Possible Role of Osteoblasts in Regulating the Initiation of Endochondral Repair Process during Fracture Healing
by
Yasha Amani Andabili
A thesis submitted in conformity with the requirements for the degree of Master of Science
Department of Laboratory Medicine and Pathobiology University of Toronto
© Copyright by Yasha Amani Andabili, 2012
ii
Possible Role of Osteoblasts in Regulating the Initiation of
Endochondral Repair Process during Fracture Healing
Yasha Amani Andabili
Master of Science
Department of Laboratory Medicine and Pathobiology University of Toronto
2012
Abstract
Fracture repair is a regenerative event that involves the precise coordination of a variety
of cells for successful healing process. Within the microstructure hierarchy of bone repair, the
predominant cells involved include the chondrocytes, osteocytes, osteoblasts, and osteoclasts.
Although the role of osteoblasts during fracture healing has been previously shown, their role
during the initiation phase of endochondral fracture repair remains unclear. In order to study
the role of osteoblasts during fracture repair, we used a transgenic mouse model expressing the
herpes simplex virus thymidine kinase gene in early differentiating osteoblasts, which allows
conditional ablation of cells in osteoblastic lineage upon treatment with the Gancicolvir drug.
Results from this study suggest that not only are osteoblasts required in later stages of fracture
repair as the medium for bone synthesis, and osteoclast activation during bone remodelling, but
could also be required for the initiation and advancement of the endochondral ossification
process.
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Acknowledgments
First and foremost, I would like to demonstrate my gratitude to a cast of people without
whom the completion of this project would not have been possible. I want to thank my
supervisor Dr. Benjamin Alman for his advice, guidance and infinite patience over the course of
my masters program. It has been a very interesting journey and a wonderful experience working
in his lab, especially for being given the opportunity to make a contribution in the field of bone
research. Thank you for your belief and support.
I want to thank all the past and present members of my advisory committee, Dr. Freda
Miller, Dr. Marc Grynpas and Dr. Jane Aubin, for their helpful advice, useful guidance and
constructive discussions. I have had the benefit of learning from your questions, which have
helped me mature as a scientist and allowed me test my abilities and limits. I specially want to
express my gratitude to Dr. Freda Miller for her efforts and support over the years, both as a co-
supervisor and a member of my committee. Thank you.
I also want to thank all the past and present members of Dr. Alman and Dr. Miller’s
laboratories who have helped me grow as a scientist, forge friendships, and most importantly for
their moral support and encourangement, thought-provoking conversations, technical help and
sharing of their expertise and knowlege. A special thanks to Dr. Saeid Amini Nik and Dr. Harry
Elsholtz for their guidance, encouragement and mentorship over the years as well as their belief
in me as a student.
This work was carried out in the laboratories of Dr. Benjamin Alman, Dr. Freda
Miller/Dr. David Kaplan. Special thanks to the laboratory of Dr. Marc Grynpas for making
available the equipments for histomorphometric analysis as well as their technical support and
knowledge. This work was done at the Sickkids division of the MaRS research facility and was
partially funded by a fellowship provided by the University of Toronto.
Finally I want to thank my family for their love and encourangment as well as their moral
support and unrelenting belief in my ability to grow and mature as a scientist.
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Table of Contents
Contents
Acknowledgments ........................................................................................................................... ii
Table of Contents ........................................................................................................................... iv
List of Figures ............................................................................................................................... vi
List of Abbreviations .................................................................................................................. vii
Chapter 1 General Introduction .................................................................................................. 1
1.1 Bone Formation ................................................................................................................. 1
1.1.1 Endochondral Ossification ................................................................................... 2
1.1.2 Intramembranous Ossification ............................................................................ 3
1.1.3 Fracture Repair ..................................................................................................... 4
1.2 Skeletal Cell Types .......................................................................................................... 10
1.2.1 Chondrocytes ....................................................................................................... 10
1.2.2 Osteoclasts ........................................................................................................... 11
1.2.3 Osteoblasts ........................................................................................................... 14
1.2.4 Osteocytes ............................................................................................................ 17
1.3 Osteogenic Proteins ......................................................................................................... 18
1.3.1 Vascularization in Successful Bone Healing ..................................................... 18
1.3.2 Matrix Metalloproteinases ................................................................................. 20
1.3.3 Bone Morphogenetic Proteins ............................................................................ 23
1.3.4 Transforming Growth Factor Beta ................................................................... 24
1.3.5 Wnt Signalling and Bone Formation ................................................................. 25
1.4 Thymidine Kinase/Ganciclovir System ......................................................................... 27
References .................................................................................................................................... 34
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Chapter 2 Possible Role of Osteoblasts in Regulating the Initiation of Endochondral Repair Process during Fracture Healing ............................................................................. 60
2 Summary ................................................................................................................................. 60
2.1 Introduction ..................................................................................................................... 60
2.2 Methods and Materials ................................................................................................... 63
2.2.1 Generation of fractures: ........................................................................................ 63
2.2.2 Mechanism of action of GCV: .............................................................................. 63
2.2.3 Real-time PCR: ..................................................................................................... 64
2.2.4 Staining method – Safranin-O/Haematoxylin-Eosin: ........................................... 64
2.2.5 Histomorphometric, Cell quantification and X-ray analysis: ............................... 64
2.2.6 Collagen type X and TRAP staining: .................................................................... 65
2.2.7 Statistical Analysis: ............................................................................................... 65
2.3 Results .............................................................................................................................. 65
2.3.1 Pretreatment of DTK transgenic mice with GCV leads to ablation of osteoblasts ............................................................................................................ 65
2.3.2 Osteoblast depletion delays initiation of endochondral bone repair .............. 66
2.3.3 Continuous ablation of osteoblasts hinders the progression of endochondral ossification past the soft callus stage ......................................... 67
2.4 Discussion ......................................................................................................................... 70
2.5 Figures .............................................................................................................................. 73
References .................................................................................................................................... 84
Chapter 3 Summary and Conclusions ...................................................................................... 87
Chapter 4 Future Directions ...................................................................................................... 88
Appendix ...................................................................................................................................... 90
Acknowledgements ..................................................................................................................... 92
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List of Figures
Figure 1: Genetic Control of Skeletal Cell Type Differentiation.
Figure 2: Bone Formation Occurs Through Two Different Modes of Ossification Processes.
Figure 3: Model of Fracture Healing Stages and Cellular Participants Involved During the Process of Repair.
Figure 4: Continuous GCV Treatment Leads to Non-union of the Fractured Bone.
Figure 5: In Vivo Experimental Design for Studying the Role of Osteoblasts During Fracture Repair Process.
Figure 6: GCV Pretreatment is Effective in Ablating Pre-existing Osteoblasts.
Figure 7: GCV Treatment of the DTK Transgenic Mice Leads to a Decrease in Bone-Lining Osteoblast Population.
Figure 8: Continuous GCV Treatment Leads to a Delay in Initiation and Progression of Endochondral Ossification Process.
Figure 9: Matrix Metalloproteinase 13 Gene Expression is Reduced in Absence of Osteoblasts.
Figure 10: Osteocytes are Unaffected by GCV Treatment.
Figure 11: Upon GCV Withdrawal Bones are Able to Replenish the Osteoblast Population as Early as 7 Days Post Fracture.
Figure 12: Osteoblast Ablation Leads to a Decrease in TRAP Positive Osteoclasts During the Remodeling Stage of Fracture Healing, and the Decrease in Osteoclast Population Hinders the Healing Process.
Figure 13: In Absence of Osteoblasts There is a Lack of Collagen Type X Matrix Resorption.
Figure 14: Continuous Depletion of Osteoblasts Results in Unresorbed Cortical Bone and Cartilage Matrix at the Fracture Site.
Figure 15: Fluorescent and Immunohistochemistry staining for GFP-tagged SKP-injected into fracture site of continuous GCV treated DTK mice.
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List of Abbreviations
ALP – Alkaline phosphatase
bHLH – A basic helix-loop-helix domain present in two Twist genes (Twist-1 and -2)
BMP – Bone morphogenetic protein
BSP – Bone sialoprotein
BV – Bone volume
CAT – Chloramphenicol acetyltransferase
Cbfa1 – Core binding factor 1
Col-I – Collagen type I
Col-II – Collagen type II
Col-X – Collagen type X
CV – Cartilage volume
CXCR4 – A CXC chemokine receptor (type 4) in the G-protein coupled receptor family specific
for stromal cell-derived factor-1.
DAPI – A polpular flourecent stain (4’, 6-diamidino-2-phenylindole) used for nuclear
counterstain in immunohistochemistry.
DTK – Refers to the transgenic mouse model expressing the HSV-tk gene under the control of a
2.3 kilobase fragment of the rat α1 type I collagen promoter (Col2.3∆tk)
ECM – Extra cellular matrix
EDTA – Ethylenediaminetetraacetic acid is a chelating agent used for bone decalcification.
FGF – Fibroblast growth factor
GCV – Ganciclovir
GFP – Green fluorescent protein
HE – Haematoxylin eosin
HIF – Hypoxia inducible factor
HSV-TK – Herpes simplex virus thymidine kinase
HTC – Hypertrophic chondrocyte
IGF – Insulin-like growth factor
Ihh – Indian hedgehog
IL – Interleukin
M-CSF – Macrophage colony stimulating factor
viii
MMP – Matrix metalloproteinase
MSC – Mesenchymal stromal cells
NF-κB – Nuclear factor-κB
NOD-SCID – Refers to the non-obese diabetic severe combined immunodeficiency mouse
model
OC – Osteocalcin
OG2 – Osteocalcin gene 2
OCIL – Osteoclast inhibitory lectin
OPG – Osteoprotegerin
OPN – Osteopontin
Osx – Osterix
PDGF – Platelet-derived growth factor
PGE2 – Prostaglandin E2
PTH – Parathyroid hormone
PTHrP – Parathyroid hormone related peptide
RANK – Receptor activator of nuclear factor-κB
RANKL – Receptor activator of nuclear factor-κB ligand
RFP – Red fluorescent protein
Runx2 – Runt-related transcription factor 2
SDF-1 – Stromal cell-derived factor-1
SKP – Skin derived precursors
SO – Safranin O
TGF – Transforming growth factor
TNF – Tumor necrosis factor
TRAF – TNF receptor-associated factor
TRAP – Tartrate-resistant acid phosphatase
TV – Tissue volume
VEGF – Vascular endothelial growth factor
1
Chapter 1 General Introduction
1.1 Bone Formation
Bone is composed of two distinct tissues, cartilage and bone, each of which includes
specific cell types that fulfill unique functions that are critical for the growth, maintenance and
integrity of the skeleton (Ducy, 1998; Ducy, 2000). Chondrocytes, found in cartilage, are
essential for longitudinal growth of bone from the two metaphyseal ends, and synthesize the
cartilaginous templates during development and postnatal skeletogenesis, onto which osteoblasts
can deposit the bone matrix (Ducy, 2000). Osteoblasts and osteoclasts, found in bone, closely
collaborate in basic multicellular units (BMU) to coordinate the anabolic build-up (synthesis)
and catabolic break down (resorption) of the bone tissue, which occurs regularly throughout the
skeleton at micro scales during adult life (Hadjidakis, 2006; Hill, 1998; Neumann, 2007; Proff,
2009).
Certain genes are critical for regulating the progression of skeletal formation through the
stage of chondrogenesis, angiogenesis, and osteogenesis to the process of remodelling. The
initial phase of mesenchymal condensation involves the expression of transcription factors core
binding factor 1/runt-related transcription factor 2 (Cbfa1/Runx2) and Sox9 genes, which play a
role in determining the population of cells that will occupy and contribute to the condensation
(Hall, 1992; Hall, 1995; Zhou, 2006). Expression of certain growth factors, such as transforming
growth factor beta (TGFβ) superfamily, regulate the proliferation of mesenchymal progenitors
and their differentiation into chondrocytes (Mundlos, 1997a; Mundlos, 1997b). The cartilage
anlage is developed by the secretion of various structural proteins such as syndecan-3, versican
and tenascin by cells within the condensate (Koyama, 1995; Koyama, 1996).
The master regulator, Runx2/Cbfa1, also plays important role in chondrocyte maturation,
angiogenesis and the remodelling of extracellular matrix, as well as being required for
intramembranous ossification (Ferguson, 1999; Yoshida, 2005). Furthermore, it is essential for
fetal osteogenesis (Komori, 1997; Otto, 1997), as deletion of the cbfa1 gene in homozygous null
mutants (cbfa1-/-) results in an almost complete lack of endochondral and intramembranous
2
ossification, while development of the cartilage scaffold proceeds normally (Komori, 1997;
Mundlos, 1997a; Mundlos, 1997b; Otto, 1997).
This transcription factor regulates the maturation of chondrocytes by controlling the
expression of osteocalcin and osteopontin (OPN), which despite being known as osteoblast-
specific markers, do get expressed in hypertrophic chondrocytes (Gerstenfeld, 1996; Lian, 1993;
Nomura, 1989). By controlling the expression of OPN, which is responsible for the attachment
of cells to the extracellular matrix (Miyauchi, 1991; Reinholt, 1990; Somerman, 1987),
Runx2/Cbfa1 indirectly affects terminal differentiation and apoptosis of chondrocytes. It is also
responsible for directly regulating the expression of matrix metalloproteinase 13 (MMP13), a
collagenase that is involved in remodelling of extracellular matrix that is laid down by
hypertrophic chondrocytes (Vu, 1998).
Another population of cells, the osteoblasts, synthesize a matrix rich in type I collagen
which eventually becomes mineralized, and osteoclasts function to resorb this mineralized
matrix, allowing the remodelling of the rigid bone tissue (Ducy, 2000). Aside from the
transcription factor Runx2/Cbfa1 (Maruyama, 2007), another important signalling molecule
during skeletogenesis is the Wnt-family of molecules, which functions during both embryology
and adult bone repair by regulating the differentiation of mesenchymal precursors into
osteoblasts and the subsequent osteoblastic bone formation (Chen, 2009; Topol, 2009).
Given the biologic importance of these three cell types, and the nonredundancy of their
functions, a defect in differentiation and function of any of these cells leads to severe
repercussions during development that manifests as various diseases of bone and cartilage
(Ducy, 2000). Therefore, knowledge of the genetic network controlling their differentiation and
function (Figure 1) becomes important for developing new and effective therapeutic strategies.
1.1.1 Endochondral Ossification
Endochondral ossification, an anabolic response driven by osteoblasts, includes the
formation of a cartilaginous template that is eventually replaced with bone matrix (Schindeler,
2009). It is initiated by the condensation of mesenchymal cells and their differentiation into
chondrocytes, which secrete a type-II collagen and aggrecan-rich cartilage matrix. Cells
surrounding the condensate form the perichondrium, while cells within the cartilage template
3
further differentiate into hypertrophic chondrocytes, which requires a cessation of proliferation
and exit from the cell cycle (Hartmann, 2009). These cells produce type-X collagen, as well as
the angiogenic vascular endothelial growth factor (VEGF) and various osteoblast-specific
proteins including alkaline phosphatase (ALP) and OPN, which results in recruitment of new
blood vessels to the avascular cartilage and the mineralization of the cartilage matrix (Iyama,
1991; Kronenberg, 2003). This matrix is eventually resorbed, and the degradation of this
cartilage matrix during endochondral ossification is dependent on the activity of MMPs,
particularly MMP9 (gelatinase B) and MMP13, that regulate remodeling and neovascularization
of the cartilage anlage (Johansson, 1997; Vu, 1998). The invasion of blood vessels into the
cartilage matrix leads to recruitment of osteoblast and osteoclast precursors.
Development of adequate vasculature is critical during the endochondral ossification, as
it determines the rate of bone formation by coupling the processes of chondrogenesis and
osteogenesis (Gerber, 2000). It has been determined that the coordination of metaphyseal and
epiphyseal vascularization, ossification and cartilage formation during endochondral process are
dependent on functions of VEGF isoforms (Maes, 2002). These factors are secreted by the
developing hypertrophic chondrocytes, and lead to recruitment of osteoblasts, osteoclasts and
haematopoietic cells.
Osteoblasts residing in the perichondrium will create a collar of compact bone around the
middle region of the cartilage template, referred to as diaphysis, the site of primary ossification
center. At the two ends of the developing bone, known as epiphyses, secondary ossification
centers are formed, and the cartilaginous plate separating the diaphysis and epiphysis will
become the growth plate, which regulates the longitudinal growth of the long bones (Collin-
Osdoby, 1994; Gerber, 2000; Hall, 2006) (Figure 2A).
1.1.2 Intramembranous Ossification
In contrast to endochondral ossification, no signal from cartilage is required for bone
formation via intramembranous ossification, which is responsible for the formation of flat bones
of the face and skull, as well as parts of the mandibles and clavicles. In this process, cells of the
mesenchymal condensate differentiate directly into osteoblasts (Hartmann, 2009), with the cells
at the edge of the condensates forming the periosteum, a connective tissue that wraps the bone
and holds most of the osteoprogenitor cells (Augustin, 2007). Newly differentiated osteoblasts
4
immediately start producing the bone matrix osteoid (Hartmann, 2009). Deposition of bone
matrix by the maturing osteoblasts leads to formation of bone spicules, which grow and
eventually fuse with neighboring spicules to create newly formed trabeculae. The increase in size
and number and the ultimate interconnection of these trabeculaes results in formation of a weak
and disorganized structure with high incidences of entrapped osteoblasts (Hermey, 1996a;
Hermey 1996b). This woven bone is gradually replaced with a more organized, stronger lamellar
bone. In this process, the emergence and differentiation of mesenchymal precursors directly into
mature osteoblasts leads to formation of compact bones, and osteoblasts that become entrapped
within lacunae of the matrix differentiate into osteocytes (Hartmann, 2009) (Figure 2B).
1.1.3 Fracture Repair
Fracture healing is a complex process that recapitulates the specific patterns of embryonic
development and bone regeneration and involves the interplay of numerous cell types (Mersell,
2011; Schindeler, 2008) (Figure 3). During the last few decades, various studies investigating
the biochemical and anatomical events of bone healing have rapidly advanced our understanding
of the signalling cascade and pathways that result in successful fracture repair. There are two
types of fracture healing processes, including direct (primary) and indirect (secondary)
(Gerstenfeld, 2003; Rahn, 2002). Also, many of the mechanisms involved in the skeletal repair
process recap the events of embryonic development (Einhorn, 2001; Ferguson, 1999;
Gerstenfeld, 2003).
Primary direct mode of skeletal repair is an uncommon event, which happens at regions
of biomechanical instability and requires rigid stabilization of the fracture site (Allgower, 1979;
Perren, 1979; Schatzker, 1989). This rigid stabilization suppresses the generation of a callus in
either the cancellous or cortical bone. In contrast to indirect bone healing, this process involves
generation of a weak intermediate and does not occur in anaerobic environment (Madison,
1993). Two forms of primary skeletogenesis exist, gap healing and contact healing, where bone
union is achieved without an external callus formation, fibrous tissue or cartilaginous scaffold
formation within the fracture gap (Liu, 1999; Perren, 1979; Schenk, 1967; Schenk, 1987). Gap
healing is initiated by formation of woven bone, followed by generation of parallel oriented
lamellar bone that provides support (Schenk, 1967; Schenk, 1987). This is ensued by
remodelling of the longitudinal haversian canals, reconstruction of the necrotic fracture ends and
5
formation of new bone at the fracture site that replaces and reorients the fractured bone back to
the original configuration (DeLacure, 1994). In contrast, contact healing occurs in cases where
bone fragments are in direct appositions and is not preceded by a transverse bone formation
between the fracture ends. In this form of direct healing osteons are able to grow parallel to the
long axis of the bone across the fracture site, supplemented by tunnelling osteoclastic activity
forming cutting cones across the fracture line that allow the penetration of blood vessels,
accompanied by osteoprogenitor and endothelial cells (Perren, 1969; Perren, 1979; Schenk,
1967; Schenk, 1987; Thomas, 1998). This form of bone repair will also lead to regeneration of
normal bone architecture.
Secondary indirect bone healing is characterized by spontaneous bone formation in
absence of a rigid stable fixation of the fracture site, and is the most common method of fracture
healing consisting of both endochondral and intramembranous process of bone generation
(Gerstenfeld, 2006). This form of healing is improved by micro-motion and weight-bearing,
although delayed healing or non-union has been observed in cases where too much motion
and/or load is applied (Green, 2005; Pape, 2002; Perren, 2002). It occurs in areas where
application of motion cannot be avoided, such as intramedullary nailing, external or internal
fixation of complicated fractures (Pape, 2002; Perren, 2002). It consists of several overlapping
phases that span an inflammatory phase, a reparative phase that consists of both
intramembranous and endochondral ossification resulting in formation of soft callus followed by
remodelling and establishment of hard callus, and a final remodelling phase that re-establishes
the orientation of the original bone orientation (Bolander, 1992; Frost, 1989; Madison, 1993;
Marsh, 1999; McKibbin, 1978; Nemeth, 1988; Thomas, 1998; White, 1977).
Bone formation during adult growth and regeneration has been shown to be reflected in
the processes occurring during fetal development (Ferguson, 1999). To understand the
similarities and differences between these two processes, a series of expression assays was
performed to examine the molecular signals critical for bone synthesis and repair (Ferguson,
1999). The study showed that some aspects of bone development are regulated by similar
molecular signals during fetal development and adult growth/repair. These include the
maturation of chondrocyte, invasion of vasculature and the process of ossification. Major
differences between these two developmental processes are the initial inflammation response
6
during adult healing that influences the mechanisms controlling the mesenchymal cell
condensation during adult repair process (Ferguson, 1999).
1.1.3.1 Inflammatory Response
The immediate response to injury from fracture trauma is usually associated with damage
of the local soft tissue integrity along with disruption to normal vascular function and nutrient
flow at the fracture site. The bleeding within the fracture site triggers the infiltration of
macrophages, degranulating platelets, and other inflammatory cells into the developing
haematoma that aid in clearing the degenerated cells and secrete various cytokines and growth
factors (Einhorn, 1998; Gerstenfeld, 2003; Schineler, 2008). The eventual coagulation of the
haematoma around the site of injury provides a template for the formation of future callus
(Gerstenfeld, 2003).
Current knowledge suggests that the cascades of growth factors secreted by the
inflammatory cells regulate different step in fracture bone healing. The signalling molecules
involved in this process include TGF-β, platelet-derived growth factor (PDGF), fibroblast growth
factor-2 (FGF-2), VEGF, macrophage colony stimulating factor (M-CSF), interleukins-1 and -6
(IL-1 and -6), bone morphogenetic proteins (BMPs), and tumor necrosis factor-α (TNF-α)
(Bolander, 1992; Einhorn, 1998; Gerstenfeld, 2003). This cellular response creates a positive
feedback loop that assists the recruitment of additional inflammatory cells, triggering the
migration and invasion of mesenchymal progenitor cells as well as influencing the initiation of
repair process and development of fracture callus (Barnes, 1999; Bolander, 1992; Iwaki, 1997;
Shapiro, 2008). Although prolonged or chronic expression of inflammatory cytokines is known
to have a negative effect on bone healing or any implanted materials, a transient highly regulated
response is critical for tissue regeneration (Gerstenfeld, 2003).
1.1.3.2 Formation of Soft Callus
The first 7 to 10 days of fracture healing involves the process of chondrogenesis, where
mesenchymal progenitor derived chondrocytes proliferate and produce a semi-rigid cartilaginous
template adjacent to the fracture site. It has been suggested that the fracture line in the bone may
set up the overall spatial relationships of the generated mesenchymal condensations, which is
exhibited by the development of various discrete crescent shaped centers of condensates that are
7
symmetric with respect to the fracture line (Gerstenfeld, 2003). The formation of the
cartilaginous tissue occurs in regions that are mechanically less stable, such as between the
fracture ends and external to the periosteal site, and serves as a soft callus that provides the initial
mechanical support to the fracture (Rahn, 2002). This tissue will later undergo mineralization,
resorption and is superseded by the production of the bony callus.
On the cellular level, this stage of bone healing represents the involvement of
chondrocytes and fibroblasts, with the relative proportion of these cells varying between
different fractures (Gerstenfeld, 2003). In cases where the cartilage production is absent or
delayed, the fracture site is instead infiltrated by fibroblasts that generate a fibrous tissue.
Mesenchymal condensations and the subsequent chondrogenesis occurs at multiple discrete sites
within the fracture site that will eventually proliferate and merge to form a centralized
fibrocartilaginous structure between the fracture ends (Barnes, 1999). Coordinated expression of
various growth factors, such as TGF-β2, TGF-β3, PDGF, FGF-1, and insulin-like growth factor
(IGF), stimulate the proliferation/differentiation of the emerging fibroblastic and chondrocytic
cells (Cho, 2002, Einhorn, 1998; Gerstenfeld, 2003), while chondrogenesis is promoted by
various members of the BMP family of cytokines, such as BMP-2, -4, -5, and -6 (Al-Aql, 2008).
In response to these factors, chondrocytes are able to produce extensive amounts of
extracellular matrix proteins, such as collagen type II. As chondrocytes within the condensate
mature, they undergo hypertrophy and begin to secrete type X collagen (Al-Aql, 2008). Prior to
the synthesis of hard callus, the deposited cartilage scaffold undergoes calcification, gradual
removal of soft callus, neovascularisation, and the systemic deposition of woven bone through
the interplay of an array of molecular cytokines and pro-angiogenic factors that include VEGF,
BMPs, FGF-1 and TGF-β (Al-Aql, 2008; Cho, 2002; Gerstenfeld, 2003; Marsell, 2009). Other
factors involved in the cascade that initiates resorption include the macrophage colony-
stimulating factor (M-CSF), receptor activator of nuclear factor NF-κB ligand (RANKL),
osteoprotegrein (OPG) and TNF-α (Barnes, 1999; Gerstenfeld, 2003).
1.1.3.3 Formation of Hard Callus
The maturation of prehypertrophic chondrocytes of the soft callus is accompanied by
enlargement of cellular volume and increase in extracellular matrix production. The deposited
matrix undergoes spontaneous calcification that result in formation of hydroxyapatite crystals.
8
The calcification and nucleation mechanism begins with the accumulation of calcium-containing
granules and their transport into the extracellular to the extracellular matrix where they
precipitate with the surrounding phosphate ions and form the initial mineral deposits (Ketenjian,
1975). This calcified cartilage needs to be resorbed prior to its replacement by the woven bone
that is secreted by osteoblasts (Einhorn, 1998; Giannoudis, 2011).
This hard callus formation stage of bone repair is characterized by high levels of
osteoblast activity and represents the most active period of osteogenesis, leading to formation of
mineralized bone matrix. The ability of the early differentiated osteoblasts to progress down the
osteogenic lineage in an ordered manner is a critical prerequisite for efficient fracture healing.
The progression of the hard callus stage involves the replacement of the calcified cartilage with
woven bone that provides mechanical rigidity to the healing bone (Gerstenfeld, 2006). This is
carried out primarily through the function of MMP13, which is produced by cells in the
osteoblastic lineage (Nakamura, 2004) that invade the cartilage matrix. Mice deficient in
MMP13 function exhibit a defect in removal of the deposited cartilage which cannot be rescued
with normal bone marrow transplantation (Behonick, 2007; Kosaki 2007). Osteoclast cells that
follow the early osteoblast in-growth phase synthesize MMP9 (Andersen, 2004), another
important degradative enzyme shown to be important for cartilage resorption (Colnot, 2003).
As the process of cartilage template removal continues the outer layer of the callus forms
the structure that will make up a new cortical shell (Cao, 2002; Komatsubara, 2005; Li, 1999).
The new woven bone matrix deposited by newly differentiated osteoblasts consists of both
proteinaceous and mineralized bone extracellular matrix, which is typically irregular and under-
remodelled (Gerstenfeld, 2006). The bone forming osteoblasts populate the newly formed
endosteal surface of the callus, and a thin layer of osteoclastic cells followed by a layer of
myofibroblastic cells advance toward the outer periosteal surface of the shell (Ushiku, 2010).
The outer cortical shell is formed with a distinctive dynamic appearance, which is influenced by
the mechanical load that is applied during motion (Ushiku, 2010). As the base of the shell
thickens the active region of bone formation contracts toward the apex of the shell and the
process of remodelling occurs in an inward manner from outside toward inside of the convex
cortical shell (Ushiku, 2010). The initial chondrocytes at the center of the fracture zone are
products of the progenitor population residing from the periosteum. The bone formation over the
surface of the callus to form the new periosteum and the outer cortical shell also derive from the
9
periosteal progenitors (Gerstenfeld, 2009; McDonald, 2008). However, the subsequent
remodelling of the fracture callus requires osteoclasts. Osteoclast inhibitors do not affect the
early stages of fracture healing, but in the absence of sufficient osteoclast function there is a
delay of inward remodelling and failure to resorb the mineralized cartilage core (Gerstenfeld,
2009; McDonald, 2008).
1.1.3.4 Bone Remodeling
In order to fully restore the biomechanical properties to the normal bone, the generated
rigid hard callus that is composed mostly of woven bone, requires to be remodelled back to the
original bone configuration. This is done through a second phase of resorption that replaces the
unorganized woven bone of the hard callus with a more organized lamellar bone structure and
creates the central medullary cavity (Gerstenfeld, 2003; Marsell, 2011). The orchestration of this
phase is regulated by a series of biochemical factors, such as IL-1, TNF-α, BMP-2, that show
reasonably high expression level during this stage of bone healing (Al-Aql, 2008; Marsell, 2009;
Mountziaris, 2008). This process involves the balanced coordination of hard callus resorption by
osteoclasts, followed by lamellar bone synthesis by osteoblasts (Marsell, 2009; Schindeler,
2008).
Based on histological evidence that shows the presence of large multinucleated cells at
the edge of the soft callus, along with the invading vascular endothelial cells, osteoclasts have
been suggested to play a key role in both soft callus and hard callus remodeling (Schell, 2006).
Although these cells display similar morphology to osteoclasts and stain for classic osteoclast
markers (such as TRAP staining), they have been termed chondroclasts, based on their physical
location (Cole, 1987).
Active bone resorption results in the release of various growth factors and products, such
as TGFβ, BMPs, and collagen peptides that signal the breakdown of deposited osteoid.
Concurrently, high levels of bone resorption are responsible for stimulation of a local anabolic
response, which is influenced and modulated by mechanical forces (Martin, 2005; Mohan, 1991).
This is seen in cases where lack of loading on the developing bone, such as disuse and/or rigid
fixation, leads to impaired bone healing (Rubin, 2002). Furthermore, it has been observed in
rodent animal models that the anabolic activity of osteoblasts can still be driven by the secreted
osteoclast factors, such as osteoclast inhibitory lectin (OCIL) (Nakamura, 2007), even in
10
presence of non-functional osteoclast cells (Marzia, 2000; Schaller, 2004). This suggests that the
release of factors from resorbed bone matrix may not be critical in stimulating osteoblast
function, and the exact mechanism for the anabolic/catabolic response coupling during bone
remodelling needs to be further investigated (Martin, 2005).
1.2 Skeletal Cell Types
Unlike other organs that generate scar tissue upon healing, the skeleton possesses a
unique regenerative potential where the newly formed bone is indistinguishable from the
uninjured adjacent tissue. As well, there are various conserved similarities and notable
differences between fetal skeletal tissue development and adult reparative process. For instance,
in both processes progenitor cells aggregate at site of future bone formation to form
condensations (Hall, 1988; Thompson, 2002) that proceed down a chonrogenic/osteogenic
lineage via the expression of a conserved set of molecular markers that regulate cell
differentiation and maturation (Ferguson, 1999; Karsenty, 2002; Vortkamp, 1998). Furthermore,
both fetal or adult skeletal formation require extracellular matrix remodelling and vascularisation
as prerequisite for ossification, and disruption of these events can lead to delay of subsequent
bone formation (Thompson, 2002; Vu, 1998). In contrast, mechanical forces (Carter, 1998; Le,
2001; Probst, 1997; Thompson, 2002) and inflammatory response (Simon, 2002) have
substantial influence on cartilage and bone formation during adult fracture repair, but are not
required for the initiation of chondrogenesis or osteogenesis during fetal growth. Chondrocytes,
osteoblasts, osteocytes and osteoclasts are key players in the process of bone formation and
repair, where the absence of any one of these lineages will disrupt the complex process of
skeletogenesis.
1.2.1 Chondrocytes
Chondrocytes, the cartilage forming cells, promote bone growth throughout development.
During endochondral growth plate development, four chondrocyte populations become arranged
in distinct narrow zones, which range from the resting zone closest to the joint surface to the
proliferating, prehypertrophic and hypertrophic zones at the calcified end of the growth plate
(Adams, 2002; Wagner, 2001). These cells are organized in columns and divide along the long
axis of the plate, changing in shape and phenotype as they progress through the various zones.
As the cells become hypertrophic, they become swollen and increase in volume while expressing
11
type X collagen. Secretion of this matrix acts to initiate mineralization, whereby trabeculae of
woven bones are deposited by osteoblasts onto the calcified cartilage septa (Adams, 2002).
A complex network of signalling molecules regulates the proliferation and differentiation
of these different chondrocyte subpopulations (Figure 1A). The earliest molecule to be
expressed during chondrogenesis is the transcription factor Sox9, which is required for
chondrogenesis of the mesenchymal condensate that prefigures bone formation (Wright, 1995).
Various studies, looking at either conditional knock-out or null Sox9 transgenic mouse lines,
have shown that in absence of Sox9 there was no cartilage formed in the limbs of these mice and
cells in the mesenchymal condensate do not adopt a chondrogenic morphology (Akiyama, 2002;
Bi, 1999). Sox9 is the master gene that regulates the expression of various genes involved in
chondrogenesis, such as Sox5 and Sox6, and together these genes are implicated in induction of
cartilage matrix proteins, aggrecan and type II collagen (Han, 2008; Lefebvre, 1997; Lefebvre,
1998a; Lefebvre, 1998b).
Furthermore, recent studies have shown Runx2/Cbfa1 to be one of the transcription
factors genetically downstream of Sox9 controlling the differentiation of chondrocyte
hypertrophy (Takeda, 2001; Ueta, 2001). This was shown in mice lacking Runx2/Cbfa1 that
lacked hypertophic chondrocytes in several but not all skeletal elements (Inada, 1999; Kim,
1999). Two other growth factors, parathyroid hormone related peptide (PTHrP) and Indian
hedgehog (Ihh), provide a tightly regulated feedback loop that play critical roles in controlling
chondrocyte maturation through the different zones. Cells from the perichondrium secrete PTHrP
that signal via their PTH/PTHrP receptors on prehypertrophic chondrocytes that suppress their
differentiation into hypertrophic chondrocytes (Karaplis, 1994; Lanske, 1996). Furthermore, Ihh
is secreted by prehypertrophic chondrocytes that signal to the cells in the perichondrium to
upregulate PTHrP synthesis that in turn inhibits chondrocyte hypertrophy and endochondral bone
formation (St-Jacques, 1999; Vortkamp, 1996).
1.2.2 Osteoclasts
Bone acts as a reservoir for calcium and phosphate in the body and is resorbed/ deposited
via feedback mechanisms in accordance with the stresses that are placed upon it (Robert, 2009).
Bone remodelling, which is essential for repairing microdamages and optimizing bone structures
for mechanical function, is a continuous episode that is mediated by specialized bone resorbing
12
cells, the osteoclasts (Robert, 2009). The haematopoietic stem cell-derived osteoclasts require
close interaction with osteoblasts for their activation (Figure 1B).
Receptor activator of nuclear factor-κB ligand (RANKL), a member of the TNF
superfamily, is expressed by osteoblasts and is the primary mediator of osteoclastogenesis
(Lacey, 1998; Robert, 2009). Through its interaction with its receptor RANK, which is expressed
on osteoclasts and their precursors, RANKL promotes osteoclast differentiation, fusion and
activation (Lacey, 1998). After ligand-induced trimerization, RANK promotes cytoplasmic
signaling by recruiting adaptor molecules that activate downstream signaling pathways mediated
by protein kinases that include inhibitor of NF- κB kinase (IKK), c-Jun N-terminal kinase (JNK),
p38, Src and extracellular signal-regulated kinase (ERK) pathways (Boyle, 2003; Teitelbaum,
2003). A key preliminary step in RANK signaling involves the binding of TNF receptor-
associated factors (TRAFs) to specific cytoplasmic domains of RANK, which directs activation
of transcription factor NF- κB and activator protein-1 (AP-1) (Boyle, 2003). NF- κB has been
shown to be pivotal to osteoclastogenesis and deletion of NF- κB leads to osteoclast-autonomous
osteopetrosis, characterized by absence of multinucleated bone-resorbing cells (Franzoso, 1997).
Another transcriptional influence of RANKL include the activation of osteoclastogenic
transcription factor NFATc1 (also called NFAT2), which prompts osteoclastogenesis when
expressed in precursor cells even in the absence of RANKL (Ishida, 2002; Takayanagi 2002).
Active osteoclasts attach to bone, creating a tight seal, and are identified by expression of
the phosphatase TRAP. In addition, they secrete a range of factors involved in progenitor
mobilization (Heissig, 2002a; Kollet, 2003; Pruijt, 1999) and migration of preosteoclasts to the
resorption site (Blavier, 1995), that include the cytokine IL-8 (Rothe, 1998) and the proteolytic
enzyme MMP-9 (Vu, 1998) and cathepsin K (Kollet, 2003). The role of MMP-9 in bone
remodelling has been shown in MMP-9 knockout mice, where the growth–plate cartilage suffers
delayed ossification suggesting a role for this enzyme in cartilage resorption (Vu, 1998).
Osteoclasts are distinguished by their polarization toward the bone and the formation of a
ruffled membrane structure, which acts as the resorptive organelle of the cell and contains a
villous-like complex with numerous ‘spike-like’ vacuolar H+ATPase proton pumps (Blair, 1989).
The formation of this structure is dependent on the contact of osteoclast with bone and allows the
creation of an acidic environment between the cell and the juxtaposed bone structure. Cathepsin
13
K, one of the major bone-resorbing proteases, is essential for bone remodelling and degradation
of type I collagen (Goto, 2003). Secretion of these enzymes by osteoclasts into the resorptive
pits, which are isolated by tight seals, allows the degradation of the skeletal matrix and
progression of bone remodelling.
Osteoprotegerin (OPG), a soluble decoy receptor, is secreted by osteoblast cells and
inhibits osteoclastogenesis by blocking RANKL binding to its cellular receptor RANK on
osteoclasts (Boyle, 2003). Mice overexpressing or deficient in OPG have been shown to exhibit
phenotypes of marked increased or decreased in bone density, respectively (Bucay, 1998;
Simonet, 1997). Overexpression of OPG leads to inhibition of RANKL activation of osteoclasts
and the resulting decreased osteoclastogenesis is manifested in increased bone density, as seen in
osteopetrotic pathologies (Simonet, 1997). In contrast, a deficiency in OPG is characterized by
increased bone resorption, severe trabecular and cortical proposity, leading to an osteoporotic
phenotype with decreased bone density and high occurrences of fractures (Bucay, 1998). The
cytokine M-CSF is expressed by osteoblasts and their precursors as a soluble and membrane-
bound protein and promotes survival and proliferation of osteoclast precursors (Teitelbaum,
2003). The role of M-CSF in osteoclastogenesis was established by studies of op/op mouse that
display osteoclast-deficient osteopetrosis due to nonfunctional M-CSF (Yoshida, 1990).
A chemokine, SDF-1, together with its receptor CXCR4 play pivotal role in survival,
proliferation, anchorage of stem cells to the endosteal region of bone or the endothelial
microenvironment within the bone tissue (Adams, 2006; Cottler-Fox, 2003; Lapidot, 2005). This
ligand is highly expressed by bone marrow endothelial cells as well as endosteal osteoblasts
(Adams, 2006; Kollet, 2006; Ponomaryov, 2000), which have been shown to support the
maintenance of the haematopoietic stem cell niche (Yin, 2006) and retaining stem cells in
quiescent state (Arai, 2004). An increase in SDF-1 is seen under stress situations such as injury,
chemotherapy, inflammation or treatment with granulocyte colony-stimulating factor (G-CSF),
which triggers an imbalance of steady-state homeostasis and induce massive stem cell
mobilization, proliferation and differentiation (Cottler-Fox, 2003; Heissig, 2002). Ultimately this
leads to enhanced secretion of proteolytic enzymes such as MMP-9, elastases and cathepsin K,
resulting in increased activity of osteoclasts and higher bone resorption (Takamatsu, 1998;
Watanbe, 2003). Through a feedback mechanism, following the increase in proteolytic enzymes
14
by osteoclasts there is degradation of SDF-1, increased expression of CXCR4 and mobilization
of maturing progenitors and stem cells (Levesque, 2003; Petit, 2002).
1.2.3 Osteoblasts
Osteoblasts are highly differentiated extracellular matrix-producing cells that are derived
from an undifferentiated progenitor population through a series of differentiation and maturation
events (Aubin, 1998). During their differentiation process, osteoblasts can either (1) undergo
programmed cell death, (2) become quiescent and transform into inactive bone-lining cells, or
(3) merge progressively within the bone matrix to become mature osteoblasts (Jilka, 1998;
Manolagas, 2000; Noble, 1997). Some researchers also report cases where osteoblasts
transdifferentiate into cells able to lay down chondroid bone (Li, 2004). Based on efforts by
numerous groups embarking in the research to identify osteoblast-specific transcription factors,
several aspects of osteoblast biology have been recognized. Primarily it has been noted that
throughout the differentiation process, osteoblasts remain similar to fibroblasts and the only
difference is slow to appear and seen late in the process, with the secretion of extracellular
matrix that is only produced by mature osteoblasts (Aubin, 1996). Due to absence of phenotypic
changes throughout osteoblast differentiation, it has been difficult to study osteoblast biology at
cellular levels. As a result, gene expression assays have been utilized to assess the various
aspects of osteoblast differentiation.
Cells of osteoblast origin have been shown to play essential role in orchestrating bone
formation and remodelling. They are involved in bone formation during embryonic development
and fracture repair by depositing new bone matrix known as osteoid, while playing a role in bone
resorption and remodelling by producing a wide range of compounds that regulate osteoclast
activity (Hofbauer, 2000; Lerner, 2000; Puzas, 1992; Suda, 1997). These regulating molecules
include prostaglandin E2 (PGE2), IL-1, IL-6, and RANKL (Hofbauer, 2000; Lerner, 2000;
Puzas, 1992; Suda, 1997).
Several publications have shown the involvement of mononuclear cells in phagocytosis
of collagen at sites adjacent to osteoclast resorption pits (Beertsen, 1978; Garant, 1976; Rifkin,
1979; Tran, 1982; Yajima, 1999). Prior to bone resorption, osteoblasts aid in removing any
nonmineralized osteoid from bone surface by secreting a number of MMPs (Chambers, 1985a;
Chambers, 1985b), and osteoblasts mediate the initiation of bone formation after resorption,
15
which results in a finely tuned remodelling process (Harris, 1969; Kahn, 1987; Martin, 1994;
Rodan, 1997). Quite a few studies have shown that after resorption by osteoclasts, the resorption
pit still contains non-digested demineralised bone matrix (Delaisse, 1987; de Saint-Georges,
1989; Holliday, 1997; Jones, 1986; Tran, 1982). A study by Everts and colleagues looked at the
involvement of bone lining cells in coordination with bone resorption and regulation of bone
formation after resorption by osteoclasts (Everts, 2002). By analysing mouse long bone and
calvariae they determined that after osteoclasts have exerted their activity, bone lining cells enter
the resorption pit and subsequently digest any protruding collagen fibrils that are left by
osteoclasts. Several publications have reported that osteoblasts are driven toward the resorption
pits via signalling molecules such as TGF-β (Erlebacher, 1998; Oursler, 1994; Pfeilschifter,
1990), IGF (Hayden, 1995), or OPN (Dodds, 1995).
Runx2/Cbfa1 is necessary for both endochondral and intramembranous bone formation
and is expressed in MSC during early embryonic development, where it acts as a master
regulator in commitment of these cells to osteoblastic lineage. Mice lacking in Runx2 gene fail to
form mineralized bone in any part of the skeleton, lack mature osteoblasts, and have significantly
decreased expression of osteoblast differentiation markers such as ALP, OPN, and osteocalcin
(Komori, 1997; Otto, 1997). Furthermore, other studies have shown that expression of
Runx2/Cbfa1 is cooperatively regulated by BMP-2 and TGF-β signalling, two genes that play
essential role in the process of skeltogenesis (Lee, 2003). Current knowledge indicates that most
of the proteins factors expressed by osteoblasts are also expressed by other cell types such as
fibroblasts or chondrocytes. As a membrane bound enzyme, bone ALP is a maker for
hypertrophic chondrocytes (Althoff, 1982; Habuchi, 1993), is associated with early
differentiation of osteoblasts (Stein, 1993), and plays important role in extracellular
hydroxyapatite formation (Hui, 1997). The matrix protein osteocalcin is expressed exclusively in
bone and attenuates the process of bone formation since osteocalcin-deficeint mice display
increased bone mass (Ducy, 1996; Mark, 1988; McKee 1992). Osteocalcin is present in the
beginning of the mineralization phase while ALP expression is linked to matrix maturation
(Tuckermann, 2000). The only true osteoblast-specific gene that is expressed by fully
differentiated osteoblasts and regulates their function is osteocalcin (Desbois, 1994; Ducy, 1996;
Hauschka, 1989).
16
The novel zinc finger-containing transcription factor, osterix (Osx), was originally
identified via a simple screen to isolate osteoblast-specific cDNA (Nakashima, 2002), and found
to be required for osteoblast differentiation and bone formation. Osx is expressed in osteoblasts
of all endochondral and intramembranous bones and in absence of Osx there is no cortical or
trabeculae bone formation. In Osx null mice cartilage anlagen is fully formed and mesenchymal
cells, along with blood vessels and osteoclasts, are able to invade the mineralized cartilage
matrix during endochondral ossification, but do not deposit any bone matrix (Nakashima, 2002).
Similarly, the osteogenic cells in intramembranous skeletal elements of these mice are
completely blocked in their differentiation into osteoblasts. These cells do however exhibit
Runx2/Cbfa1 expression at levels comparable to those in wildtype osteoblasts. In contrast, Osx is
not expressed in Runx2/Cbfa1 null mice, indicating that Osx is not required for Runx2/Cbfa1
expression and acts genetically downstream of Runx2/Cbfa1 pathway (Nakashima, 2002).
During mouse embryonic development Runx2/Cbfa1 expression is detected as early as
E10, however expression of molecular markers of differentiated osteoblasts is seen around E13
and replacement of the cartilaginous template by bone occurs at later E15 time point (Bianco,
1991; Chen, 1992; Ducy, 2000; Kauffman, 1992). This delay implies that other regulatory
proteins such as Osx, an activator of transcription (Nakashima, 2002), are involved in this
process. Other factors such as twist proteins, expressed transiently in osteoblast progenitors, act
as inhibitors of Runx2/Cbfa1 function. In vertebrates, two twist genes (Twist-1 and -2) exist that
code for the basic helix-loop-helix (bHLH)-containing transcription factors (Wolf, 1991; Li,
1995). Bialek and colleagues demonstrated that during skeletogenesis Twist proteins transiently
inhibit osteoblast differentiation via the interaction of a novel antiosteogenic domain within these
proteins with Runx2/Cbfa1 DNA binding domain (Bialek, 2004), thereby interfering with its
function. This Twist box, a domain distinct from the bHLH domains, is a 20 amino-acid long
domain that allows the antiosteogenic function of Twists (Castanon, 2002).
Regulation of the Runx2/Cbfa1 gene expression and protein functionality occurs at
multiple other levels, where various cytokines, growth factors, and hormones, including TGF-β,
BMP, FGF, sonic hedgehog, vitamin D3, and estrogen, control the spatial and temporal
expression pattern of Runx2/Cbfa1 (D’Souza, 1999; Ducy, 1997; Kim, 2003; Lee, 1999;
Takamoto, 2003; Tou, 2001; Tsuji, 1998; Zhou, 2000). Taken together, these data suggest that
aside from its critical role in osteoblast growth and chondrocyte hypertrophy, Runx2/Cbfa1 may
17
indirectly control osteoclast function by mediating RANKL and OPG production by osteoblasts
(Enomoto, 2003; Jonason, 2009; Mori, 2006; Usui, 2008).
1.2.4 Osteocytes
Osteocytes are the most abundant cellular components found in the cortical bone
structure, making up 95% of all cells in bone and covering close to 94% of all bone surfaces
(Frost, 1960; Marotti, 1996). An individual bone consists of ten times more osteocytes than
osteoblasts, embedded in the cortical bone tissue (Parfitt, 1990). They are formed during various
modes of ossification when advancing active osteoblasts lay down bone matrix that engulfs the
osteoblasts already at the ossification front (Knothe, 2004).
Similar to the cross-talk between osteoblasts and osteoclasts that has previously been
established (Lacey, 1998; Yasuda, 1998), there is cross-talk between osteocytes and osteoblasts
(Burger, 2003). Osteocytes form a meshwork of cell processes with one another through a
system of canaliculi that is formed in the bone matrix and allows communication between
adjacent osteocytes (Palumbo, 1990a). Once osteoblasts, which are estimated to only live up to
three month in human bones (Manolagas, 2000) or even shorter (10-20 days) in mouse alveolar
bone (McCulloch, 1988) become embedded in the developing bone, they mature into osteocytes
that have a greater lifespan than active osteoblasts.
Numerous terminologies have been used to describe the osteoblast to osteocyte transition
(Holtrop, 1990; Manolagas, 2000; Meunier, 1989), however not much research has been
conducted in the field of bone biology that focuses on the formation and biology of osteocytes.
The process of osteoblast transformation into osteocytes is dependent on a variety of factors,
ranging from the mode of ossification, the type of bone generated (woven vs lamellar bone), the
location of bone formation, the species, and on the gender/age of the model being studied (Hall,
2005). Once the osteoblasts become embedded in the bone matrix and transform to osteocytes
they cease their bone deposition activity and instead function to maintain the integrity of bone
structure by acting as strain and stress sensors (Burger, 2003; Knothe, 2004). The embedded
osteocytes are also able to deposit and resorb bone around the lacunae in which they reside, as a
result changing the shape of the lacuna in a process called osteocyte osteolysis. Although this
process is not seen in human osteocytes, there is evidence for its occurrence in other vertebrates
such as squirrels (Haller, 1978), rats (Belanger, 1977; Tazawa, 2004), snakes (Alcobendas,
18
1991), eels (Lopez, 1980), and bats (Doty, 1985; Kwiecinski, 1985; Kwiecinski, 1987), among
other species (Steinberg, 1981; Witten, 2000; Zhang, 2000).
Several hypotheses exist in the literature as to how the transition from osteoblasts to
osteocytes occurs within the bone matrix. Unpolarized osteoblasts can become entrapped by their
own secretion by laying down bone in all directions, while polarized osteoblasts can become
embedded within the matrix via their neighbouring cells (Nefussi, 1991; Palumbo, 1990a;
Palumbo, 1990b). Another scheme is that of acellular, osteocyte-deprived bone that occurs when
a group of highly polarized osteoblasts function as a unit to lay down bone matrix in a
synchronized matter, moving away from the osteogenic front and resulting in creation of
acellular bone (Ekanayake, 1987; Ekanayake, 1988; Witten, 1997). The fates of the remaining
osteoblasts include apoptosis or becoming bone-lining cells (Franz-Odendaal, 2006). The
number of osteoblasts that become osteocytes has been estimated to be 10-20% (Aubin, 1998),
whereas apoptosis is thought to eliminate approximately 60-80% of these osteoblasts (Jilka,
2007). In this fully formed bone, the osteocytes constitute 95% of the cells in the bone (Franz-
Odendaal, 2006).
1.3 Osteogenic Proteins
From a physiological perspective, both molecular and genetic regulatory mechanisms are
essential for the recruitment, differentiation and maturation of each of the cell types involved in
skeletogenesis. As the process of bone formation is a complex multistep phenomenon, requiring
the participation of a variety of cells, there is a broad spectrum of physiological regulatory
signaling proteins that manage the initiation and progression of individual stages of ossification,
ranging from neovascularization to chondro/osteogenesis and matrix remodeling (Einhorn,
1995). This introduction will explore the mechanism and function of a few of these proteins that
have been determined to be important during bone formation.
1.3.1 Vascularization in Successful Bone Healing
Adequate blood supply during development and bone repair has been viewed in the
literature as one of the key elements that warrants accurate and timely restoration of the bone
tissue (Schindeler, 2009). The primary underlying cause of poor bone repair has been proposed
to be due to vascular deficiency (Pelissier, 2003) and hypoxic stress has been shown to stimulate
19
secretion of pro-angiogenic factors from osteochondral cells that in turn promote the re-
establishment of a vascular supply to support bone cell differentiation (Wang, 2007a).
Furthermore, compiling evidence that argues for the temporal occurrence of bone
formation and vascularisation has generated an emerging concept that vascular cells, pericytes,
may contribute to bone repair. Pericytes have been shown to have myogenic differentiation
capacity (Dellavalle, 2007), and to exhibit osteogenic phenoytpes in vitro, expressing
osteoblastic markers and ability to form mineralized nodules (Brighton, 1992; Doherty, 1998;
Schor, 1990). This is further supported by data exhibiting a synergistic interplay between
vaslcular and osteoprogenitor cells. It has been shown that overexpression of the key factor
hypoxia inducible factor α (HIF1α) in osteoblasts results in development of overly dense bones,
while disruption of this signalling pathway produces the opposite phenotype of osteoporotic
bones (Wang, 2007b). Additionally, co-expression of the potent pro-angiogenic factor VEGF
with bone inducing molecule BMP4, transplanted in vivo via implanted stem cells, resulted in
increased ectopic bone formation (Peng, 2002).
Arrays of various molecular effectors that are involved in normal embryonic vascular
development also play a role in neovascularisation in adults (Carmeliet, 2003). These factors
range from VEGF, angiopoietins (Ang-1), basic fibroblast growth factor (bFGF), hypoxia
inducible factor α (HIF1α) and various members of TGFβ, to PDGF, IGF family (IGF-1, IGF-2),
and hepatocyte growth factor (HGF) (Madeddu, 2005). Interestingly, OPN-deficient mice, aside
from an increase in osteoclastic activity that leads to increased bone resorption and lower bone
density, display medial calcification of renal and aorta, which suggests a role for OPG in
correlation involving vascular calcification and osteoporosis (Bucay, 1998).
The family of VEGFs and their receptors are critical regulators of angiogenesis that play
an important role in skeletal growth and repair by initiating an array of signal transduction
cascades that eventually lead to development of vasculogenesis, or angiogenesis in the case of
tissue repair (Carmeliet, 2000; Ferrara, 2001; Rossant, 2002; Tammela, 2005; Yancopoulos,
2000). This creates a network between neighbouring cells, which by providing an adequate
system for flow of different cytokines, chemokines, and growth factors helps maintain the
homeostatis of the surrounding tissues. Any inhibitions in development of this system in
20
repairing bone tissue leads to various pathologies such as osteonecrosis (Childs, 2005),
osteomyelitis (Lazzarini, 2002), and osteoporosis (Alagiakrishnan, 2003; Burkhardt, 1987).
There are multiple stages during growth and development where vascularisation of
cartilage regions is required. During embryonic development, there is invasion of blood vessels
originating from the perichondrium, into the emerging cartilage structure. Second, during
development, rapid vascularisation of the growth plates allows for elevated postnatal growth.
Third, during adulthood, the process of angiogenesis is controlled periodically when anabolic or
catabolic responses are required due to bone trauma or pathophysiologic conditions, such as
rheumatoid arthritis (RA) and osteoarthritis (OA) (Gerber, 2000). Several prior studies involving
early block in osteoblastogenesis via genetic inactivation of Runx2/Cbfa1, Osx, Indian hedgehog
or β-catenin have suggested that osteoblast differentiation of progenitor cells, up to the stage of
Runx2/Osx expression, is required for vascular invasion of cartilage and primary ossification
(Colnot, 2005; Hill, 2005; Komori, 1997; Nakashima, 2002; Otto, 1997; St-Jacques, 1999).
1.3.2 Matrix Metalloproteinases
The matrix metalloproteinases (MMPs), a family of enzymes sharing common functional
domains and activation mechanisms (Sternlight, 1999),(243) play a role in regulating cell
migration and ECM remodelling, which is essential for morphogenesis, embryonic development
and tissue remodeling (Birkedal-Hansen, 1993; Brinckerhoff, 2002; Nagase, 1999).(244-246) Bone
formation involves extensive remodelling of the extracellular matrices (ECM), and MMPs 2, 8, 9
and 13 have been implicated in this process (Chin, 1997; Gack, 1995; Johansson, 1997; Maruya,
2003; Mattot, 1995; Sasano, 2002). In addition, MMPs have also been recognized to be
important in events such as osteoclast recruitment (Blavier, 1995; Engsig, 2000; Karsdal, 2001;
Sato, 1998), coupling of bone resorption and formation (Everts, 2002), angiogenesis (Vu, 1998)
and osteoblast survival (Karsdal, 2002).
Itagaki and colleagues examined the expression of MMPs, type I collagen and
osteocalcin during bone healing in an experimental rat calvarial defect model. Their study
determined that the expression of MMP8 was highest on day 1, while type I collagen,
osteocalcin, MMP2 and MMP13 expression increased toward week 2 (Itagaki, 2008). Type I
collagen and osteocalcin mRNA transcripts were shown to be localized in osteoblasts and
21
osteocytes, and the expression of MMPs 2, 8, and 13 in some of these cell types suggested that
they may play a role in remodelling of the ECM during bone healing (Itagaki, 2008).
During endochondral ossification, expression of various MMPs in the developing bone
allows the remodelling of cartilage anlage and the subsequent neovascularisation. Among the
members of MMPs thought to be important for skeletal development, MMP-9 and MMP-13 have
been shown to be two of the main proteinases involved in endochondral ossification (Inada,
2004; Stickens, 2004; Uusitalo, 2000). The critical role of these MMPs can be seen in knockout
transgenic animals that display dramatic expansion of the hypertrophic zone in the growth plate
of the primary ossification site.
Of the collagenases, MMP-13 is the most effective at cleaving type II collagen
(Billinghurst, 1997; Knauper, 1996) and its expression has been shown to be regulated by
transcription factor Runx2/Cbfa1 (Porte, 1999; Selvamurugan, 1998). A study by Wu and
colleagues, using bovine fetal chondrocytes isolated from physeal cartilages, showed an increase
in expression of transcription factor Runx2/Cbfa1, type X collagen and MMP-13 upon
differentiation of these cells into hypertrophic chondrocytes (Wu, 2002). This was followed by
increased incorporation of 45Ca2+ in the extracellular matrix associated with matrix calcification.
Inhibition of type II collagen degradation by a nontoxic carboxylate inhibitor of MMP-13
resulted in suppression of chondrocyte differentiation as seen by a repression in Runx2/Cbfa1
and type X collagen gene expression, as well as decreased incorporation of 45Ca2+ in the
extracellular matrix. Their results suggested that expression and extracellular proteolysis activity
of MMP-13 is required for chondrocyte differentiation, which is associated with matrix
mineralization (Wu, 2002). This is also seen in mice lacking Runx2/Cbfa1, where the absence of
MMP-13 expression in the growth plates results in impaired chondrocyte maturation and matrix
mineralization during endochondral development (Inada, 1999; Jimenez, 1999). In contrast,
over-expression of Runx2/Cbfa1 causes acceleration of endochondral ossification in vivo (Ueta,
2001) and promotes chondrocyte differentiation and maturation in vitro (Enomoto, 2000).
Together, these observations indicate that the Cbfa1/MMP-13 pathway is important during
endochondral bone formation by regulating chondrocyte differentiation and matrix turnover.
As previously mentioned, angiogenesis is a critical event that is needed for successful
bone development. VEGF has been identified as one of the important angiogenic factors
22
expressed by hypertrophic chondrocytes and blocking VEGF activity results in a phenotype
similar to the MMP-9 null phenotype. It has previously been shown that MMP-9-null mice
exhibit an increase in hypertrophic chondrocytes and the amount of VEGF mRNA expression
(Ortega, 2003), a delay in endochondral ossification and marrow cavity formation, as well as a
decrease in chondrocyte apoptosis, vascular invasion, trabecular bone synthesis and osteoclastic
employment (Ortega, 2003). Also, in vitro angiogenesis assays of MMP-9-null mice cartilage
explants show a delayed angiogenic response (Vu, 2000). Treatment of wildtype mice with a
soluble VEGF receptor to reduce the bioavailability of VEGF displayed the same phenotypes of
MMP9-null mice (Gerber, 1999). However, the VEGF-depleted phenotype can be rescued with
the introduction of exogenous VEGF, which causes rapid remodelling of the hypertrophic
cartilage and an increase in trabecular bone. Similar rescue is seen in MMP-null mice
transplanted with wildtype bone marrow. Furthermore, MMP-9 has also been shown to be a
chemoattractant for osteoclasts and invasion of osteoclasts into the hypertrophic cartilage is
dependent on VEGF expression (Engsig, 2000; Gerber, 1999). Taken together, these
observations display a link between degradation of the cartilage anlage by MMPs, expression of
VEGF and their coordination in advancement of the endochondral process.
MMP-9 and MMP-13 are expressed in non-overlapping cell types at the edge of
ossification and together carry out most of the protease activity during endochondral
ossifications (Ortega, 2003; Vu, 1998). While MMP-9 is expressed in osteoclasts, endothelial
cells and bone marrow stromal cells, MMP13 is primarily expressed in terminal hypertrophic
chondrocytes and newly recruited osteoblasts (Inada, 2004; Stickens, 2004; Uusitalo, 2000; Vu,
1998).
Nonetheless, despite their different expression patterns, MPP-9 deficient and MMP-13
deficient mice exhibit similar phonotypic characteristics such as elongation of the hypertrophic
cartilage zone in the metaphyseal growth plates (Inada, 2004; Stickens, 2004; Vu, 1998). MMP-
13 protease is a very potent enzyme secreted under physiological conditions, which has high
preference for type I and type II collagen, major components of extracellular matrix and bone,
respectively (Knauper, 1996). Andersen and colleagues utilized various methods, such as PCR,
Northern blots, in situ hybridization, and immunohistochemistry, to look at expression pattern of
MMP-13 mRNA. Their study show that MMP-13 mRNA is expressed highly in mononucleated
cells closely surrounding the osteoclasts and in the subosteoclastic resorption zones (Andersen,
23
2004). Their data strongly suggest that in the case of MMP-13, its strong affinity to type I
collagen (Gillet, 1977; Knauper, 1997) allows its accumulation in zones of collagen
demineralization and osteoclast resorptive lacunae (Delaisse, 1993). This suggests that successful
collagen matrix resorption requires the coordination between both osteoblasts and osteoclasts,
where secreted MMP-13 functions in conjunction with osteoclastic secreted enzymes such as
MMP9 for progression of the healing process.
1.3.3 Bone Morphogenetic Proteins
A family of regulatory molecules known as bone morphogenetic proteins was initially
discovered by researchers investigating the active components of the demineralized bone grafts.
With the use of rDNA technology, more highly purified forms of these proteins were isolated
and the osteoinductive capacity of these proteins was tested by injection into soft tissue. These
molecules have been used in studying the healing of fractures (Cook, 1997; den Boer, 1997;
Einhorn, 1997; Poplich, 1997; Turek, 1997), bridging critical size defects in various animal
models (Bostrom,1996; Cook, 1994; Kirker-Head, 1995; Yasko, 1992), and
enhancing/accelerating fracture healing (Cook, 1997; den Boer, 1997; Einhorn, 1997; Poplich,
1997; Turek, 1997).
To test the effect of BMP2 on fracture healing, Einhorn and colleagues performed local
percutanous injection of BMP2 into closed mid-diaphyseal femur fractures in rats (Einhorn,
1997). Data from biomechanical testing show that BMP2 treated fractures exhibited significant
increase in stiffness around 14 days post fractures and increase in strength at 28 days after
fracture. They also showed relatively earlier maturation of the osteochondrogenic line and faster
bridging of the callus, and substantially more peripheral woven bone at the fracture callus of
BMP2 treated groups as compared with the controls (Einhorn, 1997).
First to investigate the effect BMPs were Urist and colleagues, who observed induction of
ectopic bone formation upon implantation of demineralized bone in rats (Urist, 1965). Few other
studies have looked at the efficacy of injectable BMP7 for accelerating healing in small defects
or fracture gaps (Cook, 1997; den Boer, 1997; Poplich, 1997). In BMP7-treated canines new
bone formation and bridging of the defect occurred at an accelerated pace compared with the
controls. The defects treated with BMP7 also showed significantly greater strengths than those of
control limbs. In another study, den Boer investigated the effects of BMP7 on the healing of a
24
closed diaphyseal fracture in the goat. Three dimensional computerized tomography (CT) scans
and dual energy x-ray absorptiometry (DEXA) analysis revealed that at early time points BMP7
treated groups developed a larger callus volume and higher bone mineral content, respectively
(den Boer, 1997). Since their discovery, bone derived BMPs (BMP2, 4, and 7) have also been
used clinically to treat difficult cases of nonunion fractures and shown to have the capacity to
enhance bone induction in humans.
Additionally, BMPs have been shown to be important in chondrogenesis. Several BMPs
have been shown to be expressed around and within the chondrogenic condensations, have the
ability to trigger ectopic chondrogenesis in adult animal models, and cause significant
enlargement of cartilages in gain-of-function experiments (Duprez, 1996; Macias, 1997; Reddi,
1994; Urist, 1965). In developing cartilage, the function of BMPs in chondrogenesis is regulated
by Noggin, a BMP antagonist (Brunet, 1998; Capdevila, 1998; Merino, 1998). Among other
functions, BMPs regulate dorsa-ventral patterning during embryogenesis (Jones, 1992), control
cell apoptosis, patterning of limb buds (Zou, 1996), as well as influencing epithelial-
mesenchymal interactions during organogenesis (Cheifetz, 1999; Vainio, 1993).
1.3.4 Transforming Growth Factor Beta
One of the major groups of growth factors, the TGF-β superfamily of polypeptides,
which play a critical role in the induction, development and repair of bone via regulation and
stimulation of mesenchymal precursor cells for chondrocytes, osteoblasts, and osteoclasts.
During fracture healing TGF-βs are released during the initial inflammatory phase from platelets
and are thought to be integral mediators of fracture repair by playing a role in initiation of callus
formation (Canalis, 1988; Celeste 1990; Joyce, 1990a; Joyce, 1990b; Sporn, 1989). Members of
this group range from the five TGFβs (TGFβ1, TGFβ2, TGFβ3, TGFβ4, and TGFβ5) to BMPs,
activins, inhibins, and growth and differentiation factors (Centrella, 1994). TGFβs are produced
by a variety of cells in different tissues, but have a significantly higher concentration in bone and
are expressed by osteocytes, osteoblasts, osteoclasts and chondrocytes (Centrella, 1994;
Ellingsworth, 1986; Robey, 1987; Sandberg, 1993). By utilizing various experimental
techniques, such as immunohistochemistry, in situ hybridization and northern blot analysis, the
role of TGFβ in association with cartilage hypertrophy and calcification of the endochondral
ossification front has been previously determined (Joyce, 1990a; Joyce, 1990b).
25
In osteoblasts, TGFβ is stored as high molecular weight precursor molecule complexes
that need to go through post-translational processing prior to being secreted as an inactive
product, which gets incorporated into the mineralized bone matrix (Bonewald, 1994; Dallas,
1998; Miyazono, 1993). They are activated in an extremely acidic pH environment, which is
created by osteoclasts when they degrade the bone matrix by acid hydrolysis. This in turn
stimulates the new bone formation via the stimulating action of TGFβ on osteoblasts during
development or different stages of fracture healing (Oreffo, 1990). In fact, a majority of in vitro
studies have indicated that TGFβ functions on osteoblasts by increasing the expression of a
variety of osteoblast differentiation markers such as ALP, Type I collagen, and osteonectin
(Harris, 1994; Ingram, 1994; Massaque, 1990; Sandberg, 1993; Strong, 1991; Tally-Ronsholdt,
1995; Wergedal, 1992). TGFβ also acts to induce production of cartilage components including
Type II, III, V, VI, and X collagen, fibronectin, and proteoglycans (Harris, 1994; Massaque,
1990; Sandberg, 1993), while preventing the digestion of these extracellular matrix
macromolecules by having inhibiting effects on the action of metalloproteinases (Harris, 1994).
After secretion TGFβs are incorporated into the mineralized bone matrix where through a
feedback mechanism they regulate the presence of proteases/plasminogens that activate latent
TGFβs (Yee, 1993). Overall, TGFβ acts as a potent chemotactic agent during skeletal
development and the net effect of TGFβ can be seen in different stages of bone formation such as
its stimulation of collagen synthesis, enhancement of osteoblast proliferation and regulation of
bone matrix production.
1.3.5 Wnt Signalling and Bone Formation
Wnt molecules, a family of secreted glycoproteins, play a central role in countless
embryonic developmental processes as well as regulating homeostatsis of different tissues in
later stages of growth. During embryonic development they play a role in determining patterning
and wnt signaling regulates homeostasis during growth (Cadigan, 1997). Precise regulation of
wnt-mediated effects is necessary for successful development since fluctuations of this pathway
leads to a variety of diseases and developmental defects (Cadigan, 1997). The signalling pathway
is initiated by binding of the wnt ligand to one of its 10 Frizzled (Fz) receptor family members
on the cell surface, inducing one of the three distinct pathways that have been previously
elucidated (Glass, 2006a; Katoh, 2002). The most studied canonical wnt/β-catenin pathway plays
a crucial role in bone formation and homeostasis. This pathway is regulated at multiple steps
26
including modulations at the extracellular level, at the cell membrane, in the cytosol and in the
nucleus, which ultimately results in the intracellular accumulation and consecutive translocation
of the β-catenin protein to the nucleus. Once in the nucleus, association of β-catenin with the
lymphoid-enhancer binding facter (Lef)/T-cell specific transcription factors (Tcf’s) leads to
expression of target genes (Westendorf, 2004). This pathway is regulated extracellularly by
secreted Dickkopf (DKK) protein inhibitors (Davidson, 2002; Niehrs, 2006) or secreted frizzled-
related proteins (sFRPs)/Wnt inhibitory factor-1 (Wif-1) that prevent the induction of the wnt
signalling by binding to the coreceptor LRP5/6 or binding to the wnt ligands themselves,
respectively (Bafico, 2001; Mao, 2002; Schweizer, 2003).
Another alternative wnt pathway includes the non-canonical pathway that relies on
intracellular release of calcium for regulating cell migration, dorso-ventral patterning of the
embryo, as well as heart development (Kuhl, 2004). The planar cell polarity (PCP) is the third
wnt pathway that regulates cytoskeletal architecture, actin dynamics and cellular migration via
coactivation of two small GTPases, Rho and Rac, as well as the stress-activated protein kinase
JNK (Habas, 2003; Kaltschmidt, 2002; Katoh, 2005). The activation of these signaling
transduction pathways is known to contribute to regulation of cell proliferation and apoptosis,
cell-cell adhesion, as well as being involved in embryonic morphogenesis (Davis, 2000).
The non-canonical Wnt5a and Wnt5b are expressed in chondrogenic regions of the limb
during development where they exhibit opposite activities in regulating longitudinal growth
(Bradley, 2011; Guo, 2008; Yang, 2003). Wnt5a regulates the transition from articular/resting
chondrocytes zone to the highly proliferating and hypertrophic chondrocyte zones, while Wnt5b
counteracts the activity of Wnt5a by inhibiting chondrocyte hypertrophy (Yang, 2003). These
opposite effects of Wnt5a and Wnt5b may result from their modulation of cell cycle effectors
cyclin D1 and p130, and Sox9-controlled Col2a1 expression (Yang, 2003). Therefore, the wnt
signalling pathways have been established as a dominant regulator of the stages of bone
formation, ranging from regulating the differentiation of precursor cells down either
chondrogenic or osteogenic lineages, and controlling the process of matrix degradation during
the remodelling phase of soft callus (Glass, 2006a, Glass, 2006b) to being involved in normal
bone homeostasis via expression by osteocytes (Kramer, 2010).
27
1.4 Thymidine Kinase/Ganciclovir System
A predominant component of the extracellular matrix, type I collagen is synthesized by
an array of connective tissue cells that display characteristic regulation of these genes under a
variety of different circumstances (Pavlin, 1992). Analysis of the regions responsible for
regulation of the collagen promoters by local growth factors, systemic hormones and viral
transformation (Choe, 1987; Liau, 1985; Rossi, 1988; Walsh, 1987), have identified a number of
cis-acting elements and trans-acting factors involved in regulating the tissue-specific expression
of type I collagen (Ramirez, 1990; Vuorio, 1990). Among these, the COL1A1 and COL1A2
promoter sequences have been shown to regulate the tissue-specific collagen expression in
transfected cells (Boast, 1990; Karsenty, 1990; Rippe, 1989; Schmidt, 1986) and in transgenic
animals (Khillan, 1986; Slack, 1991).
Numerous studies have provided evidence for regulatory mechanisms that are unique for
the osseous tissue producing cells such as osteoblasts and odontoblasts, as compared to many
fibroblastic cells. For example, the rate of collagen synthesis measured in primary cell cultures
(Luben 1976; McCarthy 1988; Wong 1982), fetal rat calvariae in organ culture (Canalis 1980;
Rowe, 1982), and embryonic tissues (Diegelmann, 1971; Moen 1979) show a higher basal rate of
collagen production in osteoblasts than in fibroblastic cells. Secondly, collagen synthesis in
osteoblasts can be regulated by several hormones such as vitamin D, PTH (Kream 1980;
Lichteler 1989; Rowe, 1982), insulin and insulin-like growth factor-I (Canalis, 1980; Kream
1985), while fibroblasts are unresponsive to these hormones. Furthermore, fibroblastic cells
produce a variety of collagen types (I, III, IV, V and VI), whereas fully differentiated osteoblasts
almost exclusively synthesize type I collagen (Fessler, 1981; Liau, 1985; Olsen, 1989;
Pihlajaniemi, 1989). Finally, a retroviral insertion in the first intron of the COL1A1 gene of
MOV13 mouse, does not affect collagen transcription in osteoblasts while resulting in a
complete block of collagen transcription in fibroblasts (Kratochwil, 1989). This ability to
override the inhibitory effect of the retroviral insertion suggest the presence of a cis-active DNA
sequence within the COL1A1 gene in osteoblast and odontoblasts that differ from those used in
fibroblastic cells.
The modular organization of the Col1a1 promoter allows selection of specific domains
for use in different type I collagen-producing tissues (Bedalov, 1995; Bogdanovic, 1994;
28
Rossert, 1995). For expression of the transgene in the osteoblast layer lining newly formed
calvarial bone, a homeodomain binding TAAT sequence localized between the -1670 and -1683
base pairs have been shown to be important (Dodig, 1996). The 3.6-kilobase (kb) rat Col1a1
promoter have previously been used in conjunction with the chloramphenicol acetyl transferase
(CAT) gene, and shown high level of transgene expression in bone, tendon, and developing tooth
germ with lower levels in skin (Pavlin, 1992). Truncation of the 3.6-kb promoter to 2.3-kb
fragment caused no change in the level of CAT activity in calvaria while a 2- to 4-fold decrease
in the transgene expression was observed in tendon and a greater loss in the skin (Bogdanovic,
1994; Krebsbach, 1993). In other studies, similar cell-specific pattern of expression was detected
in transgenic mice harboring a 2.3-kb human COL1A1 promoter driving the expression of
bovine growth hormone (Liska, 1994), as well as in mice carrying a murine 2.3-kb Col1a1
promoter fragment fused to either the β-galactosidase or luciferase gene (Rossert, 1995).
One method for studying the lineage relationships and cell functions of a population of
differentiating cells is the use of tissue-specific transgenic expression of herpes simplex virus
thymidine kinase (HSV-tk) to conditionally deplete a specific cell population (Heyman, 1989).
Targeted cell ablation using this strategy requires high level synthesis of enzyme in a subset of
rapidly proliferating cells. The expression of the transgene can be directed to restricted cell types
via the use of tissue-specific promoters and enhancers that allow the physiological manipulation
of the cell and organs of interest (Hammer, 1985; Palmiter, 1982). The HSV-tk, although not
deleterious in mammalian cells by itself, becomes toxic in the presence of nucleoside analogs
like ganciclovir (GCV), leading to inhibition of DNA synthesis and cell ablation (Heyman,
1989). This is a useful approach in evaluating the contribution of a cell type to a particular
developmental program, while allowing one to control the precise timing of the toxic insult, the
degree of ablation, and the potential for recovery upon termination of drug-induced toxicity
(Heyman, 1989). Upon administration and delivery of specific nucleoside analogs to target cells,
the HSV-tk is capable of monophosphorylating the benign substrate. The nucleoside
monophosphate is further phosphorylated by cellular kinases to nucleoside triphosphate,
producing a toxic product that becomes incorporated into DNA during differentiation and leads
to inhibition of DNA synthesis and cell ablation (Elion, 1977; Furman, 1980; Fyfe, 1978). This
ablation technique has been utilized previously using an immunoglobulin promoter/enhancer to
target the expression of HSV-tk to the lymphoid system (Heyman, 1989). In transgenic animals
29
the activity of the enzyme was restricted to the spleen, lymph nodes, bone marrow and thymus,
where the expression of HSV-tk resulted in massive depletion of B and T lymphocytes after drug
treatment (Heyman, 1989).
Following the initial use of this method for the haematopoietic and immune cell
pathways, strategies have been developed to study the relationship of different cell types in bone
tissue. In regards with osteoblasts, the use of a highly tissue specific promoter, such as
osteocalcin, leads to the expression of the tk gene late in the osteoblast lineage when cell division
has ceased and the rate of cell proliferation is reduced (Corral, 1998). Previous use of the mouse
osteocalcin gene 2 (OG2) promoter, a marker of mature osteoblasts, to drive expression of the
HSV-tk gene, showed a nearly complete absence of cells with morphologic features of
osteoblasts (Corral, 1998). Due to late tk expression in the OG2 mice, osteoclast activity was still
persistent and bone resorption remained unaffected despite a reduction in the osteoblast
population. This would suggest that the surviving osteoblast progenitors that are unaffected by
the GCV treatment in OG2-tk mice could express signals that would regulate osteoclast function
(Corral, 1998).
In contrast to the osteocalcin promoter, the modular design of the Col1a1 promoter
allows the creation of promoter-reporter constructs that can be used in various type I collagen-
producing tissues (Dodig, 1996). In an in vitro study by Dacic and colleagues (Dacic, 2001)
different Col1a1 promoter fragments were used to drive the chloramphenicol acetyltransferase
(CAT) gene. In this model, the activity of a 3.6 Col1a1 fragment was detected in osteoblast
progenitors concurrent with type I collagen mRNA and ALP expression. In contrast, the 2.3
Col1a1 fragment became active coincident with expression of the early bone-specific marker
bone sialoprotein (BSP) but distinctly prior to osteocalcin mRNA, which is produced at a later
stage in the osteoblast lineage (Dacic, 2001). In another study, Visnjic and colleagues generated
transgenic mice that express the HSV-tk gene under 2.3-kb fragments of the collagen type I
promoter, which was found to be active in concurrent with other genetic markers of early
differentiating osteoblast (Visnjic, 2001). Treatment of these mice with GCV resulted in an
extensive destruction of the bone lining cells, decreased osteoclast numbers and a decrease in
bone marrow elements. The reduction in the bone marrow cellularity was more prominent in the
metaphyseal regions and areas adjacent to the endosteal surface of the diaphyseal bone, which
normally house cells of osteoblastic lineage. Termination of the GCV treatment resulted in a
30
pronounced response of new cortical and trabecular bone formation as well as the replenishment
of the bone marrow cells. Their findings suggest that early differentiating osteoblasts are
necessary for the maintenance of haematopoiesis and osteoclastogenesis (Visnjic, 2001).
Therefore, in contrast to the other osteoblast specific promoters mentioned, the 2.3-kb fragment
of type I collagen promoter provides a useful osteoblast-specific model to define and analyze the
interrelation of osteoblasts with the other bone cell types during the different modes of bone
formation and fracture healing.
31
Figure 1: Genetic Control of Skeletal Cell Type Differentiation.
(A) Differentiation of mesenchymal progenitor cells down an osteogenic or chondrogenic lineage and expression of the transcription factors and cytokines regulating each pathway is shown. Transcription factor Runx2/Cbfa1 is the earliest marker of osteoblast differentiation and is also involved in promoting hypertrophic differentiation of chondrocytes. Sox9 transcription factor, along with Sox5 and Sox6 regulate the expression of various genes involved in chondrogenesis and induction of cartilage matrix proteins such as type II collagen. (Refer to ‘Abbreviation List’, for acronyms).
(B) Molecular regulation of osteoclast differentiation from haematopoietic stem cells and expression of enzymes, secreted molecules, and transcription factors acting at different stages of differentiation/activation are depicted. The activation of osteoclast involves the formation of ruffled membrane and secretion of catabolic enzymes such as cathepsin K, TRAP and H+ into resorption pits for degradation of bone matrix. (Refer to ‘Abbreviation List’ for acronyms).
32
Figure 2: Bone Formation Occurs Through Two Different Modes of Ossification Processes.
(A) The endochondral bone formation begins with the condensation of mesenchymal progenitor cells at sites of future skeletal development, a process that is recapitulated during the early stages of fracture repair. The cells at the central region of the aggregate then differentiate along a chondrogenic lineage and produce a cartilaginous template. In the region adjacent to the hypertrophic chondrocyte (HTC), the first osteoblast precursors differentiate and secrete a non-mineralized extracellular matrix that becomes organized into compact mineralized bone. During this process osteoblasts that become entrapped in the bone differentiate into osteocytes. Osteoclasts are required for remodelling and resorption of the bone matrix.
(B) Intramembranous ossification involves the direct differentiation of osteoblasts from the condensed mesenchyme at the site of bone formation such as the flat bones of the skull. In this process bone is made without the need for an intermediate cartilaginous template.
(Fig 2A adapted from Hartmann, 2006 and Fig 2B adapted from Hartmann, 2009)
33
Figure 3: Model of Fracture Healing Stages and Cellular Participants Involved During the Process of Repair.
(A) Representative images of a fracture healing model. The process of fracture healing is partitioned into four overlapping stages. Upon damage to the bone there is an immediate inflammation response followed by the formation of a soft callus, which undergoes remodeling prior to the hard callus formation stage. The final stage of repair involves the remodeling of the hard callus back into the original cortical bone configuration.
(B) Diagram showing the corresponding cellular contributors to the healing process at various stages.
(Adapted from Schindeler, 2008. 19; 460).
34
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Chapter 2 Possible Role of Osteoblasts in Regulating the Initiation of
Endochondral Repair Process during Fracture Healing
2 Summary
Fracture healing is a regenerative process requiring precise coordination of a variety of
skeletal, haematopoietic, vascular and immune cells which are necessary for successful repair
process and union of damaged bone. Previously, osteoblasts have been shown to be important in
osteoclastogenesis during the remodelling phase of fracture repair. However, their role during the
initiation phase of endochondral fracture repair has been unclear. Therefore, we hypothesized
that ablation of osteoblasts would lead to a delay in the initiation of chondrogenesis and
progression of the endochondral bone formation. The objective for this project was to analyze
the various stages of endochondral repair process, in order to determine the relative progression
of the fracture repair in the absence of osteoblasts. A transgenic mouse model that expresses a
truncated form of herpes simplex virus thymidine kinase (HSV-tk) gene under the control of a
2.3-kilobase fragment of rat α1 type I collagen promoter (Col2.3∆tk; DTK), was used to drive
the expression of the gene in early differentiating osteoblasts. A nucleoside analog, ganciclovir
(GCV), was used to ablate the cells in osteogenic lineage and fracture calluses were examined at
3, 7 and 21 days post fracture. Our results show that osteoblast depletion delays the initiation of
endochondral bone repair and the process of chondrogenesis. Continuous ablation of osteoblasts
for 21 days post fracture hindered the progression of endochondral ossification at the soft callus
stage with abundant deposition of cartilage matrix at the fracture site of DTK transgenic mice.
Upon cessation of the drug treatment, the bone was able to heal uneventfully similar to the
wildtype controls.
2.1 Introduction
Fracture healing is a complex physiological process that recapitulates certain aspects of
normal skeletal development and growth. It is a regenerative event that involves the coordination
of a variety of cells, ranging from haematopoietic and immune cells within the bone marrow in
conjunction with vascular and skeletal cell precursors that are recruited to the fracture site, to
ensure proper repair of the damaged bone. Within the microstructure hierarchy of fracture repair,
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the predominant cells involved include the chondrocytes, osteocytes, osteoblasts, and osteoclasts.
Thus understanding the interplay between these different cell types during the healing process
would be useful for improving the development of cell-based therapies.
The repair process is comprised of four overlapping phases, initiated by (1) an
inflammatory response that results in the recruitment of mesenchymal progenitor cells that
differentiate into chondrocytes, which produce cartilage and osteoblasts, the bone forming cells
(Schindeler, 2008). Thereafter, (2) the fracture is bridged by soft callus and (3) later hard callus
until mechanical instability is restored to the fracture site (Schindeler, 2008). The final stage of
fracture repair involves the (4) remodeling of the hard callus by osteoclasts, the bone resorbing
cells, and osteoblasts into the original cortical and/or trabecular bone configuration (Gerstenfeld,
2003).
During development, bone tissue is formed by two distinct ossification processes.
Intramembranous bone formation is mediated through the inner periosteal osteogenic layer with
bone made initially without the intervention of a cartilage phase (Shapiro, 2008). Most fractures
possess some level of mechanical instability and heal by the process of endochondral
ossification, which occurs in unstable regions external to the periosteum immediately adjacent to
the fracture site (Dimitriou, 2005). The eventual bridging of the hard callus across the fracture
gap provides the initial stabilization and regaining of biomechanical function.
Previous works have shown that close coordination and interplay between the different
cell types involved in fracture repair is important for successful repair process. For example,
expression of RANKL by osteoblasts coordinates bone remodeling by stimulating bone
resorption by local osteoclasts through binding of RANKL to its cellular receptor RANK on
osteoclasts (Lacey, 1998; Li, 2000; Nakagawa, 1998). This close coordination between the two
cell types is further regulated through the expression of the decoy receptor osteoprotegerin by
osteoblasts, which binds RANKL and prevents its interaction with RANK receptors present on
osteoclasts (Roodman, 2006; Udagwa, 2000).
This coordination between the different cell types that are involved in fracture healing is
further evident by the role of various matrix metalloproteinases (MMPs) that degrade most
components of the extracellular matrix. Besides participating in degradation of ECM to allow
cell migration, MMPs are involved in modulation of biologically active molecules by direct
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cleavage and regulation of the activity of other proteases (Ortega, 2003). Two critical MMPs that
are highly expressed during endochondral ossification include MMP9 and MMP13.
Preosteoclasts and other chondroclastic cells express MMP9 during the start of
neovascularization of the cartilage anlage (Holmbeck, 1999; Zhou, 2000), and MMP13 is
expressed in the terminal hypertrophic chondrocytes and in the newly recruited osteoblasts (Vu,
1998). Together, these two MMPs account for most of the protease-dependent steps of
endochondral ossification (Engsig, 2000) and along with the angiogenic factor, VEGF, are key
regulators of the remodeling of the skeletal tissue (Ortega, 2003).
Long bone fracture has been used for many years as the model for evaluating the
induction of osteogenic and chondrogenic lineages for the initial skeletal stabilization as well as
the subsequent coordination of the osteoblast and osteoclast lineages to remodel the immature
callus back to a cortical bone (Ushiku, 2010). Although the role of osteoblasts during the
remodeling phase of fracture repair has been previously studies, their role during chondrogenesis
has not been well investigated. Few studies investigating the interplay between these two cell
populations have shown that soluble factors expressed by chondrocytes selectively promote
osteogenesis of mesenchymal stromal cells (Gerstenfeld, 2003a), and in co-culturing of
osteoblasts and chondrocytes the differentiation level of osteoblasts influenced the proliferation
and differentiation levels of chondrocytes (Nakoka, 2006). Therefore, we hypothesized that
ablation of osteoblasts would lead to a delay in the initiation of chondrogenesis and progression
of the endochondral bone formation. The objective for this project was to analyze the various
stages of endochondral repair process, in order to determine the relative progression of the
fracture repair in the absence of osteoblasts.
One method for studying the lineage relationships and cell functions of a population of
differentiating cells is the use of tissue-specific transgenic expression of herpes simplex virus
thymidine kinase (HSV-tk) to conditionally deplete a specific cell population. As such, in order
to study the role of osteoblasts during bone fracture repair, we used a transgenic mouse model
expressing the HSV-tk gene under the control of a 2.3-kilobase fragment of the rat α1 type I
collagen promoter (Col2.3∆tk; DTK), which is active in early differentiating osteoblasts.
Expression of the tk gene renders the cells susceptible to a variety of nucleoside analogs such as
GCV, which leads to ablation of cells in osteoblastic lineage. In our study, fractures were
generated in the tibia of three-month-old male DTK mice that were pretreated with GCV for two
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weeks. After surgery, one set of DTK transgenic animals were given GCV treatment for an
additional 3, 7 and 21 days, while another set received saline treatments (Figure 4). Control
animals negative for the DTK transgene were treated with GCV for above time point as controls.
Furthermore, in order to evaluate the potential side effects of the GCV drug during the healing
process, additional untreated animals (both transgenic and wildtype) were analyzed for the same
timepoints (data not shown). Our results show that osteoblast depletion delays the initiation of
endochondral bone repair and the process of chondrogenesis, with continuous ablation of
osteoblasts for 21 days post fracture hindering the progression of endochondral ossification past
the soft callus stage fracture site of DTK transgenic mice. Upon cessation of the drug treatment,
the bone was able to heal uneventfully similar to the wildtype controls.
2.2 Methods and Materials
2.2.1 Generation of fractures:
All animal procedures were approved by the animal care committee of Hospital for Sick
Children. Tibial fracture of control and DTK transgenic mice was performed as previously
described (Chen, 2007). Briefly, after anaesthetizing the animals with isoflurane, a small incision
was made close to the left leg to expose the head of the tibia. A suture needle was used to
puncture the head of the tibia to allow the insertion of a non-corrosive metal pin in the marrow
cavity in order to stabilize the leg prior to fracture generation. The fracture was generated using a
surgical scissor after the skeletal muscles, juxtaposed to the tibia, were pulled away from the
bone to prevent unnecessary damages to this tissue. The skin was then closed using silk sutures
and metal staples and the animals were allowed to recover. After recovery, an analgesic
(ibumorphine) was administered for the first three days post surgery. The fractured left tibia and
the intact right tibia were then harvested at 3, 7 and 21 days time-points post surgery (Figure 5).
2.2.2 Mechanism of action of GCV:
The DTK transgenic mice express a truncated form of HSV-tk gene whose expression is under
the control of the rat α1 type I collagen gene promoter that restricts TK expression in early
differentiating osteoblasts. Upon treatment of animals with GCV, a nucleoside analogue of
guanosine, and a homologue of acyclovir, the thymidine kinase present in target cells
monophosphorylates the GCV drug. The drug is further phosphorylated by endogenous kinases
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and competitively inhibits the incorporation of dGTP into DNA, resulting in inhibition of DNA
replication and cell ablation.
2.2.3 Real-time PCR:
Total RNA was isolated from the fracture callus at the 7 days time point and from intact
unfractured tibias at 3 and 7 days post-surgery. cDNAs were made from the extracted RNA
samples. Real-time PCR was performed to determine the level of osteoblastic (Type I collagen
and ALP), chondrogenic (Sox5 and Aggrecan) and MMP13 gene expression as compared with
GAPDH (a housekeeping gene control). At least 3 animals in each treatment group were
examined. PCR primers (Type I Collagen, ALP, Sox5, Aggrecan and Matrix metalloproteinase
13) were purchased from Applied Biosystems.
2.2.4 Staining method – Safranin-O/Haematoxylin-Eosin:
For histological analysis, the tissue at the fracture site was harvested, fixed in 4%
paraformaldehyde, decalcified in 20% EDTA (pH 8.0), dehydrated in progressive concentrations
of ethanol, cleared in xylene and embedded in paraffin. The entire tibia was sectioned 5 µM
thick and the sections from the center of the tibia with the widest diameter were selected. The
center sections were deparaffinized and hydrated in descending concentrations of ethanol. These
sections were then stained with haematoxylin-eosin (HE) and safranin-O (SO) for analysis of
gross morphology and cartilage deposition at fracture repair respectively.
2.2.5 Histomorphometric, Cell quantification and X-ray analysis:
For histomorphometric measurements, safranin-O stained tissue sections were used for
evaluation of bone and cartilage volumes as a percentage of total callus tissue volume. Bone
volume (BV) and cartilage volume (CV) were measured using the Bioquant system, and these
measurements were normalized to the overall tissue volume (TV) measured. Using the same
safranin-O stained sections from various time points, at least 5 fields were randomly selected
from the healing fracture callus for quantification of the osteoblast-like cells. In absence of a
callus, areas near the metaphyseal zone of the tibia were chosen as alternatives. Radiographic
picture were obtained by using Faxitron MX20 X-ray system for radiographic appearance of
fractured tibias (Figure 4).
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2.2.6 Collagen type X and TRAP staining:
To determine remodeling parameters of the harvested fracture calluses, sections were stained for
tartrate-resistant acid phosphatase (TRAP) and counterstained with haematoxylin. Osteoclasts
were identified by their multinucleated morphology and close proximity to the bone surface at
sites of bone resorption.
2.2.7 Statistical Analysis:
Histomorphometric data are expressed as mean +/- standard deviation. Statistical differences
were calculated using Student’s t test. Number of animals for each group at the 7 day time point
are as follows (Bone Volume; Cartilage Volume): Wildtype GCV injected = 1n;1n, DTK
continuous GCV treated = 4n;4n. Number of animals for each group at 21 day time point are as
follows (Bone Volume; Cartilage Volume): Wildtype untreated control = 4n;3n, DTK untreated
control = 3n;3n, Wildtype GCV injected = 8n;6n, DTK non-continuous GCV treated = 6n;4n,
DTK continuous GCV treated = 4n;3n. A p-value below 0.05 was considered statistically
significant.
2.3 Results
2.3.1 Pretreatment of DTK transgenic mice with GCV leads to ablation of osteoblasts
To study the role of osteoblasts during bone fracture repair DTK transgenic animals and
control mice were pretreated with GCV for two weeks prior to fracture generation. In order to
examine if the GCV pretreatment of the DTK transgenic animal was effective in ablation of the
osteoblast cell lineage, tibial fractures were collected at 3 days post fracture. Histological
analysis of safranin O stained sections of these bones showed the presence of bone lining cells at
the metaphyseal area near the site of longitudinal growth adjacent to the growth plate in the
control animals (Figure 6A’). These cells were absent in the DTK transgenic animals in both the
non-continuous and continuous GCV treated groups (Figure 6B’, C’). The effect of the GCV
treatment on osteoblasts was found to be consistent in all the time points tested (3, 7, and 21 days
post-fracture). A marked decrease in mono-nucleated bone-lining cell population was observed
around the trabecular bones with the GCV drug treatment, and the cessation of the treatment was
followed by a repopulation of these cell types around the metaphyseal trabecular niche (Figure
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7). This finding is consistent with the original study done by Vinsnjic et al that showed a strong
expression of the thymidine kinase protein localized in osteoblasts lining the endosteal,
periosteal, and trabecular surfaces (Visnjic, 2004). Furthermore, similar to their study, transgenic
mice treated with GCV had a marked loss of bone marrow cellularity as compared to the non-
transgenic controls that showed no evidence of GCV-induced toxicity (Visnjic, 2004) (data not
shown).
2.3.2 Osteoblast depletion delays initiation of endochondral bone repair
During development, the onset of endochondral ossification begins with the condensation
of mesenchymal progenitor cells at sites of future skeletal development (Hall, 1995), a process
that is recapitulated during the early stages of fracture repair (Hiltunen, 1993) Shortly after the
condensation stage, cells in the central region of the aggregation begin to differentiate along a
cartilaginous lineage (Iwasaki, 1997) in response to growth factors and cytokines released by
platelets, inflammatory cells, and neighboring cells and tissues (Bolander, 1992; Bruder, 1994).
In order to establish a baseline for comparison of repair process at a stage when
osteoblasts have not yet been recruited to the fracture callus, fractured tibias were harvested at 7
days post surgery from GCV treated controls as well as continuous and non-continuous GCV
treated DTK mice. As previously reported (Visnjic, 2004) it was hypothesized that at 7 days
following the fracture, most of the callus would be composed of cartilage. As expected,
histological analysis of the safranin-o stained sections showed an extensive collagen matrix
production in the fractures of the wildtype controls (Figure 8D). However, there was a lack of
collagen deposition at the fracture site of continuous GCV treated transgenic mice (Figure 8F).
The growth plate collagen staining was used as an internal control and collagen matrix
production was identified by the red staining of proteoglycans at the site of fracture.
In addition, fracture calluses from a separate group of GCV-treated control and
continuous GCV-treated DTK mice were harvested at 7 day time point for RNA analysis. To
control for any contamination that might occur during the harvesting of the calluses, mainly from
the muscle tissue surrounding the fracture site that would need to be collected along with the
fracture callus, intact tibias from the same animals were also collected as a reference. The muscle
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tissue was easily removed from these bones and the entire tibia containing the growth plate was
flash frozen in liquid nitrogen for further processing.
Since the expression of the tk transgene is under type I collagen promoter, and GCV
treatment should lead to ablation of dividing cells expressing type I collagen, the expression level
of this gene along with ALP (another early osteoblast marker) was examined in the fracture
calluses and intact tibia of both the control and DTK transgenic animals. Real-time PCR analysis
shows a decrease in the expression of these genes in the fracture callus of the continuous GCV
treated mice as compared to the wildtype controls (Figure 9).
Type I collagen is also expressed in osteocytes (Kamiya, 2001), but since these cells are
not dividing the GCV treatment should not lead to death of osteocytes in the cortical bone
(Figure 10). Furthermore, it has been previously reported that the distance between osteocytes
can be calculated (Sugawara, 2005). Thus it was assumed that there would be similar number of
osteocytes in each of the intact tibias collected, thereby setting a baseline threshold, where any
decrease in type I collagen in continuous GCV-treated transgenic mice could be hypothesized to
be due to the ablation of osteoblasts. Similar decrease in type I collagen expression was observed
in the intact tibias of the continuous GCV-treated mice as compared to the wildtype controls
(data not shown).
Other genes that were examined in the 7 day old calluses included genes involved in
chondrogenesis and cartilage matrix production (Sox5 and aggrecan, respectively), as well as
collagenase MMP13, which is expressed by hypertrophic chondrocytes and osteoblasts during
endochondral ossification. A significant decrease was seen in all above genes in the continuous
GCV-treated groups as compared to wildtype controls (Figure 9). These results, along with the
histology data suggests that the continuous ablation of osteoblasts with GCV leads to a lack of
cartilage deposition in these animals at the chondrogenic stage of fracture healing.
2.3.3 Continuous ablation of osteoblasts hinders the progression of endochondral ossification past the soft callus stage
To determine the role of osteoblasts during endochondral repair process DTK transgenic
animals were pretreated with GCV for duration of two weeks prior to fracture generation. One
set of transgenic animals was injected with GCV continuously for 21 days, whereas another set
68
of DTK animals received saline for the same duration (Figure 5). Wildtype control mice
receiving GCV for 21days were used as controls. Additionally, to examine the natural fracture
repair process in absence of GCV treatment, calluses were harvested from untreated wildtype
controls and DTK transgenic mice at 21 day time point (data not shown).
To verify the effect of GCV treatment on osteocytes, various locations in the cortical
bone were examined in the different treatment groups at all time points. Osteocytes were
identified by their elongated morphology and their parallel orientation to the long axis of cortical
bone. In the representative pictures (Figure 10) osteocytes are seen present in round to oval
shaped lacunae spaces in the bone (arrow). Adjacent lacuanes are linked by multiple small canals
known as canaliculi that house the osteocyte cell processes (Palumbo, 1990). This lacunar-
canalicular intraosseous system plays a key role in providing the bone cells with nutrients from
blood vessels and allows intercellular communication of bone cells for biophysical control
mechanisms essential to tissue development and maintenance (Shapiro, 2008). Any injury to the
bone that leads to a break of this communication system would result in the death of osteocytes
that lie in close proximity to the damaged site. This is apparent by the empty lacuanae spaces
found in the cortical bone adjacent to fracture site of the different treatment and control groups,
where the osteocytes closest to the damaged area have died off (Figure 11E).
Safranin-O staining was performed on paraffin-embedded tibial sections of 21 day old
fractures from the different treatment group for detection of osteoid and cartilage deposition at
the site of fracture (Figure 8G-I). Representative fracture were selected from each treatment
group and the red staining of cartilage at the growth plate was used a reference control. Other
than the continuous GCV-treated animals (Figure 8I), the callus of the various control and
treatment groups was primarily composed of new bone at 21 days post fracture. Multiple
condensation sites were still present in the fracture callus of continuous GCV-treated animals,
suggesting that osteoblast ablation delays the endochondral repair process.
In addition, HE staining of selected 21 day old fracture callus section of the various
treatment groups showed osteoblasts lining the periosteum or along the trabecular bone within
the interim callus of the untreated control animals (data not shown) as well as the GCV-treated
wildtype and non-continuous GCV-treated DTK mice (Figure 11D-F). Osteoblasts were
identified by their mononucleated morphology and their single array lining of the surface of
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newly synthesized bone at the site of fracture. Active osteoblasts were identified by their
polarized orientation at sites of bone mineralization.
Endochondral bone formation was observed at the site of fracture repair on mineralized
cartilage remains as would be seen in normal metaphyseal bone formation. The initially
synthesized woven bone at the site of repair can be seen surrounded by darker staining more
organized lamellar bone that is then immediately covered with a layer of surface osteoblasts.
Higher power view of the fracture callus (Figure 11D’-F’) shows the presence of active
osteoblasts at sites of osteoid deposition (arrows). In addition, areas of osteoblast-to-osteocyte
transformation can be observed in the fracture callus as some osteoblasts have become embedded
within the bone matrix produced by neighboring osteoblasts (Figure 11E’).
To determine the level of remodeling at 21 days post fracture, sections from the various
treatment groups were stained with TRAP. Almost no TRAP staining was observed in the
continuous GCV-treated group whereas GCV-treated control animals and non-continuous GCV-
treated transgenic animals exhibited TRAP positive multinucleated cells scattered among the
newly formed trabeculae at the fracture site (Figure 12). Osteoclasts, which are a member of the
monocyte/macrophage family that involved in bone resorption during normal bone turnover as
well as in the remodeling phase of fracture repair, can be seen along the newly deposited bone.
They are multinucleated cells that lie in close proximity to the bone surface at site of bone
resorption and stain positive for tartrate-resistant acid phosphatase (TRAP).
Staining for collagen type X showed that at 7 day time point, the fracture callus of the
continuous GCV-treated DTK mice lacked the hypertrophic chondrocyte specific type X
collagen matrix production, while in control animals abundant matrix deposition was observed
(Figure 13A-C). At 21 day time point there was still an abundant amount of collagen type X
matrix present in the fracture callus of the continuous GCV-treated DTK mice (Figure 13D-F).
These data taken together with the safranin O staining showing no new bone deposition at the
fracture site, suggest that the endochondral repair process of these callus is delayed at 7 day time
point and is still in the early chondrogenic stages at the 21 time point stage.
Finally, to quantify the level of bone and cartilage deposition at the fracture site,
histomorphometric measurements were performed on safranin O stained section of 7 day and 21
day old tibial fractures of the different treatment groups (Figure 14). Tissue areas selected for
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analysis were determined by the two farthest edges of the callus where the periosteal new bone
formation meets the adjacent cortical bone. Various bone and cartilage parameters were
examined and the data shows lower cartilage matrix synthesis in 7 day old fractures of
continuous GCV treated transgenic mice in comparison to wildtype controls (Figure 14A).A
significant decrease in new osteoid formation and a significantly higher cartilage deposition was
observed in continuous GCV-treated group as compared to the GCV-treated control animals and
non-continuous GCV-treated DTK mice at the 21 day time point (Figure 14B). Overall bone
volume (mineralized bone plus the newly synthesized osteoid) did not change across the three
treatment groups, however a significantly higher mineralized bone volume per tissue volume was
observed in the continuous GCV-treated DTK mice (Figure 14B).
2.4 Discussion
The data gathered from the 7 day continuous group taken together with how the process
of chondrogenesis progresses at the site of fracture, as observed in the 21 day fractures, suggests
that osteoblasts may play a role in the initiation and advancement of endochondral ossification.
The initiation of endochondral repair seems to be delayed, and as expected, in the absence of
osteoblasts the process does not appear to be able to advance to the next phase of hard callus
formation. One explanation for the delay and eventual cessation of this process could be because
the calcified cartilage matrix at the site of the fracture is not being resorbed to allow for the
advancement of vasculature into the fracture callus. As already mentioned, bone development
requires a complex remodeling of the extracellular matrix, which is in large part mediated by the
function of MMPs. Osteoblastic cells have been shown to express several MMPs implicated in
bone morphogenesis (Ortega, 2003), unmineralized matrix degradation (Uchida, 2001),
osteoblast and chondrocyte migration (Blavier, 1995) and cell invasion (Javed, 2005).
In addition, angiogenesis requires locally restricted extracellular proteolysis, which is
achieved by a tight balance of MMPs and MMP inhibitors. MMP2 and MMP9 are of particular
interest for angiogenesis (Bergers, 2000; Itoh, 1998). MMP9 is specially required for the
invasion of osteoclasts and endothelial cells into the mineralized hypertrophic cartilage that is
deposited within the fracture callus, whereas other MMPs, principally MMP13, are expressed in
early osteoblastic cells and required for the passage of cells through the unmineralized type I
collagen and play a role in the resorption of mineralized matrix (Itagaki, 2008). Therefore, a
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finely tuned coordination between these various MMPs that are expressed by the different cell
types involved during fracture healing is required for successful bone development and
remodelling (Engsig, 2000). In addition, other osteoblastic factors such as TNF-α, have been
shown to regulate the expression of specific angiogenic factors and MMPs during fracture
healing (Lehmann, 2005).
In this study, the ablation of osteoblasts with GCV is accompanied by delayed
mesenchymal condensation and hypertrophic chondrocyte type X collagen matrix production at
the site of injury at 7 days post-fracture. In the absence of osteoblasts there is lower production
of the necessary MMP13 (Figure 9), which is required for the degradation of the collagen
matrix. The process of endochondral ossification was found to be delayed in the absence of
osteoblasts, where the production of soft callus was observed to occur at a time when the
wildtype fractures have already advanced to the stage of new osteoid production along with bone
remodelling. Furthermore, in the continuous GCV treatment model, there is an accumulation of
the collagen matrix at the fracture site at the 21 day time point. This can be due to the continued
ablation of osteoblasts and a lower availability of MMPs required for collagen degradation.
Furthermore, decreased osteoclastic activity was seen with continuous ablation of
osteoblasts. As osteoclasts also produce the necessary MMP9 involved in matrix degradation,
this would lead to further depletion of the MMP pools that are available for extracellular matrix
resorption. In an uneventful healing process, the necrotic dead cortical bone adjacent to the
fracture site is normally resorbed by osteoclasts. However, with continuous administration of the
GCV drug, the resulting lower osteoclastic activity due to osteoblast ablation leads not only to
unresorbed cartilage matrix, but also to decreased removal of the damaged cortical bone. This is
evident by the significantly higher mineralized bone and cartilage volume in the continuous
GCV treated mice as compared to wildtype and non-continuous GCV treated groups (Figure
14B). The activity of osteoclasts during bone repair has been previously explored using various
mouse models. Strain-dependent mouse knockout models with defective osteoclast function
show an osteopetrotic phenotype where a defect in either the regulation of osteoclast activation
(Lacey, 1998), their attachment to the bone at resorption sites (Kollet, 2006), or the secretion of
degradative enzymes (Frattini, 2000), leads to reduced bone resorption.
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Moreover, since the longitudinal bone growth occurs at the two metaphyseal ends of the
bone, majority of the active osteoblast can be assumed to reside at the site of active
skeletogenesis. In contrast, the central diaphyseal region of the bone contains mostly quiescent
bone-lining cells that are not associated with mineralization (Ushiku, 2010). Upon damage to the
bone, the first response is seen in the population of osteoblasts present near the vicinity of the
fracture site, which become active and migrate toward the site of injury (Ushiku, 2010). In our
study, the pretreatment of the DTK transgenic mice with GCV leads to ablation of osteoblasts
throughout the tibial bone. Upon cessation of the drug at the time of fracture generation, the bone
is seen to be able to repopulate the osteoblast pool within the bone and progress toward
formation of soft callus, resorption of the deposited cartilage matrix, deposition of new osteoid as
well as exhibiting similar osteoclastic activity compared to the wildtype model. From these
observations, along with the notion that a correct homeostasis needs to be achieved for
successful recovery from pathologic conditions, it can be hypothesized that the repopulation of
the osteoblasts would occur at the site of injury in order to accelerate the healing process. Also,
from the shear number of osteoblasts seen at the site of fracture compared to the population of
pre-existing osteoblast present at the metaphyseal zone, along with the accelerated rate of the
healing process versus the rate of normal longitudinal growth, the involvement and influence of
pre-existing osteoblast toward fracture repair needs to be further studied.
Overall, from the data gather in our study, it can be argued that perhaps due to the lack of
resorption of the calcified matrix at the site of fracture, there is a lack of blood vessel invasion
and as a result the endochondral process does not advance to the hard callus stage. However,
osteoid deposition at the site of fracture site would not be possible in the absence of osteoblasts
in the continuous GCV-treated transgenic animals. It is nonetheless interesting that at the early
phase of endochondral repair process, the presence of osteoblasts (whether pre-existing or newly
differentiated osteoblasts) is required for regulating the event of chondrogenesis. This would
suggest that osteoblasts are not only required in the later stages of fracture repair as the medium
for osteoid deposition and osteoclast activation, which is necessary for remodelling, but also for
the initiation of the endochondral ossification process.
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2.5 Figures
Figure 4: Continuous GCV Treatment Leads to Non-union of the Fractured Bone.
Radiographic analysis from GCV treated wildtype control mice (A,D,F) as well as non-continuous (B,G) and continuous (C,E,H) GCV-treated DTK mice at various time points post fracture. Arrows indicate the fracture site. At least 3 animals were generated for each treatment group. Only one animal was examined for 7 days non-continuous GCV-treated transgenic group.
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Figure 5: In Vivo Experimental Design for Studying the Role of Osteoblasts During Fracture Repair Process.
DTK transgenic and wildtype animals were pretreated with GCV for duration of two weeks prior to fracture generation. After surgery, one set of transgenic animals was injected with GCV continuously for 3, 7 and 21 days, whereas another set of DTK animals received saline for the same duration. Wildtype littermate mice receiving GCV were used as controls.
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Figure 6: GCV Pretreatment is Effective in Ablating Pre-existing Osteoblasts.
(A-C) Safranin O staining of metaphyseal area near the growth plate showing a marked reduction in the bone marrow cellularity with GCV pretreatment. GP: growth plate.
(A’-C’) Higher magnification images showing the ablation of bone lining cells in the DTK transgenic animals with GCV pretreatment at 3 days time point. Arrows (A’) indicate the osteoblasts lining bone near the growth plate.
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Figure 7: GCV Treatment of the DTK Transgenic Mice Leads to a Decrease in Bone-Lining Osteoblast Population.
Quantification of mononucleated bone-lining cells for the three treatment groups at 3days (A), 7 days (B), and 21 days (C) post-fracture. The cessation of the GCV treatment leads to repopulation of the osteoblast in the fractured tibia.
Error bars represent standard deviation.
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Figure 8: Continuous GCV Treatment Leads to a Delay in Initiation and Progression of Endochondral Ossification Process.
Safranin-O staining of fracture calluses at 3 days (A-C), 7 days (D-F) and 21 days (G-I) time points. Growth plate staining of selected sections as an internal reference control. CB: cortical bone. At 7 days post-fracture, the wildtype callus shows abundant proteoglycans deposition at the fracture site (red staining), while in the continuous GCV treated group this collagen matrix deposition is absent. At 21 days post-fracture, which traditionally marks the hard callus phase of healing, the continuous GCV treated group exhibits multiple condensation sites (red staining), while in the wildtype and non-continuous GCV treatment groups the cartilage matrix is gradually resorbed.
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Figure 9: Matrix Metalloproteinase 13 Gene Expression is Reduced in Absence of Osteoblasts.
Real-time PCR analysis of the fracture callus at 7 days post surgery shows a decrease in osteoblastic and chondrogenic gene expression. Type I collagen (Col1), Alkaline phosphatase (ALP), MMP13, Sox5 and Aggrecan (Agcn) gene expression in fracture callus of continuous GCV-treated DTK mice is shown compared to wildtype control mice. The data is shown as fold change normalized to GAPDH housekeeping gene expression.
* is for p<0.05, error bars represent standard deviation (3 animals in each treatment group were examined).
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Figure 10: Osteocytes are Unaffected by GCV Treatment.
Random sites of cortical bone were examined at 3 days (A-C), 7 days (D-F) or 21 days (G-I). 400X magnification of cortical bone. Osteocytes are shown with arrows.
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Figure 11: Upon GCV Withdrawal Bones are Able to Replenish the Osteoblast Population as Early as 7 Days Post Fracture.
(A-C) 200X magnification showing osteoblasts lining along the newly synthesized bone (arrows) in wildtype GCV treated mice and their absence in the non-continuous and continuous treated animals.
(D-F) HE staining of the fracture callus and metaphyseal area (F) from the various treatment groups. 100X maganification, white box (E) shows areas of necrotic bone where osteocytes have fallen off.
(D’-F’) 400X magnification images of selected areas. Arrows point to osteoblasts, gray asterix (E’) shows areas of osteoblast-to-osteocyte transition.
GP: growth plate; CB: cortical bone; WB: woven bone; LB: lamellar bone.
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Figure 12: Osteoblast Ablation Leads to a Decrease in TRAP Positive Osteoclasts During the Remodeling Stage of Fracture Healing, and the Decrease in Osteoclast Population Hinders the Healing Process.
(A-C) TRAP staining was done on 21 days post fracture sections to look at the parameters of remodeling and to identify TRAP positive cells (arrows). By 21 days post-fracture the decrease in TRAP activity leads to a reduction in osteoclast-mediate degradation of the hypertrophic chondrocyte deposited type X collagen matrix.
(A’-C’) 400X magnification images of selected areas.
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Figure 13: In Absence of Osteoblasts There is a Lack of Collagen Type X Matrix Resorption.
Staining for type X collagen deposition at the site of fracture at 7days (A-C) and 21 days (D-F) post surgery. Type X collagen in only produced by hypertrophic chondrocytes. Hypertophic matrix remodelling is delayed in continuous GCV treated DTK mice. Arrows show sites of staining.
(A’-F’) 100X magnification images of selected areas.
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Figure 14: Continuous Depletion of Osteoblasts Results in Unresorbed Cortical Bone and Cartilage Matrix at the Fracture Site.
Histomorphic analysis shows a significant decrease in bone regeneration and an increase in collagen deposition in DTK mice after continuous GCV treatment as compared to control groups. (A) 7 days and (B) 21 days old fractures. Measurements of bone volume (BV), mineralized volume (Md.V), osteoid volume (OV) and cartilage volume (CV) are expressed as a percentage of total callus tissue volume (TV).
* is for p<0.05, error bars represent standard deviation.
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Chapter 3 Summary and Conclusions
Endochonral ossification is a regenerative process that is seen in most of the generated
bone traumas. Previous studies on the mechanisms of activation and differentiation have
elucidated the interplay between osteoclasts and chondrocytes during the matrix degradation
stage of bone formation, as well as the interaction between osteoblasts and osteoclasts during the
hard callus remodelling phase of bone healing. However, the relations between osteoblast and
chondrocytes have not been previously studied in vivo. Our study show that in the absence of
osteoblasts, there is a delay in the initiation of endochondral ossification process at the soft callus
formation, and an obstruction in the progression toward the hard callus formation. This can be
argued to be in part due to a lack of matrix metalloproteinase enzymes that help degrade the
cartilage matrix during the soft callus remodelling stage, which allow the invasion of vasculature
and the recruitment of osteoblastic and osteoclastic precursors. Due to the close interaction
between osteoblasts and osteoclasts, this phenotype is exacerbated with the continuous ablation
of osteoblasts, which also leads to decreased osteoclastic activity and reduced production of
necessary osteoclastic enzymes. Furthermore, our data also suggest that osteoblasts also play a
role, either directly or indirectly, during chondrogenesis.
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Chapter 4 Future Directions
Several questions that arise from this study include:
1. What are the contributions of pre-existing osteoblasts versus the newly
differentiated osteoblasts? In this study the HSV-tk was expressed in osteoblastic
lineages under the control of the type I collagen gene promoter. Therefore, the
administration of the GCV drug resulted in ablation of both pre-existing osteoblasts and
the newly differentiated osteoblasts at the stage when they begin to express type I
collagen. Upon damage to the bone, the number of newly differentiated osteoblasts
greatly exceeds those of pre-existing osteoblasts present at the growth plate regions of the
bone. Our study shows that in the absence of pre-existing osteoblasts, the contribution of
newly differentiated osteoblast at the site of fracture is sufficient for normal endochondral
ossification process. However, in order to examine the contribution of pre-existing
osteoblasts versus the newly differentiated osteoblasts, a mouse model in which the latter
population of cells is ablated without affecting the pre-existing osteoblasts needs to be
established.
2. Can administration of exogenous sources of MMP13 or MMP9 aid in
revascularization of the fracture callus at the site of injury? The importance of these
two MMPs produced by osteoblasts and osteoclast has been already established in the
literature, and in our model the delayed ossification process with continuous osteoblast
ablation can be attributed to a decreased pool of available MMPs required for matrix
degradation. Therefore, the contribution of vasculature to the endochondral ossification
process can be studied by administration of exogenous sources of MMPs that would aid
in cartilage degradation and allow the invasion of endothelial cells into developed soft
callus.
3. What are the interactions and interplay between osteoblasts and chondrocytes that
contribute to soft callus formation? From our study the requirement of osteoblasts for
the initiation of endochondral ossification process at the stage of soft callus formation
suggest that osteoblasts play a role, either directly or indirectly, in the process of
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chondrogenesis. The exact mechanism for this interaction is still not known and needs to
be further studied.
4. Can transplantation of stem cells capable of osteogenic/chondrogenic differentiation
rescue the phenotype seen with continuous ablation of osteoblasts? A recent study by
Lavoie and colleagues demonstrated that rodent and human foreskin-derived SKPs (Skin
Derived Precursors) are able to differentiate into ALP-positive, collagen type-I positive,
mineralizing osteoblasts, as well as into collagen type-II positive chondrocytes in culture
(Lavoie, 2009). In addition, this study showed that upon transplantation into a NOD/SCID
tibial bone fracture model, GFP-tagged rat SKPs behaved, morphogenically and
phenotypically, similar to the endogenous mesenchymal cells during bone healing and were
able to participate in bone healing process (Lavoie, 2009). To build on this study, we were
interested in whether SKPs are able to contribute functionally to fracture healing in animals
that lack the necessary osteogenic lineage cells. Sections from preliminary work on GFP-
tagged SKPs injection into fracture of GCV-treated DTK transgenic mice were used to
stain for GFP protein. Cells positive for GFP were observed at the site of fracture
(Figure15). To show SKPs are able to differentiate into osteoblasts, even in the harsh
microenvironment that is created by the ablation of osteoblasts, sections were stained for
osterix. The experiment was repeated twice, but since the antibody was found to be non-
specific, no conclusions could be made (data not shown). Staining for SKP markers are
needed to identify these cells as SKP cells. This preliminary data could suggest that the
microenvironment niche within the callus is beneficial but not necessary for the survival of
the SKPs, as was previously thought. Further optimization of the experimental design is
needed to maximize the survival and differentiation of SKPs after injection. Scaffolds may
be used to increase the chance of survival of injected cells while decreasing the spillage of
cells into surrounding tissues (muscle, skin, etc..). GFP+ SKPs and RFP+ MSCs can be
used separately or in combination in order to show and compare the degree of contribution
of each cell type to fracture healing.
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Appendix
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Figure 15: Fluorescent and Immunohistochemistry staining for GFP-tagged SKP-injected into fracture site of continuous GCV treated DTK mice.
Arrows point to GFP positive SKP cells within the fracture callus.
(A-B) Fluorescent staining for GFP positive SKP cells. (Panels – TopBottom) DAPI statining showing nuclei; GFP stain; Red Channel showing background autofluorescence; and merged DAPI images with Red and GFP flourescence images.
(C-E”) Immunohistochemistry staining of GFP positive cells. (C) Positive GFP staining of wound tissue. (D) Safranin O staining of the fracture site.(E) 50X magnification of the selected region in fracture area (D) showing GFP positive cells in the center (box). (E’-N) 200X magnification of negative control of the selected region.
(E’-P) 200X magnification and (E”) 400X magnification of selected area at the fracture site. GFP positive cells are shown with arrows.
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Acknowledgements
For their helpful discussion and valuable guidance, both technically and scientifically, I
want to thank members of Kaplan/Miller lab’s SKP group, especially Jean-Francois Lavoie, Jeff
Biernaskie, Maryline Paris, Karen Jones, Smitha Paul, Sibel Naska, Hiroyuki Jinno and Shallee
Dworski. I also want to mention a few other members of the Kaplan/Miller lab, namely Joseph
Antony, Loen Hansford, Tatiana Lipman and Tania Morano for their technical and intellectual
support. I am very greatfull for all your help in everything SKP related and for your friendships
through the course of my masters. It was a pleasure working with all of you.
To my collegues and friends in Ben Alman’s lab, some of whom became important
during the planning, analysis and generation of the data presented here, I want to say thanks.
Special gratitude to Saeid Amini Nik and Puviindran Nadesan for their help with mouse work;
Heather Whetstone for her technical assistance in processing of countless sections, her teachings
and her support; Gurpreet Baht, Ronak Ghanbari, Amanda Ali and Jason Rockel for their
valuable help with the real-time PCR work; and Kirsten Bielefeld, Claire Hsu, Louisa Ho, Quin
Xia Wei and Simon Kelley for their helpful discussions and technical aids. I want to thank Yan
Chen for the sections; Adeline Ng and Thomas Willett from Marc Grynpas lab for their help with
the histomorphometry.
Thank you all for your help, support and guidance during the span of my time in the lab. I
greatly appreciate the work and input you provided which helped with the completion of my
thesis project.
