Bone Structure, Development and Bone Biology: Bone Pathology

Abstract

The skeleton serves as an internal structural support system for vertebrates. It has mechanisms to grow and change in shape and size to suit varying stressors including the ability to resist the mechanical forces. In addition, bone is a major source of inorganic ions, and actively participates in the body’s calcium/phosphate balance.
Bone tissue is continuously formed and remodeled throughout life. Initially, the bone achieves its increase in size and shape through growth (increase in size) and a complicated process known as skeletal modeling. In late childhood and adulthood there is continuous renewal of the skeleton via a process termed remodeling. Both modeling and remodeling require two separate processes namely bone resorption and bone formation to occur simultaneously to be effective. This requirement is known as “coupling”.

Full text

Keywords Runx-2 (runt-related transcription factor 2)
s cbfa-1 (core binding factor alpha1) s Pebp2aA (Polyoma
enhancer binding protein 2aA) s Osterix s cleidocranial
dysplasia s osteopontin s Leptin s osteoblast specific factor-1,
N-syndecan s osteoblast/osteocyte factor-45 (OF45) s den-
tin matrix protein 1 s fibroblast growth factor 23 s sclerostin
s Sclerosteosis s SOST gene s osteocyte s osteoclastogen-
esis s parathyroid hormone s 1, 25 dihydroxyvitamin D3
s transforming growth factor alpha s epidermal growth factor
s tartrate resistant acid phosphatase s osteoprotegerin s integ-
rins s integral membrane proteins s fibronectin s collagen type I
s bone sialoprotein II s osteopontin s suppressor of cytokine
signaling-1 s osteoclast-associated receptor s apposition s
growth plate s drosophila s sarcolemma s myofilaments s motor
end plate s somites s skeletogenesis s osteoactivin s biglycan
s decorin s calcitonin s calcitriol s bone morphogenetic pro-
teins s connective tissue growth factor
Introduction
The skeleton serves as an internal structural support system
for vertebrates. It has mechanisms to grow and change in
shape and size to suit varying stressors including the ability
to resist the mechanical forces. In addition, bone is a major
source of inorganic ions, and actively participates in the
body’s calcium/phosphate balance.
Bone tissue is continuously formed and remodeled
throughout life. Initially, the bone achieves its increase in
size and shape through growth (increase in size) and a
complicated process known as skeletal modeling. In late
childhood and adulthood there is continuous renewal of
the skeleton via a process termed remodeling. Both mod-
eling and remodeling require two separate processes
namely bone resorption and bone formation to occur
simultaneously to be effective. This requirement is known
as “coupling”.
Overview Bone forms the skeletal framework of all
vertebrates. It is a composite tissue consisting of organic
matrix, inorganic minerals, cells, and water. Bone is formed by
the hardening of this matrix entrapping osteoblasts which then
become osteocytes.
The inorganic portion of bone matrix is composed mainly
of crystalline calcium phosphate salts, present in the form of
hydroxylapatite. This allows bone to serve as a reservoir of
calcium and phosphate that can be stored or mobilized in a
controlled fashion. Bone also contains carbonate, fluoride,
acid phosphate, magnesium, and citrate. Hydroxyapatite crys-
tals also form in tissues that are not normally calcified, includ-
ing in atherosclerotic plaque, in soft tissues of some patients
with abnormally high circulating calcium or phosphate, and in
articular cartilage of some patients with degenerative joint dis-
eases. Crystals in these situations are often distinctly larger.
The organic component of bone matrix comprises 40% of
the dry weight of bone. Most of the organic component is Type
I collagen, which is synthesized intracellularly as tropocolla-
gen and then exported as collagen fibrils. Pathological disor-
ders of the bone matrix exist, such as osteogenesis imperfecta,
a disorder caused by a defect in Type I collagen. This defect
results in less organized bone with loss of normal osteon struc-
ture. With loss of normal osteons, which function to withstand
deformation, the bone fails (fractures) with only minimal
amounts of force. In addition to collagen, bone matrix is com-
posed of proteoglycans, glycoproteins, phosopholipids and
phosphoproteins, as well as various growth factors including
osteocalcin, osteonectin, and bone sialoprotein.
Bones are fashioned in the form of a hollow tube or a
bilaminar plate of bone, each commonly termed compact
bone. Additionally, the architecture is strengthened by inter-
nal “struts” of trabecular bone that follow the lines of stress.
Trabecular or cancellous bone is a metabolically active com-
ponent of bone and has about nine times greater turnover
than the outer compact bone. This kind of design is known in
engineering terms as “composite” and allows bone to take
Chapter 1
Bone Structure, Development and Bone Biology
Fayez F. Safadi, Mary F. Barbe, Samir M. Abdelmagid, Mario C. Rico, Rulla A. Aswad,
Judith Litvin, and Steven N. Popoff
J.S. Khurana (ed.), Bone Pathology, DOI 10.1007/978-1-59745-347-9_1, 1
© Humana Press, a part of Springer Science+Business Media, LLC 2009

2 F.F. S afad i et a l.
advantage of the strength of components. This type of design
also allows bone to resist mechanical compression and able
to deform significantly before failing (i.e. breaking).
Part 1 Bone Structure
Macroscopic Features of Bone
At the gross level, bone can be broadly categorized into five
types: long bones (femur, tibia, ulna and radius), short bones
(carpal bones of the hand), flat bones (skull, sternum and
scapula), irregular shaped bones (vertebra and ethmoid), and
sesamoid bones (bones embedded in tendons). These bones
form through different mechanisms during embryonic devel-
opment. The long bones form by endochondral mechanisms,
while the flat bones form by intramembranous mechanisms.
These processes are discussed later in this chapter. Both long
and flat bones are organized with a hard, but relatively thin,
outer region composed of dense, compact bone called the
cortex or cortical bone. Inside the cortex is the marrow cav-
ity containing hematopoietic elements, fat and spicules of
bone. The bone spicules are also referred to as trabecular,
spongy, or cancellous bone (Fig. 1).
Types of Bones
Long-bones: Macroscopic examination of long bone shows
two extremities (epiphysis) and a cylindrical tube in the middle
(diaphysis) and a transitional zone between them (metaphy-
sis) (Fig. 2). In growing long bone, the epiphysis and the
diaphysis originate from independent ossification centers
and are separated by a layer of cartilage, termed the epiphy-
seal or growth plate (see more details later in this chapter).
Short bones: Carpal and tarsal bones of the hand and foot,
respectively, are examples of short bones. These bones are
typically cube-shaped, and have only a thin layer of compact
bone surrounding a spongy (trabecular bone) interior.
Flat-bones: Excellent examples are the bones of the skull,
which consist of inner and outer tables of compact bone with
spongy (trabecular) bone (diploe) between them.
Fig. 1 Adult long bone. Sagittal section through long bone showing the
internal structure of the bone. Note the outer dense compact bone (also
called cortical bone) and the inner cancellous bone filled with spicules (tra-
beculae), these latter small bundles of bone traverse the inner substances of
bone and are usually interconnected with one another.
Fig. 2 Schematic diagram of a tibia. The interior of a typical long bone
showing middle diaphysis, a growing proximal end (epiphysis) with a
still active epiphyseal growth plate and a distal end with the epiphysis
fused to the metaphysis. The diaphysis (shaft) of a long bone contains a
large marrow cavity surrounded by thick-walled tube of compact bone.
A small amount of spongy bone lines the inner surface of the compact
bone. The proximal and distal ends, or epiphyses, of the long bone con-
sist of spongy bone with a thin outer shell of compact bone. The outer
surface of the bone is covered by a fibrous layer of connective tissue
called the periosteum.

1 Bone Structure, Development and Bone Biology 3
Irregular bones consist of an outer thin layer of compact
bone covering an inner region of spongy bone. The scapula
is an example of an irregular bone.
Sesamoid bones: These bones are a subtype of short bones
that are embedded in tendons. The patella and pisiform are
examples of sesamoid bones.
Individual Bone Structure
Cortical (compact) bone refers to the dense hard, calcified
bone that forms the hard outer “shell” of bone that surrounds
the marrow cavity (Figs. 1, 2). This type of bone has few
gaps or spaces. In the adult, cortical bone (or the cortex) is
composed of dense aggregations of lamellar type bone (see
below). Compact bone also contains within it Haversian (2)
and Volkmann canal systems for vascular supply (Fig. 3).
Cortical bone is surrounded externally and internally by a
periosteum and endosteum, respectively.
Epiphysis: This term refers to the end of a tubular bone,
lying between the epiphyseal (growth) plate (in developing
bone) and the articular cartilage (Fig. 2). In adults, the growth
plate is absent. The place it is thought to have occupied is
arbitrarily selected to define the portion referred to as the
epiphysis. The epiphyses consist mostly of spongy bone
inside a thin sheet of dense bone.
Physis or Epiphyseal Plate: This term refers to the growth
plate in children (before cessation of growth). Injuries or
other disruptions such as infections can seriously affect the
subsequent size and shape of the bone. For example, increased
vascularity around the epiphysis, occurring during the repair
process after a fracture at this site, can cause elongation of
the limb. Inflammatory destruction of the physis may cause
shortening while its partial destruction may cause an angular
deformity. Fractures of the physeal plate have been classified
based on the amount and kind of disruption caused. This is
the basis for the Salter-Harris classification (3).
Metaphysis: This refers to the widened portion of bone
occupying the area between the cylindrical diaphysis and the
physis/epiphysis (Fig. 2). Remodeling and modeling defects
in this region are frequent in conditions such as multiple
osteochondromatosis. Several tumors have an epicenter in
the metaphysis.
Diaphysis (shaft): This refers to the middle, cylindrical
portion of a tubular bone (Fig. 2). There is a thick cortex sur-
rounding a marrow space.
Bone Marrow: The medullary cavity is filled with varying
proportions of hematopoietic marrow, fat and trabecular
bone. The marrow is most prevalent in younger age groups
and in the metaphyseal region of long bones. The diaphysis
contains mainly fat in adults. In comparison to the appen-
dicular (limb) skeleton, the axial skeleton has a greater pro-
portion of bone marrow.
Periosteum: The periosteum is a thick fibrous membrane
that covers the entire surface of a bone, with the exception of
the articular cartilage. It is composed of an outer fibrous
layer and an inner cambium (cellular) layer. The outer layer
is a connective tissue layer containing fibroblasts as well as
nerves and blood vessels supplying the underlying bone. The
inner cambium layer contains osteoprogenitor cells capable
of forming new bone, and is thus an osteogenic layer. When
tendons insert into bone, the collagen fibers (Sharpey’s
fibers) pass through the periosteum and then into the bone
lamellae. The Sharpey’s fibers contribute to the appositional
growth of bone (see intramembranous bone formation).
Endosteum: The endosteum is composed of a resting
layer of marrow at its interface with bone. This is not a mor-
phologically recognizable layer of tissue at the light or elec-
tron microscopic level. However, it is a convenient concept
which exists to explain the functional changes seen in physi-
ologic and pathologic alterations in bone.
Fig. 3 Diagram of immature and mature bone.
Immature (woven) bone displays a disorganized
lamellar appearance because of the interlacing
arrangement of collagen fibers. The cells
(osteoblasts and osteocytes) tend to be
randomly arranged, whereas the cells in the
mature bone are organized in circular fashion
that reflects the lamellar structure of the
Haversian system. Resorption canals in mature
bone have their long axes in the same direction
as the Haversian canals.

4 F.F. S afad i et a l.
Trabecular bone, also called spongy or cancellous bone,
consists of slender spicules and trabeculae of bone that are
separated by marrow spaces. Trabecular bone fills the inte-
rior of long bones, the metaphyseal region of long bones, and
epiphyseal ends of bones. Trabeculae form a network of rod-
and plate-like elements that act as scaffolding for the marrow
cavity, lighten bone, and allowing room for blood vessels
and marrow. The spicules of trabeculae usually consist of
several lamellae of bone tissue.
Microscopic Features of Bone
Bone tissue can be classified based on collagen fiber arrange-
ments into two different types: woven bone and lamellar bone.
To repeat from above, bone is also classified into compact
bone and trabecular bone.
Woven Bone: This form of bone consists of randomly ori-
ented collagen fibers, with large numbers of osteoblasts and
osteoprogenitor cells alongside (Fig. 3). Under polarized light,
it has a haphazard structure which is in great contrast to lamel-
lar bone (see below). Woven bone contains relatively more
cells per unit area than mature bone. Although woven bone is
the major bone type in the developing fetus, and lamellar
(mature) bone is the major bone type in the adult, areas of
immature bone are also present in adults, especially where
bone is being remodeled. Areas of woven bone are also seen
regularly in the alveolar socket of the adult oral cavity and
where tendons insert into bones. Except for the above exam-
ples, woven bone is generally considered pathologic if seen in
adults. It occurs in regions of rapid growth, such as in the
growing skeleton especially in the embryo, fracture callus,
fibrous dysplasia, areas of remodeling osteosarcoma, and sev-
eral other tumors. The molecular signals that are required to
trigger woven bone synthesis are thought to include platelet
derived growth factor (PDGF A and B), insulin like growth
factor (IGF I and II) and perhaps others. It is likely that a com-
bination of growth factors may be required (for more details
see growth factors in this chapter).
Lamellar bone: Lamellar bone is the mature form of adult
bone. It is readily identified on polarized light microscopy as
parallel lines of deposited bone (Fig.3). Studies have shown
that lamellar bone has a well-organized arrangement of colla-
gen fibers. Lamellar bone is formed when the rate of deposi-
tion is slow. In general, it is formed only on pre-existing bone,
either woven or lamellar. The control mechanisms involved in
the formation of lamellar bone are still under investigation.
Secondary organization is a hallmark of lamellar bone. In
the cortex, the lamellae are arranged in circumferential as
well as tubular arrangements (Figs. 3, 4). The tubular arrange-
ment is called an osteon (Fig. 4). Under the microscope,
these tubes can look like circles or parallel sheets depending
on how they are sectioned during histologic preparation. The
central part of the tube is the Haversian canal (Figs. 4, 5),
which contains blood and lymphatic vessels and nerves. The
osteons play an important role in the mechanical properties
of cortical bone, since the long axis of an osteon is parallel to
the long axis of a long bone. Each osteon acts as a fiber that
resists failure (fracture) with deformation (stretch).
Types of Lamellae:Outer circumferential lamellae are sev-
eral lamellae that lie next to the periosteum and are oriented
parallel to it. Inner circumferential lamellae lie next to the
endosteum (Fig. 5). The circumferential lamellar bone resists
compressive forces. The interstitial lamellae are remnants of
previous concentric lamellae (Fig. 5).
Haversian systems (osteons) and Volkmann’s canals: These
are cylindrical units of 5 to 15 concentric lamellae, which sur-
round a central Haversian canal. Each lamella is several
microns in thickness and its fibers run in a spiral fashion
around the canal. The Haversian canal contains capillaries,
venules, lymphatic vessels, and a loose connective tissue con-
taining osteoprogenitor cells. Since the Haversian systems are
arranged around branching blood vessels, it is easy to see how
the Haversian systems comprise a branching system of cylin-
ders that are oriented in the long axis of the bone. Volkmann’s
canals are vascular channels that connect Haversian canals to
each other as well as connect the Haversian system with the
blood vessels in the periosteum. Canaliculi containing the pro-
cesses of osteocytes (see below) are largely arranged in a radial
pattern with respect to the canal. The system of canaliculi that
opens to the Haversian canal also serves for the passage of
substances between the osteocytes and blood vessels.
Compact versus Trabecular Bone
Compact bone: To summarize from above, compact bone
usually consists of concentric lamellae arranged into
Haversian systems (osteons), interstitial lamellae between
the Haversian systems, and inner and outer circumferential
lamellae (Figs 4 and 5). Located in spaces between these
lamellar are bone cells called osteocytes (see below and Fig.
5). Because of this organization, compact mature bone is
also called lamellar bone.
Trabecular bone:This type of bone refers to the spongy
spicules of bone found within the marrow space and is also
called spongy, cancellous or medullary bone (Fig. 1 and 6).
Each spicule of trabecular bone is composed of several
lamellae and is usually not more than 0.2-0.4 mm in thick-
ness to allow for diffusion of nutrients to the osteons. If they
were thicker, they would need osteonts in order to insure
adequate vascular perfusion (Fig. 6). In trabecular bone the
lamellae are normally arranged in a longitudinal fashion, and
osteons are usually not formed.

1 Bone Structure, Development and Bone Biology 5
Fig. 4 Schematic drawing of the
cortical bone. Sectioned cortical bone
showing the tubular and circumferen-
tial arrangement of osteon. In the
center of each osteon is a canal, called
the Haversian canal. Each Haversian
canal contains blood vessels, nerve
endings and lymphatic vessels.
Fig. 5 Cartoon and a microscopic photograph depicting the lammelar organization of bone. A. Cartoon representing a Haversian system (osteon)
with interstitial lamellae, osteocytes and canaliculi (cellular processes). B. Histological representation of Volkmann’s canal connecting Haversian
canals of adjacent osteons.

6 F.F. S afad i et a l.
Separating the trabeculae from the marrow is an endos-
teum. Under electron microscopy, the endosteal layer has a
rich supply of osteoclasts and osteoblasts. Trabecular bone is
more metabolically active than compact bone. Consequently,
metabolic bone studies are best carried out on this compo-
nent of bone. Radiologic studies (such as dual and single
energy computerized tomography (CT) scan methods as well
as micro-CT developed for the study of osteoporosis) have
devised methods to exclusively study the trabecular compo-
nent and to exclude the cortex. Some amount of success has
been achieved using these approaches with the ability of
obtaining a “region of interest” by modern computer soft-
ware. Microscopically this is done using bone histomor-
phometry with image analysis software such as Bioquant
Osteo (www.bioquant.com), Osteomeasure (www.osteo-
metrics.com), and SkyScan CTAn (www.skyscan.be).
Bone Matrix
The organic component of bone makes up 40% of the dry
weight and is composed of collagen, proteoglycans, glyco-
proteins, phosopholipids and phosphoproteins. The inor-
ganic component makes up the remaining 60% of the dry
weight of bone and is composed primarily of calcium
hydroxyapatite Ca10(PO4)6(OH)2. At an ultrastructural level,
bone is organized to maximally resist applied mechanical
forces. Calcium hydroxyapatite crystals are arranged paral-
lel to collagen fibers (59). This orientation maximizes the
collagen’s resistance to tensile (stretch) forces and the cal-
cium hydroxyapatites resistance to compressive forces.
Diseases characterized by abnormal bone collagen content
result in weak bone matrix and lowered abilityof the bone
to resist mechanical forces. A more detailed discussion on
the bone matrix is given below in Part III, the section on
Bone Biology.
Blood Supply of Bone
Bone has a rich vascular supply. It receives 10-20% of the
cardiac output. Blood supply varies with different types of
bones. Blood vessels are especially rich in areas containing
red bone marrow. The extent to which the periosteal and
endosteal vascular supplies meet the metabolic needs of
bone is controversial. Some authors feel that the periosteal
and endosteal supplies are able to meet the needs of the
outer and inner halves of the cortex. Others contend that the
periosteal supply is able to meet only the ends of the outer
third of the cortex. The question however is an important
one, especially in situations of operative fracture repair and
in the surgical technique of bone elongation called distrac-
tion osteogenesis.
Vasculature and Nerve Supply in Long Bones
The diaphyseal nutrient artery is the most important supply
of arterial blood to a long bone. One or two principal diaphy-
seal nutrient arteries first pass through the cortical bone
obliquely. These arteries then divide into ascending and
descending branches and supply the inner two thirds of the
cortex and medullary cavity. There are also numerous meta-
physeal and epiphyseal arteries supplying the ends of bones.
They arise mainly from the arteries that supply the adjacent
joints. They anastomose with the diaphyseal capillaries and
terminate in bone marrow, cortical bone, trabecular bone,
and articular cartilage. In growing bones, these arteries are
separated by the epiphyseal cartilaginous plates. Finally,
periosteal arterioles are vessels that supply the outer layer of
cortical bone.
Arterial Supply of Large Irregular Bones,
Short Bones, and Flat Bones
These bones receive a superficial blood supply from the
periosteum and frequently from large nutrient arteries that
penetrate directly into the medullary bone. These two arterial
systems anastomose freely.
Venous and Lymphatic Drainage of Bone
Blood is drained from bone through veins that accompany
the arteries and frequently leave through foramina near the
articular ends of the bones. Lymph vessels are abundant in
the periosteum.
Fig. 6 Cartoon of spongy bone. Spongy bone (also called trabecular
bone) is composed of trabeculae surrounded by bone marrow. Each tra-
becula consists of lamellar bone, osteocytes and mineralized matrix.
Bone marrow fills the space between trabeculae.

1 Bone Structure, Development and Bone Biology 7
Nerve Supply of Bones
Nerves are most rich in the articular extremities of the long
bones, vertebrae, and larger flat bones. Many nerve fibers
accompany the nutrient blood vessels to the interior of the
bones and to the perivascular spaces of the Haversian canals.
Accompanying the arteries inside the bones are vasomotor
nerves, which control vascular constriction and dilation. The
periosteal nerves are sensory nerves, some of which are pain
(nociceptive) fibers. Therefore, the periosteum is especially
sensitive to tearing or tension. Nerve endings have also been
demonstrated adjacent to bone trabeculae and in proximity
to bone cells.
Bones are also innervated by sympathetic fibers. In the
upper limb, the sympathetic fibers destined for bone originate
from the sympathetic ganglion. In the lower limb, selective
peripheral neuroctomy studies revealed that the sympathetic
nerves, with reference to the tibia, descend in the sciatic
nerve, and thereafter principally in the medial popliteal nerve,
and enter bone alongside the nutrient vessels (4).
Neurotransmitters and Bone
Neurotransmitters released by these nerve endings within
bone are the subject of increasing interest in the field of bone
biology since they appear to have a role in bone formation
and remodeling. For example, mice homozygous for deletion
of the dopamine transporter gene DAT (-/-) demonstrated
reduced bone mass and strength. Cancellous bone volume in
DAT (-/-) proximal tibial metaphysis was significantly
decreased with reduced trabecular thickness. The ultimate
bending load (femoral strength) for the DAT (-/-) mice was
30% lower than the wild-type mice. Thus, deletion of the
DAT gene resulted in deficiencies in skeletal structure and
integrity (5).
Cannabinoids and Bone
Recent studies have shown that the endogenous cannabinoid
system plays a role in regulating bone remodeling. The
endogenous cannabinoids bind to and activate two G pro-
tein-coupled receptors, the predominantly central cannabi-
noid receptor type 1 (CB1) and peripheral cannabinoid
receptor type 2 (CB2). Whereas CB1 mediates cannabinoid
psychotropic and analgesic effects, CB2 has been impli-
cated recently in the regulation of liver fibrosis and athero-
sclerosis. Genetically engineered mice null for CB2
receptors showed accelerated age-related trabecular bone
loss and cortical expansion, although cortical thickness
remains unaltered. These changes are reminiscent of human
osteoporosis and may result from differential regulation of
trabecular and cortical bone remodeling. The CB2 null
mouse phenotype is also characterized by increased activity
of trabecular osteoblasts (bone forming cells), increased
osteoclast (bone resorbing cell) number, and a markedly
decreased number of diaphyseal osteoblast precursors. CB2
is expressed in osteoblasts, osteocytes, and osteoclasts (6).
A CB2-specific agonist enhances endocortical osteoblast
number and activity and restrains trabecular osteoclastogen-
esis, apparently by inhibiting proliferation of osteoclast pre-
cursors and receptor activator of NFKB ligand expression in
bone marrow-derived osteoblasts/stromal cells. This same
agonist attenuates ovariectomy-induced bone loss in ani-
mals and markedly stimulates cortical thickness through the
respective suppression of osteoclast number and stimulation
of endocortical bone formation. These results demonstrate
that the endocannabinoid system is essential for the mainte-
nance of normal bone mass by osteoblastic and osteoclastic
CB2 signaling. Hence, CB2 offers a molecular target for the
diagnosis and treatment of osteoporosis, and other bone-
loss associated diseases (6,7).
Bone Cells
The cells important in bone biology are osteoblasts, osteocytes
and osteoclasts. Osteoblasts are the primary cells responsible
for bone formation (osteogenesis) and mineralization, while
osteoclasts are primarily responsible for bone resorption.
Osteoblasts and osteocytes are derived from mesenchymal
stem cells, while osteoclasts are derived from hematopoeitic
stem cells and are related to monocyte/macrophages.
Osteoblasts
Osteoblasts originate from mesenchymal stem cells that have
the potential to proliferate and the capacity to differentiate
into several connective tissue cell types. These pluripotent
mesenchymal cells can differentiate into osteoblasts, chond-
roblasts, bone marrow stromal cells, fibroblasts, muscle cells
or adipocytes depending on the nature of the stimulus within
their local microenvironment. Given the appropriate stimuli
to differentiate into osteoblasts, they will first give rise to
osteoprogenitor cells, cells still capable of proliferating yet
committed to the osteoblast lineage. Osteoprogenitor cells
can be found in the inner layer of the periosteum, the endos-
teum lining marrow cavities, osteonal (Haversian) canals,
perforating (Volkmann’s) canals, and in perivascular tissue
adjacent to bone. Osteoprogenitor cells can also be found in

8 F.F. S afad i et a l.
the bone marrow where they are indistinguishable from mar-
row stromal cells to which they are related.
Osteoblasts are generally cuboidal or columnar in shape,
and are found lining bone surfaces at sites of active bone
formation such as during bone development (see below) or
fracture repair (Fig. 8). Osteoblasts are responsible for the
production of type I collagen and the proteoglycans (glyco-
soaminoglycans) that largely comprise the organic compo-
nent of bone matrix, also known as osteoid. Osteoid can be
visualized using specific stains as shown in Fig. 9. Osteoblasts
are also involved in the subsequent mineralization of osteoid
via the liberation of matrix vesicles and the deposition of
calcium and phosphate (8,9). Osteoblasts are joined by adhe-
rens type junctions, including desmosomes and tight
junctions. Cadherins are transmembrane proteins that are
integral to these adherens junctions and function to join cells
through their cytoskeleton.
The phenotypic characteristics of osteoblasts depend on
their stage of differentiation. Ultrastructurally, osteoblasts
are typical protein producing cells with an extensive amount
of rough endoplasmic reticulum, a large Golgi apparatus and
numerous mitochondria. Alkaline phosphatase enzyme activity
is one of the earliest markers of the osteoblast phenotype. In
addition to the production of type I collagen and proteoglycans,
osteoblasts also produce a variey of other non-collagenous
proteins including osteocalcin, osteopontin, bone sialoprotein
and osteonectin (see below). These proteins are also markers
of the osteoblast phenotype and each has a unique temporal
pattern of expression during osteoblast differentiation.
Osteoblasts secrete a variety of cytokines and colony stimu-
lating factors (CSF), such as interleukin-6, interleukin-11,
granulocyte-macrophage colony stimulating factor (GM-CSF)
and macrophage colony stimulating factor (M-CSF), and
Fig. 7 Bone lining cells. Histological microphotographs of femurs
stained with Saffranin-O and counter stained with Goldner stain show-
ing metaphyseal bone with bone lining cells (black arrows). These cells
are inactive but can redifferentiate into active, bone forming osteoblasts
in response to the appropriate stimuli.
Fig. 8 Osteoblasts lining trabecular bone surface. Photomicrographs of
metaphyseal trabecular bone, showing active cuboidal osteoblasts lining
the trabecular bone surfaces (black arrows). Note also some osteocytes
encased within the bone matrix (blue arrows).
Fig. 9 Osteoid synthesis by active osteoblasts. Photomicrographs of
undecalcified metaphyseal trabecular bone stained with von Kossa (for
mineral, black stain) and toluidine blue. Note the osteoid, unmineral-
ized bone matrix (red arrows), compared to the mineralized matrix
(black).

1 Bone Structure, Development and Bone Biology 9
thus play a role in myelopoesis. Osteoblasts also secrete
numerous growth factors including transforming growth
factor-beta (TGFB), bone morphogenetic proteins (BMPs),
platelet derived growth factors (PDGFs), and insulin-like
growth factors (IGFs). Mature osteoblasts possess receptors
for parathyroid hormone (PTH) and 1,25-dihydroxyvitamin
D, two hormones that play important roles in regulating bone
metabolism and mineral homeostasis (see below). Osteoblasts
also secrete receptor activator of nuclear factor kappa B
(RANK) ligand, a protein that plays an essential role in the
differentiation of osteoclasts (see below).
Constant mechanical stress is essential for the mainte-
nance of bone mass and strength, which is achieved through
the cooperative functions of osteoblasts, osteocytes and
osteoclasts. Osteoblasts respond to mechanical stimuli to
mediate changes in bone size and shape. This effect may be
modulated by a piezo-electric effect of the calcium hydroxy-
apatite crystals (see section on mechanosensory systems and
stretch studies).
Bone lining cells are flattened, squamous cells found
lining bone surfaces in areas where there is no active bone
formation. They are particularly prevalent in adult and
aging bone where many of the bone surfaces are inactive.
These cells can be thought of as quiescent osteoblasts and
are similar to osteoprogenitor cells in that they can be
reactivated to become functional osteoblasts under condi-
tions that warrant active bone formation such as during
remodeling, fracture repair and in certain types of bone
pathology (Fig. 7).
Regulation of Bone Formation and Osteoblast
Differentiation
Osteoblast differentiation is regulated by numerous secreted
growth factors including transforming growth factor-B (TGFB),
bone morphogenetic proteins (BMPs), fibroblast growth fac-
tors (FGFs), and others (see matrix proteins of bone below)
(10,11). Furthermore, various transcription factors also play
important roles in osteoblast differentiation. Runx-2 (runt-
related transcription factor 2)/cbfa-1 (core binding factor
alpha 1) and osterix are essential transcription factors for
osteoblast differentiation (12,14). Experimental animal mod-
els with targeted deletion of the Runx-2 gene demonstrated
that mice develop to term but have a skeleton consisting
exclusively of cartilage that does not ossify. There is no evi-
dence of osteoblast differentiation or bone formation in these
mice. In addition, Runx-2 null mice lack osteoclasts (see
regulation of osteoclast differentiation). Although null muta-
tions for Runx-2 have not been identified in humans, muta-
tions in the Runx-2 gene cause a disease known as
cleidocranial dysplasia (CCD) (12,14). CCD is characterized
by hypoplastic clavicles and delayed ossification of cranial
sutures. Runx-2 target genes include osteocalcin, bone sialo-
protein, osteopontin and collagen A1 (15,16). Osterix is
another transcription factor that was more recently shown to
play a role in osteoblast differentiation presumably by acting
downstream of Runx-2 (13).
Leptin is a peptide synthesized by adipocytes with bind-
ing affinity to its receptor in the hypothalamus. This protein
regulates bone formation via a central mechanism. Mice
deficient for leptin or its receptor have considerably higher
bone mass than normal wild-type mice. It has been shown
that patients with generalized lipodystophy (absence of adi-
pocytes and white fat) exhibit osteosclerosis and accelerated
bone growth (17). Although details of the leptin-hypothalamic
control mechanism are not fully understood, additional
experimentation on the role of the central nervous system
and the regulation peripheral leptin production will enhance
our understanding of how the leptin-hypothalmic axis regu-
lates bone metabolism.
Other transcription factors, secreted proteins and recep-
tors have been reported to have significant effects on osteo-
blast differentiation and bone formation. Many of these
effects were identified or confirmed in studies involving
genetically modified animals (for list of these animal models
refer to Table 1).
Due largely to the mineralized matrix of bone, it has
proven difficult to study osteoblasts experimentally in vivo.
Many studies of osteoblast differentiation and function uti-
lize organ or cell culture systems. Some studies employ pri-
mary osteoblast cultures in which osteoblasts are
enzymatically digested from the endocranial surface of neo-
natal rodent (rat and mouse) calvaria. Primary cultures of
human osteoblasts are generally established by enzymatic
digestion of osteoblasts from the surfaces of cancellous
bone samples. When cultured under conditions that favor
the osteogenic lineage (i.e. ascorbic acid and B-glycero-
phosphate supplemented media), many of the cells in these
primary cultures differentiate into mature osteoblasts capa-
ble of producing a mineralized bone matrix. One major
drawback to these cultures is the heterogeneity of cells that
can be isolated from calvaria or cancellous bone resulting in
significant contamination of non-osteogenic cells as well as
heterogeneity in the stage of differentiation of the osteo-
genic cells. To eliminate the potential heterogeneity of cell
types inherent in primary culture systems, some investiga-
tors have utilized osteosarcoma cell lines. However, trans-
formed cells may not behave the same as non-neoplastic
cells prompting questions about the physiological relevance
of data generated using transformed osteoblast cell lines.
Another approach has been to establish permanent cell lines
from primary osteoblast cultures such as the widely used
MC3T3-E1 cell line derived from mouse calvaria. There
have also been many studies of osteogenic differentiation
from less differentiated (osteoprogenitor) stromal or

10 F.F. S afad i et a l.
mesenchymal cells (isolation of primary cells and estab-
lished cell lines have been employed for these types of cul-
tures). These types of cultures have been particularly useful
to study osteogenic commitment and early differentiation.
As opposed to cell culture studies, bone organ cultures
utilizing whole calvaria obtained from fetal rats or mice
have been used to study bone responses to exogenous fac-
tors such as vitamin D and cortisol. These organ cultures
allow one to examine the effects of systemic (hormones)
and locally-produced factors on the multitude of cell types
within the context of their normal microenvironment.
However, the major drawback is that it is often not possible
to attribute primary versus secondary effects due to the dif-
ferent cell types that coexist in these organ cultures. From
this description of cell/organ culture systems employed to
study osteoblast differentiation and bone formation, it is
Table 1 Skeletal phenotype of selected genetically engineered mice
Gene Name Skeletal phenotypes of the null, mutant and transgenic animals
Runt-Related Transcription Factor 2;
Runx2, Cbfa1
Null mice: Complete lack of ossification of the skeleton. Mice died just after birth (14) and
showed marked changes in tooth morphogenesis (312). Transgenic mice: Normal skeleton
at birth but developed an osteopenic phenotype thereafter (313).
Secreted Phosphoprotein 1; Spp1,
Osteopontin
Null mice: Normal and viable with altered wound healing (314). These animals also showed
resistance to ovariectomy-induced bone resorption (315). These knockout animals demon-
strated an increase in ectopic calcification especially in the medial layer of the arteries (316).
Gamma-Carboxyglutamic Acid Protein, Bone;
Osteocalcin
Null mice: Develop higher bone mass and bones of improved functional quality.
Histomorphometric studies showed an increase in bone formation without impairing bone
resorption (317).
Secreted Protein, Acidic, Cysteine-Rich;
Osteonectin
Null mice: After six months of age, the animals developed severe osteopenia, cataracts, rupture
of the lens capsule and accelerated closure of dermal wounds (318). These animals also
have greater deposits of subcutaneous adipose tissue (319).
Connective Tissue Growth Factor; CTGF Transgenic mice: Just a few months after birth the animals showed dwarfism, decreased bone
density, alterations in endochondral ossification and affected fertility (202).
Null Mice: CTGF deficiency leads to skeletal dysmorphisms as a result of impaired chondrocyte
proliferation and extracellular matrix composition within the hypertrophic zone (204).
Thrombospondin I; Tsp1 Null mice: Abnormal curvature of the spine, lung abnormalities and increase in the number of
circulating leukocytes (320).
Thrombospondin II; Tsp2 Null mice: Increase cortical bone thickness and density and abnormal long bleeding times (321).
Thrombospondin III; Tsp3 Null mice: Young adult TSP3-null mice are heavier than controls, and analyses of the
geometric and biomechanical properties of long bones show increases in the moments of
inertia, endocortical and periosteal radii, and failure load (322).
Transcription Factor Sp7; Osterix Null mice: Absent bone formation and no deposition of bone matrix in these animals (13).
Parathyroid Hormone; PTH Null mice: These animals showed reduced cartilage matrix mineralization and decreased
metaphyseal osteoblasts and trabecular bone (323).
Parathyroid Hormone-Like Hormone;
PTHrP
Trangenic mice: Chondrocyte-specific overexpression of PTHrP causes a profound delay in the
developmental program of chondrocyte differentiation and endochondral ossification (324).
Sry-Box 9; Sox9 Tissue specific null mice: Inactivation of Sox9 in limb buds before mesenchymal condensa-
tions resulted in a complete absence of both cartilage and bone, but markers for the
different axes of limb development showed a normal pattern of expression (325).
Vitamin D Receptor; VDR Null mice: After weaning, animals showed severe impairment of bone formation such as the
phenotype observed in vitamin D-dependent rickets type II. Animals also exhibit alopecia,
hypocalcemia and infertility. Animals died within 15 weeks after birth (326).
Peroxisome Proliferator-Activated
Receptor-Gamma; PPARg
Heterozygous mice: PPARgamma-deficient mice exhibited high bone mass with increased
osteoblastogenesis, but normal osteoblast and osteoclast functions. The osteogenic effect of
PPARgamma haploinsufficiency became prominent with aging but was not changed upon
ovariectomy (327).
V-Src Avian Sarcoma (Schmidt-Ruppin A-2)
Viral Oncogene; Src
Null mice: These animals demonstrated severe osteoclast dysfunction resulting in osteopetro-
sis. Animals survived for only a few weeks (328)
Serum Response Factor; Srf, c-Fos Null mice: Embryos failed to gastrulate and died due to cardiac insufficiency during chamber
maturation (329). Mice lacking Fos (encoding c-Fos) develop osteopetrosis due to an early
differentiation block in the osteoclast lineage (330).
Spleen Focus Forming Virus Proviral
Integration Oncogene; Spi1, PU.1
Null mice: Exhibit the classic hallmarks of osteopetrosis, a family of sclerotic bone diseases.
Animals were rescued by marrow transplantation, with complete restoration of osteoclast
and macrophage differentiation, verifying that the PU.1 is intrinsic to haematopoietic
cells (331).
Dickkopf; Dkk1 Heterogygous mice: Progressive Dkk1 reduction increases trabecular and cortical bone mass
and even a 25% reduction in Dkk1 expression could produce significant increases in
trabecular bone volume fraction. Thus Dkk1 is a negative regulator of normal bone
formation in vivo (332).

1 Bone Structure, Development and Bone Biology 11
clear that each particular culture model has certain advan-
tages as well as limitations compared to the others. It is par-
ticularly important to be aware of the limitations when
choosing a culture model for an experimental approach, and
also when interpreting and extrapolating data generated
using these culture models.
The osteoblast cell culture systems are widely used to
study the effects of growth factors, secreted proteins, extra-
cellular matrix components and transcription factors on dif-
ferentiation and function (reviewed in Aubin and Triffit,
2002 (18) (Fig. 10). Many of the osteoblast culture models
have been well characterized identifying the temporal
sequence of gene/protein expression associated with osteo-
blast differentiation. In general, after the cells are plated in
osteogenic medium, there are three stages of differentiation;
proliferation, matrix production/maturation, and mineraliza-
tion. The proliferative phase involves the expression of cell
cycle and histone genes. This is followed by expression of
genes associated with the formation of bone matrix such as
type I collagen and alkaline phosphatase. In the final stage,
genes associated with mineralization, such as osteocalcin
and bone sialoprotein, are expressed at the highest levels.
Osteocytes
In contrast to surface cells, such as osteoblasts, osteocytes
are bone cells that live within the substance of bone. These
cells comprise 90%–95% of all bone cells. They are derived
from osteoblasts (Fig. 10) that became trapped and sur-
rounded by bone matrix which they themselves produced.
Fig. 10 Temporal pattern of expression of markers during osteoblast
differentiation in culture. As osteoblasts proliferate and differentiate
from mesenchymal stem cells they express/produce various proteins
including growth factors, transcription factors and extracellular matrix
(ECM) proteins, each of which has a distinct temporal pattern of
expression. Runx2 (Cbfa1), c-fos and c-myc are transcription factors;
ALPH, alkaline phosphatase; Col-1, Collagen type I; OCN, osteocal-
cin; OPN, osteopontin; CTGF, connective tissue growth factor; OA,
Osteoactivin; TGF-B, transforming growth factor-B

12 F. F. S afad i et a l.
The spaces which they occupy are known as lacunae. They
are said to be involved in cell signaling and maintaining the
viability of bone matrix. The processes of osteocytes com-
municate with each other and with osteoblasts through a
network of canaliculi. The processes of adjacent osteocytes
are joined together by gap junctions thereby allowing this
vast network of cells within the bone matrix to communicate
with one another and with cells outside of the bone matrix. It
is also believed that osteocytes are important in the transla-
tion of mechanical loads to cellular events such as bone for-
mation and remodeling.
Osteocytes have the ability to stimulate osteoblasts and
the accompanying matrix by expressing osteoblast specific
factor-1 (OSF-1) (19). OSF-1 accumulates on bone sur-
faces near the osteocytes and binds to its receptor,
N-syndecan (20) located on osteoblast progenitor cells.
Dentin matrix protein 1 (termed DMP1) is highly expressed
in osteocytes and, when deleted in mice, results in a
hypomineralized bone phenotype. A recent study investi-
gated the potential role of this gene to direct skeletal min-
eralization as well as to regulate phosphate (Pi) homeostasis.
Both Dmp1-null mice and individuals with a newly identi-
fied disorder, autosomal recessive hypophosphatemic rick-
ets, manifest rickets and osteomalacia with isolated renal
phosphate-wasting associated with elevated fibroblast
growth factor 23 (FGF23) levels and normocalciuria
(21,22). Mutational analyses revealed that an autosomal
recessive hypophosphatemic rickets family carried a muta-
tion affecting the DMP1 start codon, while a second family
carried a 7-base pair deletion disrupting the highly con-
served C terminus of DMP1. Studies using the Dmp1-null
mice demonstrated that the absence of DMP1 resulted in
defective osteocyte maturation and increased fibroblast
growth factor 23 (FGF23) expression, leading to patho-
logical changes in bone mineralization. These findings
suggest a bone-renal axis that is central to guiding proper
bone mineral metabolism (21,22).
Osteocyte-derived signals have remained largely enigmatic,
but it was recently reported that human osteocytes secrete
sclerostin, an inhibitor of bone formation. Sclerosteosis, a
skeletal disorder characterized by high bone mass due to
increased osteoblast activity, is caused by a loss of the SOST
gene product, sclerostin (23). Osteocytes possess receptors for
parathyroid hormone (PTH), a known regulator of mineral ion
homeostasis (24). Osteocytes also express molecules typically
associated with nerve cells, such as NMDA receptors, which
are involved with glutamate neurotransmission.
Finally, osteocytes act as mechanosensory cells. Their cell
body and processes are surrounded by a thin layer of unminer-
alized matrix, which allows a loading-derived flow of intersti-
tial fluid over the osteocyte surface. Their mechanosensory
receptor ability has been demonstrated in studies examining
loading-facilitation of macromolecule diffusion (25).
Osteoclasts
Osteoclasts are multinucleated cells generally containing
3-25 nuclei per cell. Osteoclasts are related to the monocyte/
macrophage lineage, with both cell types being derived from
hematopoietic progenitor cells. Although the lineages of
osteoclasts and osteoblasts are independent of one another,
the genesis of osteoclasts requires the presence of osteoblasts
along with a variety of hematopoetic cytokines, such as inter-
leukins 1, 3, 6, and 11, tumor necrosis factor (TNF), colony
stimulating factors (CSF), stem cell factor and others (26).
Colony stimulating factors are required for the prolifera-
tion and differentiation of osteoclast progenitor cells. After
the osteoclast is formed, other cytokines are required for
their activation and bone resorption. Parathyroid hormone
(PTH), 1,25 dihydroxyvitamin D3, transforming growth fac-
tor alpha (TGFA), and epidermal growth factor (EGF) act to
stimulate osteoclastogenesis, whereas calcitonin inhibits the
formation of osteoclasts (26).
Osteoclasts are the primary bone resorbing cells.
Osteoclasts are found at sites where resorption is taking
place, or if active resorption has already occurred, within
“eaten out” pits or cavities known as Howship’s lacunae
(Fig. 11). Osteoclasts can also tunnel through cortical bone
creating channels. Osteoclasts are highly polarized cells and
the nuclei congregate away from the resorbing bone surface.
The cell surface in direct apposition to the bone has an exten-
sive infolding of the plasma membrane called the ruffled bor-
der. When the cell is in the resting state or in certain pathological
conditions in which the osteoclast is dysfunctional, the ruf-
fled border disappears or is absent (28). There is a three
dimensional ring-like area of the cell membrane around the
perimeter of the ruffled border called the clear zone or seal-
ing zone. This zone contains abundant microfilaments (actin
filaments) but lacks other organelles. It is here in the clear
Fig. 11 Osteoclasts in actively growing bone. Osteoclasts are multi-
nucleated cells (arrows) associated with a shallow pits or concavities
(Howship’s lacunae, *) along the bone surface.

1 Bone Structure, Development and Bone Biology 13
zone that the osteoclasts attach to the bone matrix, a process
involving the participation of actin filaments and the AvB3
integrin. The actin reorganization or actin ring within the
clear zone can be visualized microscopically by staining for
actin filaments (28,29). The area of cytoplasm between the
nuclei and the ruffled border is rich in carbonic anhydrase
and in tartrate resistant acid phosphatase (TRAP) (27). By
electron microscopy, there is also an abundance of mitochon-
dria, lysosomes, vesicles and free ribosomes in the
cytoplasm.
Molecular Mechanism of Osteoclast-mediated
Bone Resorption
The mechanism of bone resorption is complex and involves
an initial trigger by osteoblasts (see below). Once osteoclasts
have been formed, bone resorption requires the secretion of
hydrogen ions by an ATP driven proton pump in the ruffled
border. This results in acidification of the extracellular space
between the bone surface and the ruffled border which is
sealed off from the general extracellular compartment by
the clear zone forming a tight seal around the perimeter of
the ruffled border region. The enzyme carbonic anhydrase
II is essential in the generation of hydrogen ions.
Simultaneously, acid hydrolases are released from lyso-
somes into the acidified extracellular space thus creating an
extracellular lysosome. Osteoclasts can move over the bone
surface, creating many resorption pits in their path. These
pits are easily visualized by scanning electron microscopy,
and correspond to the Howship’s lacunae in routine sec-
tions. It is believed that osteoclasts must be stationary for
resorption to occur, and therefore they do not resorb bone
during the motile phase. Instead they cycle between motility
and resorption, moving about, attaching to and resorbing
bone, then releasing and moving to another region for
resorption.
A combination of the acid created by the hydrogen ions
and the proteolytic enzymes released from the lysosomes
provide optimal conditions for the resorption of bone and
degradation of collagen. This environment also results in the
release and activation of certain growth factors and cytokines
such as TNF-A and TGF-B. It is possible that oxygen-derived
free radicals are also important in this process.
Osteoclastic stimulation may also be influenced by inter-
actions of the integral membrane proteins (integrins) present
on the osteoclast cell membrane and proteins in the bone
matrix that contain the amino acids RGD (arganine-glycine-
aspargine). These bone matrix proteins include fibronectin,
collagen type I, bone sialoprotein II and osteopontin, all of
which bind integrins and initiate out-side-in signaling pathways
that can regulate the bone resorption process. Much of the
data regarding factors that are important for the development,
differentiation and function of osteoclasts have come from
genetically modified animal models (see Table 1.1).
Osteoclasts have calcitonin, but not PTH or Vitamin D
receptors. They are stimulated by IL-6 (perhaps in combina-
tion with IL-1, IL-3 and IL-11) and RANK-ligand. These
cytokines are produced locally by cells of the osteoblast lin-
eage under the influence of PTH, Vitamin D3, TGF-B, IL-1
and TNF-A. It is interesting to note, that giant cell tumor
(osteoclastoma) cells respond to IL-6 (30, 31). Anti IL-6
antibodies have been shown to inhibit osteoclastic activity in
these cells. However, in the physiologic state, the evidence
for the action of IL-6 in osteoclastogensis is lacking. Perhaps
threshold levels or additional cytokines play a role (32, also
reviewed in 26).
Regulation of Osteoclast Differentiation
Macrophage colony-stimulating factor (M-CSF) is a secreted
protein that is produced by osteoblasts and bone marrow
stromal cells. M-CSF is required for the proliferation and
survival of osteoclast precursors, cells of the monocyte-
macrophage lineage (Fig. 12). Both osteoblasts and stromal
cells produce RANK-ligand that has a high affinity for bind-
ing to the RANK receptor on osteoclast precursors. Treatment
of osteoblasts or stromal cells with PTH, vitamin D, PGE2,
or IL-11 stimulates the expression of RANK-ligand mRNA
(Fig. 12)(33).
The interaction between RANK (on osteoclast precur-
sors) and RANK-ligand (on osteoblasts and stromal cells)
requires cell-cell contact for further maturation of osteoclast
precursors (34–36). Osteoblasts also secrete osteoprotegerin
(OPG), a member of the TNF-A receptor superfamily (37).
This protein lacks the transmembrane domain and is pre-
sented as a secreted form. OPG is a soluble protein and
acts as a decoy receptor that binds RANK-ligand and pre-
vents RANK/RANK-ligand interaction. Through this mech-
anism, OPG can modulate the process of osteoclastogenesis.
Macrophage-colony stimulating factor (M-CSF) is also essential
for osteoclastogenesis. M-CSF induces cells of the monoc-
tye/macrophage lineage to proliferate and become osteoclast
precursors. RANK-ligand stimulates M-CSF-induced cells
to differentiate into functional osteoclasts. Animal models of
genetically modified RANK, RANK-ligand and OPG have
been generated to understand the role of these proteins in
osteoblast biology (see below). Mouse knockouts for RANK
and RANK-ligand share similar phenotypes (38–40). Both
models develop severe osteopetrosis, a disease associated
with the absence of osteoclasts and failure of tooth eruption.
Transgenic mice that over-express OPG in the liver also
resulted in severe osteopetrosis. Therefore, the RANK/
RANK-ligand/OPG axis appears to play a key physiological
role in osteoclast differentiation and function. Clinically,

14 F. F. Saf adi e t al .
it has been reported that two mutations of heterozygous
insertion were detected in the first exon of RANK in families
with familial expansile osteolysis or familial Paget’s disease
of bone (41).
Signaling Pathway of RANK
The cytoplasmic tail of RANK interacts with TNF receptor-
associated factor (TRAF) TRAF family members. TRAF2-
mediated signals appear important for inducing osteoclast
differentiation, and TRAF6-mediated signals are indispens-
able for osteoclast activation. Activation of NF-KB, JNK and
ERK pathways, all induced by RANK–ligand in osteoclast
precursors and mature osteoclasts, appear to be involved in
the differentiation and function of these cells (42,43). OPG
strongly blocks osteoclastic bone resorption in vivo, suggest-
ing that inhibiting the RANK-ligand/RANK interaction or
RANK-mediated signals are promising targets to prevent
increased bone resorption in metabolic bone diseases such as
rheumatoid arthritis, periodontitis and osteoporosis. Studies
have also shown that TNF-A and IL-1 can substitute for
RANK-ligand in inducing osteoclastogenesis in vitro. These
studies suggest that signals other than these induced by RANK
may also play important roles in osteoclastic bone resorption
under pathological conditions.
Positive Regulation of Osteoclastogenesis
Suppressor of cytokine signaling-1 (SOCS-1) is an inhibitor
of cytokine signaling and may play a positive role in the regu-
lation of T-cell-mediated osteoclastogenesis by counteracting
inhibitory cytokines such as interferon-gamma (IFN- G) (44).
It has been reported that (SOCS)-1-deficient osteoclast pre-
cursor cells are more susceptible to the inhibitory effects of
IFN-G on osteoclastogenesis when compared to wild-type
cells (44). SOCS-1 has been shown to be induced by RANK-
ligand stimulation during osteoclastogenesis (45), indicating
that these precursor cells are resistant to IFN-G-mediated inhi-
bition if they are first stimulated by RANK-ligand. It is likely
that the fate of osteoclast precursor cells is determined not
only by the balance of cytokines, including IFN-G and RANKL
(46), but also by the cytokine first encountered (47).
Fig. 12 Regulation of osteoclast differentiation. 1. Monocytes derived
from vessels in the bone marrow, reach an area of bone formation or
remodeling. Monocytes express M-CSF receptor on their cell surface.
2. Monocytes differentiate into macrophages. M-CSF binds to M-CSF
receptor and induces the expression of RANK. 3. RANK ligand
(RANKL) is produced by stromal cells/osteoblasts and binds to the
receptor, RANK, on mononuclear osteoclast progenitors (bone marrow
macrophages). 4. OPG, a decoy receptor that binds to RANKL and
inhibits osteoclast differentiation. 5. Following the interaction between
RANK and RANKL, mononuclear osteoclast progenitors fuse to form
a resting (non functional) multinucleated osteoclast that uncouples
from the osteoblast. 6. Multiple factors including transcription factors,
hormones, locally produced cytokines/growth factors and matrix pro-
teins, mediate the activation (bone resorption) of osteoclasts.

1 Bone Structure, Development and Bone Biology 15
Negative Regulation of Osteoclastogenesis
by Interferon-b
Using a genomewide screening of RANK-ligand-inducible
genes, several interferon (IFN)-A/B-inducible genes were
identified in RANKL-stimulated osteoclast precursor cells.
The bone phenotype of mice deficient in a subunit of the
interferon- A/B receptor, IFN- A receptor type I (IFN-A-R1),
was analyzed (48). These mice exhibited marked osteopenia
accompanied by enhanced osteoclastogenesis in vivo.
Detailed molecular analyses showed that RANK-ligand
induces interferon-B in osteoclast precursor cells, and IFN-B
inhibits the expression of c-Fos, an essential transcription
factor for osteoclastogenesis. RANK-ligand-mediated induc-
tion of interferon- B is dependent on c-Fos, constituting a
negative feedback loop in which RANK-ligand-induced
c-Fos induces its own inhibitor. Thus, although type I inter-
ferons were originally characterized as critical antiviral fac-
tors, these studies situate the interferon system in a novel
context and provide a compelling example of osteoimmuno-
logic regulation (49).
Transcriptional Regulation of Osteoclastogenesis
Genomewide screening of RANK-ligand-inducible genes
identified NFATc1 (nuclear factor of activated T-cells) to be
the most highly induced transcription factor in osteoclast
precursor cells. NFATc1 binds to the Nfatc1 promoter and
induces itself (50). This strategy is often observed in hema-
tologic cells that undergo irreversible differentiation (51).
Nfatc1-/- embryonic stem cells cannot differentiate into osteo-
clasts in vitro and overexpression of NFATc1 induces osteo-
clastogenesis. These results suggest that NFATc1 is an
essential regulator of osteoclastogenesis (52), but it has
proven difficult to show that this transcription factor is indis-
pensable for osteoclast differentiation in vivo due to the
embryonic lethality of Nfatc1-/- mice.
Immunoreceptors in Osteoclastogenesis
The close relationship between bone and immune system
extends beyond the cytokines and transcription factors they
share. Activation and nuclear localization of NFAT are
dependent on its dephosphorylation by the phosphatase cal-
cineurin, which is activated by calcium (Ca2+) signaling.
Ca2+ oscillation is observed during osteoclastogenesis, and
the calcineurin inhibitors cyclosporine A and FK506
strongly inhibit osteoclastogenesis (52). It is not clear,
though, how Ca2+ signaling is activated during osteoclasto-
genesis. DNAX-activating protein 12 (DAP12) is an adap-
tor molecule that associates with immunoglobulin-like
receptors and harbors an immunoreceptor tyrosine-based
activation motif (ITAM), which is known to be crucial for
the activation of Ca2+ signaling in the immune cells. The
osteopetrotic phenotype in DAP12-deficient mice made
evident the importance of ITAM for osteoclastogenesis
(53). Mice deficient in both DAP12 and the Fc receptor
common G subunit (FcRG) have been shown to exhibit
severe osteopetrosis, indicating that immunoglobulin-like
receptors also provide important signals for osteoclasto-
genesis in addition to the RANK and M-CSF receptors
(54,55). However, immunoreceptor signaling alone cannot
induce osteoclastogenesis, suggesting that these receptors
provide co-stimulatory signals for RANKL. FcRG-
associating receptors include osteoclast-associated recep-
tor (OSCAR) and paired immunoglobulin-like receptor-A,
while DAP12-associating receptors include triggering
receptor expressed on myeloid cells (TREM-2) and signal-
regulatory protein (SIRP-B) (54). The ligands for these
immunoreceptors on osteoclasts are yet to be identified.
OSCAR expression is upregulated significantly during
osteoclast differentiation and its induction is mediated by
NFATc1 (56, 57). Thus, OSCAR-NFATc1 constitutes a
positive feedback loop in osteoclast precursor cells.
Physiological versus Pathological Bone Resorption
Under physiological conditions, osteoclast formation
requires cell-to-cell contact with osteoclast/stromal cells
which express RANK-ligand as a membrane-bound factor
in response to a number of factors that have been shown to
stimulate bone resorption. In contrast, under pathological
conditions that stimulate bone resorption, such as in rheu-
matoid arthritis, macrophages and/or T cells secret inflam-
matory cytokines such as TNF-A and IL-1. These cytokines
act directly on osteoclast progenitors and mature osteo-
clasts without cell-to-cell contact. This paradigm is charac-
terized by the uncoupling of bone resorption and bone
formation.
Model Systems Used to Study Osteoclasts
Given the technological challenges associated with isolating
sufficient numbers purified osteoclasts, it has been difficult
to study these cells experimentally. The models developed to
study osteoclasts in culture are more cumbersome than those
for osteoblasts. The recent discovery of the role for RANK-
ligand and M-CSF in osteoclastogenesis has allowed investi-
gators to differentiate large numbers of osteoclasts from bone
marrow progenitors or splenic macrophages (58) in the
presence of recombinant forms of these essential factors.
Large numbers of osteoclasts can also be generated from the

16 F.F. S afad i et a l.
RAW cell line, a macrophage cell line that has the ability to
differentiate into osteoclasts when treated with RANK-ligand
and M-CSF. Another alternative is the osteoclast-osteoblast
co-culture system in which primary osteoblasts are cultured
with bone marrow hematopoietic cells containing osteoclast
progenitors (monocytes/macrophages). This culture system
requires both vitamin D and PGE2(26).
Osteopetrosis (see section on metabolic bone disease) in
humans and animals has been useful in providing models to
study osteoclast development and function. One form of the
disease is associated with carbonic anhydrase II deficiency.
Osteoclasts are present but functionally incompetent in CAII
deficiency, and children afflicted with this disease also suffer
from renal tubular acidosis. In the op/op osteopetrotic mouse
and the tl/tl toothless rat models, there are abnormalities in
the coding region of the CSF-1 gene resulting in the produc-
tion of trauncated and functionally incompetent CSF-1
protein. In these forms of osteopetrosis, osteoclasts are
absent or greatly reduced in number, and treatment with
exogenous CSF-1 results in normal osteoclastogenesis and
correction of the disease. There are other mouse models in
which targeted disruption of certain proto-oncogenes, such
as src and fos, causes osteopetrosis (for a more comprehen-
sive list, see Table 1).
Part 2 Bone Development
The bones of the axial and appendicular skeleton are formed
by one of two processes, intramembranous or endochondral
bone formation. The primary difference between these two
processes is the absence or presence of a cartilaginous inter-
mediary. In intramembranous bone formation, bone is formed
in the absence of a cartilage model while in endochondral
bone formation, a cartilage model is first formed and then
replaced by bone tissue.
Intramembranous Bone Formation
(Membranous)
The flat bones of the skull and face are formed by intramem-
branous ossification (Fig. 13). Osteoprogenitor cells (which
will give rise to osteoblasts and osteocytes) are present within
the mesenchyme. These cells aggregate at the sites where
new bone is to be formed and differentiate into osteoblasts
that actively synthesize new bone matrix. Growth of
intramembranous bones occurs by apposition (deposition
upon prior bone) of osteoblasts lining the surfaces of the
growing bones. Ossification centers develop within the
bone and enhance the rates of mineralization. As the growth
rate slows down, the bones take on a lamellar character. In
the adult, Haversian remodeling occurs.
Chondroid bone has also been described within the skull,
occurring in association with suture closure. This type of
bone has a histological intermediate between cartilage and
bone, containing both types I and II collagen. Chondroid
bone forms a scaffold upon which lamellar bone is depos-
ited. It is not replaced by bone as occurs in the endochondral
model (see below).
There are many factors that play potential roles regulating
bone formation, and their functions and interactions are
complex. For instance, core binding factor-alpha1 (cbfa-1) is
responsible for osteoblast differentiation, and binds the
osteocalcin promoter, resulting in osteocalcin expression.
Mutation of cbfa-1 causes cleidocranial dysplasia, a disorder
in which there is delayed ossification of cranial sutures.
Endochondral Bone Formation
(Cartilage Model)
Endochondral ossification involves the formation of cartilage
tissue from aggregated mesenchymal cells and the subse-
quent replacement of this cartilage tissue by bone tissue (60).
All of the skeletal components of the vertebral column, the
pelvis, and the appendicular skeleton (limbs) develop via
endochondoral ossification. The process of endochondral
ossification is divided into five stages (Fig. 14). First, the
mesenchymal stem cells are committed to become cartilage
cells through expression of two transcription factors,
Fig. 13 Intramembranous Ossification. Mesenchymal stem cells con-
dense to produce osteoblasts, which deposit osteoid and mineralize the
bone matrix. These osteoblasts line the surfaces of the developing bone
and continue to produce bone matrix by apposition. Osteoblasts that
become trapped within the bone matrix become osteocytes. There is no
cartilage that precedes the formation of bone in this type of bone forma-
tion. Permission granted from Sinauer Associates (Gilbert, 6th edition,
1997).

1 Bone Structure, Development and Bone Biology 17
Pax1 and Scleraxis. These factors are thought to activate
cartilage-specific genes (61–63). During the second phase of
endochondral ossification, the committed mesenchymal stem
cells condense into compact nodules and differentiate into
chondrocytes. N-cadherin appears to be crucial for the initia-
tion and maintenance of these condensations (64,65). In
humans, the SOX9 gene (sex reversal Y-related high-mobil-
ity group box protein) and chondrocyte commitment, that
encodes a DNA-binding protein, is expressed in the precarti-
laginous condensations. Mutation of the SOX9 gene alters
skeletal development and results in deformities of most of
bones of the body. Infants with specific mutations of the
SOX9 gene die from respiratory failure due to poorly formed
tracheal and rib cartilages (66). During the third phase of
endochondral ossification, chondrocytes proliferate rapidly
to form the cartilage model that will eventually be replaced
by bone tissue. As they divide, chondrocytes secrete a carti-
lage-specific extracellular matrix. In the fourth phase, the
chondrocytes stop dividing and become hypertrophic.
Hypertrophic chondrocytes have increased production of
collagen type X and fibronectin, thus altering the remaining
cartilage matrix so that it can be mineralized by calcium car-
bonate. Finally, in the fifth phase, blood vessels begin the
invasion of the cartilage model. The hypertrophic chondro-
cytes undergo apoptosis and the spaces are invaded by
ingrowing blood vessels. As the cartilage cells die, osteopro-
genitor cells differentiate into osteoblasts and begin to lay
down bone matrix on the partially-degraded, mineralized
cartilage remnants (67,68). The site at the center of the carti-
laginous model where ossification first occurs is known as
the primary center of ossification (the eventual diaphysis of
the long bone). Eventually, all the cartilage is replaced by
bone so the cartilage tissue serves as an intervening model
for the bone that will eventually replace it. In experimental
models, the precise sequence of events has been worked out
reasonably well (for a detailed review see 85).
In the long bones of mammals, this process of endochon-
dral ossification spreads along the vertical axis of the devel-
oping bone in both directions from the primary ossification
center (Fig. 14). As the bones grow in length, secondary
centers of ossification form at the ends of each bone. Once
these secondary ossification centers form, there remains an
area of cartilage between the primary and secondary ossifi-
cation centers. The secondary ossification center becomes
the epiphysis and the primary center becomes the diaphysis.
The intervening cartilage is the epiphyseal growth plate and
it is here that continued growth in length occurs at both ends
of the developing bone. The epiphyseal plates contain three
regions: a region of chondrocyte proliferation, a region of
chondrocyte maturation, and a region of hypertrophic
chondrocytes (reviewed in 69). A complex series of events
occur in the growth plate as the resting chondrocytes
Fig. 14 Endochondral Ossification. (A, B) Long bones such as
humerus, femur and tibiae develop through endochondral ossification
where the mesenchymal stem cells condense and differentiate into
chondrocytes to form the cartilaginous model of the bone. (C)
Chondrocytes in the center of the shaft undergo hypertrophy and
apoptosis and their death allows blood vessels to enter that region
(primary ossification center). (D, E) Blood vessels bring in osteo-
blasts, which lay down new bone matrix on the remants of the calci-
fied cartilage (scaffold). (F-H) Secondary ossification centers also
form as blood vessels enter near the ends of the bone. Bone formation
and growth in length continue at the growth (epiphyseal) plates with
ordered arrays of resting, proliferating, hypertrophic (mineralizing)
chondrocytes. Permission granted from Sinauer Associates (Gilbert,
6th edition, 1997).

18 F.F. S afad i et a l.
proliferate, mature and become oriented in columns. As the
cells hypertrophy at the expense of the intervening cartilagi-
nous matrix, the cartilage matrix becomes calcified. It is
these calcified cartilage remnants that then serve as a scaf-
fold for the deposition of bone matrix by osteoblasts. The
spaces left behind by the apopototic hypertrophic chondro-
cytes are invaded by blood vessels, a critical event in the
formation of new bone tissue. Abnormalities in chondrocyte
function can disrupt this sequence and produce abnormally
short and misshapen bones. One illustration of this is achon-
droplasia, a condition causing short stature and thought to
be caused by a mutation in fibroblast growth factor (FGF).
Without the normal cellular processing of the signal from
the fibroblast growth factor, the chondrocytes do not prolif-
erate normally (70). This results in a disorganized and mal-
functioning growth plate.
Molecular Regulation of Growth Plate
(Chondrocytes)
Chondrocytes of the growth plate behave very differently
than chondrocytes of articular cartilage. Chondrocytes within
different parts of the growth plate are markedly different
from each other. Investigators have been studying the factors
that are important in each region. Indian hedgehog, is a fac-
tor that plays a role in the normal differentiation of growth
plate chondrocytes. The study of factors that activate Indian
hedgehog (Ihh) and other associated factors are advancing
our understanding of the molecular biology of the growth
plate and its disorders (69).
There are systemic and local mediators that regulate
growth plate chondrocyte proliferation and differentiation.
Systemic factors include insulin growth factor-1 (IGF-1)
(71), growth hormone (72), thyroid hormone (73), estrogens
(74), vitamin D (75) and glucocorticoids (76). All of these
factors have been reported to have an effect on the linear
growth of bone both prenatally and postnatally. Other factors
act locally to regulate growth plate chondrocytes, include
TGF-B, PTHrP (78), Ihh (79) and FGF-receptor type 3
(FGFR3) (80,81). It has been reported that TGF-B has the
ability to inhibit chondrocyte proliferation, hypertrophic dif-
ferentiation and matrix mineralization (82).
Control of the growth plate is also an area of intense
research. Indian hedgehog (Ihh), a protein of the same family
as sonic hedgehog, regulates the rate of hypertrophic chon-
drocyte differentiation. It is produced by the pre-hypertrophic
chondrocytes and induces the expression of parathyroid hor-
mone related protein (PTHrP) in the perichondrium, which
blocks chondrocyte differentiation. The Ihh/PTHrP axis acts
as a negative feedback loop modulating chondrocyte differ-
entiation. One can imagine that abnormalities in this control
loop can alter growth plate function or lead to inhibition of
chondrocyte proliferation (83).
The overlapping expression of FGFR3 and PTH-
receptor-1 (PTHR1) in the growth plate suggested that these
signaling pathways interact. Genetic inactivation of either
PTHrP or the PTHR1 in mice resulted in a marked decrease in
the size of the proliferative zone of the growth plate, a pheno-
type that is seen with the constitutive activation of the FGFR3
signaling. Other in vivo studies have shown that FGFR3 sig-
naling can repress Ihh and PTHR1 expression in the growth
plate (84). These studies suggest a link between Ihh/PTHrP
signaling and the FGFR3 pathway in the growth plate.
Limb Development
The development of the human limb begins at day 24 of ges-
tation and is orchestrated by the expression of a sequence of
genes expressed in specific regions of the developing limb.
Three zones typify the growing limb bud: a thickened ectoderm
located distally at the periphery, called the apical ectodermal
ridge (AER), proliferating mesoderm just deep to the AER
called the progress zone (PZ); and a zone of polarizing activ-
ity (ZPA) located posterior to the PZ (86).
The AER is responsible for general limb and bone devel-
opment. Grafting the AER from one animal to the PZ of
another, patterns the growing limb after the donor AER. In
contrasts, the ZPA orients the limb in an anterior-posterior
direction. Grafting the ZPA to the anterior border of a devel-
oping limb, results in a duplicated limb in opposite orienta-
tion. The AER maintains continuous limb bud outgrowth
along the proximal-distal (P-D) axis (shoulders to digits).
Concomitant to its elongation along the P-D axis, the limb
becomes flattened along the dorso-ventral (D-V) axis (back
of hand to palm) and is asymmetric along the antero-posterior
(A-P) axis (thumb to little finger). Differentiation of mesen-
chymal stem cells becomes morphologically apparent as
these cells condense to form the primordia of individual
skeletal elements. The most proximal elements (stylopod)
begin to differentiate first, followed by the progressive
differentiation of more distal structures (zeugopod and
autopod) (87, 88).
Limb patterning also plays a role in how bones develop
(89). Genes such as sonic hedgehog, and the homeobox
genes, organize segmentation, anterior-posterior, medial, lateral
and longitudional limb patterning during fetal development.
Abnormalities in patterning genes may lead to extra or
deficient digits, short or long limbs, or congenital amputa-
tions. The growth plate is altered in many of these malforma-
tions, and thus, the patterning genes likely modify factors
involved in chondrocyte function and differentiation in the
growth plate.

1 Bone Structure, Development and Bone Biology 19
Molecular Biology of Limb Formation
As the limb bud grows, the proximal-distal, dorso-ventral
and antero-posterior axes are apparent and development is
mediated by multiple signaling molecules. Members of the
fibroblast growth factor (FGF) family produced by AER
cells are required for P-D outgrowth. FGF signals are respon-
sible for keeping the underlying undifferentiated mesenchy-
mal cells in the progress zone, in an undifferentiated, rapidly
proliferating stage. At least five FGFs (FGF 2, FGF 4, FGF
8-10) and two FGF-receptors (FGFR1 and FGFR2) are
expressed during limb bud initiation. FGF 2, 4, 8, 9 and
FGFR 2 are found in the ectoderm and AER while FGF 10
and FGFR 1 are present in the underlying mesenchyme
(90,91). Sonic hedgehog (Shh) is an important molecule in
the ZPA, and is responsible for duplication of limb structures
as demonstrated in grafting experiments. FGF maintains Shh
expression in the ZPA and FGF 4 is largely responsible for
maintenance of its expression as the limb elongates (92).
Select FGFs in conjunction with Shh regulates expression of
the bone morphogenetic (BMP2 and 7) and Hox genes,
mostly Hoxd-12 and Hoxd-13. These genes are members of
the Hoxd complex and are expressed in the distal wrist (Hoxd
12), within the hand and fingers (Hoxd 12 and 13). These
genes regulate proximal-distal differentiation of limb seg-
ments and mutation of Hoxd-13 in the human transforms
metacarpals to carpals and metatarsals to tarsals. On other
hand, overexpression of Hoxd13 in chick limb bud in vivo
resulted in the transcriptional repression in the proximal part
of the limb of Meis, the vertebrate ortholog of an homeo-box
containing gene in drosophila called homothorax (hth) that
is required for proximal leg development (92).
Wnt proteins regulate many events in limb development,
from patterning to controlling cell proliferation, differentia-
tion and survival. The Wnt family signals through ten differ-
ent transmembrane frizzled receptors. These receptors also
function together with the LDL-related protein receptor
(LRP). Two LRP receptors bind Wnt, LRP 5 and 6.
Wnt signals through three pathways, the B-catenin path-
way, the JNK (planer polarity) pathway and the Wnt/Ca+
pathway (93). The B-catenin pathway is also called the
canonical Wnt signaling pathway and is well characterized
(94,95). Wnt signaling is antagonized by different factors,
such as members of the TGFB and FGF families, frizzled
related proteins (Sfprs), cerberus, dickkopfs (DDKs) and
members of the CCN family of proteins (93). Wnt proteins
are expressed in the AER which controls limb outgrowth
(97) and the dorsal ectoderm which controls dorso-ventral
patterning. Wnts are also expressed in the AER and in lateral
plate mesoderm where their overexpression leads to ectopic
limb formation (96). Mutation in Wnt proteins and their
downstream signaling molecules has been linked to human
limb malformations. Wnt3a and 7a regulate the expression
of Csa1, mutated in human pre-axial polydactyly (98). Sponta-
neous, naturally occurring mutations in Wnt7a has also been
associated with postaxial hemimelia characterized by dupli-
cation of the sesamoid bones and foot pads (93). It is evident
that Wnt and associated signaling pathways play a major role
in limb development and pathogenesis of limb deformities
and our knowledge of the Wnt signaling pathways and their
role in limb development will enhance our understanding of
the genetic basis of limb deformities.
Skeletal Muscle
Type(s) of Muscle
Muscle is characterized as either striated or non-striated. The
term striated originated from the appearance of the ordered
structure of this muscle type under the microscope. As
opposed to this, non-striated muscle does not show the same
regimented pattern of order. Striated muscle includes two
types of muscle: skeletal and cardiac. Smooth muscle is clas-
sified as non-striated. Here we will focus on only one type of
striated muscle: skeletal muscle. In longitudinal section,
these cells appear tubular with multiple nuclei/cell located at
the periphery of cells. In cross-section the cells are polygo-
nal with the nuceli located at the periphery. They are charac-
terized as striated, voluntary muscles with the ability to
undergo strong; quick contractions.
Organization of Skeletal Muscle at the Light
and Electron Microscope Level
A muscle cell or fiber is surrounded by a plasma membrane
referred to as the sarcolemma. In normal muscle, an impor-
tant component of the sarcolemma is dystrophin, where it
forms a complex with the sarcolemmal cytoskeletal net-
work. Dystrophin is absent in patients with muscular dystro-
phy. Muscular dystrophy is a general term used to describe a
group of inherited myogenic disorders. The cytoplasm is
referred to as the sarcoplasm. The sarcoplasm is filled with
filamentous myofibrils which run parallel to the long axis of
the cell. The myofibrils are composed of myofilaments which
are made up of polymers of primarily myosin and actin, pro-
teins responsible for contraction. A view of a myofilament
shows thick filaments made up of myosin and thin filaments
of actin. The filaments interdigitate and have the appearance
of light and dark bands. The light bands are comprised of
actin and are termed the I band (isotropic). The dark bands
are made up of myosin and the overlapping region of actin
and are called A bands (anisotropic). These alternating light

20 F.F. S afad i et a l.
and dark bands give skeletal (and cardiac) muscle their stri-
ated appearance. The actin filaments are attached to Z lines.
The actin filaments extend on either side of the Z line (disc)
to interdigitate with the myosin molecules. The Z disc is
composed of a variety of proteins which anchor the actin fila-
ments to the Z line and which extend from one myofibril to
another in cross-section. Alpha-actinin is one of those pro-
teins involved in anchoring actin to the Z disc. Another more
recently identified giant (300kD) protein ‘titin’ spans the
distance between the Z disc and the M line of the sarcomere.
It is involved in providing elasticity to the sarcomere and a
main contributor to ‘passive tension’ (passive or resting ten-
sion is that which is present in a muscle even before contrac-
tion is initiated and is a result of the elastic forces in the
muscle). Although not discussed here many other proteins
are part of the contractile apparatus including C protein,
Myomesin, and Nebulin. The region between Z lines is called
a sarcomere, a unit repeated throughout the structure of the
myofibril. The A band is bissected by the M line. This is the
region where lateral connections are made between the thick
filaments. The main component of the M line is creatine
kinase, an enzyme that catalyzes the transfer of a phosphate
group from phospho-creatine (storage form of high energy
phosphate) to ADP. This provides the energy in the form of
ATP necessary for muscle contraction.
Characteristics of the Contractile Filaments
The myosin thick filament is made up of many myosin mol-
ecules. The tail region is made up of two polymers of myosin
heavy chain molecules twisted together as a double helix
forming the body of the thick filament. At the end of the tail
is found two pairs (four) of myosin light chain molecules
which form the head region of the myosin polymer. The head
region has an ATPase, an enzyme essential to the production
of energy for contraction. The head region and a portion of
the helix of the myosin molecule (which makes up the arm:
can move away or towards the helix) extends outward to
form cross bridges that make contact with the thin actin fila-
ments as described in the “sliding filament theory”. These
cross bridges that extend outward can bend and have hinges
at two points: one, where the head attaches to the arm and the
second, where the arm attached to the axial body of the myo-
sin molecule. These hinges facilitate movement. Cross
bridges project around the entire thick.
The actin filament is a double stranded helix of F-actin
protein. Polymerized G- actin molecules make up the F-actin
filament. Each G-actin molecule has a myosin head binding
site. The thin actin filament also contains two strands of the
tropomyosin molecule which cover the myosin interactive
sites on actin, preventing interaction of actin and myosin.
Also, along the tropomyosin molecules is found the globular
troponin complex. Troponin I has a strong affinity for
actin, Troponin T for tropomyosin and Troponin C for cal-
cium ions.
Innervation of Muscle
Myelinated motor nerves which originate from spinal cord
neurons give rise to several terminal branches which make
contact with multiple individual muscle cells. At the site of
innervation the nerve terminal sits in a trough (motor end
plate) at the surface of the muscle cell. The space between
the axon and the muscle is the synaptic cleft. An action
potential at the neuromuscular junction (or motor end plate)
causes the release of acetylcholine from the axon which
binds to receptors on the highly folded sarcolemma (junc-
tional folds). In patients with myasthenia gravis, acetylcho-
line receptors on the sarcolemma are blocked by antibodies
generated as an autoimmune response. The muscle cell can-
not respond to the nerve stimulus resulting in muscle weak-
ness. After acetylcholine binds to its receptor, the sarcolemma
becomes permeable to sodium ions resulting in membrane
depolarization. Depolarization is propagated along the mus-
cle cell into the cell via the transverse tubule system. This is
a network of tubules arising as invaginations of the sarco-
lemma and surrounds the A-I interface of each sarcomere in
each myofibril. These tubules associate with two terminal
cisternae (expanded regions of the SR) of the sarcoplasmic
reticulum and together are known as triads. At the triad the
depolarization signal is transmitted to the SR resulting in
the release of stored calcium ions and initiating contraction.
Calcium is released near the thick and thin filaments.
When depolarization ends; calcium reenters the SR and
contraction ceases.
Mechanism of Contraction
The actin filament is inhibited from interacting with the
myosin cross bridges by the troponin-tropomyosin complex.
Before contraction can take place this inhibitory interaction
must be removed. It is the introduction of large amounts of
calcium ions that changes this inhibitory effect. Calcium ions
released from the sarcoplasmic reticulum combine with tro-
ponin C which changes the configuration of the troponin
complex and moves the tropomyosin molecules further into
the grooves between the actin filaments. This makes the
active sites on the actin filament available for interaction
with the cross bridges of myosin and is the binding step in
the contraction process. Once the cross bridges are attracted

1 Bone Structure, Development and Bone Biology 21
to the active site on actin the process of contraction begins.
The hypothesized mechanism is referred to as the “walk-
along” theory of contraction.
Remember that the head of the myosin molecule binds
ATP. ATPase activity in the head cleaves the ATP but the
ADP remains attached. In this conformation the head is
extended towards the actin filament. When the inhibitory
effect of the tropomyosin-troponin complex is lifted, the
myosin head binds to the actin molecule. This causes a con-
formational change in the myosin head causing it to tilt
towards the arm and this process causes a power stroke. The
energy for this is already stored in the head from the cleavage
of ATP. ADP and Pi is released allowing for a new ATP to
bind to its site on the myosin head. This binding causes the
myosin to detach from the actin and return to its original
perpendicular position, the new ATP is cleaved and the myo-
sin forms a new bond further down the actin filament to again
perform a power stroke and consequently move another step.
These steps occur again and again as the actin filament pulls
the Z disc towards the ends of the myosin filament. Remember
that contraction is not a result of shortening of individual
filaments but an increase in overlap between the actin and
myosin filaments. Therefore, as sarcomere length decreases,
tension in the muscle and strength of contraction increase.
The Neuromuscular Spindle
The sensors that keep the central nervous system informed of
the state of contraction and position of voluntary muscle are
referred to as neuromuscular spindles (NMS). This structure
consists of modified muscle fibers encased in a fluid filled
sheath of connective tissue. The fibers comprising the neuro-
muscular spindle are referred to as intrafusal fibers. The
fibers we have discussed earlier (making up the bulk of skel-
etal muscle) are extrafusal fibers. Intrafusal fibers come in
two varieties: nuclear bag fibers (nuclei accumulated in the
midregion) and nuclear chain fibers (nuclei arranged in a
central chain). Nuclear bag fibers extend beyond the confines
of the connective tissue sheath and are attached to the extra-
fusal fibers (i.e. skeletal muscle proper)
Innervation is a very complex subject. Anterior horn cells
may be either large alpha motor neurons that give rise to
motor nerve fibers that innervate extrafusal fibers or even
smaller gamma motor neurons that give rise to fibers that
innervate the small intrafusal fibers. Afferent sensory fibers
transmit information from the NMS to cell bodies in the sen-
sory posterior horn of the spinal cord. Therefore, these
encapsulated proprioceptors are a mechanism by which the
CNS remains informed as to the state of your muscles allow-
ing the CNS to better coordinate the function of voluntary
muscle.
Repair of Skeletal Muscle
Satellite cells are stem cells found between the plasma mem-
brane and the basal lamina of muscle fibers. These cells can
differentiate to form myotubes. If damage includes disrup-
tion of the basal lamina satellite cells are not responsible for
the repair that results. Instead fibroblast repair results in scar
tissue.
The Origins of Skeletal Muscle
The development and maturation of skeletal muscle has been
studied since the early 1900’s and much is know compared to
either cardiac or smooth muscle. It is fashioned during
embryogenesis from the mesoderm which is generated after
the process of gastrulation. On either side of the neural tube,
that lies dorsal to the notochord, extends a band of mesoderm
called the paraxial mesoderm (Fig. 15). This band of meso-
derm separates into blocks referred to as somites. This sepa-
ration and the subsequent development of somites proceeds
in a rostral to caudal direction, with the rostral somites bud-
ding off and developing before those located caudal. The
number of somites is often used as an indicator of the stage
of embryonic development, i.e. how far development has
progressed. For example, a chicken embryo is defined as
being at stage 11 (40–45 hours of incubation after laying) by
the presence of thirteen pairs of somites (99,100).
Somites are transient structures that supply cells which
populate the vertebrae and ribs, the dermis of the dorsal skin,
the skeletal muscles of the back, body wall and limbs
(101,102). They form as mesenchymal structures but convert
to an epithelial block with tall columnar cells arranged
Fig. 15 Diagram of cross-sectional area of a vertebrate embryo that
shows organization of mesoderm tissue. Somites which comprise the
paraxial mesoderm generate skeletal muscle, the focus of this section in
the chapter. Drawing by James O.H. Montgomery

22 F.F. Sa fadi et a l.
around a central cavity, a process referred to as epithelializa-
tion. This process is accompanied by changes in cell-cell and
cell-matrix interactions and the signals regulating the seg-
mentation and epithelialization of somites has been studied
extensively (103–105). Although all somites look very much
alike they form different structures along the anterior-posterior
axis and this specification is dictated by the expression of
Hox genes (106,107).
Somites contribute cells to different structures and the
commitment of cells to their ultimate fate takes place during
the early stages of somite development. As the somite
matures its various regions become destined (committed) to
form certain cell types. Cells in the ventral/medial region,
those which are furthest from the back and closest to the neu-
ral tube change their shape, become mesenchymal and
migrate away to form the sclerotome. These cells ultimately
form the chondrocytes of the vertebrae and the ribs. The
remaining epithelial somite becomes organized into three
regions. The region closest to, and furthest from, the neural
tube (epaxial and hypaxial myotome, respectively) is the
myotome. The cells in the myotome proliferate and delami-
nate to produce a lower layer of myoblasts, precursor cells
committed to the skeletal muscle lineage. This double lay-
ered structure is referred to as the dermamyotome. Myoblasts
formed from the region of the dermamyotome closest to the
neural tubed will form the muscles of the back (epaxial mus-
cles), whereas the myoblasts in the region farthest from the
neural tube will form the muscles of the body wall, limbs and
tongue (hypaxial muscles). The dermatome located in the
central region of the dermamyotome will form the dermis of
the skin of the back (Fig. 16). Signals that direct the forma-
tion of the sclerotome, dermatome and myotome are well
understood. Briefly, formation of the sclerotome from the
ventral-medial cells of the somite is directed by Sonic
Hedgehog secreted from the notochord and the floor plate of
the neural tube. The sclerotomal cells which become chon-
drocytes of the vertebrae and ribs themselves express Pax 1,
a transcription factor necessary for the formation of cartilage
(108,111). The dermatome forms under the direction of two
factors secreted by the neural tube: Neurotropin 3 and Wnt 1
(112,113). The epaxial portion of the myotome is induced by
factors from the neural tube (Wnt 1 & 3a and Sonic hedge-
hog) whereas the hypaxial portion is specified by proteins
from the epidermis (Wnt) and from the lateral plate meso-
derm (bone morphogenetic protein 4). It should be noted that
in this brief discussion of muscle, the inhibitory or negative
signals are not discussed. Nonetheless their role is vital to
normal somite development (114,116). After formation of
the double layered dermamyotme, a subset of cells located in
the central region of the dermamyotome, identified as being
Pax 3 and 7 positive, were found to proliferate and give rise
to skeletal muscle cells upon activation of muscle specific
transcription factors. Later in development, these cells make
a major contribution to the embryonic and fetal muscle mass.
These cells also give rise to dermal cells. In addition, the
dermamyotome gives rise to endothelial and smooth muscle
cells of the dorsal aorta.
Techniques used to Study Skeletal Muscle
Development
The development and maturation of skeletal muscle is most
often studied in vertebrate animals: amphibians, chicken,
quail and mice, but the avian model system is most com-
monly used. This is attributed to the ease with which avian
embryos can be manipulated and dissected, cultured either in
vitro or in ovo during embryonic development observed
under a dissecting scope or sectioned and then analyzed.
Chicken-quail chimeras have been used to trace the origin of
muscles from specific somites. A natural marker that differ-
entiates quail nucleoli from chicken is used to identify tissues
Fig. 16 Maturation of the somite in vertebrate embryos. (a) In the
3 day old chicken embryo cells detach from the ventral-lateral somite
and begin to migrate away to form the sclerotome. (b) In the late 4 day
old chicken embryo a layer of myoblasts (muscle precursor cells)
are formed beneath the overlying dermatome. Drawing by James
O.H. Montgomery. Adapted after Developmental Biology, 7th, edition,
Scott F. Gilbert

1 Bone Structure, Development and Bone Biology 23
of chicken versus quail origin. Consequently, when quail
somites are transplanted into recipient chicken embryos and
the embryos studied after a period of incubation, the muscles
derived from the quail somite can be easily identified and
studied. This same model has been used to study Notch sig-
naling and somite formation (101,117). Genetic manipula-
tions to determine the function of a protein by knock-out or
transgenic methods are most often performed in mice
(118,119).
Development of Skeletal Muscle
Signals that induce the formation of muscle differ from the
epaxial and hypaxial regions of the dermamyotome. In the
hypaxial portion, factors from surrounding tissues induce
expression of the transcription factor Pax 3, which in turn
induces the expression of MyoD. However, in the epaxial
portion MyoD is induced by the Myf5 protein. Both MyoD
and Myf5 are basic helix-loop-helix (bHLH) transcription
factors that belong to the myogenic bHLH family of myo-
genic regulatory factors (MRFs). These transcription factors
bind to similar sites (-CANNTG-) to turn on transcription of
muscle-specific genes.
Cells in the epaxial and hypaxial regions of the dermamy-
otome that produce MyoD and Myf5 are myoblasts, cells
committed to the myogenic lineage. How these cells progress
from stellate-appearing single cells to tube-like myofibers in
mature skeletal muscle has been studied in culture as well as
in chimeric mice. Myoblasts proliferate when cultured in
media containing serum. As serum is withdrawn the cells
withdraw from the cell cycle and fuse to ultimately form
multinucleated myofibers or mature skeletal muscle cells.
Once growth factors, specifically fibroblast growth factor,
are withdrawn from the culture media (by lowering the serum
concentration) the myoblasts begin to produce fibronectin
and interact with this extracellular matrix protein through the
integrin receptor A5B1.The myoblasts then line up, a process
mediated by plasma membrane proteins such as CAMs and
cadherins and the cells fuse. Fusion of myoblasts to form
myotubes also referred to as myofibers is calcium mediated
process and appears to require metalloproteinases, enzymes
involved in remodeling of the extracellular matrix. Just prior
to fusion the cells expresses another MRF, myogenin.
Myogenin mediates differentiation of these cells. Differen-
tiation is the process whereby cells become specialized; in
this case they express muscle-specific contractile proteins
and acquire the ability to contract. In vivo, the force of con-
traction of skeletal muscle is transmitted via tendons to
bones, allowing these structures to move. During adult muscle
regeneration, satellite cells (adult skeletal muscle stem cells)
that are present under the basal lamina become activated,
proliferate to generate additional satellite cells and differentiate
into muscle fibers. As during embryonic development, both
Pax and MRFs are involved in this process (109,111,116).
Part 3 Bone Biology
The structure and function of bone can best be understood
with an insight into the composition of the bone matrix, its
mineral, its cells, the mechanism of turnover and remodeling
and the unique responses that the mechanical environment
evokes in bone. These studies are forming the basis of a
rapidly evolving field of bone biology.
Collagen
Classification of Collagens
Connective tissues contain varying amounts of collagen,
elastin (a related fibrous protein), glycosaminoglycans and
proteoglycans. Of these, collagen is the most abundant. The
details of collagen synthesis and function have been exten-
sively reviewed in several specialized texts, and only some of
the relevant aspects will be discussed.
Collagens are a class of proteins with common features
such as a unique triple helix composed of three component
polypeptide alpha chains. However, there are several sub-
types (types I to XIII) . Each of these are a product of a
different gene and differ from each other in their biochemi-
cal structure. Several different types of fibrillar, basement
membrane-associated, fiber-associated, and short chain
collagens are recognized. Type I collagen is the most abun-
dant type of collagen in most connective tissues.
Type I Collagen, The Primary Component
of Bone
Type I collagen is a fibrillar type collagen and is found in
bone, skin, meniscus, tendon, ligaments, annulus fibrosis and
joint capsules. About 90% of bone matrix is composed of
type I collagen. There are several subtypes of type I collagens.
The bone type I collagen appears to have predominantly
galactosyl-hydroxylysine as opposed to glucosyl-galactosyl-
hydroxylysine, the predominant amino-acid configuration
found in the skin. Hydroxylation and glycosylation are post-
translational modifications of collagen and are specific to
bone. These modifications, partially explain why mineraliza-
tion only occurs in bone and not in other sites (120).
The basic structure of type I collagen is composed of a
repeating tripeptide sequence that form a left handed helix.

24 F. F. Saf adi e t al.
Type I collagen is a hetero-trimer of two pro-AI and one pro-
A2 chains. The helix (the A chain) is highly coiled. The A chain
corresponds to the basic chemical structure of Gly-X-Y where
X and Y represent various amino acids; in practice however, X
and Y are rich in proline and hydroxyproline and to a lesser
extent, lysine and hydroxylysine. The helix is supertwisted,
which provides enormous strength. Collagen fibers can support
10,000 times their own weight and are said to have greater
tensile strength than steel wire of equivalent cross section.
Other Collagen Types
Types II, III, V and XI are also fibrillar type collagens. Type II
collagen is located mainly in articular cartilage, fibrocartilage,
the vitreous humor of the eye and the nucleus pulposus of the
intervertebral disk. The other fibrillar collagens are the “minor”
collagens. Type III is present in large blood vessels (30%), and
in other tissues in association with Type I. Type III collagen is
also present in tissues undergoing repair. Type V collagen is
present in large blood vessels (5%), cornea, bone, and a few
other connective tisues, while Type XI is present only in artic-
ular cartilage, comprising 5-20% of articular cartilage.
Basement membrane-associated and fiber-associated type
collagens include Types IV, VII, IX and XII. Type IV collagen
is the prototype and major component of the basement
membrane (95%). Type VII forms the anchoring filament in
epithelial basement membranes, while Type IX comprises
5-20% of cartilage. Small amounts of Type XII collagen are
associated with Type I. The least understood class of collagens
is the short chain collagens and are comprised of the Types VI,
VIII and X. Short chain collagens may function in association
with other collagens and have a role in cartilage physiology.
Collagen Synthesis and Cross-linking
Collagen synthesis is under the control of over 20 genes
(120). The biosynthesis of collagen, its secretion and aggre-
gation is a complicated process, and has been the subject of
several reviews (121,122). Aspects directly applicable to
musculoskeletal pathology will be mentioned in other chap-
ters of this book.
The promoters for synthesis of many of the collagen
chains have been identified. Many growth factors and hormones
also exert their effect on collagen synthesis at the transcrip-
tional level. Collagen mRNAs usually contain a large number
of introns. Once the precursor mRNA is transcribed, the introns
are removed and the mRNA is transported from the nucleus
to the cytoplasm for translation. At this point, additional
translational control can be exercised. The N and C propep-
tidases (see later) are thought to act at this level. Like many
other proteins, a precursor form (procollagen) is first synthe-
sized, with peptide extensions at each end. It is at this point
that the A chain of collagen is formed and is transfered into
the endoplasmic reticulum. At this stage of synthesis, several
amino acids are modified posttranslationally (such as hydrox-
ylation of proline residues and lysine residues, forming
hydroxyproline and hydroxylysine, respectively), the addition
of sugars (such as glucose and galactose to the hydroxy-
lysines), and the formation of hydroxylysine and lysine alde-
hydes. Co-factors for these processes include atmospheric
oxygen, ascorbic acid, ferrous ion and several required
enzymes. Once the translation is complete, the triple helix
forms, from the C terminal end, and intra- and inter- molecu-
lar disulfide bonds are formed. The completed procollagen is
then secreted via vesicles into the extracellular space.
Glycosylation may be important to facilitate this final step.
As stated earlier, the fundamental units of collagen fibrils
are three polypetide chains arranged in a helical fashion. The
polypeptide chains aggregate in units of threes to form tropo-
collagen. The tropocollagen, in turn, aggregates in a staggered
fashion in a collagen microfibril. Collagen fibers are made up
of several of these microfibrils. The process of collagen fibril
formation is not fully understood. Removal of the N and C
propeptides may be important. After their removal, the mol-
ecules aggregate in a head-to-tail fashion with a characteristic
stagger, resulting in a 64 nm banding pattern seen under the
electron microscope. The ultrastructurally dark area between
two tropocollagen molecules is termed a “hole”. It measures
about 41 nm and is the site where mineralization is thought
to first occur (121).
The collagenous scaffolding is stabilized by cross linking
and perhaps by interaction with proteoglycans. The process
of cross-linking is important for stabilization and structural
integrity. The first step is the enzymatic production of aldehydes
by the removal of terminal amino groups of lysyl or hydrox-
ylysyl groups of tropocollagen (123). These then can either
condense with a lysyl or hydroxyl group to form a cross link
and produce a Schiff base, or condense with a similar aldehyde
in an aldol reaction (a stronger bond). The amino-oxidase
enzyme that catalyzes the aldehyde formation is susceptible
to blockage by nitriles. Nitriles are alkyl cyanide substances
involved in the disorder called Lathyrism. Lathyrism is
characterized by spinal deformities, demineralized bone,
dislocations, aortic aneurysm, and various nervous system
manifestations.
Cross-linking of collagen occurs in the extra-cellular
space. Collagen molecules are cleaved in this space at both
the N- and the C- terminal ends by specific peptidases. Cross-
linking then occurs and the collagens are packed into a
one-quarter stagger array. Specific interactions also occur
among collagen and other extracellular macromolecules
such as fibronectin, osteonectin and the proteoglycans.

1 Bone Structure, Development and Bone Biology 25
Extensive “cross-linking” between A component chains
results in a rigid, brittle character to the connective tissue.
This type of cross-linking is found in aging individuals.
Defects in the process of forming cross links can render the
collagen susceptible to collagenases (discussed more below).
Penicillamine prevents collagen cross-linking; and is admin-
istered to patients with scleroderma, a disorder of excessive
collagen deposition. Genetic defects in collagen can also
result in several lethal and non-lethal conditions. Examples
include Ehlers-Danlos syndrome (loose joints, characterized
by a Gly to Serine change) or Osteogenesis imperfecta
(brittle bones, characterized by a Gly to Cystine change).
Osteogenesis imperfecta (see section on metabolic diseases)
is a heritable disorder of Type I collagen. It is due to a variety
of point mutations in either the pro-A1 or pro-A2 collagen
chains. Over 100 point mutations have been found in
probands with osteogenesis imperfecta. In a few cases, the
mutations cause a decrease in the synthesis of pro-A1 and
pro-A2 chains. In the majority of cases, however, there is a
production of structurally abnormal collagen chains (124). A
mouse model has also been developed which demonstrates
the relationship between abnormal collagen genes and osteo-
porosis in heterozygous animals. In homozygous animals,
osteogenesis imperfecta develops (125,126).
Collagenases are enzymes that catalyze the hydrolysis of
collagen. Several collagenases have been isolated, purified
and synthesized. Collagenase levels are increased in rheuma-
toid arthritis nodules and in synovial fluid from patients with
rheumatoid and septic arthritis. Colchicine and heparin
increase collagenase synthesis. Collagenases and several
other enzymes, such as cathepsin B, are capable of degrading
collagen and have been implicated in the pathogenesis of
collagen-vascular diseases.
Urinary excretion of hydroxyproline (found exclusively
in collagen) and other products of collagen degradation
(cross-linked products such as pyridinoline and deoxypyridi-
noline), act as markers of collagen breakdown. The level of
collagen degradation byproducts in urine or serum reflect the
amounts of bone turnover (see section on the laboratory in
orthopaedic practice).
Non Collagenous Matrix Proteins
Calcium Binding Proteins
(The Glyco- and Phosphoproteins)
This is not an easily classified group, since some glycopro-
teins are also phosphorylated. In the former class are three
“sialoproteins” (bone sialoprotein or BSP), including BSP I
or osteopontin, BSP II, and bone acidic glycoprotein-75 or
BAG-75. There is also a dentin sialoprotein which is found
in the jaw. These compounds play a role in the control of
extracellular calcium, regulation of crystal growth and shape,
and cell adhesion to bone surfaces. Another important phos-
phorylated glycoprotein is osteonectin.
Osteopontin
The amino acid sequence has been determined, and its gene
localized to chromosome 4. Osteopontin is a sialated and
highly phosphorylated phosphoprotein, which exists in mul-
tiple forms, due to both alternate splicing as well as post-
translational variations in the degree of phosphorylation.
This protein is transcriptionally regulated by substances such
as 1,25 dihydroxyvitamin D, TGF-B, dexamethasone and
parathyroid hormone, at least in experimental models.
Osteopontin contains a GRGDS cell attachment sequence
similar to binding proteins such as fibronectin (see below).
Osteopontin in particular is important in that it has been
shown to bind to the integrin receptor on osteoclasts. This
binding leads to the activation of the phospholipase C path-
way in osteoclasts, and a resultant increase in intracellular
calcium. This process may involve the src tyrosine kinase.
Immunolocalization of osteopontin reveals high amounts
in the extracellular matrix of developing intramembranous
and endochondral bones. Its localization within cells reveals
a broad pattern, including osteoblasts, osteocytes, osteo-
clasts, precursor cells, chondrocytes and fibroblasts. In situ
hybridization studies have also shown the presence of mRNA
in mononuclear marrow cells, proximal convoluted tubules
of the kidney, neuronal cells within the brain and inner ear as
well as (murine) placenta (127,128).
Bone Sialoprotein-II (BSP II)
The gene for this sialoprotein has been localized to chromo-
some 4. Northern blot studies have suggested that BSP II is
fairly bone specific. Fetal bone studies have indicated that
the initial translation product may differ significantly from
the mature form. Like BSP I, this protein has cell attachment
properties due to its RGD sequence. However, osteopontin is
more active than BSP II in this regard, and maintains cell
attachment for more prolonged periods (129).
Bone Acidic Glycoprotein (BAG -75)
This protein binds to the small bone proteoglycans. There is
cross-reaction of antibodies with osteopontin in some species;
however there are significant differences between this protein
and BSP I at the N-terminal end. Complete characterization
of this protein is not clearly understood (130,131).

26 F. F. S af ad i et a l.
Phosphoproteins (Example: Osteonectin,
SPARC or BM-40)
These proteins have a role in regulating the extracellular cal-
cium hydroxyapatite formation and mineralization. Examples
include phosphorylated glycoproteins like osteonectin. This
protein also called secreted protein, acidic, rich in cysteine
(SPARC), culture shock protein or basement membrane-40
(BM-40). Osteonectin binds to Ca2+, collagen type I, hydroxy-
apatite and thrombospondin. It promotes and initiates crystal
growth. The gene for osteonectin has been localized to chro-
mosome 5. Several tissues express osteonectin, however, its
concentration is extremely high in bone (up to 10,000 times
that of other connective tissues). In fact, in bone it may be the
most abundant non-collagenous protein. The concentration
of osteonectin in bone increases with maturity. Other tissues /cells
having osteonectin include skin fibroblasts, tendon cells (but
not tendon matrix) and odontoblasts. Interestingly, when
osteonectin was activated by the use of blocking antibodies
during tadpole development, there was a disruption of somite
formation and malformation in the head and trunk (132).
Mice lacking osteonectin develop severe cataracts (133) and
low turnover osteopenia. In vitro studies of osteonectin-null
osteoblastic cells showed that osteonectin supports osteo-
blast formation, maturation and survival (134). Osteonectin
also plays role in cell attachment, migration, proliferation
and differentiation.
Mineralization Proteins: Gamma-
carboxyglutamic acid proteins
(“Gla” proteins)
Osteocalcin (also called bone Gla protein) is an example of
this group. Osteocalcin contains three G-carboxyglutamic acid
(Gla) residues. It comprises about 20% of the non collagenous
proteins in human bone. There is also a matrix Gla protein
found in bone, cartilage, lung, heart and kidney (135).
Osteocalcin is made by osteoblasts and odontoblasts in
response to 1,25 dihydroxyvitamin D3. It is secreted into the
osteoid after the initiation of mineralization. The bone localiza-
tion of osteocalcin has been confirmed by several different
methods including Northern blotting, immunohistochemistry
and electron microscopy. It therefore serves as marker for
mineralized tissue. In fact, both osteocalcin and alkaline
phosphatase are valuable markers in the repertoire of the
surgical and clinical pathologist. Osteocalcin serves as a
marker of increased bone turnover, in particular of enhanced
osteoblastic activity. Serum osteocalcin levels do not always
correspond well with the levels of serum alkaline phos-
phatase, suggesting that these two markers may be synthe-
sized by osteoblasts at different stages of development. These
substances can be used for following the progress of patients
with osteosarcoma and may be can be used as a marker for
recurrences or metastases in this situation (135).
The role of osteocalcin in the body is unclear, but it may
function in regulating mineralization and remodeling. It may
also act as a chemoattractant for osteoclast progenitors (also
see section on mineralization). Its secretion is under the con-
trol of many factors including Vitamin D, TGF-B, PTH and
others. Serum levels reflect bone turnover. Osteocalcin has
an affinity for Ca2+ that is dependent on the presence of Gla
residues and an intact disulfide bond. It therefore may have a
role in the regulation of crystal growth and recruitment of
osteoclasts. Developmentally, low levels are found in the
early stages of bone development while maximal levels are
reached at maturity. The entire primary structure of osteocal-
cin has been determined (amino acid sequencing, cDNA
clone sequencing, etc.) and the gene is localized to chromo-
some 1 in humans. The promoter region has a TATA box and
a CCAAT sequence. There is a NF1 site, and a binding site
for two other nuclear factors AP1 and AP2. There is a cAMP
responsive region as well as a 1,25-dihydroxyvitamin D3
enhancer element. Genetic studies showed that osteocalcin
acts as an inhibitor of osteoblast function. Osteocalcin knock-
out mice were reported to have increased bone mineral den-
sity compared to normal controls, but the changes in mineral
properties that occur with age were not observed in osteocal-
cin deficient mice compared to age-matched normal control
mice (136,137). Collectively, the published literature pro-
vides evidence that osteocalcin is required to stimulate bone
mineral maturation.
Adhesion Proteins (Osteopontin, Fibronectin,
Sialoproteins and Thrombospondin)
These proteins contain an arginine-glycine-aspartic acid
(RGD) amino acid sequence in their composition. This
sequence mediates the attachment to certain integral mem-
brane proteins or integrins, which are located on cell
surfaces.
Osteopontin: Discussed earlier (see section
on Calcium binding proteins)
Fibronectin (FN)
FN is a multifunctional glycoprotein present in the extracel-
lular matrix as an insoluble component or in circulating
plasma as a soluble protein. FN mediates the adhesion,
migration, differentiation, and proliferation of cells and has
been implicated in wound healing and embryonic development

1 Bone Structure, Development and Bone Biology 27
(138). FN is one of the most prevalent and versatile of the
extracellular matrix proteins. Disruption of the FN gene in
mice results in an embryonic lethality, confirming the impor-
tance of FN in embryonic development (139,140). The mol-
ecule is a dimer, its subunits being held together by two
disulfide bonds. The subunits contain binding domains for
fibrin, heparin, bacteria, gelatin, collagen, other extacellular
matrix proteins, DNA and cell surfaces. The primary
sequence of fibronectin has been determined, and the gene
localized to chromosome 7. Fibronectin is characterized by
several repeat sequences, for fibrin, collagen and integrin
receptor binding. The latter is composed of the Gly-Arg-
Gly-Asp-Ser cell attachment consensus sequence known as
the GRGDS sequence.
There is heterogeneity associated with fibronectin mRNA,
both dependent on origin (plasma versus tissue) and on stage
of development (fetal versus adult). This results from alter-
native splicing of the primary transcript. This may allow the
cell to utilize the form more suited to its needs. FNs exhibit
molecular heterogeneity arising from alternative splicing of
the primary transcript at three distinct regions termed EDA,
EDB, and IIICS (141–144). Alternative splicing at the EDA
and EDB regions is regulated in a tissue specific and devel-
opmental stage-dependent manner. Despite accumulating
evidence for the regulated expression of EDA- and/or EDB-
containing FNs in vivo, the biological functions of these iso-
forms are poorly understood. Recent studies have shown that
the EDA segment regulates the binding affinity of FNs for
integrin A5B1 and thereby stimulates integrin-mediated sig-
nal transduction and subsequent cell cycle progression.
Unlike the EDA segment, the EDB segment does not enhance
FN binding to integrin A5B1 (145,146).
Fibronectin is synthesized during bone development.
During embryonic development, fibronectin is present at
high levels during mesenchymal condensations and plays a
crucial role in the overt differentiation of these cells into
chondrocytes (65). It is also present around osteoblasts dur-
ing osteogenesis. Osteoblasts can utilize fibronectin as a cell
attachment protein. The synthesis of fibronectin from osteo-
blasts is probably under the control of TGF-B.
Mice deficient for the EDB domain of FN were appar-
ently normal and fertile, although the fibroblasts obtained
from the homozygous mice exhibited reduced potential for
cell growth and FN matrix assembly in vitro (147). Skeletal
characterization of EDB null mice revealed no changes in
any cartilage elements of skeletal development when com-
pared to the wild type mice.
Thrombospondin (TSP)
This is a 450 kilo Dalton trimeric glycoprotein. It is com-
posed of identical subunits that are disulfide-bonded to each
other. It is the predominant protein of the A granules of plate-
lets, but is synthesized in several connective tissues. Like
fibronectin, there are “domains” for binding to a host of con-
nective tissues and serum proteins. The molecule also binds
Ca2+ to hydroxyapatite and to osteonectin. Thrombospondin
and osteonectin co-localize in the A granules of platelets,
where they bind to one another.
The structure of thrombospondin reveals a homology to
fibrinogen with binding sites to collagen, thrombin, fibrino-
gen, laminin, plasminogen activator and plasminogen. There
are areas with homology to A (1) chains of types I and III
collagen, von Willebrand factor and epidermal growth factor.
There is a region for activating platelet aggregation, as well
as sequences with homology to calmodulin and paralbumin.
In addition there is an RGD sequence in the middle of a Ca2+
binding region. Thrombospondin is distributed in a variety of
tissues, including the dermo-epidermal junction of skin, in
small blood vessels, surrounding skeletal muscle and beneath
glandular epithelium.
Temporally, there is an orderly increase in amounts dur-
ing organogenesis, followed by a reduction as differentiation
proceeds. There is evidence to suggest that TGF-B may be
involved in the modulation of thrombospondin biosynthesis.
The proposed functions of this molecule include mediation
of platelet aggregation, organization of the extracellular
matrix (by its multiple binding sites) and action as an auto-
crine growth factor (148).
TSP is expressed by bone cells such as osteoblasts as well
as chondrocytes and this protein is usually deposited into the
matrix and regulates other extracellular matrix proteins. There
are different types of TSP including TSP1, 2, 3 and 4, some of
which have common physiological roles while others do not.
Genetically targeted mouse models have been used to define
the physiological role of TSPs in bone and other tissues (149).
Mice lacking TSP1 exhibit curvature of the spine and minor
abnormalities in trabecular bone (150). TSP2-null mice dis-
play increased endocortical, but not periosteal, bone formation
rates, compared to wild-type, normal mice, as a result of a
larger pool of marrow osteoprogenitor cells (151). From the
above information it is evident that the role of TSPs in bone is
varied and is largely context-dependent.
Other Proteins, Cytokines and Growth Factors
Osteoblast cell culture studies have revealed the presence of
several bio-products. Plasminogen activator and its inhibitor
have been identified. Collagenase and tissue inhibitor of met-
alloproteinase (TIMP) have been isolated from such experi-
ments. The extrapolation of these results across species and
to in vivo situations should be treated with caution. Several
plasma products including albumin and A 2 HS-glycoprotein

28 F.F. S af ad i e t al.
can bind to bone. There is evidence in the literature suggest-
ing a role for plasminogen activators in bone remodeling.
Plasminogen activators tPA and uPA are involved in tissue
remodeling and bone metabolism. Mice lacking tPA and uPA
show increased bone formation and bone mass associated
with increase osteoblast function and delay in extracellular
matrix degradation (152).
Connective Tissue Growth Factor
Connective Tissue Growth Factor (CTGF) is a cysteine-rich
protein first discovered by Bradham and colleagues (153)
while screening a human umbilical vein endothelial cell
cDNA expression library using a polyclonal anti-PDGF anti-
body. At about the same time, two independent groups iso-
lated mouse CTGF (Fisp 12/BIG-M2) from serum-stimulated
NIH-3T3 cells and TGF-B -stimulated mouse AKR-2B cells
using differential cloning techniques (154,155). Since that
time CTGF has been isolated, cloned and sequenced in other
species including the cow, pig (156), frog (157), and most
recently in the rat (158). The CTGF gene belongs to a larger
CCN gene family that also includes Cyr61/CEF10 and nov.
Cyr61 and CEF10 were isolated by differential cloning tech-
niques from mouse and avian fibroblasts, respectively, and
nov was identified from myeloblastosis-associated virus-
induced avian nephroblastomas (159). More recent additions
to this protein family include ELM-1 (WISP-1) (160,161),
WISP-3 (161) and COP-1 (WISP-2) (161), bringing the total
to six distinct members. With the exception of nov, CTGF
family members are immediate early growth-responsive
genes that regulate the proliferation and differentiation of
various connective tissue cell types (159,162). Most of the
functional information on CCN proteins has emerged within
the last 5 years, including the identification of receptors (i.e.
integrins) and the elucidation of potential mechanisms of
action, the field is poised for major advances in understand-
ing the activities and functions of these proteins. For reviews
of CTGF and the CCN family see 163–166.
All members of the CTGF gene family share 30-50%
amino acid sequence identity overall, possess a secretory
signal peptide at the N terminus, and contain 38 cysteine
residues that are largely conserved (165,166). The CCN
proteins are organized into four discrete and conserved
structural domains, each encoded by a separate exon.
Domain I shares significant sequence homology with the
N-terminal region of the insulin-like growth factor binding
proteins (IGFBPs) (167), although only low levels of IGF
binding activity have been demonstrated for CTGF (168).
Since the affinity of CTGF for IGF is much lower than that
of the IGFBPs, the physiological significance of this bind-
ing is unclear. Domain II includes a von Willebrand factor
type C repeat followed by a variable region that is highly
charged and devoid of cysteine residues. This variable region
may serve as a hinge connecting the N- and C-terminal
halves of the protein. The central hinge region located
between domains II and III of CTGF and other CCN family
members is highly susceptible to enzymatic cleavage with
additional sites of proteolysis between other domains (169).
Domain III contains a region that is homologous to the
thrombospondin type I repeat and may be involved in bind-
ing to the extracellular matrix via sulfated glycoconjugates
(170). Domain IV is the C-terminal (CT) module resem-
bling the CT domains of several other extracellular proteins
believed to mediate protein-protein interaction or dimeriza-
tion (171). Within this domain are six cysteines forming a
motif called a cysteine knot. Cysteine knots are also found
in other growth factors (TGF-B, PDGF and NGF) and are
involved in their dimerization.
CTGF Effects on Cellular Functions and Role
in Biological and Pathological Processes
In general, CTGF (as with most other members of the CCN
family) is a secreted, extracellular matrix-associated protein
that regulates a diversity of cellular functions including
adhesion, proliferation, migration, differentiation, matrix
production, and survival. In vivo, CTGF mRNA is expressed
in many tissues with highest levels in the kidney and brain
(155,165). In bone, CTGF has been reported to be expressed
in normal rat bone and overexpressed in osteopetrotic bone
(158). To date, CTGF mRNA expression and protein pro-
duction has been demonstrated in endothelial cells, fibro-
blasts (169,172) and chondrocytes (173). It is believed that
CTGF acts as an autocrine or paracrine regulator of various
cellular processes with its specific effects being target cell-
dependent (169). Although CTGF is mitogenic for various
cell types, it also promotes the differentiation of fibroblasts
and chondrocytes in culture (173–175). CTGF has been
shown to up-regulate the expression and production of
extracellular matrix (ECM) proteins, such as type I collagen
and fibronectin in fibroblasts (174–175) and osteoblasts
(176), and collagen types II and X and aggrecan in chondro-
cytes (177) Since secreted CTGF is an ECM-associated
heparin-binding protein, it is able to mediate cell-matrix
interactions (178). CTGF also stimulates the migration/
chemotaxis of fibroblasts, mesenchymal stem cells, endothe-
lial cells and vascular smooth muscle cells (179,180). CTGF
has also been shown to enhance cell survival or block apop-
tosis under conditions where cell adhesion is prevented
(179,181) but induces apoptosis in vascular smooth muscle
cells (182,183).
In addition to the cellular activities discussed above,
CTGF has been implicated in more complex biological pro-
cesses including embryonic development, angiogenesis,

1 Bone Structure, Development and Bone Biology 29
endochondral ossification and wound healing. Based on the
angiogenic activity of CTGF, it has been proposed that CTGF
is involved in neovascularization of the mineralized hyper-
trophic cartilage during endochondral ossification (184,186)
CTGF expression is induced during cutaneous wound heal-
ing. Its effects on fibroblast chemotaxis, extracellular matrix
production and angiogenesis suggest that it contributes to
wound repair (174,185,187). It has been postulated that
CTGF family members play a role in various pathological
processes including tumorigenesis such as in cartilaginous
tumors (188), atherosclerosis, and various fibrotic diseases
(189). It is interesting to note that several different mutations
of WISP3, another CCN family member, have been associ-
ated with the autosomal recessive disorder progressive pseu-
dorheumatoid dysplasia in which patients experience
continued cartilage loss and destructive bone changes around
synovial joints (190). This is the first study establishing a
definitive link between a CCN family member and the patho-
genesis of a disorder affecting bone and cartilage.
CTGF and Skeletogenesis–CTGF and MSC Condensation
During embryonic development there two types of mesen-
chymal skeletal condensations, pre-cartilaginous condensations
that develop into primary cartilage (endochondral ossification
during limb development), and pre-osseous condensations
that develop into membranous bones (65, 191, 192). During
endochondral ossification, mesenchymal stem cells (MSC)
aggregate and undergo condensation and subsequent
chondrogenic differentiation to form a cartilaginous core
(193). This cartilaginous core is then shaped to become the
cartilaginous anlage of the future skeleton (192). The cellular
condensation process is dependent on signals initiated by
cell-matrix and cell-cell adhesion, and these signals are
modified by a cell’s response to growth and differentiation
factors in the extracellular milieu. The hallmarks of cellular
condensation include changes in cell adhesion and cytoskel-
etal architecture (194). The roles of adhesion molecules
including N-cadherin, neural cell adhesion molecule
(N-CAM), syndecans, and ECM proteins (fibronectin), and
other signaling molecules, such as focal adhesion kinase
(FAK), paxillin and Wnt have been reported in mesenchymal
condensations (65,195). Perturbation of the functions of
these molecules leads to disruption in MSC condensation
and inhibition of normal cartilage formation (194). It has
been reported that CTGF mRNA expression in the newly
forming cartilage is high during the initial stage of condensa-
tion (193), and deceases as the chondrocytes mature (193,196)
Studies by others has shown that CTGF mRNA is expressed
strongly in mesenchymal condensations during Meckel’s
cartilage development, decreases in newly differentiated
chondrocytes, and surges again in hypertrophic chondrocytes
(193,197). In mice at embryonic stage E10.5, the CTGF pro-
tein is highly expressed in mesenchymal condensations of
the developing vertebral column and is associated with strong
expression of the condensation-matrix protein, fibronectin.
In a model of fracture repair, CTGF mRNA and protein are
expressed early in the developing fracture callus suggesting
that CTGF plays a role in tissue repair. Primary high-density
chick and murine limb bud micromass cultures are ideal
methods of analyzing in vitro the process of mesenchymal
condensation (198). Micromass cultures of the mouse cell
line, C3H10T1/2, treated with TGF-B results in the forma-
tion of a three-dimensional spheroid structure (mesenchymal
condensation) (191) Another study showed that Cyr-61, a
closely related member of the CCN family, is expressed during
mesenchymal condensation in vivo. Treatment of mesenchymal
cells with recombinant Cyr-61 (199) or rCTGF induced mes-
enchymal condensation. CTGF is highly expressed during
in vitro mesenchymal condensation, and that its expression is
associated with the expression of condensation-related ECM
proteins including fibronectin and N-cadherin. Together,
these data suggest that CTGF plays an important role in mes-
enchymal condensation.
CTGF and Chondrogenesis
During development and in newly formed cartilage, CTGF
expression is localized in mesenchymal condensation and
disappears in mature cartilage. This coincides with the strong
proliferative effects of CTGF on chondrocytes (193) and the
down regulation of the expression of this factor during
chondrocyte re-differentiation (200) Whole mount in situ
hybridization of CTGF mRNA in E17 mouse embryos
showed that CTGF is selectively expressed in the hypertro-
phic, but not, proliferative chondrocytes, and in cells of the
zone of calcifying cartilage (193) which correspond to the
region of chondrocyte cell death. CTGF has been shown to
induce apoptosis in a number of cell types including smooth
muscle cells, mesangial cells and mammary cells (193).
CTGF has also been shown to stimulate chondrocyte prolif-
eration, and and late differentiation associated with increased
expression of type X collagen (201). Important functional
insights may also be gained from developmental approaches
such as targeted gene disruption or cell/tissue-specific over-
expression. Transgenic mice that over-expressed CTGF
under the control of type XI collagen promoter were gener-
ated and their embryonic and neonatal growth was normal
(202,203). However, they showed dwarfism within a few
months of birth associated with decreased bone mineral
density. These results suggest that CTGF over-expression
in the growth plate can affect the process of endochondral
ossification. The CTGF null mouse has provided great in-
sight into the role of CTGF during chondrogenesis and

30 F.F. Sa fadi et a l.
osteogenesis (204). These mice have an obvious skeletal
phenotype involving both cartilage and bone. CTGF -/- mice
die shortly after birth due to respiratory distress and cyanosis
caused by severe malformation of the rib cage. CTGF defi-
ciency resulted in impairment of chondrocyte proliferation
and ECM composition within the hypertrophic zone, includ-
ing aggrecan and link protein, suggesting a role of CTGF in
acquisition of tensile strength in cartilage (204). It has been
shown that there is an inverse relationship between CTGF
and Sox-9 expression in MSC. TGF-B treatment inhibits
Sox-9 expression, but this effect is completely abolished
when the cells are transfected with CTGF siRNA. High lev-
els of CTGF correlate with low levels of Sox-9 and vice
versa, suggesting that CTGF is a negative regulator of early
chondrocyte commitment/differentiation from MSC.
CTGF and Osteogenesis
Using the osteopetrotic (op) rat as a model to examine dif-
ferential gene expression in bone from normal and osteo-
petrotic rats, CTGF mRNA and protein expression was
discovered in bone (158). In situ hybridization and immuno-
histochemical analyses demonstrated that CTGF mRNA and
protein are localized in osteoblasts lining metaphyseal trabe-
culae. Examination of CTGF expression in the fracture callus
of a fracture repair model demonstrated that CTGF was pri-
marily localized mesenchymal cells and in osteoblasts lining
active, osteogenic surfaces. In primary osteoblast cultures,
CTGF mRNA levels demonstrated a bimodal pattern of
expression, being high during the peak of the proliferative
period, abating as the cells became confluent, and increasing
to peak levels and remaining high during mineralization
(205). This pattern suggests that CTGF may play a role in
osteoblast proliferation and differentiation. Treatment of
primary osteoblast cultures with anti-CTGF neutralizing
antibody caused a dose-dependent inhibition of nodule for-
mation and mineralization. Treatment of primary osteoblast
cultures with rCTGF caused an increase in cell proliferation,
alkaline phosphatase activity and calcium deposition, thereby
establishing a functional connection between CTGF and
osteoblast differentiation (205). In vivo delivery of rCTGF
into the femoral marrow cavity induced osteogenesis that
was associated with increased angiogenesis. These studies
clearly showed that CTGF is important for osteoblast devel-
opment and function in vitro and in vivo. Studies of CTGF
null mice showed an impairment of endochondral ossifica-
tion associated with decreased vascular endothelial growth
factor (VGEF) in the ossification zone of the growth plates.
CTGF null mice have decreased bone mineral density and
osteopenia (204). Another group of investigators identified
and cloned a CTGF-like cDNA from human osteoblasts
encoding a protein of 250 amino acids (26 kDa) and sharing
approximately 60% homology with other members of the
CCN protein family (206). This CTGF-like protein is secreted
in primary human osteoblast cultures and it promotes osteo-
blast adhesion. It was recently shown that CYR61, another
member of the CCN family that is closely related to CTGF,
is produced and secreted in cultures of human osteoblasts,
suggesting that it may also function as an extracellular
signaling molecule in bone (207). Furthermore, CYR61 has
been shown to be up-regulated during fracture repair particu-
larly in proliferating chondrocytes and osteoprogenitor cells
suggesting that it may serve as an important regulator of
fracture healing (208).
Mechanisms of Action of CTGF in Skeletogenesis
The mechanisms of action of CTGF involved in mesenchy-
mal stem cells, chondrocytes, osteoblast differentiation and
bone formation are not well understood. One recent study
reported synergistic or antagonistic roles for CTGF with
TGF-B1 or BMP-4, respectively (209). In this study, CTGF
enhanced TGF-B1 signaling through receptor and cell-sur-
face binding, Smad-2 phosphorylation, activation of gene
expression and cell differentiation. Since TGF-B1 has been
shown to have a proliferative effect on osteoblasts (210) and
to stimulate CTGF gene expression, it is possible that CTGF
may mediate some of TGF-B1 effects on osteoblasts. This
same study also demonstrated that CTGF binds directly to
BMP-4 through its chordin-like domain, and that CTGF
inhibited alkaline phosphatase activity induced by BMP-4 in
C3H10T1/2 mesenchymal cells and Smad1 phosphorylation
in human 293T cells. Another study showed that CTGF
inhibits Wnt-3a and BMP-9-induced osteogenic differentia-
tion of MSC, suggesting that CTGF may act as a negative
regulator of early osteogenic differentiation of MSC (211).
The inhibitory effect of CTGF in osteogenic differentiation
of MSC is interesting.
In chondrocytes, another group has shown that CTGF
mediates its effect on chondrocyte proliferation via the ERK
pathway and on chondrocyte maturation via the p38 MAK
kinase pathway (201) In separate studies by Nishida and
colleagues (212,213), they identified a 280-kDa binding
protein complex for CTGF on chondrocytic and osteoblastic
cells lines, but the nature of the protein(s) contained in this
binding complex has not been determined. CTGF has also
been shown to bind low-density lipoprotein receptor-related
protein (Lrp), such as Lrp5 and 6 and antagonizes Wnt
signaling (214). However, the downstream signal transduc-
tion pathways in response to this interaction are not fully
understood. Clearly, the association between CTGF and
MSC is an interesting one that requires further investigation.
For other cytokines and growth factors please see section on
Regulation of Bone and Cartilage.

1 Bone Structure, Development and Bone Biology 31
Osteoactivin
Identification and Characterization
of Osteoactivin (OA) in Bone
The initial identification of OA came from studies using
an animal model of osteopetrosis (op) in rats. This model
was used to examine the differential gene expression in
bone from normal rats and op mutant osteopetrotic rats.
Osteopetrosis is a sclerosing bone disease characterized by
generalized increase in the bone mass and has severe skeletal
phenotype resulting from abnormalities during development
(215). Using the technique of mRNA differential display, a
novel cDNA that is highly up-regulated in op compared to
normal bone was identified.
The over-expression of osteoactivin in op mutant bone
compared to normal one was confirmed by Northern blot
analysis and found to be increased 4 fold in op versus normal
long bones and calvaria (216). Subsequent cloning and
sequencing of the full-length OA cDNA revealed sequence
homology with the previously reported human NMB (217),
DC-HIL (dendritic cell heparan sulfate proteoglycan integrin
dependent ligand) (218), HGFIN (hematopoietic growth fac-
tor inducible neurokinin) (219) and PMEL17 (melanocyte
specific gene) (220).
Osteoactivin has an open reading frame of 1716 bp with
a short 5’- untranslated region, encoding a protein sequence
of 572 amino acids (a.a.) with a native M.W of 65 kDa.
The first 22 a.a. represent a potential signal peptide, so OA
is potentially a secreted protein (216). Bioinformatic anal-
ysis of the full length OA a.a. sequence revealed a poten-
tial polycystic kidney domain and an RGD integrin
recognition site at position 556 that constitutes a potential
site for cell attachment. OA protein sequence analysis
revealed 11 N-linked and 19 O-linked potential glycosyla-
tion sites suggesting that OA is a highly glycosylated pro-
tein. A potential transmembrane hydrophobic sequence
has been identified in OA protein sequence from a.a. 499
to a.a. 522 suggesting that OA may have a transmembrane
isoform.
Characterization of OA Expression in Primary
Osteoblast Cultures
OA expression in primary osteoblast cultures showed that
the level of OA increased markedly as the cells differentiate
with maximal expression during matrix maturation and
mineralization as correlated with the early differentiation
marker, alkaline phosphatase and the late differentiation
markers, osteopontin and osteocalcin (216). By RT-PCR
analysis, OA showed the highest level in normal bone (long
bones and calveria) and primary osteoblast cell cultures
compared to much lower expression levels in brain, heart
and skeletal muscles (216). The high level of OA expres-
sion during osteoblast terminal differentiation in culture
and the preferential expression in bones compared to other
tissues are consistent with a gene that has a functional role
in osteoblast cell biology.
OA is highly expressed in various malignant tumors such
as in glioma (221) and hepatocellular carcinoma (222). It has
been shown that over-expression of OA in glioma cell lines
(223), as well as in hepatoma cell lines (222), permits tumor
invasiveness. OA also has been found to function as an acti-
vator of fibroblasts infiltrated into denervated skeletal mus-
cles (224). Treating mouse NIH-3T3 fibroblast cell cultures
with recombinant mouse osteoactivin increased the amounts
of fibroblast markers, MMP-3 and MMP-9, suggesting that
OA has a pathophysiological role in skeletal muscles atro-
phied by degeneration (224).
Enzyme: Alkaline Phosphatase
Although not generally thought of as a matrix component,
this enzyme is an osteoblast product (such as osteocalcin and
osteonectin) and would best be discussed here. There are
three related isozymes that are tissue related and are associ-
ated with three separate genes (reviewed in 225). These are
the placental, intestinal and tissue nonspecific forms. The
last is seen at high levels in a variety of tissues such as bone,
liver, kidney and skin. All three isozymes require Zn2+ and
Mg2+ and catalyze the hydrolysis of monoester phosphates
(such as pyridoxal-5’-phosphate) at a pH between 8 and 10
(alkaline pH). The isozyme important in bone is the tissue
non-specific form.
Although the tissue non-specific isozyme is associated
with a single gene (on chromosome 1), there are electro-
phoretic and other subtle differences with the other isozymes
from different tissues. These differences are probably due
to altered glycosylation. The enzyme is attached to the cell
membrane via phosphotidyl inositol and can be removed
from the cell surface by phospholipase C. This may be a
mechanism whereby it enters the serum. The enzyme can
be demonstrated by a variety of histochemical and immu-
nohistochemical methods. It has been seen that the bone
form of the enzyme can be localized to osteoblasts and
young osteocytes, but mature osteocytes are consistently
negative for its presence. Vitamin D and thyroxin increase
the biosynthesis of alkaline phosphatase. Glucocorticoids
and parathyroid hormone inhibit it. The expression of alka-
line phosphatase is cell cycle dependent with its activity
increasing through the G1 and S phases, and decreasing in
G2 and M phases.

32 F. F. Saf adi e t al.
The role on alkaline phosphatase in biomineralization is
still speculative. It has been found in matrix vesicles, but its
exact role is unclear. Serum alkaline phosphatase activity is
increased in growing children, pregnancy, healing fractures,
Paget’s disease, rickets, osteomalacia, hyperparathyroidism,
bone-forming tumors and certain skeletal metastases. It is
reduced in hypophosphatasia.
The Hydrated (Muco)Polysaccharide Gels
The functional units of bone extracellular matrix “gels”
are the Glycosaminoglycans (GAGs). Of the several GAGs,
four main groups are present. Hyaluronic acid, chondroitin
and dermatan sulfates, heparan sulfate and heparin, and
keratan sulfate. The majority are aggregated to a protein
core to form a proteoglycan. Prior to release from the syn-
thesizing cell, GAGs (except hyaluronic acid) are sulfated.
This step imparts a net negative charge on these molecules.
In turn, this charge serves two functions. First, it keeps the
molecules extended, increasing the volume to weight ratio.
Second, by attracting osmotically charged cations, it
attracts water. This mechanism creates a gel with very high
swelling pressures and tremendous resistance to
compression.
The highly viscous hyaluronic acid is a major constituent
of synovial fluid, where it helps in lubrication. Additionally,
it may impede bacteria, by its physical and chemical proper-
ties. Its synthesis is thought to involve a pathway located in
the cell membrane, thus hyaluronic acid is not sulfated. The
remaining GAGs (mentioned above) are synthesized within
the Golgi.
The kind of GAG present within bone is different than those
in cartilage. Bone has longer chains, and is rich in chondroitin-
4-sulfate. Cartilage on the other hand contains chondroitin-6-
sulfate. Developing bone has been shown to contain a large
chondroitin sulfate proteoglycan and a small chondroitin sulfate
proteoglycan.
Further characterization of the small proteoglycan in turn
reveals two proteoglycans (PG I or biglycan and PG II or
decorin). Biglycan is rich in Leu, whereas Decorin is rich
in Glu/Gln. Each is demonstrable by non-cross reacting
antibodies. In bone, both biglycan and decorin can be immu-
nolocalized within the matrix and cells of new bone formation.
In mature bone, biglycan (but not decorin) is detected in
lacunae and canaliculi.
Both biglycan and decorin have central proteins of 45 kilo
Daltons. Biglycan contains two chondroitin sulfates, whereas
decorin contins a single chondroitin sulfate which occurs in
a periodic manner within collagen fibrils. In fact decorin was
so named because of its peculiar localization around colla-
gen (“decoration” of collagen fibers). For more details on
biglycan and decorin refer to Table 2.
Biglycan Decorin
The gene for biglycan is located on
chromosome X, the mRNA contains
sites of homology to two morpho-
genic proteins (Toll and Chaoptin) as
well as von Willebrand factor.
The mRNA for decorin
contains a sequence of
epidermal growth
factor.
Rate of synthesis is faster, but reduced by
vitamin D
Slow synthesis
Rate of degradation slows with age Remains the same with age
Mouse null for biglycan has increased
osteoclast differentiation due to
defective osteoblasts (225) and
hypomineralization of dentin (227).
Mouse null for decorin
has hypomineraliza-
tion of dentin (227).
The rates of synthesis as well as degradation are faster for
biglycan as compared to decorin. The rates for degradation
of biglycan however, significantly slow down with advancing
age. This leads to a relative accumulation of biglycan.
Whether this reflects an added need for biglycan is speculative.
Additionally, the rate of synthesis of biglycan is significantly
reduced by 1,25 dihydroxyvitamin D, whereas for decorin it
is unaffected.
The role of compressive forces and its role in proteogly-
can removal prior to mineralization are controversial.
Similarly, their role in mineralization and in the developing
bone is unclear. There is a probable role in “space capture”,
hydroxyapatite precipitation and the development of colla-
gen scaffolding.
Mineralization
Calcium hydroxyapatite is the main form of mineral in the
human. In invertebrates the mineral most prevalent is cal-
cium carbonate, whereas in plants it is the oxalate. The
body’s “biologic” hydroxyapatite crystal however differs
from the form found in igneous rocks in its crystallographic
structure and its size.
Calcification in the body occurs in several different situa-
tions. Firstly, there is the physiologic process of cartilage and
osteoid mineralization. Secondly, there is the pathologic
extracellular or intracellular “dystrophic” calcification, which
occurs in association with tissue damage. Thirdly, there is a
“metastatic” calcification, which occurs in association with
altered serum levels of calcium and phosphate, and finally,
there are the diseases of abnormal crystal deposition.
Most cases of calcium deposition within the soft tissues
are caused by calcium hydroxyapatite - trauma, fat necrosis,
scleroderma, hyperparathryroidism, familial hyperphos-
phatemia, sarcoidosis, myeloma, metastases, and others. These
encompass both dystrophic and metastatic calcification.
Punctate or linear calcification seen radiographically along

1 Bone Structure, Development and Bone Biology 33
menisci, articular cartilage or intervertebral disks is gener-
ally due to calcium pyrophosphate deposition (CPPD disease).
This deposition is rarely massive and simulates tophaceous
deposits. In such cases the term tophaceous pseudogout may
be appropriate (228). In the latter, tissue processing may
remove the crystals making the diagnosis difficult.
Additionally, areas of chondroid metaplasia and chondrocyte
cellular atypia may raise the possibility of chondrosarcoma.
The identification of areas of granulomatous inflammation
may be the only clue to the real nature of the process.
Calcification of osteoid (bone mineralization) differs from
soft-tissue calcification in being an orderly process. It is dis-
tributed within the hole-zones of collagen molecules. This
method does not disrupt the spatial organization of collagen.
The process is tightly regulated, but poorly understood. The
mineral is initially deposited as amorphous calcium phos-
phate. The initial solid phase is formed at neutral pH. This
phase is randomly and poorly oriented. Subsequently a series
of solid phase transformations occur that lead to crystalline
hydroxyapatite as the final stable solid phase. The initiation
of mineralization is probably caused by heterogeneous nucle-
ation, the active binding of calcium, phosphate and calcium
phosphate complexes at the nucleation site in the matrix
rather than by simple precipitation. In mature bone, it is pos-
sible that crystalline calcium carbonate containing hydroxy-
apatite is deposited rather than an amorphous calcium
phosphate or hydroxyapatite.
The Process of Mineralization
The process of mineralization is complicated and has not
been satisfactorily elucidated. It is probably under genetic
control, and since the physiologic state of extracellular fluids
is supersaturated with respect to octacalcium phosphate, there
are probably crystal inhibitors present. These include sub-
stances such as pyrophosphate and serum proteins. A local
increase in concentration far beyond supersaturation is required
to overcome the energy of the reaction of crystal formation.
Additionally a local environment containing phosphatases
and proteases to remove the inhibitors is essential.
A candidate for such a local environment is the “matrix
vesicle”. These are membrane bound cell free structures
derived from chondrocytes and osteoblasts. Matrix vesicles
are eccentrically placed within these cells. They are likely to
be important in calcified cartilage and woven bone, but less
so in mature lamellar bone. Extrusion of the matrix vesicle
occurs at cell surfaces, next to mineralizing bone. These
structures are rich in alkaline phosphatases and in ATPases.
They presumably function by concentrating calcium and
phosphate, and also by removing the inhibitors of
calcification.
It must be emphasized that mineralization is also under
hormonal control. Vitamin D plays an important role. Other
exogenous factors that affect mineralization include aluminum
intoxication, fluoride intoxication (osteofluorosis), and phos-
phate deficiency. The mechanisms of action of aluminum
and fluoride are unclear. Mineralization is thus more than
just a straight-forward physico-chemical process.
Hydroxyapatite crystal formation follows two phases:
nucleation and propagation (multiplicative proliferation).
The mechanisms operative to achieve this are thought to
include: matrix vesicle mediated and collagen mediated
hydroxyapatite precipitation. There are at least two hypoth-
eses for mineralization. The first (229,230) gives primacy to
matrix vesicles. The second (231) gives primacy to factors
within the matrix. Each has been under investigation since
the 1960s.
Hypothesized Mechanisms of Mineralization
in Tissues
Mineralization in Cartilage
Mineralization occurs within “matrix vesicles” derived from
chondrocytes. These are structures 2-4 μm in size, where
hydroxyapatite is deposited. They are derived from the
plasma membrane of cells. The membrane retains its phos-
phatases, which serves to raise the phosphate level. The lipid
component of the membrane helps to concentrate calcium.
This leads to intravesicular crystal formation. Subsequently,
part of or the whole crystal is extruded. This leads to crystal
propagation. This phase of crystal growth is physico-
chemical and matrix factors play an important part. These
matrix factors include collagen, carboxyglutamate proteins
(Gla proteins), phosphoproteins, glycoproteins such as chon-
drocalcin, calcium-acidic phopholipid-phosphate complexes
and proteolipids. Inhibitors of calcification include proteo-
glycans, magnesium, pyrophosphate, ATP, ADP and several
synthetic compounds such as diphosphonates.
Mineralization in Woven bone
It occurs both within matrix vesicles and within the “holes”
of the fibers of Type I collagen. Rapid mineralization occurs
since the matrix vesicles act as a nidus for the larger deposits
in the collagen fibers. This works well for the bone to act as
repair or reactive bone, but the organizational architecture is
haphazard. The bone has a lower strength and is less rigid. It
is likely that a phosphoprotein is involved in modulating this
process. The rapid mineralization is reflected on the presence
of only a thin zone of osteoid separating the osteoblast from

34 F.F. Safa di et al.
the mineralization front. Mineralization is seen to occur
within 24–72 hours of matrix deposition.
Mineralization in Lamellar bone
Mineral is deposited in a regular fashion within the collagen
fibers, initiating from the “hole” region (see section on col-
lagen structure). It takes up to 10 days for this slow mineral-
ization to occur, consequently the osteoid zone separating
the osteoblast and the mineralization front is wider in lamel-
lar bone. This collagen mediated mineralization is thought to
be a “heterogeneous” nucleation - and non-collagenus pro-
teins may play a role in mediating the process.
Surgical Pathology of Mineralization
It is difficult to visualize the site of mineralization in routine
decalcified sections. Increased basophilia in the cartilage or
woven bone lends suspicion for its location. However, it is
reasonably straight forward to be able to do so in undecalci-
fied sections. This is especially true in the case of lamellar
bone mineralization. Fluorescent microscopy following a
flurochrome (such as a tetracycline) administered 48 hours
prior to the study aids the delineation. Osteoid mineraliza-
tion commences 7-9 days following a resorptive front, and
takes up to 200 days to complete.
Mineral Deposits
Pathologic mineralization can be of several different types
(dystrophic, metastatic or crystal deposition). Metastatic
calcification (associated with increased serum calcium) is
often intracellular. The increased Ca 2+ of the tissue fluid is
initially taken up by mitochondria in the cytosol in an
attempt to preserve the cytoplasmic homeostasis. When the
mechanism is “overwhelmed” the calcium precipitates
within the cell. With intra-cellular dystrophic calcification
the process may be similar. The plasma membrane associ-
ated Ca 2+ pump is thought to be destroyed as a result of
tissue damage. In this situation, there are increased cytoso-
lic levels of calcium and a similar process follows. Extra-
cellular dystrophic calcification is associated with a different
mechanism. It is postulated, in this situation, that tissue
damage leads to extrusion of matrix vesicles, which then
cause calcium precipitation outside the cell. Crystal
deposition disease (chondrocalcinosis or pseudogout) is the
most difficult to explain. The role of matrix vesicles is
un-established. An imbalance between the production of
intrasynovial inorganic pyrophosphate and its removal by
joint phosphatases may be more important.
Recent studies by Xiao et al. using proteomic analysis of
matrix vesicles of mineralizing osteoblasts showed these
vesicles produce different proteins. Some of these are previ-
ously known proteins such as annexins and peptidases, while
others are novel proteins including a variety of enzymes,
osteoblast-specific factors, ion channels, and signal trans-
duction molecules, such as 14-3-3 family members and Rab-
related proteins. These studies suggest that these proteins
play a role in osteoblast matrix mineralization (232).
Factors Important in Mineralization
Collagen Collagen provides oriented support for the newly
formed crystals. The post-translational changes
in collagen type I make it possible for diffusion
of large hydrated ions such as calcium
phosphate into the fibril. Collagen also has sites
that may initiate crystal precipitation. Also,
there are high energy phosphate bonds (obtained
from molecules such as ATP) which facilitate
the formation of solid phase from solution.
Collagen however is unable to initiate
mineralization.
Calcium
binding
proteins
Phosphoproteins and sialoproteins in the bone
matrix may bind calcium to promote crystal
deposition and growth, thus acting as nuclea-
tors. Crystal growth could then depend upon the
conformational change in these proteins after
deposition. The initiation of mineralization is
coincident with the deploymerization of
proteoglycan molecules. Proteoglycans may
inhibit calcification by a number of mechanisms
including shielding of collagen, chemical
interaction with collagen side chains, sequester-
ing calcium or phosphate ions or occupying
critical space in the molecule. Different
phosphoproteins have varying importance in
mineralization.
Pyrophosphate This is a naturally occurring inhibitor of calcifica-
tion. It has a short half-life due to its rapid
degradation by pyrophosphatases.
Pyrophosphates are present in body fluids and
increase the stability of the solution phase of
calcium phosphate. Diphosphonates are
pyrophosphate analogs, and are powerful
inhibitors of calcification in large doses.
Bone Gla
proteins
Osteocalcin is a highly conserved protein which
is abundant in the bone matrix. Because of the
Gla residues, it is able to bind calcium. Its
role in mineralization is controversial and
there is debate over whether it promotes or
inhibits mineralization (233). Depletion of
osteocalcin from newly formed bone by
warfarin treatment results in no impairment of
mineralization (234). (see also osteocalcin
described above).
Lipids and
proteolipids
Within bone there are acid phospholipids that form
complexes with calcium phosphate and could
thereby influence mineralization. These
substances have the capacity to bind to
calcium.

1 Bone Structure, Development and Bone Biology 35
Factors Important in Mineralization
Alkaline
phos-
phatase
This is an ectoenzyme produced by osteoblasts and is
likely to be involved with the mineralization
process. Patients with decreased amounts of
enzyme (hypophosphatasia) have impaired
mineralization (see rachitic syndromes in
metabolic bone disease). Bone alkaline phos-
phatase is present in high concentrations in matrix
vesicles, but its precise role in mineralization is
unclear. Alkaline phosphatase may be involved in
the degradation of inorganic pyrophosphate, thus
providing a sufficient level of organic phosphate
for mineralization to proceed.
Remodeling of Bone
Bone undergoes remodeling throughout life. This involves
the coupling of resorption of existing bone and the formation
of new bone. Thus the entire bony skeleton is “renewed” on
a continuous basis. This mechanism is important, because of
the cyclical loading and torsional stresses that the skeleton
undergoes. In the absence of renewal, bone would exceed its
tolerance limits within a short period of time.
Bone turnover or remodeling is thought to occur in dis-
crete foci or packets scattered throughout the skeleton. Each
packet takes 3-4 months to complete. Such foci have been
termed bone remodeling units or BRUs by Frost, who
described the process in 1964. About 25 percent of the meta-
bolically active trabecular bone and about 3 percent of the
cortical bone completely renews itself each year (32,235).
The amount of bone added in each remodeling cycle,
however, reduces slightly with age. This is probably due to a
decreased number of osteoblasts. This has been suggested as a
possible mechanism for age related (but not post-menopausal)
osteoporosis.
In the adult bone, remodeling involves activation, resorp-
tion and formation at endosteal and periosteal surfaces and
within Haversian systems. Remodeling at the endosteal and
periosteal surfaces would result in alterations in the thick-
ness and width of tubular bones. Conditions such as acro-
megaly, osteopetrosis and hypo- or hyperthyroidism alter
trabecular and cortical bone mass.
Bone formation during a remodeling process requires a
prior resorption. Resorption takes approximately 10 days. The
resorption is carried out by a “cutting cone” of osteoclasts.
The trigger for resorption includes the stimulatory cytokines
IL-1 and IL-6 produced by osteoblasts, as well as the modula-
tion of the integrin-RGD sequence interaction and other
factors such as transcription factors and membrane proteins.
The defect created after resorption, is filled in by fibrovas-
cular tissue. The vessel component is especially important.
The formation of the Haversian and the Volkmann systems
are thought to be created by these mechanisms. In addition,
the fibrovascular core contains pericytes, loose connective
tissue, macrophages, mesenchymal stem cells and undiffer-
entiated osteoprogenitor cells. The outer edge of the osteon
(where resorption ends and bone formation first starts) is
marked by an intensely basophilic line - the “cement” or the
“reversal” line. This area is poor in collagen and mineral, and
has a high content of sulfur.
Bone formation is carried out by osteoblasts. The process
takes approximately 3 months. As the remaining bone and its
osteocytes (old, partially resorbed osteons) gets cut off by
newly forming osteons, they remain as “interstitial” lamel-
lae. Osteoblastogenesis has identifiable processes of chemot-
axis, proliferation and differentiation of osteoblasts. This is
then followed by mineralization and the cessation of osteo-
blast activity.
Mediators of osteoblastic activity and bone formation
include transforming growth factor-beta (TGF-B, bone Gla
protein fragments, platelet derived growth factors A and B
(PDGF A and B), all of which are chemotactic for osteo-
blasts. The second event, proliferation of osteoblasts, is
thought to be mediated by TGF-B, PDGF, IGF I and II, and
fibroblast growth factors (FGFs). Cytokines that may play a
role in the differentiation of osteoblasts and the production
of alkaline phosphatase activity within these cells include
IGF-I and bone morphogenetic protein-2 (BMP-2).
The linking or “coupling” of bone resorption and bone
formation is complex and difficult to explain. There is emerg-
ing evidence however to suggest that “osteoclastogenic”
cytokines such as IL-6, IL-1 and IL-11 as well as “osteoblas-
togenic” cytokines such as leukemia inhibitory factor may be
stimulated together by the same signal transduction pathway.
Glycoprotein 130 is a molecule present in this pathway, and
is involved in the transduction of the signal delivered by each
of these cytokines. Sex steroids inhibit, whereas parathyroid
hormone and vitamin D increase glycoprotein 130 in experi-
mental models (32,235). This type of model would conve-
niently explain bone formation-resorption coupling as well
as the various acknowledged effects of these hormones on
bone turnover.
Another mechanism that may help explain coupling, is
the release of osteoblast stimulating factors such as IGF I
and II and TGF-B during the osteoclastic process. Another
possible mechanism to explain coupling is the RANK/
RANKL interaction in which osteoblasts regulate the devel-
opment and function of osteoclasts (please refer to regulation
of osteoclastogenesis). Coupling is the rationale for the
counterintuitive, but clinically validated method of treating
osteoporosis by giving intermittent parathyroid hormone
therapy.
Several diseases of bone are superimposed on this normal
cellular remodeling sequence. In diseases such as primary
hyperparathyroidism, hyperthyroidism and Paget’s disease,
there is osteoclast activation. However, there is also a

36 F.F. Sa fadi et al .
compensatory and relatively balanced increase in bone for-
mation, due to the coupling of these events.
Other diseases of bone are the result of abnormal cou-
pling. One example is the decreased bone formation after
extensive resorption in the osteolytic lesions of myeloma,
where there may be a defect in osteoblast maturation. In solid
tumors and in elderly patients with age-related osteoporosis
there may be similar mechanisms operating, increased bone
resorption and decreased bone formation. Osteoblastic activity
in the absence of prior osteoclastic activation is thought to
occur in some special situations such as osteoblastic metas-
tases and in the response of bone to fluoride therapy.
Regulation of Bone by Endocrine
and Paracrine Factors
Endocrine Control
Endocrine control of the bony skeleton is multifarious and
includes the need to maintain a balance between bone forma-
tion and loss, maintenance of homeostasis in calcium and
phosphate levels in the body, and maintenance of a reservoir
of phosphate required for generating energy. The major players
in the endocrine system that participate in this regulation
include parathyroid hormone, PTH-related peptide, calci-
tonin, vitamins A and D, estrogens, androgens and growth
hormone.
Parathyroid Hormone
Parathyroid hormone (PTH) viewed as catabolic for bone is
synthesized in the parathyroid gland from a biosynthetic
precursor pro-PTH. PTH, a single chain polypeptide (84
amino acids referred to as PTH 1-84) impacts bone, intestine
and kidney function. PTH mediates bone loss in older ani-
mals in its role to maintain calcium homeostasis and is
required in fetal and neonatal animals for normal trabecular
bone formation. In response to a decrease in serum calcium,
PTH is released from the parathyroid gland. It targets the
kidney to reduce calcium excretion, inhibits phosphate
resorption and stimulates 1, 25 - dihydroxy vitamin D produc-
tion which in turn targets the gastrointestinal tract to increase
dietary absorption of calcium resulting in suppression of
PTH. In addition, both PTH and 1, 25 (OH)2-vitamin D are
able to bind to osteoblasts and through RANK and RANKL
increase osteoclastic activity which results in calcium and
phosphate release from the bony skeleton returning serum
calcium levels to normal by an increase in bone resorption.
Receptors for PTH are found on pre-osteoblasts, osteoblasts
and chondrocytes. They are not, however present on osteoclasts
supporting the notion that the action of PTH on osteoclasts is
osteoblast-dependent and mediated via substances such as
IL-1, IL-6 and prostaglandins of the E series. The net result
is osteoclast activation and initiation of bone resorption lead-
ing to calcium release from bone.
Evidence suggests that in certain situations PTH stimu-
lates bone formation. When administered continuously, it
increases osteoclastic resorption and suppresses bone forma-
tion. When administered in low doses, intermittently, it stim-
ulates bone formation without resorption. This anabolic
effect, like the resorptive effect is probably indirect, and
mediated via IGF-1, TGFB, etc. High serum PTH levels,
maintained for even a few hours, initiates osteoclast forma-
tion resulting in bone resorption that overrides the effects of
activating genes that direct bone formation. Identification of
PTH-related protein (PTHrP) expression early in the osteo-
blast progenitor cells, its action through the PTH 1 receptor
(PTH1R) on mature osteoblasts, and the observation that
PTHrP+/- mice are osteoporotic, raise the possibility that PTHrP
is a crucial paracrine regulator of bone formation.
Calcitonin
Calcitonin is a peptide hormone synthesized and secreted by
thyroid parafollicular C cells, is regulated by extracellular cal-
cium levels, and gastrointestinal hormones such as gastrin. It
is encoded by a gene that undergoes alternate splicing to gen-
erate several other peptides including calcitonin gene related
peptide. Calcitonin receptors are present on osteoclasts,
preosteoclasts, monocytes and certain tumor cells and
increased levels result in a short lived fall in plasma calcium.
In bone, calcitonin blocks bone resorption probably via
mature osteoclasts, by enhancement of adenylate cyclase and
cAMP or as a mitogen acting on bone cells. It promotes renal
calcium excretion possibly to maintain normocalcemia after
a large calcium containing meal.
The physiological role of calcitonin remains controver-
sial. Calcitonin and alpha-calcitonin gene-related peptide
(alphaCGRP)-deficient mice exhibit high bone mass mediated
by increased bone formation with normal bone resorption.
The absence of significant changes in bone mineral density
caused by a decline or overproduction of calcitonin in
humans questions the physiological relevance of calcitonin
as an inhibitor of bone resorption. A recent study on the age-
dependent bone phenotype in two mouse models, one lacking
calcitonin and alphaCGRP (Calca-/-), the other lacking
alphaCGRP (alphaCGRP-/-) reported osteopenia at all ages
in AlphaCGRP-/- mice. However, the Calca-/- -mice displayed
increased bone turnover with age and at 12 months of age a

1 Bone Structure, Development and Bone Biology 37
significant increase in bone formation and resorption. These
data suggest that calcitonin may have dual actions, in bone
formation and resorption, which may explain, at least in part,
why alterations of calcitonin serum levels in humans do not
result in major changes in bone mineral density (236). In
addition, calcitonin has a role in the therapy of hypercalce-
mia of malignancy, in Paget’s disease and in osteoporosis.
Osteoclasts from Paget’s patients are hyper-responsive to
calcitonin, for longer periods of time than control cells
although the molecular mechanism(s) for this hyper-
responsivity is unknown (233).
Vitamin D
Ergosterol and 7-dehydrocholesterol are the precursors for
vitamin D, best labeled as a hormone and vitamin. These
compounds are stored in the skin, transported in the body via
an alpha-globulin binding protein/vitamin D binding protein
(DBP) and become activated by ultraviolet light. Findings
procured from gene targeting experiments in mice suggest
that DBP possibly maintains stable serum stores of vitamin
D metabolites and modulates the rate of its bioavailability,
activation, and end-organ responsiveness. These properties
may have evolved to stabilize and maintain serum levels of
vitamin D in environments with variable vitamin D avail-
ability (238).
Activation of ergosterol and 7-dehydrocholesterol in
turn generates calciferol and cholecalciferol. These sub-
stances are hydroxylated in the liver to yield 25-hydroxy-
vitamin D in the presence of magnesium, and then are
converted in the proximal tubule of the kidney to generate
metabolites of 25-hydroxy-vitamin D. The most active form
of vitamin D is 1,25-dihydroxyvitamin D. This hormone is
key to the control of calcium metabolism in the gut, proxi-
mal tubule in the kidney and bone. 1,25-dihydroxyvitamin
D production is regulated by calcium and PTH . It stimu-
lates calcium binding protein, affects osteocalcin produc-
tion, osteoclastic resorption, monocytic maturation,
myelocytic differentiation, skin growth and insulin secre-
tion. Lack of vitamin D results in impaired mineralization
of newly formed bone which results in rickets in children,
and osteomalacia in adults. These conditions are typified by
an increase in proteinaceous bone matrix which does not
mineralize. An excess of vitamin D leads to an increase in
bone resorption and hypercalcemia.
Vitamin D acts via vitamin D receptors, and receptor sites
of 1,25-dihydroxyvitamin D have been identified on several
cell types. The vitamin D receptor is a transcription factor
which forms homo- or heterodimers with members of the
steroid hormone receptor superfamily (most notably the
retinoic acid receptor RXR). Errors in genes that code for
these nuclear receptors are reported in several forms of rick-
ets. It is also suggested that postmenopausal osteoporosis
may be genetically predetermined by polymorphisms pres-
ent on the vitamin D receptor gene (237).
The vitamin D receptor type II (VDR-II) null mouse sug-
gests a role for vitamin D in bone metabolism. These mice
are phenotypically normal at birth, survive to 6 months of
age, develop hypocalcemia at 21 days of age, at which time
their parathyroid hormone (PTH) levels begin to rise. They
also develop hyperparathyroidism accompanied by an
increase in the size of the parathyroid gland with a concomi-
tant increase in PTH mRNA levels. This phenotype is also
associated with rickets and osteomalacia as early as day 15,
and there is an expansion in the zone of hypertrophic chon-
drocytes in the growth plate. Interestingly the VDR-II knock-
out mouse also develops progressive alopecia at 4 weeks of
age (239). Studies using primary calvarial cultures revealed
that ablation of VDR-enhanced osteoblast differentiation
was associated with an increase in alkaline phosphatase
activity, as well as an early sustained increase in formation of
mineralized matrix. The expression of bone sialoprotein, a
marker for mineralization, was also increased in VDR null
osteoblasts. These studies demonstrate that VDR attenuates
osteoblast differentiation in vitro, and that other endocrine
and paracrine factors may modulate the effect of VDR on
osteoblast differentiation in vivo (240).
Evidence suggests that marrow mononuclear cells and
monocytes fuse to form osteoclasts on exposure to vitamin D
(233). Vitamin D receptors are not present on mature osteo-
clasts, thus osteoblasts are needed to mediate the effects of
vitamin D to induce bone resorption and PTH may act syner-
gistically with vitamin D to mediate this activity. In addition,
it is likely that IL-1 and IL-2 play an intermediary role in
bone resorption mediated by vitamin D.
Calcitriol and Osteogenesis
Calcitriol (1A, 25(OH)2 D3), the active form of vitamin D3, is
synthesized from 25-hydroxyvitamin D3 by the action of 1A
hydroxylase which is present predominantly in the kidney
(247). Mutations in the human1A hydroxylase gene cause
pseudo-vitamin D deficiency rickets (248). Targeted ablation
of the 1A hydroxylase gene in a mouse model leads to devel-
opment of retarded growth, and skeletal abnormalities
characteristic of rickets (249). Calcitriol resorbs bone by
stimulating the formation of osteoclasts. Receptors for 1A,
25(OH)2 D
3 are found on osteoblasts and osteoprogenitor
cells but not osteoclasts (241). Stimulation of osteoclast for-
mation requires cell-cell contact between osteoblasts and
osteoclast precursor cells, and involves the upregulation of
the osteoclast-differentiating factor, RANK ligand in

38 F.F. Sa fadi et al .
osteoblasts, and downregulatation of OPG expression, an
osteoclastogenesis inhibitory factor that works as a decoy
receptor for RANK (242). Through stimulation of osteoclast
formation, 1A, 25(OH)2 D
3 is believed to mediate bone
resorption and remodeling. In addition, 1A, 25(OH)2 D3 has
been shown to inhibit osteoblast proliferation and stimulate
apoptosis through induction of tumor necrosis factor alpha
(243).
In vitro studies demonstrate that vitamin D3 stimulates
osteoblast differentiation through induction of osteocalcin
and alkaline phosphatase expression (both markers of mature
osteoblasts) (244). These findings are supported by studies
showing that the Ca+2 binding proteins osteocalcin and osteo-
pontin secreted by osteoblasts during differentiation, are
upregulated by 1A, 25(OH)2 D3 through its response element
on the osteocalcin and osteopontin promoter (245). Moreover,
1A, 25(OH)2 D
3 stimulates osteoblast differentiation by the
release of alkaline phosphatase (ALP) through the ERK-
MAPK signaling pathway. Treatment of primary osteoblast
cultures with an ERK inhibitor resulted in reduced 1A,
25(OH)2 D
3 induction of ALP, which confirms that 1A,
25(OH)2 D
3 stimulates ERK expression in primary human
osteoblasts (246).
Vitamin A
Retinoids, in excess, decrease the formation of bone and car-
tilage matrix, whereas a deficiency has the opposite effect.
Several years ago, it was discovered that an imbalance of
vitamin A during embryonic development had dramatic
teratogenic effects. These effects have since been attributed
to vitamin A’s most active metabolite, retinoic acid (RA),
which itself profoundly influences the development of mul-
tiple organs including the skeleton. After decades of study,
researchers are still uncovering the molecular basis whereby
retinoids regulate skeletal development. Retinoid signaling
involves several components, from the enzymes that control
the synthesis and degradation of RA, to the cytoplasmic
RA-binding proteins, and the nuclear receptors that modu-
late gene transcription. As new functions for each compo-
nent continue to be discovered, their developmental roles
appear increasingly complex and each has been implicated in
skeletal development. Moreover, retinoid signaling comes
into play at distinct stages throughout the developmental
sequence of skeletogenesis, highlighting a fundamental role
for this pathway in forming the adult skeleton. Consistent
with these roles, manipulation of the retinoid signaling path-
way significantly affects the expression of the skeletogenic
master regulatory factors, Sox9 and Cbfa1. In addition to the
fact that we now have a greater understanding of the retinoid
signaling pathway on a molecular level, we are able to place
retinoid signaling within the context of other factors that
regulate skeletogenesis. Here we review these recent
advances and describe our current understanding of how
retinoid signaling functions to coordinate skeletal develop-
ment. We also discuss future directions and clinical implica-
tions in this field.
Retinoic acid (RA) is an endogenous metabolite of vita-
min A that acts as potent regulator of osteoblast growth and
differentiation of (250). The actions of RA are mediated by
nuclear receptors that belong to the steroid hormone receptor
superfamily (251). Changes in levels of RA during skeletal
development result in severe abnormalities in the appendicular
and craniofacial skeleton (252–254). Several studies have
investigated the effects of RA on osteoblasts in vitro. Low
doses of RA (0.01 MM) resulted in a increased levels of
osteopontin and osteocalcin mRNA in fetal rat calvarial
osteoblasts (255). Similarly, treatment of clonal pre-osteo-
blasts with pharmacologic doses (1 μM) of RA have shown
an increase in osteopontin transcript levels and enhancement
in nuclear processing of primary mRNA transcripts (256).
While these reports suggest a direct relationship between RA
level and osteoblast differentiation, other studies have dem-
onstrated a decrease in alkaline phosphatase activity with
both low and high-doses of RA, and decreases in osteocalcin
transcription at higher doses (255,257). Thus, the actual
effect that RA has on osteoblast differentiation and matrix
mineralization remains to be determined.
An extensive literature on the role of steroid hormones
(estrogens and androgens), and growth hormone reviews
their impact on musculoskeletal development and disease
and is not covered in the introductory chapter. In brief, in
experimental situations, reduced estrogen leads to bone loss.
This may be a direct effect on osteoblasts and possibly osteo-
clasts, and may be mediated via PTH and calcitonin.
Androgens are reported to maintain bone mass via receptors
on osteoblasts and the effect of growth hormone on bone is
primarily mediated via insulin-like-growth factor (IGF).
There may however also be a direct effect through growth
hormone receptors on osteoblasts and chondrocytes.
Growth Factors
Transforming Growth Factor-Beta
Initially isolated from “transformed” neoplastic cells in tissue
culture studies. Two “factors” were isolated and named
TGFA and-B. TGFA is not found in bone and is now called
epidermal growth factor. A number of similar compounds
also exist (the TGFB supergene family) including bone mor-
phogenetic proteins (BMP). There are four known receptors
for TGFB. Additionally, there are cross effects from the stim-
ulation of similar receptors. The net effect is to increase DNA

1 Bone Structure, Development and Bone Biology 39
at low concentrations, enhance the synthesis of type I colla-
gen and non collagenous proteins (fibronectin, proteoglycans
etc.), and reduce the activity of alkaline phosphatase. There
is less information on the effect on osteoclasts. There may be
stimulation at low and inhibition at high concentrations. The
latter effect is in association with the production of prosta-
glandins. TGFB is said to have a prominent role in soft tissue
healing, in a cascade fashion. It is released from the degranu-
lation of platelets as well as from macrophages. It may help
in the deactivation of the production of hydrogen peroxide,
inhibit proteolytic enzymes and upregulate the integrin
receptors for extracellular matrix proteins allowing the pro-
duction of abundant granulation tissue (258,259). The cur-
rent hypothesis is that TGFB induces bone formation during
remodeling. Additionally, high amounts are seen in tissues
undergoing endochondral ossification. Experimental evi-
dence suggests that TGFB plays a positive role in intramem-
branous and endochondral bone formation as well as fracture
and wound healing in experimental animals (258,259). The
action of TGFB in bone induction may however be only in
conjunction with other factors such as the BMPs.
Role of TGFb-1 in Osteoblast Development in Vitro
It is well established that the members of the TGFB super-
family play a crucial role in bone development, remodeling,
and disease. However, the various TGFB members have
contradictory functions that have been documented in vitro
and in vivo models. For example, knockout of TGFB-2 has
been shown to result in bone defects, indicating a positive
role for these molecules in bone development (260). However,
transgenic mice over-expressing TGFB-2 under the control
of an osteocalcin promoter displayed an osteoporosis-like
phenotype (261). On the other hand, TGFB-1 has been dem-
onstrated to either stimulate or inhibit bone formation in
vivo, and to differentially modulate distinct osteoblast mark-
ers in vitro. It has been suggested that TGFB-1 enhances the
proliferation and early differentiation of osteoblasts in vitro,
which is characterized by a high rate of collagen synthesis,
but impairs their terminal differentiation based on osteocal-
cin production (a differentiation marker) and mineralization
of culture matrix (262). The TGFB-1 signaling pathway
begins by the binding of TGFB to TGFB specific type I and
type II receptors leading to the phosphorylation of Smads 2
and 3, complex formation with Smad 4, translocation
of Smad 2/3/4 to the nucleus, and transcriptional activation
of specific target genes (263).
TGFB-1 enhances intracellular Ca+2 transport. This is cru-
cial for osteoblast adhesion and early development in cul-
ture, since Ca+2 enhances expression of A5 integrin, which is
important in the formation of focal contact adhesions and
cytoskeletal reorganization. These early events are necessary
for osteoblast adhesion. Thus, they determine the fate of the
osteoblast cell and ultimately affect bone function (264).
TGFB-1 abrogates the steady-state levels of mRNA for
lysyl hydroxylase in human osteoblast-like cells in vitro thus
inhibiting the matrix maturation by affecting the degree of
lysyl hydroxylation in newly synthesized collagen. The
mRNA for lysyl hydroxylase was reduced by one-third under
the influence of TGFB-1. However, the mRNAs for both pro-
collagen I alpha-chains were stimulated by TGFB-1. Thus,
TGFB-1 increases collagen production and decreases its
maturation (265). TGFB-1 also stimulates osteoblast prolif-
eration indirectly through inhibition of p57 cyclin-dependent
kinase inhibitory protein (CKIs), a negative regulator of the
cell cycle acting through the ubiquitin-proteasome pathway
in newly proliferating osteoblast cells (266).
Nishimori and his colleagues, (267) found when the consti-
tutively active form of the TGFB-1 type I receptor was ectopi-
cally expressed in osteoblast cells, the p57 that had been
accumulated by serum starvation and causing the cell-cycle
arrest was rapidly degraded in a manner analogous to TGF-B1
stimulation. Moreover, Smad2 or Smad3 binding to Smad4
enhanced the proteolytic pathway of p57. All of the pathways
mediated by TGFB-1 growth factor suggest its important role
in osteoblast proliferation but not terminal differentiation.
Studies on TGFB-1 null mice have shown that growth
plates, alkaline phosphatase (ALP) activity and collagen
maturity were reduced in the tibiae at all ages compared to
age-matched wild-type (WT) control animals using Fourier
transform-infrared imaging (FTIRI) and immunohistochem-
istry (268). Also analysis of proximal tibial metaphyses
showed significant decreases in the bone mineral content of
the TGFB-1 null mice compared to TGFB-1 wild-type (WT)
control animals. However, no significant differences were
observed in bone mineral density (BMD) between the groups
of mice. Histomorphometry revealed that the width of the
tibial growth plate and the longitudinal growth rate were sig-
nificantly decreased in the TGFB-1 null mice, resulting in
shorter tibia (269).
Bone Morphogenetic Proteins (BMP)
A bone inducing principal was first postulated in 1952 by
Marshall Urist et al. (270) Since then, at least ten proteins
with this property have been extracted from demineralized
bone, the amino acid sequence has been characterized and
synthesized by recombinant DNA technology (271–276).
These have been named bone morphogenetic proteins 1-10.
BMP 3 is also called osteogenin, BMP 4 is also called BMP
2B, BMP 6 is also Vgr-1 and BMP 7 is known as osteogenic
protein-1. This clash of terminology is due to the reclassifi-
cation after characterization. These BMPs can be thought of
as three separate families. One consisting of BMP 2 and 4,

40 F.F. Sa fadi et al .
the other of BMPs 5, 6 and 7 and the last consisting of BMP 3.
These divisions are on basis of homology of structures.
The issue has become complicated by the finding that these
proteins (mostly members of the TGFB super gene family)
are found in several tissue types other than bone (277). In
fact, the developing embryo has areas such as the apical epi-
dermal ridge, which exhibit this property of bone induction,
possibly due to such factors being elaborated.
Urist’s work had suggested that demineralized bone
matrix contains biologic signals to induce endochondral
bone formation when implanted in soft tissues (osteoinduc-
tion). The relative osteoinductive contribution of bone cells
as opposed to matrix in demineralized bone is debated. In the
1970’s Japanese workers identified bone inducing activity in
certain osteosarcoma cell lines. The molecule involved in
this bone induction was later characterized as BMP 4. Certain
human osteosarcoma cell lines such as the Saos-2 have also
shown to produce several BMPs and TGFB. Current recom-
binant technology however, has allowed the synthesis of
these proteins from cDNA, obviating the need of large
amounts of demineralized bone or neoplastic cell lines. Most
of the BMPs (except for BMP 1) are basic proteins of 15
kDa, existing as dimers and belonging to the TGFB super-
family. Disulfide bonds link these dimers. BMP-1 has
recently been shown to be a protease with procollagen as its
substrate (275–276). The synthesis of most BMPs has been
performed by Wozney et al. at the Genetics Institute
(Cambridge, Massachusetts). Their approach has been to
isolate and sequence the cDNA for each BMP using a cDNA
library obtained from the U-20S human osteosarcoma line.
Following cloning, functional regions of the BMP sequences
were transfected into a second mammalian line (Chinese
hamster ovary) for expression and secretion of the mature
BMP molecules. These were then isolated and purified by a
chromatographic method developed by the Genetics Institute
and Genentech (Cambridge, Massachusetts). Osteoinductive
activity was tested using a bioassay employing rats.
Purified BMPs have been used to promote bone repair.
Several trials have shown their efficacy in experimental
models (278–280). Mixtures of BMPs have also been used,
and shown to be more effective than comparable doses of
single homodimeric BMP (281).
Bone Morphogenetic Proteins and Osteogenesis
Bone morphogenetic proteins (BMPs) are osteotrophic fac-
tors as well as members of the TGFB superfamily. The activ-
ity of BMPs was first identified in the 1960s (282), but the
proteins responsible for bone induction remained unknown
until the purification and sequence of bovine BMP-3 (osteo-
genin) and cloning of human BMP-2 and BMP-4 in the late
1980s (274,283,284).
BMP-2 induces gene expression and synthesis of osteo-
blast differentiation markers, alkaline phosphatase and
osteocalcin, in pluripotent and preosteoblast cells. BMP-2
exposure for a short duration is sufficient to induce cell dif-
ferentiation (285). Functions of bone morphogenetic proteins,
such as BMP-2, are initiated by signaling through specific
type I and type II serine/ threonine kinase receptors. It was
previously reported that BMP receptor type IB (BMPR-IB)
plays an essential and specific role in osteoblast commitment
and differentiation (285). Smad1, 5, and 8 are substrates for
BMP receptor I (BMPR-I) and mediators of the BMP signals
that inhibit myogenic differentiation and induce osteoblast
differentiation, in the mesenchymal C2C12 cell line (286).
Studies from transgenic and knockout mice and from animals
and humans with naturally occurring mutations in BMPs and
related genes have shown that BMP signaling plays critical
roles in heart, neural and cartilage development. BMPs also
play an important role in postnatal bone formation (287).
BMP-2 is known to induce osteoblast differentiation by
inducing Runx2. a global regulator for osteogenesis. Runx2
co-operates with BMP-2-induced Smad proteins to stimulate
osteoblast differentiation (288). BMP-2 receptor activated
Smad proteins induce Runx2; however, Smad does not
directly induce Runx2 expression. The mitogen-activated
protein kinase/p38 (MAPK/p38) cascade is also involved in
the induction of Runx2 by BMP-2 (289). In addition, BMP-2
induces osteoblast differentiation through activation of an
endogenous B-catenin signaling pathway thus implicating
B-catenin in early steps of BMP-2 mediated osteoblast dif-
ferentiation (290). In support, ectopic expression of stabi-
lized B-catenin in the murine embryonic mesenchymal
C3H10T1/2 cell line or activation of endogenous B-catenin
signaling with lithium chloride induced expression of
alkaline phosphatase mRNA and protein (an early osteoblast
differentiation marker). However, unlike BMP-2 protein,
stabilized B-catenin does not induce osteocalcin gene
expression (a late osteoblast differentiation marker) (291).
Insulin like Growth Factors: IGF I and II
Insulin-like growth factors are produced by many cell types
including osteoblasts and chondrocytes. They act via receptors
to promote proliferation, differentiation and matrix produc-
tion of bone and cartilage. The action of growth hormone is
closely linked with the IGFs. It is thought that growth
hormone binding with specific receptors in target tissues stim-
ulates the production of IGF-1. This, in turn, may have endo-
crine, paracrine and autocrine effects. IGF-1 is transported
via carrier proteins, such as IGF-binding proteins and
IGFBPs of which, IGFBP-3 is the most important. Deficiency
of IGF or IGFBP-3 may be responsible for certain kinds of
dwarfism, such as Laron type dwarfism.

1 Bone Structure, Development and Bone Biology 41
Other Growth Factors
Growth factors discussed earlier include Epithelial Growth
Factor, Acid and Basic Fibroblast Growth Factors and
Platelet Derived Growth Factors A and B (PDGFA and
PDGFB). PDGFs, in particular, are potent mitogens of osteo-
blasts in vitro and have a chemotaxic effect on them. PDGFs
are thought to be particularly important in bone remodeling.
They are heterodimers of A and B chains, and function via
specific receptors. Mutations in fibroblast growth factors are
thought to play a role in certain kinds of skeletal deformities,
including achondroplasia, Apert’s syndrome, Cruzon syn-
drome, Pfeiffer syndrome, and Jackson-Weiss syndrome.
Cytokines: Prostaglandins and Interleukins
Prostaglandins: Prostaglandins have multiple effects on
bone cells, and sometimes opposite effects in different spe-
cies. Their role is therefore difficult to discern. They are
powerful bone resorbers in certain culture studies, yet they
are potent anabolic (bone forming) agents when adminis-
tered in vivo. (especially true of the E series). Prostaglandins
are produced by monocytes under appropriate stimuli. It is
possible that some effects of interleukins are mediated by
prostaglandins.
Interleukin 6 (IL-6): This cytokine is produced by many cell
types, including osteoblasts and bone marrow stromal cells.
Bone cells produce IL-6 in response to PTH, Vitamin D3,
TGFB, IL-1 and TNFA, to name a few. Human osteoclas-
toma cells respond to this cytokine; however, it is still unclear
whether normal mature osteoclasts respond to IL-6. It is
known though that IL-6 has a pathogenetic role in diseases
such as multiple myeloma, Paget’s disease, rheumatoid
arthritis and Gorham’s disease (vanishing bone disease).
Experimentally, estrogens and androgens inhibit the produc-
tion of IL-6 by osteoblasts. Additionally, there is evidence to
suggest that osteoclastic activity may be inhibited by anti-
IL-6 antibodies (32).
Mechanosensory Systems and Stretch
Studies (Wolff’s Law)
Wolff’s Law - Every change in form and function of bones,
is followed by changes in the internal architecture and exter-
nal conformation, in strict accordance with mathematical
laws (Julius Wolff, 1882).
Wolff’s law has been confirmed by experimental studies.
However, only recently have studies been performed to
investigate the basis of this law at a molecular level. It is
clear that mechanical forces effect skeleton morphology. For
example, individuals who lift weights tend to develop bigger
and stronger bones. If use of a limb is stopped, it undergoes
“disuse” osteoporosis. Children with malunited limb frac-
tures frequently remodel into almost normal appearing bones.
If, though, they are unable to bear weight or use the limb
(such as with poliomyelitis), then the limbs stay malunited.
Paraplegics or quadriplegics with a spastic form of paresis
often have exuberant callus formation. In contrast, patients
with flaccid paresis fail to develop such an exaggerated
response. Weightlessness in space causes rapid decrease in
bone mass reflecting the need for constant force in maintain-
ing skeletal bone.
All these examples illustrate the close link of mechanical
forces with skeletal response and bone formation. What is
still under investigation, however, is how these mechanical
forces are translated into cellular events. It is likely, that
signaling mechanisms, such as electricity or chemical mes-
sengers, such as certain cytokines, mediate these responses.
Computer controlled membranes holding tissue cultures
of osteoblasts and fibroblasts have been used to alter the
amounts of “stretch” provided to the cells. These studies have
suggested that there is an altered metabolism and DNA syn-
thesis under conditions of load. It has been suggested
(292–294) that there may be two components to this system:
The Cell Network: This consists of osteocytes and their
processes in communication with surface cells. Stretch sen-
sitive ion channels are thought to exist on osteoblasts and
fibroblasts.
The Mineralized Matrix: Stream generated potentials are
created when fluid flowing through the matrix carries along
a species of ion (in the presence of another species attached
to the matrix).
These two mechanisms may be responsible for the signal
for altered cellular metabolism observed. A “piezo-electric”
effect as a result of compression of the hydroxyapatite crys-
tal is also theoretically possible, but unlikely to be responsi-
ble for the coupling of mechanical-electric phenomena in
bone.
The mechanism by which strain induces osteoblast prolif-
eration in strain studies has thought to be mediated by the
inositol 1,4,5 triphosphate system (295–296). Inhibition of
phospholipase C (by neomycin) blocks inositol triphosphate
production and subsequent proliferation. Additional signal-
ing pathways (such as by cyclic AMP) may co-exist.
There is a hypothesis that cells maintain a basal equilib-
rium stress state that is a function of the number and quality
of focal adhesions, the polymerization of the cytoskeleton
and the amount of extrinsic applied mechanical deformation
(297). A load stimulus detected by a mechano-electrochemi-
cal sensory system (including stretch sensitive ion-channels,
integrin cytoskeletal machinery, and load-conformational
sensitive receptor tyrosine kinase) activates G proteins,
induces second messengers, and activates another kinase
cascade to allow a respose.

42 F. F. S afad i et al.
It is also possible that integrins serve as important compo-
nents of the mechanical sensory system. The RGD sequence of
matrix proteins undergoes conformational changes with ten-
sion. This allows matrix tension to be “communicated” to bone
cells. Signaling molecules such as nitric oxide (NO) have also
been postulated in this process (298). Some studies have
suggested that molecules such as osteopontin might be interme-
diary in this signalling process. For example, the binding of the
AvB3integrin with the RGD sequence of osteopontin might trig-
ger osteoclastic resorption (299).
References
1. Cowin SC. Tissue growth and remodeling. Annu Rev Biomed Eng
2004;6:77–107.
2. Havers. http://www.biology-online.org/dictionary/Haversian_
system. 1691.
3. Brown JH, DeLuca SA. Growth plate injuries: Salter-Harris clas-
sification. Am Fam Physician 1992;46(4):1180–4.
4. Duncan CP, Shim SSJ. Edouard Samson Address. The autonomic
nerve supply of bone. An experimental study of the intraosseous
adrenergic nervi vasorum in the rabbit. J Bone Joint Surg Br
1977;59(3):323–30.
5. Bliziotes M, Gunness M, Eshleman A, Wiren K. The role of dop-
amine and serotonin in regulating bone mass and strength: studies
on dopamine and serotonin transporter null mice. J Musculoskelet
Neuronal Interact 2002;2(3):291–5.
6. Tam J, Ofek O, Fride E, et al. Involvement of neuronal cannabinoid
receptor CB1 in regulation of bone mass and bone remodeling. Mol
Pharmacol 2006;70(3):786–92.
7. Idris AI, van’t Hof RJ, Greig IR, et al. Regulation of bone mass,
bone loss and osteoclast activity by cannabinoid receptors. Nat
Med 2005;11(7):774–9.
8. Landis WJ, Song MJ, Leith A, McEwen L, McEwen BF. Mineral
and organic matrix interaction in normally calcifying tendon visu-
alized in three dimensions by high-voltage electron microscopic
tomography and graphic image reconstruction. J Struct Biol
1993;110(1):39–54.
9. Hohling HJ, Barckhaus RH, Drefting ER, Quint P, Athoff J.
Quantitative electron microscopy of the early stages of cartilage
mineralization. Metab Bone Dis Res 1978;1:109–14.
10. Khan SN, Bostrom MP, Lane JM. Bone growth factors. Orthop
Clin North Am 2000;31(3):375–88.
11. Rodan GA. Control of bone formation and resorption: biological
and clinical perspective. J Cell Biochem Suppl 1998;30/31:55–61.
12. Otto F, Thornell AP, Crompton T, et al. Cbfa1, a candidate gene for
cleidocranial dysplasia syndrome, is essential for osteoblast dif-
ferentiation and bone development. Cell 1997;89(5):765–71.
13. Nakashima K, Zhou X, Kunkel G, et al. The novel zinc finger-
containing transcription factor osterix is required for osteoblast dif-
ferentiation and bone formation. Cell 2002;108(1):17–29.
14. Komori T, Yagi H, Nomura S, et al. Targeted disruption of Cbfa1
results in a complete lack of bone formation owing to maturational
arrest of osteoblasts. Cell 1997;89(5):755–64.
15. Ducy P. Cbfa1: a molecular switch in osteoblast biology. Dev Dyn
2000;219(4):461–71.
16. Ducy P, Karsenty G. The family of bone morphogenetic proteins.
Kidney Int 2000;57(6):2207–14.
17. Ducy P, Amling M, Takeda S, et al. Leptin inhibits bone formation
through a hypothalamic relay: a central control of bone mass. Cell
2000;100(2):197–207.
18. Aubin JE, Triffit JT. Mesenchymal Stem Cells and Osteoblast
Differentiation In: J. P. Bilezikian, L. G. Raisz, G. A. Rodan, Eds.
Principles of Bone Biology. 2nd ed. San Diego: Academic Press;
2002:59–81.
19. Tezuka K, Takeshita S, Hakeda Y, Kumegawa M, Kikuno R,
Hashimoto-Gotoh T. Isolation of mouse and human cDNA clones
encoding a protein expressed specifically in osteoblasts and brain
tissues. Biochem Biophys Res Commun 1990;173(1):246–51.
20. Raulo E, Chernousov MA, Carey DJ, Nolo R, Rauvala H. Isolation
of a neuronal cell surface receptor of heparin binding growth-
associated molecule (HB-GAM). Identification as N-syndecan
(syndecan-3). J Biol Chem 1994;269(17):12999–3004.
21. Lorenz-Depiereux B, Bastepe M, Benet-Pages A, et al. DMP1
mutati