Application of a human bone engineering platform to an in vitro and in vivo breast cancer metastasis model

Application of a Human Bone Engineering
Platform to an In Vitro and In Vivo Breast
Cancer Metastasis Model
Verena Maria Charlotte Reichert, MD
Faculty of Built Environment and Engineering, School of Engineering
Systems, Queensland University of Technology

Thesis submitted for:
Doctor of Philosophy (PhD)

2011



Keywords
Breast cancer, bone metastasis, human osteoblast matrix, breast cancer
related bone disease, adhesion, single cell force spectroscopy, migration,
invasion, tissue engineering, scaffold, polycaprolactone, osteoblasts, human
mesenchymal stem cells

I


II


Abstract
Breast cancer in its advanced stage has a high predilection to the skeleton.
Currently, treatment options of breast cancer-related bone metastasis are
restricted to only palliative therapeutic modalities. This is due to the fact that
mechanisms regarding the breast cancer celI-bone colonisation as well as

the interactions of breast cancer cells with the bone microenvironment are
not fully understood, yet. This might be explained through a lack of
appropriate in vitro and in vivo models that are currently addressing the
above mentioned issue.
Hence the hypothesis that the translation of a bone tissue engineering
platform could lead to improved and more physiological in vitro and in vivo
model systems in order to investigate breast cancer related bone
colonisation was embraced in this PhD thesis.
Therefore the first objective was to develop an in vitro model system that
mimics human mineralised bone matrix to the highest possible extent to
examine the specific biological question, how the human bone matrix
influences breast cancer cell behaviour. Thus, primary human osteoblasts
were isolated from human bone and cultured under osteogenic conditions.
Upon ammonium hydroxide treatment, a cell-free intact mineralised human
bone matrix was left behind. Analyses revealed a similar protein and mineral
composition of the decellularised osteoblast matrix to human bone. Seeding
of a panel of breast cancer cells onto the bone mimicking matrix as well as
reference substrates like standard tissue culture plastic and collagen coated
tissue culture plastic revealed substrate specific differences of cellular
behaviour. Analyses of attachment, alignment, migration, proliferation,
invasion, as well as downstream signalling pathways showed that these
cellular properties were influenced through the osteoblast matrix.
The second objective of this PhD project was the development of a human
ectopic

bone

polycaprolactone

model

in

NOD/SCID

tricalcium phosphate

mice

using

medical

grade

(mPCL-TCP) scaffold. Human

osteoblasts and mesenchymal stem cells were seeded onto an mPCL-TCP
III


scaffold, fabricated using a fused deposition modelling technique. After
subcutaneous implantation in conjunction with the bone morphogenetic
protein 7, limited bone formation was observed due to the mechanical
properties of the applied scaffold and restricted integration into the soft tissue
of flank of NOD/SCID mice. Thus, a different scaffold fabrication technique
was chosen using the same polymer. Electrospun tubular scaffolds were
seeded with human osteoblasts, as they showed previously the highest
amount of bone formation and implanted into the flanks of NOD/SCID mice.
Ectopic bone formation with sufficient vascularisation could be observed.

After implantation of breast cancer cells using a polyethylene glycol hydrogel
in close proximity to the newly formed bone, macroscopic communication
between the newly formed bone and the tumour could be observed.
Taken together, this PhD project showed that bone tissue engineering
platforms could be used to develop an in vitro and in vivo model system to
study cancer cell colonisation in the bone microenvironment.

IV


Table of contents
Keywords ..................................................................................................... I
Abstract ..................................................................................................... III
Table of contents ........................................................................................ V
List of illustrations and diagrams ............................................................. XIII
List of abbreviations ................................................................................ XIX
Statement of original authorship ............................................................ XXV
Acknowledgements.............................................................................. XXVII
Chapter 1 - Overview .................................................................................... 1
1. Overview .............................................................................................. 3
Chapter 2 - Literature Review - Mechanisms of breast cancer-related bone
metastasis – overview of currently used in vitro and in vivo models .............. 5
2.1

Epidemiology – facts about breast cancer ........................................ 7

2.2

Clinical presentation of bone metastases and treatment options ...... 8

2.3

Tumour cell metastasis – a multistep process – from the primary site

to the skeleton .......................................................................................... 11
2.4

Characteristics of the “the congenial soil” – bone ........................... 13

2.5

How the “seeds” find their “congenial” soil ...................................... 18

2.6

Attachment at the metastatic site - interaction of integrins with bone

matrix constituents .................................................................................... 19
2.7

More factors involved in the promotion of bone metastasis ............ 23

2.8

Osteomimicry of breast cancer cells ............................................... 26

2.9

Osteolytic bone metastasis ............................................................. 27

2.10

Models investigating bone-cancer cell interaction in vitro ............ 29

2.11

Models investigating bone-cancer cell interaction in vivo ............ 37

2.12

Summary ..................................................................................... 46

Chapter 3 - Establishment of a human primary osteoblast matrix model .... 49
3.1

Introduction ..................................................................................... 51
V


3.2

Material and Methods ...................................................................... 53

3.2.1

Characterisation of type I collagen coated tissue culture plastic ...
................................................................................................. 53

3.2.1.1

Collagen type I coating of tissue culture polystyrene or

thermanox coverslips.......................................................................... 53
3.2.1.2

Anti-col I staining of col I coated coverlips ......................... 53

3.2.1.3

Scanning electron microscopy analysis of col I coated

thermanox coverslips.......................................................................... 54
3.2.2

Generation and characterisation of primary human osteoblast

matrices ................................................................................................. 54
3.2.2.1

Isolation

of

human

primary

osteoblasts

and

matrix

production........................................................................................... 54
3.2.2.2

Confocal laser microscopy to confirm completeness of

decellularisation .................................................................................. 55
3.2.2.3

Morphology - SEM ............................................................. 56

3.2.2.4

Morphology - Transmission Electron microscopy ............... 56

3.2.2.5

Mineralisation - Alizarin red S ............................................ 56

3.2.2.6

Mineralisation - Wako HRII calcium assay ......................... 56

3.2.2.7

Mineralisation - X-ray photoelectron spectroscopy (XPS) .. 57

3.2.2.8

Mineralisation - RAMAN analysis ....................................... 57

3.2.2.9

Composition - Immunohistochemistry ................................ 58

3.2.2.10 Composition - Growth factor analysis................................. 59
3.2.2.11 Composition - 2D PAGE with MALDI or LC/MS/MS ........... 59
3.2.2.12 Composition - Western blot analysis .................................. 59
3.2.3

Image analysis .......................................................................... 60

3.2.4

Statistical analysis .................................................................... 61

3.3

Results ............................................................................................ 62

VI


3.3.1

Presence and Morphology of collagen I on coated thermanox

coverslips .............................................................................................. 62

3.3.2

Characterisation of the composition and morphology of the

human decellularised osteoblast matrix ................................................ 63
3.4

Discussion ...................................................................................... 75

3.5

Conclusion ...................................................................................... 79

Chapter 4 - Interactions of human breast cancer cells with the OBM.......... 81
4.1

Introduction ..................................................................................... 83

4.2

Materials and Methods.................................................................... 85

4.2.1

Characteristics and growth conditions for cell lines used in this

study

................................................................................................. 85

4.2.1.1

MCF10A ............................................................................ 85

4.2.1.2

T47D .................................................................................. 86

4.2.1.3

SUM1315 ........................................................................... 86

4.2.1.4

MDA-MB-231 ..................................................................... 86

4.2.1.5

MDA-MB-231SA ................................................................ 87

4.2.1.6

Gene expression profile ..................................................... 88

4.2.2

Morphology .............................................................................. 89

4.2.2.1

Flow cytometry................................................................... 89

4.2.2.2

Morphology - Transmitted light microscopy ....................... 90

4.2.2.3

Morphology – Fluorescent staining .................................... 90

4.2.2.4

Morphology - SEM ............................................................. 91

4.2.3

Cell proliferation ....................................................................... 92

4.2.4

Cell adhesion ........................................................................... 92

4.2.5

Cell migration ........................................................................... 95

4.2.6

Cell Invasion ............................................................................. 96

4.2.6.1

SEM image analysis .......................................................... 96

4.2.6.2

Confocal microscopy image analysis ................................. 98
VII


4.2.7

Cell signalling pathways ........................................................... 98

4.2.8

Gene expression .................................................................... 100

4.2.8.1

RNA isolation ................................................................... 100

4.2.8.2

cDNA synthesis ................................................................ 100

4.2.8.3

qRT-PCR ......................................................................... 101

4.2.9
4.3

Statistical analysis .................................................................. 102

Results .......................................................................................... 103

4.3.1

Characteristics of utilised cell lines ......................................... 103

4.3.1.1

MCF10A ........................................................................... 103

4.3.1.2

T47D ................................................................................ 104

4.3.1.3

SUM1315 ......................................................................... 105

4.3.1.4

MDA-MB-231 ................................................................... 106

4.3.1.5

MDA-MB-231-SA ............................................................. 107

4.3.2

Cell morphology of different cell lines on TCP, col I and OBM 113

4.3.2.1

Morphology on TCP, col I and OBM assessed with

transmitted light and confocal laser scanning microscopy ................ 113
4.3.2.2
4.3.3

Image analysis ................................................................. 116

Proliferation of MCF10A, T47D, SUM1315, MDA-MB231 and

MDA-MB-231SA on TCP, col I and OBM ............................................ 120
4.3.4

Assessment of established detachment forces of each cell line

to disconnect from col I and OBM ........................................................ 121
4.3.5

Migratory features of the 5 BC cell lines on the three

investigated substrates ........................................................................ 123
4.3.6

Invasive potential of MCF10A, T47D, SUM1315, MDA-MB-231

and MDA-MB-231SA cells ................................................................... 127
4.3.6.1

SEM image analysis......................................................... 127

4.3.6.2

Confocal microscopy image analysis ............................... 127

VIII


4.3.7

Western blot analysis of five cell lines on different substrates to

examine various cell signalling pathways ............................................ 131
4.3.7.1

FAK .................................................................................. 132

4.3.7.2

MAPK .............................................................................. 133

4.3.8

mRNA expression profile of MCF10A, T47D, SUM1315, MDA-

MB231 and MDA-MB-231SA cells on TCP, col I and OBM ................. 135
4.3.8.1

Attachment....................................................................... 135

4.3.8.2

Invasion ........................................................................... 138

4.4

Discussion .................................................................................... 140

4.5

Conclusion .................................................................................... 157

Chapter 5 - Human bone and marrow derived progenitor cells and their
potential for human bone tissue engineering using a medical grade
polycaprolactone-tricalcium phosphate scaffold in NOD/SCID mice .......... 161
5.1

Introduction ................................................................................... 163

5.2

Materials and Methods.................................................................. 166

5.2.1

Cells ....................................................................................... 166

5.2.1.1

Isolation of human osteoblasts ........................................ 166

5.2.1.2

Human mesenchymal precursor cells .............................. 166

5.2.2

Characteristics of human osteoblasts and human mesenchymal

precursor cells under osteogenic conditions on TCP (2D) .................. 167
5.2.2.1

Cell culture ....................................................................... 167

5.2.2.2

Mineralisation - Alizarin red S .......................................... 167

5.2.2.3

Mineralisation - Wako HRII calcium assay ....................... 167

5.2.3

In vitro characterisation of hOBs and hMSCs on a medical grade

polycaprolactone tricalcium phosphate (mPCL-TCP) scaffold............. 167
5.2.3.1

Scaffold fabrication using fused deposition modelling

technique ......................................................................................... 167
5.2.3.2

Cell seeding ..................................................................... 168

IX


5.2.3.3

Seeding efficiency and proliferation ................................. 168

5.2.3.4

Cell viability ...................................................................... 169

5.2.3.5

Cell morphology ............................................................... 169

5.2.3.6

RNA isolation, primer design and qRT-PCR .................... 170

5.2.4

Ectopic bone assay ................................................................ 172

5.2.4.1

Implantation ..................................................................... 172

5.2.4.2

Calcein injection and imaging of live animals ................... 173

5.2.4.3

Micro-CT analysis ............................................................ 173

5.2.4.4

Biomechanical testing ...................................................... 174

5.2.5

Histology and immunohistochemistry ..................................... 174

5.2.5.1

Paraffin Embedding.......................................................... 174

5.2.5.2

Histochemistry

and

immunohistochemistry

on

paraffin

embedded samples .......................................................................... 174
5.2.5.3

Tartrate resistant acid phosphatase (TRAP) staining of

paraffin embedded samples ............................................................. 176
5.2.5.4

Resin Embedding ............................................................. 176

5.2.5.5

Histomorphometry ............................................................ 177

5.2.6
5.3

Statistical analysis .................................................................. 177

Results .......................................................................................... 178

5.3.1

Characteristics of human osteoblasts and human mesenchymal

precursor cells under osteogenic conditions on TCP (2D) ................... 178
5.3.1.1

Mineralisation ................................................................... 178

5.3.1.2

Proliferation ...................................................................... 179

5.3.1.3

qRT-PCR ......................................................................... 180

5.3.2

Characterisation of human marrow and bone derived cells on

the scaffold .......................................................................................... 181
5.3.2.1

Morphology and viability ................................................... 181

5.3.2.2

Proliferation ...................................................................... 183
X


5.3.2.3
5.3.3

5.4

qRT-PCR ......................................................................... 185

Ectopic bone assay ................................................................ 186

5.3.3.1

Live imaging..................................................................... 186

5.3.3.2

Micro-CT analysis ............................................................ 186

5.3.3.3

Biomechanical testing ...................................................... 187

5.3.3.4

Histological analysis ........................................................ 188

Discussion .................................................................................... 192

Chapter 6 - Human bone derived progenitor cells and their potential for
human bone tissue engineering using an electrospun polycaprolactone
scaffold with calcium-phosphate coating in NOD/SCID mice ..................... 201
6.1

Introduction ................................................................................... 203

6.2

Material and Methods ................................................................... 203

6.2.1

Cells ....................................................................................... 203

6.2.1.1
6.2.2

Isolation of human osteoblasts ........................................ 203

In vitro characterisation of the electrospun PCL scaffold ........ 203

6.2.2.1

Scaffold fabrication .......................................................... 203

6.2.2.2

Scaffold characteristics and testing in biological applications

......................................................................................... 203

6.2.2.3

Calcium phosphate coating ............................................. 205

6.2.2.4

Cell seeding ..................................................................... 206

6.2.2.5

Cell viability ...................................................................... 206

6.2.2.6

Cell morphology ............................................................... 206

6.2.3

Ectopic bone assay ................................................................ 206

6.2.3.1

Implantation ..................................................................... 206

6.2.3.2

Implantation of PEG gels incorporated with human breast

cancer cells ...................................................................................... 207
6.2.3.3

Micro-CTanalysis ............................................................. 208

6.2.3.4

Histology .......................................................................... 208
XI


6.3

Results .......................................................................................... 209

6.3.1

In vitro characterisation of hOBs on the electrospun scaffold . 209

6.3.1.1
6.3.2

Morphology and viability ................................................... 209

Morphology assessment of BC cells in PEG gels cultured in vitro
............................................................................................... 209

6.3.3

Ectopic bone assay ................................................................ 210

6.3.3.1

Implantation ..................................................................... 210

6.3.3.2

Micro-CT-analysis ............................................................ 211

6.3.3.1

Histological analysis ......................................................... 211

6.4

Discussion ..................................................................................... 215

6.5

Conclusion – Chapter 5 and 6 ....................................................... 218

Chapter 7 - Future directions ..................................................................... 221
Chapter 8 – Bibliography ........................................................................... 227

XII


List of illustrations and diagrams
Chapter 2
Figure 1:

Schematic of bone structure and types of metastases
visualized by imaging modalities

9

Figure 2:

Main steps of tumour progression and metastasis

13

Figure 3:

Description of the basic multicellular unit

17

Figure 4:

Potential role of the plasminogen activators and
plasmin in the pericellular activation cascade for MMPs

26

Figure 5:

Vicious cycle of bone metastases

28

Figure 6:

Illustration of the compartmentalised bioreactor model

32

Figure 7:

Illustration of the acellular bone matrix model utilised
by Pathi et al.

36

Figure 8:

Image of intracardiac injection

41

Chapter 3
Figure 9:

Immunohistochemical
thermanox coverslips

analysis

Figure 10:

SEM image of collagen I coated thermanox coverslip

63

Figure 11:

Human primary osteoblast explant culture

64

Figure 12:

Light microscopy
decellularised OBM

and

64

Figure 13:

Confocal microscopy image of OBM before and after
decellularisation

65

Figure 14:

SEM image of OBM before and after decellularisation

66

Figure 15:

SEM image of cortical human bone

66

Figure 16:

TEM images of OBM

67

Figure 17:

TEM reference image of synthetically produced
mineralised rat tail collagen fibrils

67

Figure 18:

Alizarin red staining of OBM

68

Figure 19:

Bar graph presenting calcium content of the OBM

69

Figure 20:

XPS analysis of the OBM

70

Figure 21:

XPS analysis of human native bone

70

Figure 22:

RAMAN spectra of non-mineralised and mineralised
OBM in addition to human native bone

71

image

XIII

of

of

col

I

coated

cellularised

62


Figure 23:

Immunohistochemistry for human bone ECM

72

Figure 24:

Protein analysis of OBM

73

Figure 25:

Western blot analysis of OBM

73

Figure 26:

Bar graph presenting the presence of different growth
factors in the OBM

74

Figure 27:

Adhesion of cells on an AFM cantilever

94

Figure 28:

SCFC measurement

95

Figure 29:

Exemplary SEM images for the invasion study

97

Figure 30:

98

Figure 31:

Exemplary confocal microscopy image for invasion
study
Light microscopy image of MCF10A cells

103

Figure 32:

Flow cytometry profile of MCF10A cells

104

Figure 33:

Light microscopy image of T47D cells

104

Figure 34:

Flow cytometry profile of T47D cells

105

Figure 35:

Light microscopy image of SUM1315 cells

105

Figure 36:

Flow cytometry profile of SUM1315 cells

106

Figure 37:

Light microscopy image of MDA-MB-231 cells

106

Figure 38:

Flow cytometry profile of MDA-MB-231 cells

107

Figure 39:

Light microscopy image of MDA-MB-231SA cells

107

Figure 40:

Flow cytometry profile of MDA-MB-231SA cells

108

Figure 41:

E-cadherin expression

109

Figure 42:

Vimentin expression

110

Figure 43:

Expression of IL-6 and IL-8

111

Figure 44:

Expression of Ck8 and Ck18

112

Figure 45:

Light microscopy images of MCF10A, T47D,
SUM1315, MDA-MB-231 and MDA-MB-231SA cells
cultured on TCP, col I and OBM

114

Figure 46:

Confocal laser microscopy images of MCF10A,
T47D,SUM1315, MDA-MB-231 and MDA-MB-231SA
cells cultured on TCP, col I and OBM

115

Figure 47:

Box plots demonstrating the shape factor of MCF10A,
T47D, SUM1315, MDA-MB-231 and MDA-MB-231SA
cells cultured on TCP, col I and OBM

117

Figure 48:

Box plots demonstrating the spreading area of
MCF10A, T47D, SUM1315, MDA-MB-231 and MDA-

118

Chapter 4

XIV

MB-231SA cells cultured on TCP, col I and OBM
Figure 49:

Polar blots demonstrating the orientation angel of
MCF10A, T47D, SUM1315, MDA-MB-231 and MDAMB-231SA cells cultured on TCP, col I and OBM

119

Figure 50:

Proliferation rate of MCF10A, T47D, SUM1315, MDAMB-231 and MDA-MB-231SA cells cultured on TCP,
col I and OBM
Box plots of detachment forces of MCF10A, T47D,
SUM1315, MDA-MB-231 and MDA-MB-231SA from
col I and OBM

120

Figure 52:

Bar graphs demonstrating mean squared displacement
of MCF10A, T47D, SUM1315, MDA-MB-231 and MDAMB-231SA on TCP, col I and OBM

125

Figure 53:

Bar graphs representing random migratory coefficient
and persistence time of MCF10A, T47D, SUM1315,
MDA-MB-231 and MDA-MB-231SA on TCP, col I and

OBM

126

Figure 54:

SEM images to determine the invasive potential of
MCF10A, T47D, SUM1315, MDA-MB-231 and MDAMB-231SA on OBM

128

Figure 55:

Bar graphs demonstrating the percentage of invaded
cells

129

Figure 56:

Cross sections of confocal images of MCF10A, T47D,
SUM1315, MDA-MB-231 and MDA-MB-231SA cells on
OBM at d 3

130

Figure 57:

Quantification of cross sections from confocal
microscopy images to determine invasiveness

131

Figure 58:

Image of a representative western blot analysing FAK
phosphorylation

132

Figure 59:

Quantification of western blot analysis regarding FAK
phosphorylation

133

Figure 60:

Image of a representative western blot analysing
p42/44 MAPK phosphorylation

134

Figure 61:

Quantification of western blot analysis regarding p42
MAPK phosphorylation

135

Figure 62:

Bar graphs demonstrating relative mRNA expression
levels of integrin alpha 2, 3, 5, v and beta 1and 3

137

Figure 63:

Bar graphs demonstrating relative mRNA expression
levels of MMP2 and MMP9

138

Figure 64:

Bar graphs demonstrating relative mRNA expression
level of OP

139

Figure 51:

XV

122


Chapter 5

Figure 65:

Schematic of scaffold implantation procedure

173

Figure 66:

Alizarin red staining of hMSC and hOBs

178

Figure 67:

Bar graph demonstrating the calcium content of hMSC
and hOBs

179

Figure 68:

Proliferation of hMSCs and hOBs in 2D

180

Figure 69:

Bar graphs representing quantitative RT-PCR analysis
of 2D cultures

182

Figure 70:

Images of morphology and viability of hMSCs and
hOBs in 3D

183

Figure 71:

SEM images of hMSCs and hOBs in 3D

184

Figure 72:

Graph indicating the proliferation of hMSCs and hOBs
in 3D

184

Figure 73:

Bar graphs displaying results from comparative qRTPCR analysis of 2D and 3D cultures

185

Figure 74:

Images displaying live imaging of mice after calcein
injections as well as scaffolds before and after
implantation

186

Figure 75:

Bar graphs demonstrating micro-CT analysis as well as
results from biomechanical testing

188

Figure 76:

Quantification
mineralisation

determined

189

Figure 77:

Representative images from micro-CT analysis, von
Kossa/van Gieson and H&E stained sections

190

Figure 78:

Images of TRAP, OC, and vWF stained sections

191

Figure 79:

Image of electrospun PCL tubes

203

Figure 80:

SEM image of electrospun sheets

204

Figure 81:

Image of viability assay displaying hMSCs on e-spun
meshes

205

Figure 82:

Schematic of scaffold implantation procedure

207

Figure 83:

Images displaying the morphology and viability of
hOBs on e-spun PCL-CaP scaffolds

209

Figure 84:

Confocal images of four cell lines incorporated in PEG
gels

210

of

histologically

Chapter 6

XVI


Figure 85:

Images of PCL-CaP scaffold and PEG gels before and
after implantation

211

Figure 86:

Micro-CT images as well as images of H&E and TRAP
stained histology sections

213

Figure 87:

Image displaying bone marrow-like features within the
human tissue engineered bone construct

214

List of Tables
Chapter 2
Table 1:

Major components of human bone and their overall
function

15

Table 2:

Main integrins and their ECM ligands that participate in
bone metastasis and tumour growth in bone

20

Table 3:

In vitro models investigating the mutual influence of
breast cancer cells with the bone microenvironment

31

Table 4:

Examples for in vivo models investigating cancer
metastasis to bone

38

Sequences of oligonucleotides used for qRT-PCR in
Chapter 4

102

Sequences of oligonucleotides used for qRT-PCR in
Chapter 5

172

Chapter 4
Table 5:

Chapter 5
Table 6:

XVII


XVIII


List of abbreviations
2D

Two dimensional

3D

Three dimensional

AFM

Atomic force microscopy

AIWH

Australian Institute of Health and Welfare

ALP

Alkaline phosphatase

AM

Adrenomedullin

ANOVA

Analysis of Variance

BC

Breast Cancer

BMP

Bone morphogenetic protein

BMSC

Bone marrow derived mesenchymal stem
cells

BMU

Basic multicellular unit

BSA

Bovine serum albumin

BSP

Bone sialoprotein

BV

Bone volume

BrdU

Bromdesoxyuridin

CaP

Calcium phosphate

Cbfa 1

core binding factor

CCN

Cyr61, CTGF, nov

CDM

Cellular derived matrix

Col I

Collage type I

CO2

Carbon dioxide

CSF

Colony stimulation factor

CT

Computed Tomography

CTGF

Connective tissue growth factor

CXC

Chemokine

CXCL

Chemokine ligand

CXCR

Chemokine receptor

Cyr 61

Cystein-rich protein 61

DAB

3,3-diaminobenzidine

dd

double distilled

DAPI

4’,6-diamidino-2-phenylindole
XIX

1 a.k.a. Runx-2


DIC

Differential interference contrast

DMEM

Dulbecco’s modified eagle medium

DNA

Deoxyribonucleic acid

DTC

Disseminated tumour cell

DTT

Dithiotreitol

ECL

Enhanced chemiluminescence

ECM

Extracellular matrix

EDTA

Ethylene-di-amine-tetra-acetic acid

EDS

Energy dispersive spectroscopy

EGFR

Epidermal growth factor receptor

ELISA

Enzyme linked immunosorbent assay

EMT

Epithelial to mesenchymal transition

ER

Oestrogen receptor

ERK

Extracellular signal-regulated kinase

FAK

Focal adhesion kinase

FBS

Foetal bovine serum

FDA

Fluorescein diacetate

FDA

Food and drug administration

FDM

Fused deposition modelling

FDG

Fluorodeoxyglucose

FGF

Fibroblast growth factor

FN

Fibronectin

Gla

-carboxylated

GRB2

Growth-factor-receptor-bound-2

HA

Hydroxyapatite

HCl

Hydrogen chloride

HE

Haematoxylin-eosin

HGF

Hepatocyte growth factor

Hh

Hedghog

H2O

Water

HRP

Horseradish peroxidase

HSC

Hematopoietic stem cell

hTEBC

Human tissue engineered bone construct

ICAM

Inter-cellular adhesion molecule
XX


IGF

Insulin-like growth factor

IgG

Immunoglobulin

IL

Interleukin

IP3

Phosphatidylinositol γ’-kinase

MALDI

Matrix-assisted laser desorption/ionisation

MAPK

Mitogen activated kinase

M-CSF

Macrophage colony stimulating factor

Micro-CT

Micro-computed tomography

MMA

Methyl-methacrylate

MMP

Matrix metalloproteinase

MMTV

Mouse mammary tumour virus

mPCL-TCP

Medical grade polycaprolactone tricalcium
phosphate

MRI

Magnetic resonance imaging

MSC

Mesenchymal stem cell

MSD

Mean squared displacement

NaOH

Sodium-hydroxide

NOD/SCID

non-obese diabetic/severe combined
immunodeficient

Nov

Nephroblastoma overexpressed

NPT

Sodium dependent phosphate transporter

OB

Osteoblast

OBM

Osteoblast matrix

OC

Osteoclast

OCPC

O-cresolphthalein complexion

OD

Optical density

ON

Osteonectin a.k.a SPARC

OP

Osteopontin

OPG

Osteoprotegerin

OPN

Osteopontin

Pa

Pascal

PAA

Polyacrylamide

PAGE

Polyacrylamide gel electrophoresis

PBS

Phosphate buffered saline
XXI


PCL

Polycaprolactone

PD

Photodiode

PDGF

Platelet derived growth factor

PEG

Polyethylene glycol

PET

Positron emission tomography

PFA

Paraformaldehyde

PI

Propidium iodide

PI3K

Phosphatidylinositol γ’-kinase

PLGA

poly(lactide-co-glycolide) acid

PMMA

Poly-Methyl methacrylate

Prkdc

DNA-dependent protein kinase catalytic
subunit

PTH

Parathyroid hormone

PTHrP

Parathyroid hormone related protein

PUR

Polyurethane

RANKL

Receptor activator nuclear factor kappa B
ligand

Rh

Recombinant human

RNA

Ribonucleic acid

ROCK

Rho associated kinase

RT

reverse transkriptase

RT-PCR

Real time polymerase chain reaction

Runx-2

Runt related transcription factor 2 a.k.a
Cbfa1

SBF

Simulated body fluid

SCFS

Single cell force spectroscopy

SD

Standard deviation

SDF-1

Stromal cell derived factor a.k.a. CXCL12

SDS

Sodium dodecyl sulfate

SEM

Scanning electron microscopy

SEM

Standard error of the mean

SFK

Src-family kinase

SOS

Son-of-sevenless

XXII


SPARC

Secreted protein, acidic cysteine-rich, a.k.a
osteonectin

SPECT

Single

photon

emission

computed

tomography
SS

Skeletal scintigraphy

TBST

Tris buffered saline and Tween 20

TCP

Tri-calcium phosphate

TCP

Tissue culture plastic

TE

Tissue engineering

TEBC

Tissue engineered bone construct

TEM

Transmission electron microscopy

TGF

Transforming growth factor

TIMP

Tissue inhibitor of metalloproteinase

TNF-

Tumour necrosis factor

TOF

time-of-flight

TRAP

Tartrate resistant acid phosphatase

TSP-1

Thrombospondin 1

VCAM

Vascular cell adhesion molecule 1

VEGF

Vascular endothelial growth factor

VN

Vitronectin

Wnt

Wingless

XPS

X-ray photoelectron spectroscopy

XXIII