Characterisation of bone defect models in immunodeficientanimals
Author:Gan, Jade Ho Yue
Publication Date:2005
DOI:https://doi.org/10.26190/unsworks/22500
License:https://creativecommons.org/licenses/by-nc-nd/3.0/au/Link to license to see what you are allowed to do with this resource.
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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
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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
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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
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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
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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
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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
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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.
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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
Background and Literature Review
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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.
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Figure 2-1 Radiographs of long bones in different species (a) mouse (b) estrogen deficient rat (courtesy
of Rema Rajaratnam) (c) human
Background and Literature Review
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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
Background and Literature Review
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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.
Background and Literature Review
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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
Background and Literature Review
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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.
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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.
Background and Literature Review
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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
Background and Literature Review
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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
Background and Literature Review
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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.
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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
Background and Literature Review
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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.
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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
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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.
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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].
Background and Literature Review
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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
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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].
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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].
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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].
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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
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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.
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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
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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]
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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
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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.
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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
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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
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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
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(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.
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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
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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
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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
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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].
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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].
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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.
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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]
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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]
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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
Macrophages, NK cells
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].
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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
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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
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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
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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.
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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
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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.
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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).
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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.
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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
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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.
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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.
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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
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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.
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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.
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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.
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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.
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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.
Methodology
- 75 -
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
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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
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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
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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
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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
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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
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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
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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
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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).
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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.
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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.
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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 - + ++ ++ - + ++
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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
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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.
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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
4.1.4. Critical size defect (CSD)
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.
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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.
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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).
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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
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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.
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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.
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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.
5.1.4. Critical size defect (CSD)
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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Reference
1. Oklund, S.A., D.J. Prolo, R.V. Gutierrez, and S.E. King, Quantitative
comparisons of healing in cranial fresh autografts, frozen autografts and
processed autografts, and allografts in canine skull defects. Clin Orthop Relat
Res, 1986(205): p. 269-91.
2. Boyan, B.D., J. McMillan, C.H. Lohmann, D.N. Ranly, and Z. Schwartz, Bone
Graft Substitutes: Basic information for successful clinical use with special
focus on synthetic graft substitutes, in Bone Graft Substitutes, C.T. Laurencin,
Editor. 2003, ASTM International: West Conshohocken, PA. p. 231-259.
3. Younger, E.M. and M.W. Chapman, Morbidity at bone graft donor sites. J
Orthop Trauma, 1989. 3(3): p. 192-5.
4. Rocha, L.B., G. Goissis, and M.A. Rossi, Biocompatibility of anionic collagen
matrix as scaffold for bone healing. Biomaterials, 2002. 23(2): p. 449-56.
5. Tomford, W.W., H.J. Mankin, G.E. Friedlaender, S.H. Doppelt, and M.C.
Gebhardt, Methods of banking bone and cartilage for allograft transplantation.
Orthop Clin North Am, 1987. 18(2): p. 241-7.
6. Tomford, W.W., Bone allografts: past, present and future. Cell Tissue Bank,
2000. 1(2): p. 105-9.
7. Betz, R.R., Limitations of autograft and allograft: new synthetic solutions.
Orthopedics, 2002. 25(5 Suppl): p. s561-70.
8. Solheim, E., Osteoinduction by demineralised bone. Int Orthop, 1998. 22(5): p.
335-42.
9. Friedlaender, G.E., D.M. Strong, and K.W. Sell, Studies on the antigenicity of
Reference
- 140 -
bone. I. Freeze-dried and deep-frozen bone allografts in rabbits. J Bone Joint
Surg Am, 1976. 58(6): p. 854-8.
10. Yu, Y., J.B. Chen, J.L. Yang, W.R. Walsh, R. Verhuel, N. Johnson, and D.A.F.
Morgan, In Vivo Asesssment of Gamma Irradiated Bone:Osteoconductivity
and Osteoinductivity. Adv Tissue Banking, 2003. 7: p. 321-37.
11. Prolo, D.J., P.W. Pedrotti, K.P. Burres, and S. Oklund, Superior osteogenesis in
transplanted allogeneic canine skull following chemical sterilization. Clin
Orthop Relat Res, 1982(168): p. 230-42.
12. Balaburski, G. and J.P. O'Connor, Determination of variations in gene
expression during fracture healing. Acta Orthop Scand, 2003. 74(1): p. 22-30.
13. Bucholz, R.W., Clinical Issues in the Development of Bone Graft Substitues in
Orthopedic Trauma Care, in Bone Graft Substitutes, C.T. Laurencin, Editor.
2003, ASTM International: West Conshohocken, PA. p. 289-297.
14. Sandhu, H.S. and S.N. Khan, Animal models for preclinical assessment of bone
morphogenetic proteins in the spine. Spine, 2002. 27(16 Suppl 1): p. S32-8.
15. Bonnarens, F., Einhorn, T. A., Production of a standard closed fracture in
laboratory animal bone. J Orthop Res, 1984. 2(27): p. 97-101.
16. Brighton, C.T., Hunt, R.M., Early histological and ultrastructural changes in
medullary fracture callus. J Bone Joint Surg Am, 1991. 73A: p. 832-847.
17. Walsh, W.R., P. Sherman, C.R. Howlett, D.H. Sonnabend, and M.G. Ehrlich,
Fracture healing in a rat osteopenia model. Clin Orthop, 1997(342): p.
218-27.
18. D'Aoust, P., C.A. McCulloch, H.C. Tenenbaum, and P.C. Lekic, Etidronate
(HEBP) promotes osteoblast differentiation and wound closure in rat calvaria.
Cell Tissue Res, 2000. 302(3): p. 353-63.
Reference
- 141 -
19. Richards, M., J.A. Goulet, J.A. Weiss, N.A. Waanders, M.B. Schaffler, and S.A.
Goldstein, Bone regeneration and fracture healing. Experience with
distraction osteogenesis model. Clin Orthop, 1998(355 Suppl): p. S191-204.
20. Perren, S.M., Physical and biological aspects of fracture healing with special
reference to internal fixation. Clin Orthop Relat Res, 1979(138): p. 175-96.
21. Heiple, K.G., V.M. Goldberg, A.E. Powell, G.D. Bos, and J.M. Zika, Biology of
cancellous bone grafts. Orthop Clin North Am, 1987. 18(2): p. 179-85.
22. Lind, M., Growth factor stimulation of bone healing. Effects on osteoblasts,
osteomies, and implants fixation. Acta Orthop Scand Suppl, 1998. 283: p.
2-37.
23. Le, A.X., T. Miclau, D. Hu, and J.A. Helms, Molecular aspects of healing in
stabilized and non-stabilized fractures. J Orthop Res, 2001. 19(1): p. 78-84.
24. Porter, W.P., Rats and mice [kit]: Introduction and use in research, Parts I and
II. Laboratory Animal Medicine and Science - Series II. 1993, Seattle, WA:
Health Sciences Center for Educational Resources, University of Washington.
25. Aspenberg, P. and E. Andolf, Bone induction by fetal and adult human bone
matrix in athymic rats. Acta Orthop Scand, 1989. 60(2): p. 195-9.
26. Tsai, C.H., M.Y. Chou, M. Jonas, Y.T. Tien, and E.Y. Chi, A composite graft
material containing bone particles and collagen in osteoinduction in mouse. J
Biomed Mater Res, 2002. 63(1): p. 65-70.
27. Lane, J.M. and H.S. Sandhu, Current approaches to experimental bone
grafting. Orthop Clin North Am, 1987. 18(2): p. 213-25.
28. Asaumi, K., T. Nakanishi, H. Asahara, H. Inoue, and M. Takigawa, Expression
of neurotrophins and their receptors (TRK) during fracture healing. Bone,
2000. 26(6): p. 625-33.
Reference
- 142 -
29. Nakase, T., S. Nomura, H. Yoshikawa, J. Hashimoto, S. Hirota, Y. Kitamura, S.
Oikawa, K. Ono, and K. Takaoka, Transient and localized expression of bone
morphogenetic protein 4 messenger RNA during fracture healing. J Bone
Miner Res, 1994. 9(5): p. 651-9.
30. Yamagiwa, H., K. Tokunaga, T. Hayami, H. Hatano, M. Uchida, N. Endo, and
H.E. Takahashi, Expression of metalloproteinase-13 (Collagenase-3) is
induced during fracture healing in mice. Bone, 1999. 25(2): p. 197-203.
31. Hashimoto, J., H. Yoshikawa, K. Takaoka, N. Shimizu, K. Masuhara, T. Tsuda,
S. Miyamoto, and K. Ono, Inhibitory effects of tumor necrosis factor alpha on
fracture healing in rats. Bone, 1989. 10(6): p. 453-7.
32. Thompson, Z., T. Miclau, D. Hu, and J.A. Helms, A model for
intramembranous ossification during fracture healing. J Orthop Res, 2002.
20(5): p. 1091-8.
33. Manigrasso, M.B. and P. O'Connor J, Characterization of a closed femur
fracture model in mice. J Orthop Trauma, 2004. 18(10): p. 687-95.
34. Bourque, W.T., M. Gross, and B.K. Hall, A reproducible method for producing
and quantifying the stages of fracture repair. Lab Anim Sci, 1992. 42(4): p.
369-74.
35. Hiltunen, A., E. Vuorio, and H.T. Aro, Regulation of extracellular matrix
genes during fracture healing. Clin Orthop, 1993. 297: p. 23-27.
36. Lee, J.Y., H. Peng, A. Usas, D. Musgrave, J. Cummins, D. Pelinkovic, R.
Jankowski, B. Ziran, P. Robbins, and J. Huard, Enhancement of bone healing
based on ex vivo gene therapy using human muscle-derived cells expressing
bone morphogenetic protein 2. Hum Gene Ther, 2002. 13(10): p. 1201-11.
37. Aalami, O.O., R.P. Nacamuli, K.A. Lenton, C.M. Cowan, T.D. Fang, K.D.
Reference
- 143 -
Fong, Y.Y. Shi, H.M. Song, D.E. Sahar, and M.T. Longaker, Applications of a
mouse model of calvarial healing: differences in regenerative abilities of
juveniles and adults. Plast Reconstr Surg, 2004. 114(3): p. 713-20.
38. Lee, J.Y., D. Musgrave, D. Pelinkovic, K. Fukushima, J. Cummins, A. Usas, P.
Robbins, F.H. Fu, and J. Huard, Effect of bone morphogenetic
protein-2-expressing muscle-derived cells on healing of critical-sized bone
defects in mice. J Bone Joint Surg Am, 2001. 83-A(7): p. 1032-9.
39. Kirkeby, O.J., L. Nordsletten, and S. Skjeldal, Healing of cortical bone grafts
in athymic rats. Acta Orthop Scand, 1992. 63(3): p. 318-22.
40. Raschke, M., S. Kolbeck, H. Bail, G. Schmidmaier, A. Flyvbjerg, T. Lindner,
M. Dahne, I. Roenne, and N. Haas, Homologous growth hormone accelerates
healing of segmental bone defects. Bone, 2001. 29(4): p. 368-73.
41. Cheng, M.H., E.M. Brey, A. Allori, W.C. Satterfield, D.W. Chang, C.W.
Patrick, and M.J. Miller, Ovine model for engineering bone segments. Tissue
Eng, 2005. 11(1-2): p. 214-25.
42. Street, J.T., McGarth M., O'Regan K., Wakai A., McGuiness A., and Redmond
P., Thrimboprophylaxis Using a Low Molecular Weight Heparin Delays
Fracture repair. Clin Orthop Rel Res, 2000. 2000(381): p. 278-289.
43. Schmitz, J.P. and J.O. Hollinger, The critical size defect as an experimental
model for craniomandibulofacial nonunions. Clin Orthop, 1986(205): p.
299-308.
44. Einhorn, T.A., Clinically applied models of bone regeneration in tissue
engineering research. Clin Orthop, 1999(367 Suppl): p. S59-67.
45. Tamura, S., H. Kataoka, Y. Matsui, Y. Shionoya, K. Ohno, K. Michi, K.
Takahashi, and A. Yamaguchi, The effects of transplantation of osteoblastic
Reference
- 144 -
cells with bone morphogenetic protein (BMP)/carrier complex on bone repair.
Bone, 2001. 29(2): p. 169-75.
46. Bosch, C., B. Melsen, and K. Vargervik, Importance of the critical-size bone
defect in testing bone-regenerating materials. J Craniofac Surg, 1998. 9(4): p.
310-6.
47. Kleinschmidt, J.C. and J.O. Hollinger, Animal Models in Bone Research, in
Bone Grafts & Bone Substitutes, M.B. Habal and A.H. Reddi, Editors. 1992,
W.B. Saunders Company: Philadelphia, PA. p. 133-146.
48. Stubbs, D., M. Deakin, P. Chapman-Sheath, W. Bruce, J. Debes, R.M. Gillies,
and W.R. Walsh, In vivo evaluation of resorbable bone graft substitutes in a
rabbit tibial defect model. Biomaterials, 2004. 25(20): p. 5037-44.
49. Cheung, K.M., K. Kaluarachi, G. Andrew, W. Lu, D. Chan, and K.S. Cheah, An
externally fixed femoral fracture model for mice. J Orthop Res, 2003. 21(4): p.
685-90.
50. Brand, R.A., Fracture Healing, in Surgery of the muscoloskeletal system, C.M.
Evarts, Editor. 1983, Churchill Livingstone Inc.: New York. p. 65-87.
51. Tielinen, L., M. Manninen, P. Puolakkainen, M. Kellomaki, P. Tormala, J. Rich,
J. Seppala, and P. Rokkanen, Inability of transforming growth factor-beta 1,
combined with a bioabsorbable polymer paste, to promote healing of bone
defects in the rat distal femur. Arch Orthop Trauma Surg, 2001. 121(4): p.
191-6.
52. Uusitalo, H., J. Rantakokko, M. Ahonen, T. Jamsa, J. Tuukkanen, V. KaHari, E.
Vuorio, and H.T. Aro, A metaphyseal defect model of the femur for studies of
murine bone healing. Bone, 2001. 28(4): p. 423-9.
53. Campbell, T.M., W.T. Wong, and E.J. Mackie, Establishment of a Model of
Reference
- 145 -
Cortical Bone Repair in Mice. Calcif Tissue Int, 2003. 73: p. 1.
54. Street, J., M. Bao, L. deGuzman, S. Bunting, F.V. Peale, Jr., N. Ferrara, H.
Steinmetz, J. Hoeffel, J.L. Cleland, A. Daugherty, N. van Bruggen, H.P.
Redmond, R.A. Carano, and E.H. Filvaroff, Vascular endothelial growth factor
stimulates bone repair by promoting angiogenesis and bone turnover. Proc
Natl Acad Sci U S A, 2002. 99(15): p. 9656-61.
55. Hausman, M.R., M.B. Schaffler, and R.J. Majeska, Prevention of fracture
healing in rats by an inhibitor of angiogenesis. Bone, 2001. 29(6): p. 560-4.
56. Hankemeier, S., S. Grassel, G. Plenz, H.U. Spiegel, P. Bruckner, and A. Probst,
Alteration of fracture stability influences chondrogenesis, osteogenesis and
immigration of macrophages. J Orthop Res, 2001. 19(4): p. 531-8.
57. O'Driscoll, S.W. and J.S. Fitzsimmons, The role of periosteum in cartilage
repair. Clin Orthop, 2001(391 Suppl): p. S190-207.
58. Takayanagi, H., S. Kim, K. Matsuo, H. Suzuki, T. Suzuki, K. Sato, T. Yokochi,
H. Oda, K. Nakamura, N. Ida, E.F. Wagner, and T. Taniguchi, RANKL
maintains bone homeostasis through c-Fos-dependent induction of
interferon-beta. Nature, 2002. 416(6882): p. 744-9.
59. Kong, Y.Y., W.J. Boyle, and J.M. Penninger, Osteoprotegerin ligand: a
regulator of immune responses and bone physiology. Immunol Today, 2000.
21(10): p. 495-502.
60. Alliston, T. and R. Derynck, Medicine: Interfering with bone remodelling.
Nature, 2002. 416(6882): p. 686-7.
61. Horowitz, M.C. and G.E. Friedlaender, Immunologic aspects of bone
transplantation. A rationale for future studies. Orthop Clin North Am, 1987.
18(2): p. 227-33.
Reference
- 146 -
62. Fox, A. and L.C. Harrison, Innate immunity and graft rejection. Immunol Rev,
2000. 173: p. 141-7.
63. MacDonald, H.R., Phenotypic and functional characteristics of 'T-like' cells in
nude mice. Exp Cell Biol, 1984. 52(1-2): p. 2-6.
64. Flanagan, S.P., 'Nude', a new hairless gene with pleiotropic effects in the mouse.
Genet Res, 1966. 8(3): p. 295-309.
65. Holub, M., Immunology of nude mice. 1989, Boca Raton, FL: CRC Press, Inc.
13-52.
66. Li, J.Z., H. Li, T. Sasaki, D. Holman, B. Beres, R.J. Dumont, D.D. Pittman, G.R.
Hankins, and G.A. Helm, Osteogenic potential of five different recombinant
human bone morphogenetic protein adenoviral vectors in the rat. Gene Ther,
2003. 10(20): p. 1735-43.
67. Varady, P., J.Z. Li, M. Cunningham, E.J. Beres, S. Das, J. Engh, T.D. Alden,
D.D. Pittman, K.M. Kerns, D.F. Kallmes, and G.A. Helm, Morphologic
analysis of BMP-9 gene therapy-induced osteogenesis. Hum Gene Ther, 2001.
12(6): p. 697-710.
68. Alden, T.D., D.D. Pittman, G.R. Hankins, E.J. Beres, J.A. Engh, S. Das, S.B.
Hudson, K.M. Kerns, D.F. Kallmes, and G.A. Helm, In vivo endochondral
bone formation using a bone morphogenetic protein 2 adenoviral vector. Hum
Gene Ther, 1999. 10(13): p. 2245-53.
69. Musgrave, D.S., P. Bosch, S. Ghivizzani, P.D. Robbins, C.H. Evans, and J.
Huard, Adenovirus-mediated direct gene therapy with bone morphogenetic
protein-2 produces bone. Bone, 1999. 24(6): p. 541-7.
70. Esses, S., P. Halloran, M. Kliman, and F. Langer, Bone allografts in mice:
determinants of immunogenicity and healing. Transplant Proc, 1981. 13(1 Pt 2):
Reference
- 147 -
p. 885-7.
71. Vignery, A., A. Silverglate, M. Horowitz, L. Shultz, and R. Baron, Abnormal
bone remodeling activity in the immunodeficient nude (Nu/Nu) and Motheaten
(me/me) mutant mice. Calcif Tissue Int, 1981. 33: p. 301 (abstract).
72. McCauley, L.K., T.J. Rosol, C.C. Capen, J.E. Horton, and P.F. Piguet, A
comparison of bone turnover in athymic (nude) and euthymic mice:
biochemical, histomorphometric, bone ash and in vitro studies. Change in the
humoral response of athymic nude mice with ageing. Bone, 1989. 10(1): p.
29-34.
73. Kirkeby, O.J., Bone metabolism and repair are normal in athymic rats. Acta
Orthop Scand, 1991. 62(3): p. 253-6.
74. Baron, R., R. Tross, and A. Vignery, Evidence of sequential remodeling in rat
trabecular bone: morphology, dynamic histomorphometry, and changes
during skeletal maturation. Anat Rec, 1984. 208(1): p. 137-45.
75. Postlethwaite, A.E. and A.H. Kang, Advantages and limitations of in vitro
models of wound healing and tissue repair. Prog Clin Biol Res, 1988. 266: p.
237-42.
76. Bostrom, M.P., A.L. Boskey, J.K. Kaufman, and T.A. Einhorn, Form and
function of bone, in Orthopaedic Basic Science: Biology and Biomechanics of
the Musculoskeletal System, J.A. Buckwalter, T.A. Einhorn, and S.R. Simon,
Editors. 2000, American Academy of Orthopaedic Surgeons. p. 320-369.
77. Tencer, A.F. and K.D. Johnson, Biomechanics of Bone and Fracture, in
Biomechanics in Orthopaedic Trauma: bone fracture and fixation. 1994, J.B.
Lippincott: Philadelphia. p. 1-17.
78. Currey, J.D., The mechanical adaptations of bones. 1984, Princeton, New
Reference
- 148 -
Jersey: Princeton University Press. 294.
79. Gray, H., Gray's Anatomy. 16th ed, ed. T.P. Pick and R. Howdern. 1994,
Moscow, Russia: Krasny Proletary Printing House. 159-345.
80. Einhorn, T.A., The bone organ system: form and function, in Osteoporosis, R.
Marcus, D. Feldman, and J. Kelsey, Editors. 1996, Academic Press: San Diego,
CA. p. 3-22.
81. Eriksen, E.F., A. Vesterby, M. Kassem, F. Melsen, and L. Mosekilde, Bone
remodelling and Bone structure, in Physiology and Pharmacology of Bone, M.
G.R. and M. T.J., Editors. 1993, Springer-Verlag: Berlin Heidelberg. p. 1993.
82. Parfitt, A.M., The Physiologic And Clinical Significance of Bone
Histomorphometric Data, in Bone Histomorphometry: Techniques and
Interpretation, R.R. Recker, Editor. 1983, CRC Press Inc.: Boca Raton, FL. p.
143-223.
83. Huiskes, R. and B.V. Rietbergen, Chapter 4: Biomechanics of bone, in Basic
Orthopaedic Biomechanics and Mechano-biology, V.C. Mow and R. Huiskes,
Editors. 2005, Lippincott Williams & Wilkins: Philadelphia. p. 123-179.
84. Mandracchia, V.J., S.C. Nelson, and E.A. Barp, Current concepts of bone
healing. Clin Podiatr Med Surg, 2001. 18(1): p. 55-77.
85. Nade, S.M.L., Biology of fracture repair, in Principles of Fracture
Management, C.S. Galasko, Editor. 1984, Churchill Livingstone: Edinburgh. p.
1-24.
86. Bianco, P., Structure and mineralization of bone, in Calcification in Biological
Systems, E. Bonucci, Editor. 1992, CRC Press: Boca Raton, FL. p. 243-268.
87. Sommerfeldt, D.W. and C.T. Rubin, Biology of bone and how it orchestrates
the form and function of the skeleton. Eur Spine J, 2001. 10(Suppl 2): p.
Reference
- 149 -
S86-95.
88. Walsh, W.R., M. Walton, B. Warwick, Y. Yu, R.M. Gillies, and M. Svehla, Cell
Structure and Biology of Bone and Cartilage, in Handbook of Histology
Methods for Bone and Cartilage, Y.H. An and K.L. Martin, Editors. 2003,
Human Press Inc.: Totowa, NJ. p. 35-58.
89. Weiner, S., W. Traub, and H.D. Wagner, Lamellar bone: structure-function
relations. J Struct Biol, 1999. 126(3): p. 241-55.
90. Remedios, A., Bone and bone healing. Vet Clin North Am Small Anim Pract,
1999. 29(5): p. 1029-44, v.
91. Marieb, E.N., Human Anatomy & Physiology. Fourth ed. 1998, California:
Benjamin/Cummings Science Publishing.
92. Rubin, C.T. and L.E. Lanyon, Limb mechanics as a function of speed and gait:
a study of functional strains in the radius and tibia of horse and dog. J Exp Biol,
1982. 101: p. 187-211.
93. Rahn, B.A., Bone healing: Histological and physiological concepts, in Bone in
Clinical Orthopaedics, G. Sumner-Smith, Editor. 2002, AO Publishing:
Stuttgart. p. 286-325.
94. Steiniche, T. and E.M. Hauge, Normal Structure and Function of Bone, in
Handbook of Histology Methods for Bone and Cartilage, Y.H. An and K.L.
Martin, Editors. 2003, Humana Press Inc.: Totowa, NJ. p. 59-72.
95. Szczesny, G., Molecular aspects of bone healing and remodeling. Pol J Pathol,
2002. 53(3): p. 145-53.
96. Murakami, H. and M.A. Emery, The role of elastic fibres in the periosteum in
fracture healing in guinea pigs. I. Histological studies of the elastic fibres in
the periosteum and the possible relationship between the osteogenic cells and
Reference
- 150 -
the cells that form elastic fibres. Can J Surg, 1967. 10(3): p. 359-70.
97. Ham, W.H., Harris, R.H., Repair and Transplantation of Bone, in The
Biochemistry and Physiology of Bone: Development and Growth, G.H. Bourne,
Editor. 1971, Academic Press, Inc: New York. p. 337-399.
98. Knudson, C.B. and W. Knudson, Cartilage proteoglycans. Semin Cell Dev
Biol, 2001. 12(2): p. 69-78.
99. Mow, V.C. and A. Ratcliffe, Structure and function of articular cartilage and
meniscus, in Basic Orthopaedic Biomechanics, V.C. Mow and W.C. Hayes,
Editors. 1997, Lippincott-Raven: Philadelphia PA. p. 113-177.
100. Maroudas, A.I., Balance between swelling pressure and collagen tension in
normal and degenerate cartilage. Nature, 1976. 260(5554): p. 808-9.
101. Mow, V.C. and R. Huiskes, Chapter 1: A Brief History of Science and
Orthopaedic Biomechanics, in Basic Orthopaedic Biomechanics and
Mechano-biology, V.C. Mow and R. Huiskes, Editors. 2005, Lippincott
Williams & Wilkins: Philadelphia. p. 1-27.
102. Burstein, A.H. and V.H. Frankel, A standard test for laboratory animal bone. J
Biomech, 1971. 4: p. 155-158.
103. Adams, J.C. and D. Hamblen, Pathology of fractures and fracture healing, in
Outline of fractures including joint injuries. 1999, Churchill Livingston:
London. p. 3-18.
104. Wolff J.: translated by Maquet P.G.J. and Furlong R, The law of bone
remodelling. 1986, Berlin; New York: Springer-Verlag. 126.
105. Hattner, R., B.N. Epker, and H.M. Frost, Suggested sequential mode of control
of changes in cell behaviour in adult bone remodelling. Nature, 1965. 206(983):
p. 489-90.
Reference
- 151 -
106. McBroom, R.J., E.J. Cheal, and W.C. Hayes, Strength reductions from
metastatic cortical defects in long bones. J Orthop Res, 1988. 6(3): p. 369-78.
107. Edgerton, B.C., K.N. An, and B.F. Morrey, Torsional strength reduction due to
cortical defects in bone. J Orthop Res, 1990. 8(6): p. 851-5.
108. The Burden of Brittle Bones: Costing Osteoporosis in Australia. 2001, Access
Economics Pty Limited: Canberra ACT.
109. Blauvelt, C.T. and F.R.T. Nelson, A Manual of Orthopaedic Terminilogy. Sixth
Edition ed. 1998, St. Louis, Missouri, USA: Mosby Inc. 463.
110. Moucha, C.S. and T.A. Einhorn, Bone Morphogenetic Proteins and Other
Growth Factors to Enhance Fracture Healing and Treatment of Nonunions, in
Bone regeneration and Repair: Biology and Clinical Applications, J.R.
Lieberman and G.E. Friedlaender, Editors. 2005, Humana Press Inc.: Totowa,
New Jersey, USA. p. 169-195.
111. Campbell's Operative Orthopaedics. Eighth ed, ed. A.H. Crenshaw. Vol.
Volume 2. 1992, St. Louis: Mosby-Year Book, Inc. 1461.
112. Knighton, D.R., T.K. Hunt, H. Scheuenstuhl, B.J. Halliday, Z. Werb, and M.J.
Banda, Oxygen tension regulates the expression of angiogenesis factor by
macrophages. Science, 1983. 221(4617): p. 1283-5.
113. Schultz, R.J., The Language of Fractures. 1972, Baltimore, Md: The William
& Wilkins Company. 394.
114. Adams, J.C. and D.L. Hamblen, Outline of Orthopaedics. 13th ed. 2001,
London: Churchill Livingstone. 461.
115. Australian Orthopaedic Association National Joint Replacement Registry.
Annual Report. 2004, AOA: Adelaide.
116. Simmons, D.J., Fracture healing perspectives. Clin Orthop, 1985(200): p.
Reference
- 152 -
100-13.
117. Einhorn, T.A., The Cell and Molecular Biology of Fracture Healing. Clinical
Orthopaedics and Related Research, 1998. 355S: p. S7-S21.
118. White, A.A., 3rd, M.M. Panjabi, and W.O. Southwick, The four biomechanical
stages of fracture repair. J Bone Joint Surg Am, 1977. 59(2): p. 188-92.
119. Tomford, W.W. and H.J. Mankin, Bone banking. Update on methods and
materials. Orthop Clin North Am, 1999. 30(4): p. 565-70.
120. Cook, S.D. and D.C. Rueger, Preclinical models of recombinant BMP induced
healing of orthopedic defects, in Bone Morphogenetic Proteins: From
Laboratory to Clincial Practice, S. Vukicevic and K.T. Sampath, Editors. 2002,
Birkhauser Verlag: Basel. p. 121-144.
121. Shors, E.C., The Development of Coralline Porous Ceramic Graft Substitutes,
in Bone Graft Substitutes, C.T. Laurencin, Editor. 2003, ASTM International:
West Conshohocken, PA.
122. Boyan, B.D., Z. Schwartz, and A.L. Boskey, The importance of mineral in
bone and mineral research. Bone, 2000. 27(3): p. 341-2.
123. Thibodeau, G.A., Anatomy and Physiology. 1987, St. Louis: Times Mirror /
Mosby College Publishing. 140-154.
124. Yasui, N., M. Sato, T. Ochi, T. Kimura, H. Kawahata, Y. Kitamura, and S.
Nomura, Three modes of ossification during distraction osteogenesis in the rat.
J Bone Joint Surg Br, 1997. 79(5): p. 824-30.
125. Sato, M., N. Yasui, T. Nakase, H. Kawahata, M. Sugimoto, S. Hirota, Y.
Kitamura, S. Nomura, and T. Ochi, Expression of bone matrix proteins mRNA
during distraction osteogenesis. J Bone Miner Res, 1998. 13(8): p. 1221-31.
126. Feldman, R.S., N.S. Krieger, and A.H. Tashjian, Jr., Effects of parathyroid
Reference
- 153 -
hormone and calcitonin on osteoclast formation in vitro. Endocrinology, 1980.
107(4): p. 1137-43.
127. Chambers, T.J., N.A. Athanasou, and K. Fuller, Effect of parathyroid hormone
and calcitonin on the cytoplasmic spreading of isolated osteoclasts. J
Endocrinol, 1984. 102(3): p. 281-6.
128. Ham, W.H. and R.H. Harris, Repair and Transplantation of Bone, in The
Biochemistry and Physiology of Bone: Development and Growth, G.H. Bourne,
Editor. 1972, Academic Press, Inc.: New York. p. 337-399.
129. Bassett, C., Current concepts on bone formation. J Bone Joint Surg Am, 1962.
44A(1217).
130. Nemeth, G.G., M.E. Bolander, and G.R. Martin, Growth factors and their role
in wound and fracture healing. Prog Clin Biol Res, 1988. 266: p. 1-17.
131. Murphey, M.D., D.J. Sartoris, and J.M. Bramble, Radiographic Assessment of
Bone Grafts, in Bone Grafts & Bone Substitues, M.B. Habal and A.H. Reddi,
Editors. 1992, W.B. Saunders Company: Philadelphia, PA. p. 9-36.
132. Sedlin, E.D., H.M. Frost, and A.R. Villanueva, AGE CHANGES IN
RESORPTION IN HUMAN RIB CORTEX. J Gerontol, 1963. 18: p. 345-9.
133. Jaworski, Z.F.G., Histomorphometric Characteristics of Metabolic Bone
Disease, in Bone Histomorphometry: Techniques and Interpretation, R.R.
Recker, Editor. 1983, CRC Press, Inc.: Boca Raton, FL. p. 241-263.
134. Mosekilde, L. and L. Mosekilde, Iliac crest trabecular bone volume as
predictor for vertebral compressive strength, ash density and trabecular bone
volume in normal individuals. Bone, 1988. 9(4): p. 195-9.
135. Epker, B.N., M. Kelin, and H.M. Frost, Magnitude and location of cortical
bone loss in human rib with aging. Clin Orthop Relat Res, 1965. 41: p.
Reference
- 154 -
198-203.
136. Mosekilde, L., Age-related changes in vertebral trabecular bone
architecture--assessed by a new method. Bone, 1988. 9(4): p. 247-50.
137. Bak, B. and T.T. Andreassen, The effect of growth hormone on fracture healing
in old rats. Bone, 1991. 12(3): p. 151-4.
138. Friedenberg, Z.B., Musculoskeletal surgery in eighteenth century America.
Clin Orthop Relat Res, 2000(374): p. 10-6.
139. Urist, M.R., Bone: formation by autoinduction. Science, 1965. 150(698): p.
893-9.
140. Weitzmann, M.N., S. Cenci, L. Rifas, J. Haug, J. Dipersio, and R. Pacifici, T
cell activation induces human osteoclast formation via receptor activator of
nuclear factor kappaB ligand-dependent and -independent mechanisms. J
Bone Miner Res, 2001. 16(2): p. 328-37.
141. Sandhu, H.S., H.S. Grewal, and H. Parvataneni, Bone grafting for spinal fusion.
Orthop Clin North Am, 1999. 30(4): p. 685-98.
142. Habal, M.B., Different forms of bone grafts, in Bone Grafts & Bone Substitutes,
M.B. Habal and A.H. Reddi, Editors. 1992, W.B. Saunders Company:
Philadelphia, PA. p. 6-8.
143. Tagil, M., The morselized and impacted bone graft. Animal experiments on
proteins, impaction and load. Acta Orthop Scand Suppl, 2000. 290: p. 1-40.
144. Lieberman, J.R., A. Daluiski, and T.A. Einhorn, The role of growth factors in
the repair of bone. Biology and clinical applications. J Bone Joint Surg Am,
2002. 84-A(6): p. 1032-44.
145. Spector, J.A., J.S. Luchs, B.J. Mehrara, J.A. Greenwald, L.P. Smith, and M.T.
Longaker, Expression of bone morphogenetic proteins during membranous
Reference
- 155 -
bone healing. Plast Reconstr Surg, 2001. 107(1): p. 124-34.
146. Bostrom, M.P., Expression of bone morphogenetic proteins in fracture healing.
Clin Orthop, 1998(355 Suppl): p. S116-23.
147. Cheung, E.V., D.S. Katti, R.N. Rosier, and C.T. Laurencin, Review of State of
the Art: Growth Factor-based systems for use as bone graft substitutes, in
Bone Graft Substitutes, C.T. Laurencin, Editor. 2003, ASTM International:
West Conshohocken, PA. p. 174-193.
148. Reddi, A.H., S. Wientroub, and N. Muthukumaran, Biologic principles of bone
induction. Orthop Clin North Am, 1987. 18(2): p. 207-12.
149. Lacroix, P., Recent investigations on the growth of bone. Nature, 1945. 156: p.
576-583.
150. Yamaguchi, A., [Recent advances in research on bone formation--BMP action
and its mechanism]. Nippon Rinsho, 2002. 60(Suppl 3): p. 40-7.
151. Bostrom, M.P., J.M. Lane, W.S. Berberian, A.A. Missri, E. Tomin, A. Weiland,
S.B. Doty, D. Glaser, and V.M. Rosen, Immunolocalization and expression of
bone morphogenetic proteins 2 and 4 in fracture healing. J Orthop Res, 1995.
13(3): p. 357-67.
152. Yu, Y., J.L. Yang, P.J. Chapman-Sheath, and W.R. Walsh, TGF-beta, BMPS,
and their signal transducing mediators, Smads, in rat fracture healing. J
Biomed Mater Res, 2002. 60(3): p. 392-7.
153. Cheline, A.J., A.H. Reddi, and R.B. Martin, Bone morphogenetic protein-7
selectively enhances mechanically induced bone formation. Bone, 2002. 31(5):
p. 570-574.
154. Cook, S.D., G.C. Baffes, M.W. Wolfe, T.K. Sampath, and D.C. Rueger,
Recombinant human bone morphogenetic protein-7 induces healing in a
Reference
- 156 -
canine long-bone segmental defect model. Clin Orthop Relat Res, 1994(301): p.
302-12.
155. Oursler, M.J., Osteoclast synthesis and secretion and activation of latent
transforming growth factor beta. J Bone Miner Res, 1994. 9(4): p. 443-52.
156. Letterio, J.J. and A.B. Roberts, Regulation of immune responses by TGF-beta.
Annu Rev Immunol, 1998. 16: p. 137-61.
157. Gospodarawicz, D., Biological Activities of Fibroblast Growth Factors, in The
Fibroblast Growth Factor Family, A. Baird and M. Klagsbrun, Editors. 1991,
Annals of The New York Academy of Sciences: New York, N.Y. p. 1-8.
158. Radomsky, M.L., A.Y. Thompson, R.C. Spiro, and J.W. Poser, Potential role of
fibroblast growth factor in enhancement of fracture healing. Clin Orthop,
1998(355 Suppl): p. S283-93.
159. Canalis, E., T.L. McCarthy, and M. Centrella, The role of growth factors in
skeletal remodeling. Endocrinol Metab Clin North Am, 1989. 18(4): p. 903-18.
160. Joyce, M.E., S. Jingushi, S.P. Scully, and M.E. Bolander, Role of growth
factors in fracture healing. Prog Clin Biol Res, 1991. 365: p. 391-416.
161. Takeda, S., J.P. Bonnamy, M.J. Owen, P. Ducy, and G. Karsenty, Continuous
expression of Cbfa1 in nonhypertrophic chondrocytes uncovers its ability to
induce hypertrophic chondrocyte differentiation and partially rescues
Cbfa1-deficient mice. Genes Dev, 2001. 15(4): p. 467-81.
162. Martin, I., A. Muraglia, G. Campanile, R. Cancedda, and R. Quarto, Fibroblast
growth factor-2 supports ex vivo expansion and maintenance of osteogenic
precursors from human bone marrow. Endocrinology, 1997. 138(10): p.
4456-62.
163. Ducy, P., R. Zhang, V. Geoffroy, A.L. Ridall, and G. Karsenty, Osf2/Cbfa1: a
Reference
- 157 -
transcriptional activator of osteoblast differentiation. Cell, 1997. 89(5): p.
747-54.
164. Karsenty, G., Role of Cbfa1 in osteoblast differentiation and function. Semin
Cell Dev Biol, 2000. 11(5): p. 343-6.
165. de Crombrugghe, B., V. Lefebvre, and K. Nakashima, Regulatory mechanisms
in the pathways of cartilage and bone formation. Curr Opin Cell Biol, 2001.
13(6): p. 721-7.
166. Ferrara, N., H.P. Gerber, and J. LeCouter, The biology of VEGF and its
receptors. Nat Med, 2003. 9(6): p. 669-76.
167. Reddi, A.H., Bone morphogenetic proteins and related cytokines, in TGF-b
and related cytokines in inflammation, S.N. Breit and S.M. Wahl, Editors. 2001,
Birkhauser-Verlag: Basel, Switzerland. p. 147-156.
168. Joyce, M.E., S. Jingushi, and M.E. Bolander, Transforming growth factor-beta
in the regulation of fracture repair. Orthop Clin North Am, 1990. 21(1): p.
199-209.
169. Ballock, R.T. and R.J. O'Keefe, The biology of the growth plate. J Bone Joint
Surg Am, 2003. 85-A(4): p. 715-26.
170. Datto, M. and X.F. Wang, The Smads: transcriptional regulation and mouse
models. Cytokine Growth Factor Rev, 2000. 11(1-2): p. 37-48.
171. Suckow, M.A., P. Danneman, and C. Brayton, The Laboratory Mouse. The
Laboratory Animal Pocket Reference Series, ed. M.A. Suckow. 2001, Boca
Raton, Fl: CRC Press Inc.
172. Tay, B.K., A.X. Le, S.E. Gould, and J.A. Helms, Histochemical and molecular
analyses of distraction osteogenesis in a mouse model. J Orthop Res, 1998.
16(5): p. 636-42.
Reference
- 158 -
173. Animal Resources Centre, personal communication: Canning Vale, WA,
Australia.
174. Shultz, L.D. and C.L. Sidman, Genetically determined murine models of
immunodeficiency. Annu Rev Immunol, 1987. 5: p. 367-403.
175. The Jackson Laboratory, Immunodeficient Model Selection: Choosing a nude,
scid or Rag1 strain, in JAX Communications No. 2. 2000, The Jackson
Laboratory: Bar Harbor, Maine, USA.
176. Askalonov, A.A., S.M. Gordienko, O.E. Avdyunicheva, A.V. Bondarenko, and
S.F. Voronkov, The role of T-system immunity in reparatory regeneration of the
bone tissue in animals. J Hyg Epidemiol Microbiol Immunol, 1987. 31(2): p.
219-24.
177. Barbul, A. and M.C. Regan, The regulatory role of T lymphocytes in wound
healing. J Trauma, 1990. 30: p. S97-100.
178. Hansen, C.T., The Nude Gene and Its Effects, in The Nude Mouse in
Experimental and Clinical Research, J. Fogh and B.C. Giovanella, Editors.
1978, Academic Press, Inc: New York. p. 1-13.
179. Institute of Laboratory Animal Resources, Guide for the care and Use of the
Nude (Thymus-Deficient) Mouse in Biomedical Research. 1976, National Acad.
Sci.: Washington D.C. p. M1-M20.
180. JAX Laboratory, personal communication: Maine, USA.
181. Pantelouris, E.M., Absence of thymus in a mouse mutant. Nature, 1968.
217(126): p. 370-1.
182. Pelleitier, M. and S. Montplaisir, The nude mouse: a model of deficient T-cell
function. Methods Achiev Exp Pathol, 1975. 7: p. 149-66.
183. Immunodeficient Model Selection: Choosing a nude, scid or Rag1 strain, in
Reference
- 159 -
JAX Communications No. 2. 2000, The Jackson Laboratory: Bar Harbor,
Maine, USA.
184. Immunodeficient Model Selection: Choosing a nude, scid or Rag1 strain. JAX
Communications No. 2, 2000.
185. Hougen, H.P., The athymic nude rat. Immunobiological characteristics with
special reference to establishment of non-antigen-specific T-cell reactivity and
induction of antigen-specific immunity. APMIS Suppl, 1991. 21: p. 1-39.
186. Rolstad, B., The athymic nude rat: an animal experimental model to reveal
novel aspects of innate immune responses? Immunol Rev, 2001. 184: p.
136-44.
187. Pantelouris, E.M., Observations on the immunobiology of 'nude' mice.
Immunology, 1971. 20(2): p. 247-52.
188. Gaertner, D.J., R.O. Jacoby, A.L. Smith, R.B. Ardito, and F.X. Paturzo,
Persistence of rat parvovirus in athymic rats. Arch Virol, 1989. 105(3-4): p.
259-68.
189. Maier, S., C. Tertilt, N. Chambron, K. Gerauer, N. Huser, C.D. Heidecke, and
K. Pfeffer, Inhibition of natural killer cells results in acceptance of cardiac
allografts in CD28-/- mice. Nat Med, 2001. 7(5): p. 557-62.
190. Rygaard, J. and C.O. Povlsen, The nude mouse vs. the hypothesis of
immunological surveillance. Transplant Rev, 1976. 28: p. 43-61.
191. Cohen, I.R. and M. Feldman, Cellular interactions controlling the immune
reactivity of T-lymphocytes. Ann N Y Acad Sci, 1975. 249: p. 106-15.
192. Miller, R.A., Biochemical and genetic analyses of T cell aging in mice.
Springer Semin Immunopathol, 2002. 24(1): p. 61-73.
193. Leunig, M., F. Yuan, D.A. Berk, L.E. Gerweck, and R.K. Jain, Angiogenesis
Reference
- 160 -
and growth of isografted bone: quantitative in vivo assay in nude mice. Lab
Invest, 1994. 71(2): p. 300-7.
194. Edwards, J.T., M.H. Diegmann, and N.L. Scarborough, Osteoinduction of
human demineralized bone: characterization in a rat model. Clin Orthop,
1998(357): p. 219-28.
195. Ripamonti, U., A. Magan, S. Ma, B. van den Heever, T. Moehl, and A.H. Reddi,
Xenogeneic osteogenin, a bone morphogenetic protein, and demineralized
bone matrices, including human, induce bone differentiation in athymic rats
and baboons. Matrix, 1991. 11(6): p. 404-11.
196. Barbul, A., T. Shawe, S.M. Rotter, J.E. Efron, H.L. Wasserkrug, and S.B.
Badawy, Wound healing in nude mice: a study on the regulatory role of
lymphocytes in fibroplasia. Surgery, 1989. 105(6): p. 764-9.
197. Heiple, K.G., C.H. Herndon, S.W. Chase, and A. Wattleworth, Osteogenic
induction by osteosarcoma and normal bone in mice. J Bone Joint Surg Am,
1968. 50(2): p. 311-25.
198. Melsen, F., L. Mosekilde, and J. Kragstrup, Metabolic bone diseases as
evaluated by bone histomorphometry, in Bone Histomorphometry: Techniques
and Interpretation, R.R. Recker, Editor. 1983, CRC Press Inc.: Boca Raton, Fl.
p. 265-284.
199. Sanchez, M., K. Lindroth, E. Sverremark, A. Gonzalez Fernandez, and C.
Fernandez, The response in old mice: positive and negative immune memory
after priming in early age. Int Immunol, 2001. 13(10): p. 1213-21.
200. Hunig, T., T-cell function and specificity in athymic mice. Immunol Today,
1983. 4: p. 84.
201. Eerola, I., H. Uusitalo, H. Aro, and E. Vuorio, Production of cartilage
Reference
- 161 -
collagens during metaphyseal bone healing in the mouse. Matrix Biol, 1998.
17(4): p. 317-20.
202. Faxitron X-ray Corporation: Wheeling, IL. p. http://www.faxitron.com.
203. Tumuluri, V., R. Markham, and G.A. Thomas, A Review of the Fundamental
Aspects of Immunohistochemistry. Aust J Med Sc, 1999. 20(3): p. 73.
204. Kiernan, J.A., Chapter 19. 3rd Ed. ed. Immunohistochemistry, Histological &
Histochemical Methods: Theory & Practice. 1999, Somerset, UK.: The Bath
Press.
205. Boenisch, T., Staining methods, in Handbook Immunochemical staining
methods, T. Boenisch, Editor. 2001, DakoCytomation: California, US. p.
26-31.
206. Yu, Y., W.R. Walsh, D.H. Sonnabend, J.L. Yang, F. Bonar, B. Markovic, W.
Bruce, L. Kohan, and M. Neil, Cytokines and matrix metalloproteinases mRNA
expression in archival human tissues from failed total hip arthroplasty using in
situ hybridization and color video image analysis. Bull Hosp Jt Dis, 1998.
57(1): p. 23-9.
207. SkyScan, SkyScan 1072 - Desktop x-ray Microtomograph (Instruction
Manual). 1998-2001: Belgium.
208. Yang, J.L., D. Seetoo, Y. Wang, M. Ranson, C.R. Berney, J.M. Ham, P.J.
Russell, and P.J. Crowe, Urokinase-type plasminogen activator and its
receptor in colorectal cancer: independent prognostic factors of metastasis
and cancer-specific survival and potential therapeutic targets. Int J Cancer,
2000. 89(5): p. 431-9.
209. Hahn, M., M. Vogel, M. Pompesius-Kempa, and G. Delling, Trabecular bone
pattern factor--a new parameter for simple quantification of bone
Reference
- 162 -
microarchitecture. Bone, 1992. 13(4): p. 327-30.
210. Brand, R.A., Fracture Healing, in Surgery of the muscoloskeletal system, C.M.
Evarts, Editor. 1983, Churchill Livingstone Inc.: New York. p. 1:65-87.
211. Ekeland, A., L.B. Engesaeter, and N. Langeland, Mechanical properties of
fractured and intact rat femora evaluated by bending, torsional and tensile
tests. Acta Orthop Scand, 1981. 52(6): p. 605-13.
212. Caplan, A.I., Bone development. Ciba Found Symp, 1988. 136: p. 3-21.
213. Chidgey, L., D. Chakkalakal, A. Blotcky, and J.F. Connolly, Vascular
reorganization and return of rigidity in fracture healing. J Orthop Res, 1986.
4(2): p. 173-9.
214. Glimcher, M.J., F. Shapiro, R.D. Ellis, and D.R. Eyre, Changes in tissue
morphology and collagen composition during the repair of cortical bone in the
adult chicken. J Bone Joint Surg Am, 1980. 62(6): p. 964-73.
215. Hansson, G.K., J. Holm, S. Holm, Z. Fotev, H.J. Hedrich, and J. Fingerle, T
lymphocytes inhibit the vascular response to injury. Proc Natl Acad Sci U S A,
1991. 88(23): p. 10530-4.
216. Leibovich, S.J. and R. Ross, The role of the macrophage in wound repair. A
study with hydrocortisone and antimacrophage serum. Am J Pathol, 1975.
78(1): p. 71-100.
217. The Jackson Laboratory, JAX Mice Data Sheet. 2005.
218. Rocha, B., D. Guy-Grand, and P. Vassalli, Extrathymic T cell differentiation.
Curr Opin Immunol, 1995. 7(2): p. 235-42.
219. Saijo, M., R. Kitazawa, M. Nakajima, M. Kurosaka, S. Maeda, and S.
Kitazawa, Heparanase mRNA expression during fracture repair in mice.
Histochem Cell Biol, 2003. 15: p. 15.
Reference
- 163 -
220. Zhang, M., R.M. Powers, Jr., and L. Wolfinbarger, Jr., A quantitative
assessment of osteoinductivity of human demineralized bone matrix. J
Periodontol, 1997. 68(11): p. 1076-84.
221. Klinman, N.R. and G.H. Kline, The B-cell biology of aging. Immunol Rev,
1997. 160: p. 103-14.
222. Radzikowski, C., J. Rygaard, W. Budzynski, J.P. Stenvang, M. Schou, A.
Vangsted, and J. Zeuthen, Strain- and age-dependent natural and activated in
vitro cytotoxicity in athymic nude mice. Apmis, 1994. 102(7): p. 481-8.
223. Leunig, M., R. Hertel, K.A. Siebenrock, F.T. Ballmer, J.W. Mast, and R. Ganz,
The evolution of indirect reduction techniques for the treatment of fractures.
Clin Orthop, 2000(375): p. 7-14.
224. Gardner, T.N. and S. Mishra, The biomechanical environment of a bone
fracture and its influence upon the morphology of healing. Med Eng Phys,
2003. 25(6): p. 455-64.
225. Gao, J., J.E. Dennis, L.A. Solchaga, A.S. Awadallah, V.M. Goldberg, and A.I.
Caplan, Tissue-Engineered Fabrication of an Osteochondral Composite Graft
Using Rat Bone Marrow-Derived Mesenchymal Stem Cells. Tissue Eng, 2001.
7(4): p. 363-71.
226. Niyibizi, C., A. Baltzer, C. Lattermann, M. Oyama, J.D. Whalen, P.D. Robbins,
and C.H. Evans, Potential role for gene therapy in the enhancement of fracture
healing. Clin Orthop, 1998(355 Suppl): p. S148-53.
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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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)
Masson’s Trichrome staining (40x magnification)
Appendix
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1.0 mm defect in 20-week-old nude mice (Masson’s Trichrome)
Masson’s Trichrome staining (40x magnification)
Appendix
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1.0 mm defect in 20-week-old normal mice (Masson’s Trichrome)
Masson’s Trichrome staining (40x magnification)
Appendix
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1.2 mm defect in 20-week-old nude mice (Masson’s Trichrome)
Masson’s Trichrome staining (40x magnification)
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)