0.3% casein,

0.01% Tween 20 (Polyoxyethylene (2) sorbitan monolaurate, AJAX Chemicals,

Australia) in PBS for 10 minutes to reduce non-specific binding to the tissues. After

blocking the slides were again rinsed with PBS-T. Primary antibody was diluted in 1%

Bovine Serum Albumin in PBS (1% BSA/PBS) and applied after bordering the

sections with a wax pen (DAKO pen S2002, DAKO Inc., CA, USA). A negative

control slide was provided by using no primary antibody, no secondary antibody, no

streptavidin and non-immunised immunoglobulin G to replace of the primary

antibody.

The slides were incubated with the primary or non-immunised IgG overnight at 4 °C in

a humidified horizontal chamber. After rinsing the sections in PBS-T to remove excess

primary antibodies and neutralising the pH, secondary antibodies was applied and

incubated for 60 minutes. Application of the secondary antibodies was dependent on

the primary antibodies; biotinylated F(ab’)2 fragments of swine anti-rabbit

immunoglobulins (DAKO E0431, DAKO Inc., CA, USA) for primary antibodies

raised in rabbits and biotinylated F(ab’)2 fragments rabbit anti-goat immunoglobulins

(DAKO E0466, DAKO Inc., CA, USA) for primary antibodies raised in goats. After

washing the slides in PBS-T, horseradish peroxidase (HRP)-conjugated streptavidin

(DAKO P0397, DAKO Inc., CA, USA) diluted in PBS was then applied to the slides

to detect and further amplify the signal from the biotinylated secondary antibodies.

Liquid diaminobenzidine (DAB+) chromogen (DAKO K3468, DAKO Inc., Denmark)

in a buffer substrate was finally applied to provide the brown colour associated with

protein expression in the tissues.

Methodology

- 77 -

Sections were counterstained with Harris’ haematoxylin to visualise morphology, then

dehydrated through increasing concentrations of ethanol. The sections were cleared

thoroughly in three changes of xylene and mounted with Eukitt solution (O. Kindler

GmbH & Co, Freiburg, Germany) with cover slips.

Staining Protocol B

Staining Protocol B is a two-step staining technique used to detect antigens present in

low concentration or low titre of primary antibodies, used for rabbit primary

antibodies. This method uses a HRP labelled polymer (does not contain avidin or

biotin), which conjugates with secondary antibodies. After dewaxing and rehydration,

the tissues were treated to Antigen Retrieval Citra HK086-09K (BioGenex, CA, USA)

at 95 °C. The tissues were quenched in freshly prepared 0.3% H2O2 in di-H2O for 10

minutes. No blocking was carried out for this protocol. Primary antibody was applied

and incubated overnight at 4 °C in a humidified chamber. Again, substituting

non-specific IgG as the primary antibody provided a negative control. EnVision™ +

Dual Link (K4003, DakoCytomation, Denmark) was applied after thorough washing

in PBS-T and incubated for one hour. After washing with PBS-T, DAB+ chromogen

was applied and allowed to develop. The sections were then counterstained with

Haematoxylin, dehydrated, cleared and mounted in Eukitt Mounting Solution as

described previously.

Primary antibodies were obtained from Santa Cruz Biotechnology Inc., CA, USA and

the corresponding secondary antibodies are listed below in Table 16. Summary of

immunostaining protocols A and B are shown in Table 17.

Methodology

- 78 -

Table 16 List of primary and secondary antibodies used in immunohistochemistry

Secondary antibody Primary antibody Species Final Concentration Protocol

A Protocol B

VEGF (147) (SC-507) Rabbit 0.67 µg/ml FGF-2 (147) (SC-079) Rabbit 0.5 µg/ml SMAD-4 (H-552) (SC-7154)

Rabbit 2 µg/ml

Swine anti-rabbit Ig (DAKO E0431)

EnVision™ + Dual Link anti-Rabbit (DAKO K4003)

SMAD-5 (D-20) (SC-7443)

Goat 4 µg/ml Rabbit anti-goat IgG (DAKO E0466)

N/A

CBFA-1/ PEBP 2αA (C-19) (SC-8566)

Goat 4 µg/ml

BMP-2 (N-14) (SC-6895)

Goat 2 µg/ml

BMP-7 (N-19) (SC-6899)

Goat 2 µg/ml

Normal IgG (SC-2028) Goat Similar to primary Ab

Rabbit anti-goat IgG

N/A

Normal IgG (SC-2027) Rabbit Similar to primary Ab

Swine anti-rabbit Ig

EnVision™ + Dual Link anti-Rabbit

Methodology

- 79 -

Table 17 Immunostaining protocols

Protocol A Protocol B Day 1 Dewax in xylene 5 min, 3 times Dehydrate in 100% Ethanol 5 min 100% Ethanol 2 min 95% Ethanol 2 min, 2 times 70% Ethanol 2 min 50% Ethanol 2 min deionised water 5 min Antigen Retrieval 20 min, 95 °C cool down to RT Quench in 0.3% H2O2 in 50% methanol 10 min, RT Wash in PBS-T Blocking in 0.3% casein, 0.01% Tween-20 in PBS, 10 min, RT Wash in PBS-T Apply primary antibodies in 1% BSA/PBS Incubate overnight, 4 °C

Day 1 Dewax in xylene 5 min, 3 times Dehydrate in 100% Ethanol 5 min 100% Ethanol 2 min 95% Ethanol 2 min, 2 times 70% Ethanol 2 min 50% Ethanol 2 min deionised water 5 min Antigen Retrieval 20 min, 95 °C cool down to RT Quench in 0.3% H2O2 in 50% methanol 10 min, RT Wash in PBS-T Apply primary antibodies in 1% Bovine Serum Albumin/PBS (1% BSA/PBS) Incubate overnight, 4 °C

Day 2 Wash in PBS-T Apply secondary antibodies in 1% BSA/PBS 15 min, RT Wash in PBS-T Apply Streptavidin/HRP diluted in PBS 15min, RT Wash in PBS-T Apply secondary antibodies in 1% BSA/PBS 15 min, RT Wash in PBS-T Apply Streptavidin/HRP 15min, RT Wash in PBS-T Develop staining by Apply DAB+ chromogen, 5-30 min, RT Rinse in PBS-T Counterstain with Haematoxylin Wash in running water 1% Acid Ethanol 1 dip Wash in running water Blueing solution using Scott’s Blue 10 dips Wash in running water Dehydrate in 70%, 80%, 95%, 100%, 100% Ethanol 10 dips Clear in xylene 10 dips, 3 times Mount using Eukitt Mounting Solution

Day 2 Wash in PBS-T Apply HRP Polymer conjugated with secondary antibodies 60 min, RT Wash in PBS-T Develop staining by Apply DAB+ chromogen, 5-30 min, RT Rinse in PBS-T Counterstain with Haematoxylin Wash in running water 1% Acid Ethanol 1 dip Wash in running water Blueing solution using Scott’s Blue 10 dips Wash in running water Dehydrate in 70%, 80%, 95%, 100%, 100% Ethanol 10 dips Clear in xylene 10 dips, 3 times Mount using Eukitt Mounting Solution

RT – Room temperature; PBS - Phosphate Buffered Saline; PBS-T - Phosphate Buffered Saline in

Tween-20; BSA/PBS - Bovine Serum Albumin/PBS; DAB+ - diaminobenzidine; HRP – horse radish

peroxidase

Methodology

- 80 -

3.3.4. Microcomputed tomography (µCT)

Microcomputed tomography (Micro-CT or µCT) is a non-destructive image acquiring

method in three-dimensions (3D) at the micron level. Micro-CT in essence is a smaller

version of the CT scanner and can provide resolution up to 10 µm depending on the

field of view and measurement time [83, 207]. The basic principle of µCT is a 3D

reconstruction of multiple two-dimensional (2D) projections. As the x-ray beam

passes through a point in an object, different absorption points are projected by the

corresponding x-ray shadow. Multiple x-ray shadow images of an object are acquired

from different angles while the object rotates on a platform. By increasing shadow

projections from different views, an image with a good definition of the absorption

position is obtained. These shadow projections are then reconstructed by a cone-beam

algorithm to create a 3D representation of the internal structure and density [207] as

illustrated in Figure 3-8.

The diminutive size of mice and rats allows them to be assessed using microcomputed

tomography. Fixed limbs from mice and rats were scanned using a Skyscan µCT

system (Skyscan-1072, Skyscan, Belgium) with a charged-coupled device (CCD)

attached to a 1024 x 1024 12-bit sensor. Each slice was taken at 20 times magnification,

resulting in a voxel (i.e. 3D pixel) size of 13.67 µm. In this instant, the resultant

resolution is twice of the voxel size (i.e. 27.34 µm) which was accurate enough to

detect the trabecular architecture in the range of approximately 50-400 µm. A 1-mm

aluminium filter on the X-ray source was used to exclude high-contrast and low-noise

images from the region of interest.

Methodology

- 81 -

Figure 3-8 (a) Reconstruction of x-ray shadows into 3D images using microcomputed tomography (b)

Acquisition of multiple x-ray shadow images of an object. Adapted from [207].

3.3.5. Quantitative analysis of histology

Investigation of stained images under light microscopy provided a general yet

subjective overview of the cellular components. H&E sections were evaluated for new

bone and cartilage formation using a colour-based histomorphometry software

(Bioquant Nova Prime, Bioquant Image Analysis Corporation, TN, US). Bioquant

Nova Prime uses a colour intensity threshold value to detect pixels with similar

numerical threshold value in a defined region of interest (ROI) as shown in Figure 3-9 .

Calculation of the total pixel count provided the absolute and percentage bone

formation. A flow chart of the process is illustrated in Figure 3-10 .

Methodology

- 82 -

Figure 3-9 Screen capture showing the use of Bioquant Nova Prime software for quantitative bone

histomorphometry. The highlighted region is the area occupied by new bone.

Figure 3-10 Flow chart of quantitative analysis process

Stain specimen with H&E

View under microscope

Obtain digital image

Import image into Bioquant

Define region of interest (ROI)

Threshold image Compute measurement

Export results into Excel file

Import into SPSS for Windows

Calculate statistics

Methodology

- 83 -

3.3.6. Semi-quantitative analysis of immunohistochemistry

A semi-quantitative analysis of the immunohistochemical staining was quantified

under a light microscope using a 40x objective and a 10x eyepiece. The whole cell

numbers (for all types of cells) in the defect area was estimated by manual counting.

The percentage of positively stained cells was calculated and the intensity was graded

as weak, moderate or strong while unstained cells were graded as negative. The

grading scale is presented in Table 18.

Table 18 Grading scale of immunohistochemical staining (adapted from [208]). Unstained cells are

graded as negative or “-”.

Intensity % of stained cells Weak Moderate Strong 0 - - - < 10% + ++ ++ 10-30% + ++ +++ 30-50% ++ +++ +++ > 50% +++ ++++ ++++

3.3.7. Statistics

Analysis of Variance (ANOVA) provides an appropriate analysis compared to a

student t-test in this thesis due to the number of groups involved. Fundamentally,

student t-test detects whether significant differences exist in the sample means of two

groups while ANOVA tests if the sample means are equal. In this thesis, one-way

ANOVA was applied to analyse groups containing one independent variable at a time.

For example, bone formation between normal and nude mice (aged 12-weeks-old) was

compared at the same time point, e.g. day 3, 7, 10, 14 or 21. The significant difference

i.e. the probability of type I error, alpha α was set at 0.05.

Results

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CHAPTER 4 Results

This section describes observations and results for the different factors under

consideration: selection of defect model, immune status (nude and normal mice), age

(nude and normal mice), defect size (nude mice) and comparison between nude mice

and nude rats.

For the purpose of this study, healing is defined as formation of woven or lamellar

bone while complete healing occurs when the defect site is completely covered with

woven or lamellar bone with closure of the cortex. Healing of the defect area was

indicated by 100% occupation of bone tissue (e.g. woven or mature bone).

4.1. Mouse models

Surgery procedure

The surgery time averaged around 5-6 minutes per animal for a unilateral defect

including performing a sham surgery on the opposite leg and 10-12 minutes per animal

for bilateral defect surgery.

Animal handling

Animals recovered immediately after the surgery without any adverse reaction.

Unhindered weight bearing capability was observed in all animals. The animals

initially lost weight in the first day after surgery (4.85% loss) but recovered to the

pre-operative weight by day 7. The weight loss was not considered significant. No

signs of infection were noted at the wounds in any animal at any time.

Results

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4.1.1. Characteristics of defect models

Tibial window (TW) and distal femur condylar defect (DFCD) models were

performed as a pilot study in 18 animals. The models were used to provide data for

subsequent power calculation and development of end point techniques. Unilateral

models were used with the contralateral limb acting as sham.

Tibial window

Histology sections revealed that the tibial window (TW) was filled with fibrous tissue

and undifferentiated mesenchymal cells at the periosteal surface at one week. By three

weeks, a thin layer of new woven bone had bridged the two opposite ends of the tibial

window at the cortex. At the subperiosteal and endosteal layers, fragments of new

woven bone were observed while the medullary canal was partially filled with new

bone marrow as in Figure 4-1(a). Numerous blood vessels were observed within the

newly formed woven bone. At six weeks, the tibial window had been healed, with

bone marrow filling the previously empty void. Woven bone had remodelled to mature

lamellar bone as indicated by the presence of numerous osteons. Directional alignment

of mature bone was observed at six weeks.

Results

- 86 -

Figure 4-1 Tibial window defect at (a) 3 weeks and (b) 6 weeks postoperative (Haematoxylin and eosin

(H&E), 40x magnification). At 3 weeks, new woven bone had bridged the opposite ends of the tibial

window. At 6 weeks, regeneration of bone marrow and cortical bone was evident.

It was noted that the proximal region of the mouse tibia tapered from the proximal end

to mid-diaphyseal region, thus presenting a non-uniform tibial window (Figure 4-2).

Furthermore, the tibial shaft was very thin, approximately 2 mm. The window area

was too small and unable to contain test materials. From this preliminary study, it was

concluded that the TW in mice was not feasible for evaluation of potential test

materials. Consequently, radiographic assessment of the tibial window was not carried

out.

Figure 4-2 Dimensions of nude mice limbs in centimetres (cm)

Results

- 87 -

Distal femur condylar defect

This is a general description of bone healing of the DFCD model using radiographs,

microcomputed tomography (µCT), histology and immunohistochemistry. Variation

and distinctive outcomes are highlighted under separate healings: immune status, age

and defect size.

Radiograph analysis

Faxitron radiographs revealed progressive healing of the defect area. Bone matrix

minerals were progressively laid down as indicated by the intensity of opacity from

day 3 to day 21. Semi-quantitative analysis of bone healing is shown in Figure 4-4

based on the scoring system mentioned in Table 15.

Figure 4-3 Faxitron radiographs showing progressive bone healing of a DFCD model in nude mice

(a) Day 3 (b) Day 7 (c) Day 10 (d) Day 14 (e) Day 21. Formation of new bone was detected from an

increase in opacity due to bone mineralization from day 10 to day 21.

Results

- 88 -

Radiographic assessment of bone healing in mice

0

0.5

1

1.5

2

2.5

3

3 7 10 14 21Time (day)

Scor

e

NudeNormal

Figure 4-4 Semi quantitative analysis of bone healing from radiographs based on scoring system in

Table 15. No bone formation was detected from the radiographs in nude mice at day 7. Error bars

represent Standard Mean Error (SEM) of each group.

Microcomputed tomography (µCT)

The use of µCT involved an acquisition of the image and a reconstruction of sequential

slices to form a 2D and 3D model (Figure 4-5 and Figure 4-6 respectively). The

average burden time was 56 minutes and 25 minutes respectively. A built-in software

CTAn (CT Analysis) and CTVol (CT Volume) calculated the image properties of each

slice in binary (black and white) and processed images (coloured) and translated the

values into 3D structural properties (Figure 4-7).

Results

- 89 -

Figure 4-5 A reconstruction of the slices to form a 2D model

Figure 4-6 A 3D rendition of a mouse knee joint using µCT

Results

- 90 -

Figure 4-7 Calculation of bone properties using CTAn and CTVol software ©

µCT results provided useful information of the trabecular structure of bone as well as

degree of mineralization [83]. The resolution of µCT data images (approximately

30µm) is comparable to the size of trabecular bone structure. Various bone properties

were extracted from the data and summarised in Table 19.

Results

- 91 -

Table 19 Parameters of the trabecular bone structure as calculated by µCT. Adapted from [83, 207, 209]

Properties Unit Explanation Relative bone volume

BV/TV

Percentage bone volume

Bone surface to volume ratio

BS/BV

1/mm

Bone specific surface. Estimate of thickness. Characterize the complexity of structures.

Trabecular pattern factor

Tb.Pf

1/mm

Indicator of trabecular bone connectivity, which takes into account the relative convexity or concavity of the total bone surface [207]. Hahn et al, who developed this histomorphometric parameter, indicated that concave surfaces represent a well-connected spongy lattice, while convex surfaces indicate a isolated, disconnected trabecular lattice [209]. A low Tb.Pf signifies well-connected trabecular lattices while a high Tb.Pf characterizes a poorly connected structure.

Structure model index

SMI

Relative prevalence of rods and plates in the trabecular bone. An SMI=3 indicates a rod-like characteristic while an SMI=0 showed a tendency to form plate-like structures [83]. This index is of importance when assessing the degree of osteoporosis in the trabecular bone where the manifestation of plate-like bone into rod-like structure occurs.

Mean trabecular thickness

Mean Tb.Th.

mm

Trabecular thickness. Indication of trabecular distribution [82].

Trabeculae number

Tb.N. 1/mm

Number of trabeculae plates and rods.

Mean trabecular separation

Mean Tb.Sp

mm

The distance between each trabecular structure.

Results

- 92 -

Histology results

Haematoxylin and eosin (H&E) staining revealed that the defect was filled with fibrin,

undifferentiated mesenchymal cells, small haemorrhages and trace amount of

lymphocytes at early time point (3 and 7 days). Dead bone (necrosis) was visible

adjacent to the defect outline indicating traumatized tissues from the surgery. This

could be due to the heat generated during drilling and local trauma. The necrotic bone

could also be remnants of bone debris during surgery, although utmost care was taken

to ensure that all bone debris were flushed out with saline. Multinucleated cells

presumed to be osteoclasts were observed at the bone – fibrous tissue interface. It was

noted that a few lymphocyte-like cells were observed at the periphery of the defect at

day 3. Presence of new vascular tissues and bone ingrowth were detected around the

periphery of the defect. New woven bone formed as finger-like projections from defect

boundary towards the centre.

Percentage of new woven bone increased with time and regeneration of bone marrow

were observed. At three weeks, continuity of bone marrow from the medullary canal

was observed. By six weeks, contents of the defect were indistinguishable from the

surrounding bone, with bone marrow and newly remodelled bone completely filling

the defect site. The presence of a defect was only indicated by a discontinuity in the

growth plate, which did not heal or regenerate. Cellular and tissue morphology are

summarised in Table 20. Pictographs showing typical bone healing in the DFCD

model are illustrated in Figure 4-8.

Results

- 93 -

Table 20 Summary of typical cell and tissue morphology in the DFCD model

Timeframe post-injury Type of cells and tissues 0-1 day No cells, fibrin layer 3 days No cells, fibrin layer 7 days (1 week) Granulation, fibroblasts, mesenchymal cells,

osteoprogenitor cells, osteoclasts 10 days Granulation, fibroblasts, osteoprogenitor cells,

osteoblasts, osteoclasts, woven bone, chondrocytes 14 days Granulation, osteoblasts, osteoclasts, woven bone,

chondrocytes 21 days (3 weeks) Osteoblasts, woven bone, lamellar bone, bone

marrow 42 days (6 weeks) Lamellar bone, bone marrow

Results

- 94 -

Figure 4-8 Histology of bone healing of a distal femoral condyle defect in nude mice (H&E staining,

100x magnification unless indicated otherwise)

New bone grew predominantly from the medullary canal aspect, which was named as

the proximal for identification purposes in this thesis. Figure 4-9 outlines the

Results

- 95 -

nomenclature used. Of the new bone growth, 61.11% initiated from the proximal

aspects with an additional 27.78% from both the proximal and distal aspects (Figure

4-10). Proximal and posterior aspects were associated with areas of abundant

vascularity and soft tissues and minimal new bone growth

Figure 4-9 Nomenclature for bone growth direction (H&E staining, 40x magnification)

Direction of bone growth in nude mice

61.11%

8.33%2.78%

27.78%

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

Proximal Distal Anterior andPosterior

Both Proximal andDistal

Direction

Per

cent

age

(%)

Figure 4-10 Graph indicating the direction of new bone growth in the DFCD model in nude mice. Note :

Data excluded specimens with no bone formation or undetermined direction of bone growth.

Distal

Proximal

Anterior

Posterior

Results

- 96 -

From the radiographs, it was possible to detect the position of the defect site; whether

it was in contact with the growth plate or beyond it. Some defects were drilled across

the growth plate, and thereby causing a discontinuity to the structure. In 85.96% of the

samples, the defect was drilled across the growth plate. One-way ANOVA results

indicated that samples with discontinued growth plate showed no significant

difference to samples with intact growth plate in the overall bone healing. Pearson’s

correlation analysis showed similar results, i.e. no correlation between interruption to

the growth plate and cartilage formation. In addition, ANOVA detected no significant

differences (p > 0.05) that the presence of chondrocytes was due to interruption to the

growth plate during surgery (Table 21). Surgical drilling into growth plates presented

no significant difference (p > 0.05) whether healing occurred through cartilage

formation or otherwise.

Table 21 Statistical results to investigate influence of drilling into growth plate on bone healing

n % with cartilage p Drilling through growth plate 114 7.89 0.743 Touching growth plate 4 0.00 Intact growth plate 12 0.00

Immunohistochemistry results

Protein expression of VEGF, BMP-2, BMP-7, Smad-4, Smad-5, FGF-2 and CBFA-1

in nude mice are tabulated and summarised in Table 22. At early time point i.e. day 3

and 7, there was no expression of BMP-2, BMP-7, FGF-2, Smad-4, Smad-5, CBFA-1

or VEGF in nude mice. Low levels of BMP-2 and BMP-7 were detected respectively

at day 10 and day 14. Expression of FGF-2 was first detected at day 10 and gradually

Results

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increased at day 14 before dropping down at day 21. Smad-4, first detected at day 10,

increased in intensity at day 14. CBFA-1 was weakly present from day 10 to day 21. A

high level of VEGF expression was detected in osteoblasts at day 10 before levelling

off at day 14 and day 21 (Figure 4-11(a)). FGF-2 expression was elevated at day 14

and VEGF expression peaked at Day 10 (Figure 4-11(b)). Smad-5 was undetected at

any time point.

Table 22 Immunostaining data for BMPs, VEGF, Smads, FGF-2 and CBFA-1 detected in nude mice

Time BMP-2 BMP-7 FGF-2 Smad-4 Smad-5 CBFA-1 VEGF

Day 3 - - - - - - - Day 7 - - - - - - - Day 10 + - ++ + - + ++++ Day 14 - + +++ ++ - + ++ Day 21 - - + ++ - + +++

Figure 4-11 (a) (left) Expression of VEGF in nude mice at Day 21 (400x magnification) (b) (right)

Expression of FGF-2 in nude mice at Day 14 (400x magnification).

4.1.2. Immune status

A typical section of histological differences between nude and normal mice is

Results

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displayed in Figure 4-12. As early as day 3, normal mice showed ingrowth of new

bone covering less than 1% of total defect area. At day 7, the percentage of new bone

formed was similar in both nude and normal mice. From day 10 to day 14, hyaline

cartilage was observed predominantly in apposition to the new woven bone in nude

mice. Only one normal mice sample showed presence of cartilage at any time point. In

nude mice, replacement of fibrous tissues by cartilage and later new woven bone

resembled a propagating effect, while in normal mice it resembled that of an

intramembranous healing. By day 21, bone remodelling was clearly evident with bone

marrow and neovascularity clearly noted at the defect area.

Figure 4-12 Histology of bone defect healing in nude and normal mice at Day 10 (H&E staining, 100x

magnification). (a) Cartilage was observed in nude mice. (b) No cartilage was observed in this section of

normal mice defect.

Normal mice showed earlier bone formation and greater quantities at day 21 than nude

animals (Figure 4-13). The amount of new bone formed was quantified as described in

Section 3.3.5. The raw data obtained from Bioquant Nova Prime was converted to

percentage values to provide direct comparison between samples. Statistical analysis

Results

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(ANOVA) showed no significant difference in percentage bone formation between

nude and normal mice (p > 0.05).

New bone formation in 12 week-old mice

0

10

20

30

40

50

60

70

80

90

100

3 7 10 14 21Time (day)

Perc

enta

ge n

ew b

one

%

Nude

Normal

Figure 4-13 Graph showing percentage bone formed in normal and nude mice. Percentage new bone

formation was not significantly different between 12-week-old nude and normal mice at any time point.

Error bars represent standard deviation (SD).

In nude mice, cartilage formation was not observed in the first 7 days. The amount of

cartilage formed from day 10 to day 21 amounted to less than 5% in total. In normal

mice, cartilage was observed in one sample only at day 14. No cartilage was observed

at any other time points in normal mice (Figure 4-14).

Results

- 100 -

Cartilage formation in 12 week-old mice

0

2

4

6

8

10

12

14

16

18

20

3 7 10 14 21Time (day)

Perc

enta

ge c

artil

age

form

atio

n %

Nude

Normal

Figure 4-14 Graph showing percentage cartilage formed in 12-week-old normal and nude mice. Error

bars represent standard deviation (SD). Large SD represents large variation between individuals in the

group.

Again, majority of new bone grew from the proximal and posterior region as shown in

Figure 4-15. This is associated with abundant vascularisation and soft tissue

contribution to spur new bone growth.

Results

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Direction of bone growth in DFCD model

61.11%

8.33%2.78%

27.78%25.81%

48.39%

3.23%

22.58%

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

Proximal Distal Anterior andPosterior

Both Proximaland Distal

Direction

Perc

enta

ge (%

)Nude mice

Normal mice

Figure 4-15 Graph comparing the direction of new bone growth in nude and normal mice. Majority of

new bone grew from the proximal and posterior region where vascular tissues are abundant in addition

to soft tissue contribution.

Immunohistochemistry data revealed some similarity and differences in growth factor

expression between nude and normal mice. Expression of BMP-2 and BMP-7 were

shown to be weak and peaked at Days 7 and 14 in both strains. Expression of VEGF

peaked at Day 10 in nude mice and at Day 14 in normal mice. Smad-4 was detected in

nude and normal mice at day 10 whereas Smad-5 was not detected in nude mice but

was weakly expressed in normal mice. Figure 4-16 highlights the stained cells

showing Smad-4 and Smad-5 expression in normal mice.

Results

- 102 -

Figure 4-16 Expression of Smad 4 (left) and Smad 5 (right) in normal mice at Day 10 (400x

magnification). The arrows depict stained cells.

Semi quantitative analysis of the immunostaining results were tabulated and are

presented in Table 23. Graphical representation of VEGF and Smad-5 expressions are

featured in Figure 4-17 and Figure 4-18.

Table 23 Immunostaining data for BMPs, VEGF, Smads, FGF-2 and CBFA-1 detected in normal mice

Time BMP-2 BMP-7 FGF-2 Smad-4 Smad-5 CBFA-1 VEGF

Day 3 - - - - - - - Day 7 + + - - + + - Day 10 - ++ ++ ++ + ++ +++ Day 14 + NA ++ + ++ ++ +++ Day 21 - + ++ ++ - + ++

Results

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Expression of VEGF-B in nude and normal mice

0

0.51

1.52

2.53

3.54

4.5

3 7 10 14 21Time (day)

Scor

e NudemiceNormalmice

Figure 4-17 Graph showing VEGF expression in nude and normal mice from Day 3 to Day 21.

Expression of Smad-5 in nude and normal mice

0

0.5

1

1.5

2

2.5

3 7 10 14 21Time (day)

Scor

e NudemiceNormalmice

Figure 4-18 Graph showing Smad -5 expression in nude and normal mice from Day 3 to Day 21.

The CTAn and CTVol program calculated the structural properties from the collated

µCT slices. Data obtained from µCT scans are summarised in Table 24. Normal mice

had a larger percentage bone volume and bone specific surface compared to nude mice.

The trabecular structure in normal mice showed a more prominent plate-like

Results

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characteristic as indicated by a near zero SMI. A lower trabecular pattern factor

(-2.85454) in normal mice signified a more well-connected trabecular lattices

compared to nude mice (-1.99395). The mean tracebular thickness and separation

distance in nude mice were 0.20689 mm and 4.95837 mm respectively. In normal mice,

these values were lower at 0.18026 mm and 4.10531 mm respectively.

Table 24 µCT data of the trabecular structure in Balb/c nude and normal mice

Nude

Balb/c nu/nu Normal Balb/c

BV/TV 0.01788 0.02025 BS/BV 1/mm 25.48472 30.20063 Tb.Pf 1/mm -1.99395 -2.85454 SMI 1.07 0.524 Mean Tb.Th. mm 0.20689 0.18026 Tb.N. 1/mm 0.08641 0.11232 Mean Tb.Sp. mm 4.95837 4.10531

Legend : BV/TV = Bone volume /Total volume = Percentage bone volume; BS/BV = Bone surface /

Bone volume = Bone specific surface; Tb.Pf = Trabecular pattern factor; SMI = Structure model index;

Tb.Th = Trabecular thickness; Tb.N = Trabecular number; Tb.Sp = Trabecular separation.

4.1.3. Age

Aging is generally associated with the deterioration of bodily function and numerical

healthy cell counts. In this thesis, 12 and 20-week-old nude and normal mice were

examined for their bone formation capability. For easy identification, 12-week-old

mice are categorized as young and 20-week-old mice are adult mice. Typical bone

healing histology is shown in Figure 4-19. Additional images are located in the

Appendix.

As expected, histology sections displayed a decrease in amount of new bone formed

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with age in normal mice. Surprisingly, nude mice displayed no difference in the

amount of bone formed between the young and adult. The adult normal mice showed a

substantial decrease in the amount of new bone formation in the first 10 days but the

results were only significant at day 7 (p < 0.05) when compared to their younger

counterparts. By day 21, similar amount of new bone was detected with cortical

closure. Osteoclasts were observed at all time intervals, either at the defect perimeter

or adjacent to the dead bone.

Nude mice, both young and adult showed new bone formation at day 7 with cartilage

formation at day 10. Multinucleated cells were observed at bone-tissue interface of

adult nude mice at all time intervals, similar to adult normal mice. By day 21,

approximately 83.81% of the defect areas in adult nude mice were filled with the

newly formed bone.

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Figure 4-19 H&E sections of typical bone healing at 14 days (a) 12-week-old nude mouse (b)

12-week-old normal mouse (c) 20-week-old nude mouse (d) 20-week-old normal mouse (10x

magnification)

Figure 4-20 illustrates the percentage of bone formation between young and adult nude

and normal mice at five different time points. ANOVA tests revealed no significant

differences in bone healing between young and adult nude mice and young and adult

normal mice. On the other hand, comparison between adult nude and normal mice

detected a significant difference in percentage bone formation at day 7 (p <0.05) while

the comparison at other time points showed no significant differences.

Results

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New bone formation in mice

0102030405060708090

100

3 7 10 14 21 3 7 10 14 21Time point (days)

Perc

enta

ge n

ew b

one

%

NudeNormal

Figure 4-20 Graph showing percentage bone formed in 12-week-old (young) and 20-week-old (adult)

normal and nude mice at day 3, 7, 10, 14 and 21. ANOVA was performed between nude and normal

mice for the same time point. Percentage new bone formation was significantly different at day 7

between adult nude and normal mice (** p < 0.05). There was no significant difference between young

and adult nude mice or young and adult normal mice at all time points. Error bars = SD

Bone healing via cartilage formation was not evident in adult nude mice as shown in

Figure 4-21. In adult normal mice, cartilage was only observed at day 21 i.e. final

stages of bone healing. In adult nude mice, bone healing appeared to bypass the

cartilaginous intermediate pathway as shown as the absence of cartilage. The large SD

depicts large variation between individuals in each group.

**

12 week-old 20 week-old

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Cartilage formation in mice

02

46

81012

1416

1820

3 7 10 14 21 3 7 10 14 21Time point (days)

Perc

enta

ge c

artil

age

form

atio

n %

NudeNormal

Figure 4-21 Graph showing cartilage formation in nude and normal mice at 12 (young) and 20 (adults)

weeks. Error bars represent standard deviation (SD). The large SD depicts large variation between

individuals at each time point.

Analysis from µCT data showed that normal Balb/c mice possessed higher bone

specific surface than nude Balb/c mice of the same age group. With increasing age, the

trabecular structure of nude mice showed tendency to form more plate-like

characteristic as indicated by a rapid decrease in the SMI value. This was not the case

in normal mice. There was no obvious trend in other trabeculae structure differences

between the two strains as summarised in Table 25.

12 week-old 20 week-old

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Table 25 Analysis of µCT scans using CTAn and CTVol ®

Strain Age BV/TV BS/BV (1/mm)

Tb.Pf (1/mm)

SMI Tb.N (1/mm)

Balb/c nu/nu 0.01788 25.48472 -1.99395 1.07 0.08641Balb/c

12 weeks 0.02025 30.20063 -2.85454 0.524 0.11232

Balb/c nu/nu 0.02222 26.28295 -2.78371 0.352 0.10492Balb/c

20 weeks 0.01956 30.05728 -1.77738 0.647 0.09802

Legend: BV/TV = Bone volume /Total volume; BS/BV = Bone surface / Bone volume; Tb.Pf =

Trabecular pattern factor; SMI = Structure model index; Tb.N = Trabecular number

Distribution of trabeculae thickness of the femoral condyle in nude and normal mice is

illustrated in Figure 4-22 and Figure 4-23 respectively. The thickness interval of

0.055mm was automatically determined by the CTAn and CTVol software.

84.35% and 84.13% of trabecular structures lay in the 0.082 – 0.301mm region in

young and adult nude mice respectively. In young normal mice, 95.50% of the

trabecular structures were 0.082 – 0.301mm thick while in adult mice, 90.47%

trabecular lie within the same thickness range. The trabecular thickness in normal

mice decreased with age while showing no change in nude mice.

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Trabeculae thickness distribution (Tb.Th)in nude Balb/c mice

0

5

10

15

20

25

30

0.027

- <0.0

82 m

m

0.082

- <0.1

37 m

m

0.137

- <0.1

91 m

m

0.191

- <0.2

46 m

m

0.246

- <0.3

01 m

m

0.301

- <0.3

56 m

m

0.356

- <0.4

10 m

m

0.410

- <0.4

65 m

m

0.465

- <0.5

20 m

m

Trabeculae Thickness (mm)

Perc

enta

ge %

12w k NUDE

20w k NUDE

Figure 4-22 Trabeculae thickness distribution in nude mice

Trabeculae thickness distribution (Tb.Th)in normal Balb/c mice

05

101520253035

0.027

- <0.0

82 m

m

0.082

- <0.1

37 m

m

0.137

- <0.1

91 m

m

0.191

- <0.2

46 m

m

0.246

- <0.3

01 m

m

0.301

- <0.3

56 m

m

0.356

- <0.4

10 m

m

0.410

- <0.4

65 m

m

0.465

- <0.5

20 m

m

Trabeculae thickness (mm)

Perc

enta

ge %

12wk Balb/c20wk Balb/c

Figure 4-23 Trabeculae thickness distribution in normal mice

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Figure 4-24 illustrates the separation distance between individual trabecular structures

in nude and normal mice. The separation interval was again predetermined by the

calculating software. All groups with the exception of 12-week-old normal mice

recorded maximum trabeculae separation above 6.046mm. In 12-week-old normal

mice, maximum trabeculae separation was recorded at 5.006 - 6.046 mm.

Trabeculae separation distribution (Tb.Sp)

05

1015202530354045

< 1.012mm

1.012 -< 2.052

mm

2.052 -< 3.037

mm

3.037 -< 4.021

mm

4.021 -< 5.006

mm

5.006 -< 6.046

mm

> 6.046mm

Trabecular separation (mm)

Perc

enta

ge (%

)

12week Nude20week Nude12week Normal20week Normal

Figure 4-24 Trabeculae separation distribution in mice


The relative size of DFCD defects with reference to the total condyle diameter was

measured using a built-in grid ruler in the confocal microscope as shown in Figure

4-25.

Proportion of defect size = Defect diameter / Condyle diameter * 100 %

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Figure 4-25 Calculation of relative defect size. Defect diameter (solid line) Total condyle diameter

(broken line)

A 1.0 mm defect represented 43.7% of the total condyle diameter while 1.2 mm defect

was approximately 48.7% of the diameter as shown in Table 26.

Table 26 Proportion of defect size in comparison with the femoral condyle

1.0mm 1.2mm Proportion of defect size Defect diameter x 100% Condyle diameter

43.74 ± 6.65 48.74 ± 4.62

The 1.2 mm defect showed similar cell and tissue morphology as the 1.0 mm defects at

day 21 as described earlier in Section 4.1.1. A summary of the percentage bone

formation between the two groups is given in Figure 4-26.

Results

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Comparison of defect size on bone formation

0

10

20

30

40

50

60

70

80

90

100

3 7 10 14 21

Time (days)

Perc

enta

ge n

ew b

one

%

1.0mm diameter

1.2mm diameter

Figure 4-26 Graph showing percentage bone formed in 1.0 mm and 1.2 mm defect sizes in 20 week-old

nude mice. There were no significant differences between the two defect sizes at any time points. The

error bar = SD.

4.2. Nude rat models

Overall, the healing process in nude rats was similar to nude mice albeit at a longer

time frame. A radiographic and a surgical overview of the defects are displayed in

Figure 4-27. At six weeks, the defects were not completely healed, although bone

remodelling was observed.

Results

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Figure 4-27 Overview of TW in nude rats on a Faxitron radiograph (left) and post operative (right)

4.2.1. Characteristics of defect models

Tibial window

After three weeks, H&E staining showed that the empty tibial windows were filled

with fibrous tissue with a zone of chondrocytes detected along the center of the

longitudinal axis. Apposition of new woven bone occurred from regions in contact

with the endosteal and marrow surfaces. Multinucleated cells presumed to be

osteoclasts were observed at the bone – fibrous tissue interface. By six weeks

post-operative, newly-formed bone with osteocytes and bone marrow were visible at

90% of the defect site. Blood vessels were located within the cortex area. At the

periosteal surface, fibroblast-like cells were still present. Woven bone had remodelled

to new bone, indicated by the amount of collagen Type I fibres present under polarized

light as shown in Figure 4-28.

No radiographic grading of the tibial window was carried due to reasons mentioned in

the preliminary studies in nude mice (Section 4.1.1).

Results

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(a) (b)

Figure 4-28 (a) A typical section of tibial window in nude rats at 6 weeks postsurgery (H&E staining,

100x magnification) and (b) Collagen Type I of the same section under polarized light

Distal femur condylar defect

A progression in healing was observed radiographically from one to six weeks

postoperative as illustrated in Figure 4-29. At one week, the outline of the defect was

clearly visible with limited bone formation in the defect. At four weeks, approximately

40% of the defect area was filled with mineralised bone. At six weeks, although the

defect boundary was still distinguishable, the defect area was filled with bone as

indicated by an increase in opacity.

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Figure 4-29 Radiographic representation of DFCD healing in nude rats. At 1 week, the defect was

clearly outlined with limited bone formation. At 4 weeks, more new bone formed in the defect area. At 6

weeks, the DFCD outline was blur and the defect area was filled with mineralised bone.

Histology sections are shown in Figure 4-30. Under light microscopy, the defect was

filled with hematoma and fibrin at one week. By four weeks, fibroblasts and new

woven bone filled only 10% of the defect. Active osteoblasts were found along the

perimeter of the defect. At six weeks, the defect had not healed completely.

Approximately 90% of the defect was filled with new bone and bone marrow while the

remaining 10% was still occupied by fibroblasts.

In nude rats, new bone was laid down from the periphery of the defect in a converging

manner towards the center of the defect. As a result, the defect size retained its shape

but reduced its size. This pattern is slightly different from that in nude mice which

exemplified a displacement or propagating effect similar to waves. New bone in nude

mice extended from one end with an advancing front. The differences are illustrated in

Figure 4-31.

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Figure 4-30 Typical representation of bone healing in DFCD models in nude rat (H&E, 40x

magnification). At 1 week, the defect was filled with hematoma and fibrin. At 4 weeks new bone

formation progressed from the defect border to the center. At 6 weeks, defect was almost healed with ~

90% filled with new bone and bone marrow

(a) (b)

Figure 4-31 Difference in DFCD healing pattern in (a) nude rats (H&E, 40x magnification) and (b)

nude mice (H&E, 100x magnification). (a) New bone formed in a converging manner towards the center

of the defect. (b) New bone approached from a propagating front from one end of the defect. Solid arrow

represents the direction of healing.

Bone properties in the femoral condyle of nude rats were calculated using CTAn and

CTVol software as shown in Figure 4-32. Figure 4-33 depicts a typical slice section of

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the DFCD model in nude rats using micro-CT.

µCT results showing the changes in trabecular structure are summarised in Table 27. A

decrease in trabecular pattern factor in normal rats signified a more well-connected

trabecular lattices as the healing time progressed from week one to week six. The

change in mean tracebular thickness decreased rapidly with time (from 12% (from

week one to four) to 2% (from week four to six)). Trabeculae number remained

constant (~ 2%) while the change in mean separation distance was approximately 2.5 –

5%.

Table 27 µCT data of the trabecular structure in nude rats which were sacrificed at 1 week, 4 weeks and

6 weeks.

1 week 4 weeks 6 weeks BV/TV 0.061185 0.064945 0.067905 BS/BV 1/mm 12.80242 12.72017 15.77599 Tb.Pf 1/mm -9.62371 -7.3043 -5.15906 SMI -0.931 -0.7985 -0.2035 Mean Tb.Th. mm 0.308865 0.34648 0.33937 Tb.N. 1/mm 0.19808 0.18746 0.200105 Mean Tb.Sp. mm 4.02949 4.26871 4.37693

Legend : BV/TV = Bone volume /Total volume; BS/BV = Bone surface / Bone volume; Tb.Pf =

Trabecular pattern factor; SMI = Structure model index; Tb.Th = Trabecular thickness; Tb.N =

Trabecular number; Tb.Sp = Trabecular separation

Results

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Figure 4-32 Calculation of bone properties in nude rats using CTAn and CTVol software ©

Figure 4-33 A slice rendition using Skyscan (Skyscan, Be) in colour (left) and binary form (right) at the

position shown in Figure 4-32.

Results

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Cross-sectional areas from micro-CT images provided a non-invasive technique to

observe bone healing (Figure 4-34). The defects were clearly outlined from 1 week to

6 weeks. New mineralised bone was observed at 4 weeks and 6 weeks where it grew

from the external border of the cortical bone. At 6 weeks, a thin layer of mineralised

bone formed across the defect circumference Figure 4-34(c).

Figure 4-34 µCT slices showing the cross-sectional area of the defect in nude rats at 1 week, 4 weeks

and 6 weeks.

Discussion

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CHAPTER 5 Discussion

5.1. Mouse defect models

Understanding the biology of healing in an animal model is of paramount importance

in order to obtain accurate and reliable data that may lead to further development and

human clinical trials. While immunodeficient animals have formed the backbone for

oncology and immunology research, the implication of an immunodeficient system on

bone healing and its treatment is not well known. Establishment of a

well-characterised defect model is the first step to investigate the osteoinductivity and

osteoconductivity of potential implants and bone substitutes. This thesis focused on

the establishment and characterisation of a bone defect model at a weight bearing

skeletal site in nude animals. The model has shown interesting research implications in

the use of these unique animals, particularly nude mice in bone healing.

The definition of bone healing can be defined in different contexts. Some authors

regarded the formation of rigid callus bridging the fracture segments as clinical union,

hence healing has occurred; for others, healing is symbolised by the return of

mechanical properties [210] and functional capabilities [211]. For the purpose of this

dissertation, healing is defined as formation of woven or lamellar bone with or without

regeneration of bone marrow, while complete healing occurs when the defect site is

completely covered with woven or lamellar bone.

Several techniques were utilised to quantify bone healing in a quantitative and

semi-quantitative manner. Radiographs and µCT provided a non-destructive overview

Discussion

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of bone mineralization. However, due to the small size of the defects, visualisation of

radiographs was subjective and did not necessary present an accurate view of bone

healing. Furthermore, unmineralised bone was not visible on radiographs and this

could account for the discrepancy between histology and radiographic assessment.

Histology and immunohistochemistry allowed a detailed visualisation of bone repair

from a biological aspect.

5.1.1. Selection of defect model

The location of an injured site is one of the deciding factors of bone healing. For this

thesis, both the tibial window (TW) and distal-femur-condylar-defect (DFCD) model

were successfully carried out and were shown to fulfil the criteria for a feasible defect

site as outlined in Section 3.1 i.e. weight-bearing location, no requirements for an

internal or external fixator, does not hinder mobility, location is not in apposition to

any major organs such in calvarial defects, easy identification of surface anatomy,

defect size is standardised and reproducible, the average time to create a defect was 10

minutes per animal and cost efficient. Assessment of the tibial window and models

revealed that they were healed by six weeks and did not represent a critical size defect.

Bone healing in the TW model was more rapid than the DFCD model. This can be

attributed in part, to the intact posterior cortex and surrounding bone marrow which

are rich in undifferentiated mesenchymal stem cells (MSC) and osteoprogenitor cells

[2]. MSCs have the potential to differentiate into different cell types depending on the

environment and regulatory factors [2]. The favourable conditions such as rich

vascularisation and relative stability of the bone may cause the cells to differentiate

into osteoblasts or chondrocytes, and thus result in healed bone. Some marrow cells

may also differentiate into adipocytes which were found in healing bone sections.

Discussion

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While the TW defect fulfilled the initial criteria of a feasible bone defect model, the

anatomy of the mouse tibia limits its use for further assessment of bone substitute

materials. The antero-proximal region of the mouse tibia was shaped like a prism

(Figure 4-2). Difficulty in creating a crater-like recess to insert and hold any material

rendered this model unviable. The paltry amount of material that may be used is not

reflective of the amount used clinically for healing purposes. Also, a tibial window

with accurate dimensions requires accurate measurements on the part of the surgeon or

the use of specialised instruments. The DFCD model is a more robust model because

the defect size is standardised and easily reproducible. The defect size is determined

by the burr size - similar to the use of trephine to create a calvarial model. Furthermore,

it could house an implant without “leaking” to the surrounding soft tissue. By taking

all these factors into consideration, the DFCD model was selected to investigate the

influence of other biological factors such as the influence of the immune system and

age.

The direction of new bone formation from these two models subscribe to the school of

thought that “bone cannot form in the absence of vasculature” [212]. In the TW

model, new bone was observed in apposition to the old bone where neoangiogenesis

first penetrates. In the DFCD model, healing progressed from the shaft end where the

vascular-rich bone marrow was located. Angiogenesis is essential in bone healing [55],

although its importance in different phases of healing is debatable. Disruption to

vascular supply has shown to delay, but not prevent fracture healing. Chidgey and

colleagues demonstrated that callus rigidity returned to normal with restoration of

vascularity between fragmented ends [213]. However, when angiogenesis is inhibited,

Discussion

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initial callus and periosteal woven bone formation is dramatically suppressed,

resulting in atrophic non-union [55].

The presence of chondrocytes at defect sites invited some discussion pertaining to

their source. During surgery, some defects were drilled across the growth plate due to

the minute size of the femoral condyle, and thereby interrupting the physical

continuity of the growth plate. Could the drilling inadvertently trigger the proliferation

of chondrocytes to subsequently undergo hypertrophy and differentiate into bone or is

the presence of chondrocytes a component of normal bone repair? ANOVA results

showed that interruption of the growth plate did not affect the end-point healing of

bone, nor did it contribute to cartilage formation. The presence of some cartilage

showed that endochondral ossification and intramembranous may have occurred

simultaneously in long bone defect repair in mice. This agrees with other studies

where endochondral ossification occurs as an intermediate pathway in small animals

[30, 52, 201]. A study by Glimcher found that cartilaginous intermediate stage bone

repair is absent in small animals [214]. This cartilaginous characteristic may be unique

to murine as proposed by Uusitalo [52].

5.1.2. Influence of immune system

Tissue rejection is one of the key reasons for the use of immunodeficient animals. Use

of natural, wild-type immunodeficient mice and rats prevail over that of large, immuno

suppressed animals. High cost, difficulty in applying immunosuppressed drugs such as

Cyclosporin A and complete suppression of the immune system are some difficulties

associated with immunosuppressed sheep and rabbits. T lymphocytes have been

shown to participate in the recognition and destruction of foreign materials that enter

Discussion

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the body. Apart from its defensive role, T lymphocytes are shown to modulate smooth

muscle and vascular repair [215]. In wound healing, T lymphocytes are shown to play

a dual role; an early phase stimulatory role on macrophages, endothelial cells, and

fibroblasts and a later inhibitory role where they inhibit fibroblast migration,

replication and protein synthesis [196]. The same process is thought to occur in bone

healing.

In the event of trauma or invasion of foreign bodies, influx of lymphocytes to the

particular site incite an early-phase inflammatory response to trigger a series of

cascading reactions and result in the migration of macrophages, lymphocytes and

granulocytes and production of growth factors to the defect site [90, 116].

Macrophages and lymphocytes act as scavenger cells, and produce growth factors that

stimulate cell proliferation and growth, as well as angiogenic factors for

neovascularisation [90, 216]. These cells subsequently activate and recruit proteins

and growth factors to aid the healing process. The opposite effect is expected in nude

mice. A lack of inflammatory reaction in nude mice is thought to delay the healing

process due to insufficient recruitment and activation of growth factors. However,

impairment of bone healing or delay was not apparent in this thesis. There are several

explanations for this outcome.

Firstly, the activation of growth factors required for bone healing is not governed

exclusively by the immune system or specifically by T lymphocytes. Similarity in

growth factors expressions in nude and normal mice during bone healing concurred

with this theory. Moreover, vascularity is not impaired in nude mice as shown by the

strong expression of vascular endothelial growth factor (VEGF).

Discussion

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Secondly, the role of T lymphocytes is focused on bone remodelling instead of initial

bone repair. Weitzmann et al. has shown that activated T cells enhanced

osteoclastogenesis formation and accelerate bone resorption similar to that in

osteoporotic patients [140].

From this thesis, histology results showed that normal mice experienced faster (at 3

days postoperative) and greater new bone formation (at 21 days postoperative) than

nude mice but the differences were not significant. Similar outcome in athymic rats

found by Kirkeby [73]. Esses demonstrated no impairment of allogenic bone healing

in athymic mice [70]. This indicates that T lymphocyte deficiency in nude animals

does not augment bone repair. This is a favourable indication that immunodeficient

animals can be utilised to investigate test materials and later provides the basis for

studies in immunocompetent animals. McCauley and colleagues however found that

young and adult nude mice displayed significant difference in bone turnover rate when

compared to normal immunocompetent mice [72]. The discrepancy could be attributed

to the technique used; they used bone ash calculation to evaluate bone density. In

contrast, their histomorphometry data revealed that cortical and cancellous bone area

were not significantly different between the two strains.

Background strain “leak” in nude mice was confirmed by the presence of trace

amounts of T lymphocytes during the initial stages of the bone repair (at day 3). The

“leakage” refers to an incomplete absence of T lymphocytes despite a non functional

thymus [217]. In the absence of a functional thymus, production of T lymphocytes via

extrathymic pathway occurs in the liver and gut mucosa [218]. Nevertheless, the low

Discussion

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level of T lymphocytes is insufficient to mount an immune response.

Both nude and normal mice do undergo simultaneous intramembranous and

endochondral ossification as demonstrated by strong VEGF staining. VEGF is

required for endochondral ossification, where invasion of new blood vessels signals

progress of new bone formation [219]. Disruption to the growth plate did not affect the

end healing point, nor did it indicate a preference for a cartilaginous pathway

5.1.3. Influence of age

Age or senescence is identified as a factor in bone repair [76]. The young are known to

heal at a rapid rate compared to an adult organism, both in human and animals. Mice

are considered sexually and skeletally mature at 6-8 weeks. This investigation took

this factor into consideration and looked at the healing rate in skeletally mature

animals, but at different age groups (12 weeks and 20 weeks). Results from this thesis

showed that immunocompetent normal mice experienced a delay in initial healing

with increasing age. While this is consistent with the general concept of aging, nude

mice, however, did not demonstrate such a trend. From Figure 4-20, it appeared that

20-week-old immunodeficient nude mice showed a faster healing rate than normal

mice. However, a comparison between 12-week-old and 20-week-old mice revealed

that in reality, 20-week-old normal mice showed a delay in healing while there were no

apparent changes in nude mice of the same age. In other words, age does not affect

adult nude mice while in normal mice age affected the healing rate, at least at the initial

phase. One plausible explanation for the discrepancy between adult nude and normal

mice is that T cell deficiency in nude mice might have effectively subdued the

influence of aging, predominantly the initial phases of aging. Speculation that the

Discussion

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breakdown of cell function normally associated with aging is “missing” in nude mice

due to T-cell deficiency (hence, no difference in the amount of bone formed) must be

tempered by the fact that aging is mediated by other factors such as hormones and

related growth factors.

At day 7, 20-week-old normal mice expressed significantly less new bone compared to

12-week-old normal mice, while the total bone formed at 21 days from both groups

were not significantly different (Figure 4-19 and Figure 4-20). Nude mice did not

show any significant difference in the amount of new bone formed at any time interval.

It has been shown that percentage CD4+ and CD8+ (subsets of T cells) in young and

aged Balb/c nude mice were reported to be similar [220]. Miller stated that T cells in

aged immunocompetent mice and human show signs of defects in the early stages of

the immune cascade [192]. Changes to calcium and protein kinase signal generation

were implicated in the disruption on age-dependent T cell activation [192], although

the overall cascading changes is not clear. This complemented our findings where

early-phase bone healing was impaired in normal Balb/c mice.

This thesis did not utilise aged animals (animals more than 12 months-old), but other

studies have indicated that bone healing is delayed in geriatric (aged) nude animals.

Studies by Hunig [200] and MacDonald [63] have shown that functional T cells

appeared in lymphoid tissues in nude mice by six months of age. Systemic immune

response is drastically impaired in aged normal mice [199, 221]. Aging rnu/rnu nude

rats have shown presence of T-like cells (CD3+ expressing TCR (T cell receptors))

[186]. These cells were absent in 2-4 month-old nude rats but present at 4-6 months

[186]. This gives rise to several questions: Firstly, does T cell development in nude

Discussion

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mice become complete with increasing age? If nude mice gradually increased their T

cell numbers, how do age-dependent factors affect their functions at this age? While

the first question is yet to be answered in full, other studies have found that the

acceptance rate of xenotransplanted tumours decreased with increasing age while T

cells markers increased [222]. A similar situation is expected for bone substitutes of

xenographic origins.

The cumulative results suggest that T cells have a key regulatory role associated with

bone regeneration with increasing age. While this thesis showed that T lymphocytes

were not imperative for bone healing, the effect of T lymphocytes on bone healing

whether they act as inducers or inhibitors of bone healing cannot be dismissed

completely. Aging has more profound effect on T cell and its associated functions

compared to B-cells [192, 199]. The link between T and B cells and the imbalance in

T cell/B-cell ratio may have secondary effect on bone healing.

While other studies looked at aged or senescent mice at 8-22 months, this thesis

demonstrated that effect of aging on bone repair began as early as 20 weeks in

immunocompetent mice. Moreover, The Jackson Laboratory has shown that the onset

of senescence varies for different strains of mice. Primitive hematopoietic stem cells in

C57BL/6J mice showed a distinctive delay in senescence compared to Balb/c and

DBA/2J [217]. Variation in animal strains highlights the importance of a careful

experimental design.

Micro-CT images and analysis provided detailed information on the properties of

trabecular bone. Parfitt et al. performed a structural analysis on human iliac trabecular

Discussion

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bone and found that the trabecular network is riddled with discontinued struts and rods

in the elderly [82]. While trabecular separation increased with age, there was only a

slight reduction in trabeculae thickness. This thesis found that there is a

non-significant (5.26%) reduction in the trabeculae thickness distribution in

20-week-old normal mice compared to their younger counterpart. Since there is no

variation in nude mice, this could indicate that resorption and deposition is balanced in

nude mice up to 20 weeks old. Similarity in the physical bone structure between both

strains showed that any differences in bone healing may be attributed to biological

intervention rather than mechanical properties.


Critical size defects are often desirable when evaluating the ability of bone graft

substitutes or various biological factors to promote bone healing. A critical size defect

is defined as the smallest intra-osseous wound that would not heal by bone formation

in the life-time of the animal [223]. When a defect is of critical nature, any healing

response due to the implant or treatment is unequivocal. Furthermore, a CSD may

stimulate the body to function at its maximum capacity to regenerate [47]

The 1.0 mm defect in this thesis healed, as indicated by complete marrow regeneration

and remodelled bone. The largest defect was 1.2 mm which spanned approximately

50% of the diameter of the femoral condyle. Whilst the end point of the 1.2

mm-diameter defects was truncated to Day 21, it is expected to heal as the 1.0 mm.

This is in contrast to other studies in mice where a smaller size defect was claimed to

be of critical nature [35, 201]. This could be due to variation in mice strains;

immunocompetent C57black/DBA mice were used instead of immunodeficient Balb/c

Discussion

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mice [201]. While it would have been prudent to explore the effect of an even larger

defect, it has been shown that bending and torsional strength decreases significantly

when a defect surpasses 10% of the bone diameter [107]. The effect of an even larger

defect would sharply decrease the bone strength and render the structure unstable. In

this case, fracture is highly probable and would detract from the aim of the thesis i.e. to

establish a non-fracture defect. This leads to the question of “Is a critical-sized defect

at a weight-bearing location possible in mice without compromising the structural and

mechanical function?” Hausman et al. commented that “healing in murine and

rodents are robust and failures of healing has been difficult to achieve” [55]. In a

review by Brand, he noted that non-unions in animals are difficult to produce without

“extraordinary surgical means” [210]. The rapid healing rate in lower phylogenetic

animals such as murine and rodents may seem a deterrent when it comes to producing

critical size bone defects. Even if critical size defects were possible, it is impractical to

assess the animal during its lifetime. A practical solution would be to limit any

assessment to earlier time points where spontaneous healing does not occur. Since this

model did not heal at three weeks, this bone defect model could be utilized for early

time points up to three weeks. Though this model is not of critical size, it is still useful

for studies involving gene modified stem cells in vivo expression and viability and

short term bone formation capabilities.

While other studies have shown that calvarial defects in mice and rodents may achieve

critical size, a defect at a load-bearing site such as the one chosen for this thesis again

underlined the differences that arise from different skeletal sites.

Discussion

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5.2. Nude rat models

The same defect models in larger mammals, i.e. rats showed similar healing patterns,

albeit a longer healing period. This is expected as healing period increases when the

animals go up the phylogenetic hierarchy. The TW and DFCD defects in nude rats

were more practical due to their larger sizes and may be considered as defect models to

assess osteoinductive and osteoconductive properties of selected materials.

5.3. Limitations

The precise mechanism of bone repair is yet to be understood completely. Although

this thesis looked at certain factors thought to influence bone repair in animals, it is

known that bone repair in human entail a more complex interaction between the

endocrine system [80], mechanical signals [224], and a myriad of other factors. The

model utilised for this study contained advantages and shortcomings.

The minute size of immunodeficient mice may limit its use for studies of orthotopic

bone formation (as opposed to ectopic bone formation). While the DFCD model may

be utilised to investigate osteoinductive and osteoconductive biomaterials in small

quantities, in mice, it is limited to very small quantities of materials. A critical size

bony defect (CSD) model would be ideal as any healing response due to the implant or

treatment is unequivocal [47]. However, the possibility of creating a CSD at weight

bearing sites in nude mice could not be demonstrated without compromising the

mechanical and structural integrity. All these limitations in mice are not an issue for

nude rats. The larger physical state of nude rats allows them to be utilized in models

that require application of a larger surgical site, a longer assessment period for material

properties [186] and larger volume samples.

Discussion

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While nude mice are athymic and are theoretically T-cell deficient, trace level of T

lymphocytes due to background strain “leak” has been shown in other studies [180].

The low level of T lymphocytes is insufficient to mount an immune response but the

extent of extrathymic T cell development and its functional properties are still debated.

The rapid healing rate in mice and rats was noted which resulted in non-critical size

defects. However, by selecting a shorter assessment period where spontaneous bone

healing does not occur, this model is useful. A better characterisation of the entire

healing pathway could be done by having more end points and shorter time gaps.

This thesis utilised radiographic and histological techniques to provide an analysis of

new bone formation from a morphological point of view and micro-CT analysis in

relation to microscopic structural changes but did not follow through with a

measurement of the functional changes i.e. mechanical properties. Wolff’s law states

that “form follows function” which indicates that bone is a dynamic structure where

mechanical augmentation cannot be ignored [104]. Additional mechanical data would

provide a better interpretation and understanding of bone healing instead of relying

solely on the current three techniques used in this thesis i.e. histology,

immunohistochemistry and micro-CT.

Measurement and interpretation of bone healing is subjected to the technique used and

the skills of the investigators. Histology and immunohistochemistry provided a more

detailed assessment of bone healing when compared to radiographic and micro-CT

assessments. While the latter two techniques are non-invasive, the inability of these

Discussion

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two techniques to identify non-mineralised new bone leaves room for error when

assessing the progression of bone healing. The advent of computer had undoubtedly

accelerated tedious and repetitive measurements especially in histomorphometry. The

use of manual quantitative technique versus computerised image analysis such as

Bioquant remained a personal point for debate. Objective, quantitative measurement

may not be fully realised given that computerized image analysis is still dependent on

the ability of the program to make a distinction. For example, computerized image

analysis such as Bioquant provided a degree of standardization, yet poor contrast of

the stained sections may lead to manual (human) editing which then introduces human

error.

Immunohistochemical staining of whole panel factors related to bone regeneration,

aging and inflammation may give more detail of defect healing mechanism in nude vs.

normal and aged vs. young animals. The availability and cost of mouse antibodies

limit more comprehensive studies in this field.

However, not all faults lie with the model, but on how one perceives its viability. In

spite of the limitations in this dissertation, the use of a DFCD model in larger

immunodeficient animals is a promising tool for bone defect research.

Conclusion

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CHAPTER 6 Conclusion

6.1. Conclusion

Animal models in research are pre-requisites to clinical trials, where in vivo models

mimic the living environment similar to that of human being. Immunodeficient

animals possess a unique advantage to study potential bone substitutes with minimal

tissue rejection. This thesis confirmed that bone healing can progress in the absence of

T lymphocytes as seen in immunodeficient animals. While the use of immunodeficient

animals is heralded as an invaluable research tool, its unique characteristics must be

taken into considered fully before providing a conclusive result. This thesis revealed

that bone healing in immunodeficient animals is not critically different from that of

immunocompetent animals, thus alleviating the uncertainty when using

immunocompetent animals.

The outcomes of the thesis are as follows:

1. Establishment of critical sized defects in mice at a weight-bearing location was

not feasible due to the robust healing of murine. Distal-femur-condylar defect

model is a more feasible model compared to tibial window for evaluation of

potential test materials. This DFCD model can provide a valuable tool for

investigation of bone repair in genetically manipulated mice.

2. Nude mice displayed similar healing pattern (endochondral ossification and

intramembranous ossification) as normal mice despite being T cell deficient.

3. Use of older nude mice did not affect the healing rate, in contrast to older normal

Conclusion

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mice, which showed a delay in bone healing in the initial phase. This unusual

response is attributed to the lack of developed and mature T cells in nude mice,

which has been shown to affect osteoclast activation.

4. Early bone repair was impaired in middle-aged (20-week old) immunocompetent

animals.

5. By definition of critical size defect, the defect model used in nude mice was not

viable without compromising the mechanical and structural integrity due to their

small structure. However, this model is useful for short-term assessment.

6. Both the tibial window and distal-femur-condylar-defect models were carried out

successfully in nude rats. This enhances the potential of using nude rats for future

investigations of osteoinductivity and osteoconductivity of implants and bone

substitutes.

6.2. Future Direction

There is a large scope for further investigation given the different aspects that were

addressed. The role of immunodeficient animals in research is gaining momentum,

and should be characterized in detail. The need to examine the osteoinductive and

osteoconductive properties in vivo without interference from the immune system has

been discussed.

Results from this thesis suggest a new paradigm for future work related to bone

healing in immunodeficient animals. Despite their small features, nude mice are still

practical for studies involving growth factors and gene therapy. Gene manipulation

of these mice may further contribute to genetic research in bone healing. A

well-characterised defect model provides a solid foundation upon which implants and

Conclusion

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bone substitutes (synthetic and non-synthetic) materials may be investigated. A more

comprehensive and complete task in identifying related factors i.e.

immunohistochemistry and real-time polymerase chain reaction of bone healing may

be undertaken to contribute further to the characterisation of this model.

In addition, mechanical testing could be carried out to correlate the change in

mechanical properties with changes in cell morphology, biochemical signalling

pathways and microscopic structural entities. Development of nanotechnology will no

doubt be a key component to derive the complexity in bone repair in small animals.

Nude rats are considered better animal models to host potential bone substitute

materials of xenographic origin without interspecies incompatibility than nude mice.

Given their larger sizes, it is feasible to quantify changes to bone mechanical

properties, which is one of the limitations in using nude mice. The athymic nude rats

have a longer lifespan than athymic nude mice [185] and this may allow a prolonged

assessment period for certain materials such as a biodegradable matrix.

The shift from bone substitutes to tissue engineering and gene therapy has not

diminished the use of animals in research [36, 225, 226]. The transition from an

immunodeficient animal to an immunocompetent animal is the subsequent step to be

taken into consideration. Murine and rodents play a role as initial test subjects and

subsequent testing in larger animals is recommended [43]. Though the accuracy of an

animal model to a human patient remains a debated issue, it is important to stress the

importance of pre-clinical trials prior to a human trial, which is limited in terms of

subject suitability, reproducibility, viability and financial constraint. Nevertheless,

Conclusion

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results from animal studies may not be reproduced in a human body. Only human

clinical trials will provide the ultimate answer.

Reference

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Appendix

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Appendix

Tissue Processing Protocol

1. 70% alcohol 1 hour 2. 95% alcohol 1 hour 3. 95% alcohol 1 hour 4. Absolute alcohol (100%) 1 hour 5. Absolute alcohol (100%) 1 hour 6. Absolute alcohol (100%) 2 hours 7. Absolute alcohol (100%) 2 hours 8. Xylene ½ hour 9. Xylene 1½ hours 10. Xylene 2 hours 11. Paraplast (Wax) 2 hours 12. Paraplast (Wax) 2 hours

Appendix

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Haemotoxylin and Eosin (H&E) Protocol

Dewaxing

1. Xylene 5 minutes 2. Xylene 5 minutes 3. Xylene 5 minutes

Hydration

4. Absolute Ethanol 5 minutes 5. Absolute Ethanol 2 minutes 6. 95% Ethanol 2 minutes 7. 95% Ethanol 2 minutes 8. 70% Ethanol 2 minutes 9. 50% Ethanol 2 minutes 10. di-H2O 5 minutes

Staining

11. Filtered Harris haemotoxylin for 10 minutes, wash in running tap water 12. Dip in acid ethanol once, wash in running water 13. Dip 6 times in Scott’s blue and back to tap water and wash properly

Dehydration

14. 70% Ethanol 2 minutes 15. 80% Ethanol 2 minutes 16. 95% Ethanol 2 minutes 17. Absolute Ethanol 2 minutes 18. Absolute Ethanol 2 minutes

Clearing

19. Xylene 5 minutes 20. Xylene 5 minutes 21. Xylene 5 minutes 22. Mount with Eukitt solution and cover with cover slips

Appendix

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Masson’s Trichrome Protocol

Dewaxing

1. Xylene 5 minutes 2. Xylene 5 minutes 3. Xylene 5 minutes

Hydration

4. Absolute Ethanol 5 minutes 5. Absolute Ethanol 2 minutes 6. 95% Ethanol 2 minutes 7. 95% Ethanol 2 minutes 8. 70% Ethanol 2 minutes 9. 50% Ethanol 2 minutes 10. di-H2O 5 minutes

Staining

11. Filtered Weigert’s haemotoxylin for 5 minutes, wash in running tap water 12. Acid fushsin for 2-5 minutes, wash in running water 13. 1% Phosphomolybdic acid for 5 minutes, rinse in di-H2O 14. Methyl Blue or 2% Light Green for 5 minutes, wash in running water

Dehydration

15. 70% Ethanol 2 minutes 16. 80% Ethanol 2 minutes 17. 95% Ethanol 2 minutes 18. Absolute Ethanol 2 minutes 19. Absolute Ethanol 2 minutes

Clearing

20. Xylene 5 minutes 21. Xylene 5 minutes 22. Xylene 5 minutes 23. Mount with Eukitt solution and cover with cover slips

Appendix

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Histology : Haemotoxylin and Eosin (H&E)

1.0 mm defect in 12-week-old normal mice

H&E staining (100 magnification except for (a) at 40x magnification)

Appendix

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1.0 mm defect in 20-week-old nude mice

H&E staining (100x magnification)

Appendix

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1.0 mm defect in 20-week-old normal mice


Appendix

- 170 -

1.2 mm defect in 20-week-old nude mice


Appendix

- 171 -

Histology : Masson’s Trichrome

1.0 mm defect in 12-week-old nude mice (Masson’s Trichrome)

Masson’s Trichrome staining (40x magnification)

Appendix

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1.0 mm defect in 12-week-old normal mice (Masson’s Trichrome)


Appendix

- 173 -



Appendix

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1.0 mm defect in 20-week-old normal mice (Masson’s Trichrome)


Appendix

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Appendix

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Immunohistochemistry

BMP-7 expression in normal mice at Day 10 (400x magnification)

FGF-2 expression in normal mice at Day 14 (400x magnification)

Appendix

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Smad 4 expression in nude mice at Day 10 (400x magnification)

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