Cell Lines and Primary Cell Cultures in The Study of Bone Cell Biology PDF

Molecular and Cellular Endocrinology 228 (2004) 79102
Cell lines and primary cell cultures in the study of bone cell biology
Vicky Kartsogiannisb , Kong Wah Nga,
a
Department of Endocrinology and Diabetes, St. Vincents Hospital, 4th Floor, Daly Wing, 35 Victoria Parade, Fitzroy, Vic. 3065, Australia
b St. Vincents Institute 9 Princes Street, Fitzroy, Vic. 3065, Australia
Received 8 April 2003; accepted 12 June 2003
Abstract
Bone is a metabolically active and highly organized tissue consisting of a mineral phase of hydroxyapatite and amorphous calcium phosphate
crystals deposited in an organic matrix. Bone has two main functions. It forms a rigid skeleton and has a central role in calcium and phosphate
homeostasis. The major cell types of bone are osteoblasts, osteoclasts and chondrocytes. In the laboratory, primary cultures or cell lines
established from each of these different cell types provide valuable information about the processes of skeletal development, bone formation
and bone resorption, leading ultimately, to the formulation of new forms of treatment for common bone diseases such as osteoporosis.
2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cell lines; Primary cell cultures; Bone cell biology
1. Introduction
Bone has several major functions. It forms a rigid skeleton
to provide a framework for the body, support for soft tissues,
points of attachment for skeletal muscles, protection for internal organs, housing for bone marrow as well as a central role
in mineral homeostasis, principally of calcium and phosphate
ions, but also of sodium and magnesium.
Bone is a dynamic tissue that is constantly remodeled
throughout life. During fetal development, most of the skeleton develops from cartilage anlagen which is eventually resorbed and replaced with bone by a process termed endochondral ossification. In contrast, bones which form the calvaria,
mandible and maxilla are developed from mesenchyme by
a process termed intramembranous ossification. Bone modeling is the process associated with growth and reshaping of
bones in childhood and adolescence. In bone modeling, longitudinal growth of long bones depends on proliferation and differentiation of cartilage cells at the growth plate while growth
in width and thickness is accomplished by formation of bone
at the periosteal surface with resorption at the endosteal surface. In adults, after the epiphyses close, growth in length
Corresponding author. Tel.: +61 3 9288 3568; fax: +61 3 9288 3590.
E-mail address: kongwn@unimelb.edu.au (K.W. Ng).
0303-7207/$ see front matter 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mce.2003.06.002
and endochondral bone formation cease but remodeling of

bone continues. Remodeling constitutes the lifelong renewal
process whereby the mechanical integrity of the skeleton is
preserved. It implies the continuous removal of bone (bone resorption) followed by synthesis of new bone matrix and subsequent mineralization (bone formation). The maintenance
of normal, healthy bone requires the coupling of bone formation to bone resorption, with intercellular communication
between osteoblasts and osteoclasts integral to the achievement of a balance between the two processes. Furthermore,
bone remodeling is an integral part of the calcium homeostatic system (Eriksen et al., 1993) that also involves the
parathyroid glands, intestinal system and the kidneys.
Many aspects of the processes described above can be
investigated in the laboratory using primarily cell culture.
The major cell types are the bone-forming osteoblasts, boneresorbing osteoclasts and cartilage-forming chondrocytes. A
thorough understanding of the factors regulating the differentiation of each of these cell types, the mechanisms by which
regulatory factors influence their function, and the manner in
which these cells communicate and interact with each other, is
central to the design of rational therapeutic strategies to treat
bone diseases such as osteoporosis. This review will focus on
cell lines that are established in the laboratory from these different cell types. While much information has been derived
80
V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79102
from established cell lines, particularly in osteoblast biology,

a substantial amount of work is nonetheless still being carried
out with primary cultures of osteoblasts, chondrocytes and osteoclasts, and attention will be drawn to these, where relevant.
In bone cell biology, cell cultures are used mainly to examine:
Regulation of expression of phenotypic characteristics typical of osteoblasts, chondrocytes and osteoclasts.
Regulation of differentiation of relatively undifferentiated
mesenchymal cells along different lineages, for example,
muscle, osteoblasts, chondrocytes and adipocytes.
Signaling pathways relevant to osteoblast, osteoclast and
chondrocyte functions.
Effects of over-expression and under-expression of particular gene products on cell function.
In vitro bone formation/mineralization.
Interactions between osteoblasts and osteoclasts, particularly in the regulation of osteoclast formation in vitro.
2. Osteoblasts
2.1. Osteoblast ontogeny
Osteogenic cells arise from pluripotential mesenchymal
stem cells. These stem cells have the capacity to differentiate
into lineages other than osteoblasts, including those of chondroblasts, fibroblasts, adipocytes, and myoblasts (reviewed
in Nijweide et al., 1986; Friedenstein et al., 1987; Aubin et
al., 1995). By analogy with hematopoietic cell differentiation, each of these differentiation lineages is thought to originate from a different committed progenitor, which for the
osteogenic lineage is called the osteoprogenitor.
Osteodifferentiation progresses via a number of progenitor and precursor stages to the mature osteoblast, as illustrated in Fig. 1. Pluripotent mesenchymal cell lines can be
used to study this process which is regulated by a number of
regulatory molecules such as members of the transforming
growth factor beta (TGF) superfamily, including the bone
morphogenetic proteins (BMPs). When murine C2C12 mesenchymal precursor cells are treated with TGF1, terminal
differentiation into myotubes is blocked. Treatment with bone
morphogenetic protein 2 (BMP-2) similarly blocks myogenic
differentiation of C2C12 cells but induces osteoblast differentiation (Lee et al., 2000). Further evidence implicating a
role of the BMP receptors (type IA and IB) in both the specification and differentiation of osteoblastic and adipocytic lineages comes from recent studies using a well-characterized
clonal cell line 2T3, derived from mouse calvariae (Chen
et al., 1998). In other studies (Spinella-Jaegle et al., 2001),
the proteins of the hedgehog (Hh) family which are known
to regulate various aspects of normal limb patterning, have
also been shown to influence the osteoblastic and adipocytic
commitment/differentiation of mesenchymal stem cells. For
example, recombinant N-terminal sonic hedgehog (N-Shh)
abolishes adipocytic differentiation of murine mesenchymal
Fig. 1. Origin of cells of the osteoblast and chondrocyte lineages (modified

from Nijweide et al., 1986).
stem cells C3H10T1/2 both in the presence and absence of

BMP-2, while committing these pluripotent cells into the osteoblastic lineage. Treatment of C3H10T1/2 cells with BMP7 has also been shown to induce both chondrogenesis and
osteogenesis (Gerstenfeld et al., 2002) while treatment with
the potent DNA demethylating agent 5-azacytidine has previously been known to induce differentiation to myoblasts,
adipocytes and chondrocytes (Taylor and Jones, 1979).
A variety of cell culture models and other tools such
as the use of monoclonal antibodies have been employed
by researchers to track the various stages of osteogenesis.
The murine IgM monoclonal antibody STRO-1 recognizes
a cell surface antigen expressed by stromal cells in human
bone marrow and is used to identify clonogenic bone marrow stromal cell progenitors (fibroblast colony-forming units
[CFU-F] (Simmons and Torok-Storb, 1991). Gronthos and
colleagues used dual-color fluorescence-activated cell sorting to identify cells expressing STRO-1 and ALP in primary
cultures of normal human bone cells (NHMC). They showed
that preosteoblastic STRO-1+/ALP cells did not express
bone-related markers such as bone sialoprotein, osteopontin,
and parathyroid hormone receptor and had a reduced ability
to form a mineralized bone matrix over time. The majority
of NHBCs representing fully differentiated osteoblasts, expressed STRO-1/ALP+ and STRO-1/ALP phenotypes,
while the STRO-1+/ALP+ subset represented an intermediate preosteoblastic stage of development. All STRO-1/ALP
NHBC subsets expressed the transcription factor cbfa-1, confirming that they were committed osteogenic cells (Gronthos
et al., 1999). A survey of human osteosarcoma cell lines revealed that STRO-1 was expressed by MG-63 but not SaOS2. Among murine cell lines tested, expression of STRO-1 was
detected in the bipotential line BMS-2 but not the committed osteoblast precursor MC3T3-E1 (Stewart et al., 1999).
Human fetal bone marrow STRO-1 expressing stromal cells

with weak ALP activity could be induced by dexamethasone and 1,25-dihydroxyvitamin D3 to increase their expression of ALP activity. Furthermore, when cultured in threedimensional aggregates, osteogenesis occurred along with
the expression of osteocalcin, a marker of differentiated osteoblasts (Oyajobi et al., 1999). Thus, a hierarchy of bone
cell development in vitro could be identified to facilitate the
study of bone cell differentiation and function, using STRO1 expression to identify less well-differentiated cells of the
osteoblast lineage.
Important in vitro cell culture models employed to further
study the various stages of osteogenesis, include clonal cell
lines derived either from bone tumors (ROS 17/2.8 or UMR
106; human MG-63 or SaOS-2) or from primary bone cell
cultures (MC3T3-E1, UMR 201 and RCJ cell lines). These
cell lines will be discussed in detail later. MC3T3-E1 and
UMR 201 cells have been described as representatives of preosteoblastic cells while ROS 17/2.8 as more differentiated
osteoblasts based on the profile of expression of osteoblast
phenotypic features. Extensive in vitro analyses of the various
cell lines have provided evidence for considerable flexibility
in the repertoire of osteoblast-associated genes expressed at
multiple differentiation stages as osteoprogenitors mature to
form functional osteoblasts.
2.2. Morphology and function
Mature osteoblast cells constitute a lineage of highly differentiated cells which differ substantially in their properties
at different stages of development. Thus, depending on the
stage of functional differentiation, they are represented by a
distinctive phenotype, morphology and biosynthetic activity.
Active osteoblasts are plump, cuboidal, mononuclear cells
lying on the matrix which they have synthesized. Osteocytes
are mature osteoblasts which have become trapped within the
bone matrix and are responsible for its maintenance. Bone
lining cells are flat, elongated cells that cover bone surfaces
that are undergoing neither bone formation nor resorption
(Miller and Jee, 1987). Inactive osteoblasts are much thinner and more elongated cells, yet they are morphologically
indistinguishable from the lining cells.
Actively synthesizing osteoblasts contain an extensive,
bone oriented secretory organelle apparatus, a large Golgi
complex near the nucleus and a high mitochondrial content;
all reflecting the capacity of osteoblasts for their primary
function of protein synthesis (Doty and Schofield, 1976). Osteoblasts are rich in alkaline phosphatase enzyme (Heath and
Reynolds, 1990). They synthesize and secrete type I collagen, glycoproteins such as osteopontin (rich in sialic acid;
N- and O-linked oligosaccharides) and osteocalcin (contains
glutamic and aspartic acid residues), cytokines and growth
factors into a region of unmineralized matrix (osteoid) between the cell body and the mineralized matrix. Osteoblasts
have receptors and responses to parathyroid hormone (PTH),
1,25-dihydroxyvitamin D3 [1,25(OH)2D3], prostaglandins
81
Table 1
Systemic agents that influence osteoblast function
Agent
Endocrine hormones
Parathyroid hormone (PTH)
1,25(OH)2D3
Growth hormone (GH)
Glucocorticoid hormones
Gonadal steroids (estrogen,
testosterone)
Insulin
Retinoids
Other systemic agents
Epidermal growth factor (EGF)
Transforming growth factor
(TGF)
Prostaglandins (PGs)
Parathyroid hormone-related
protein (PTHrP)
Calcitonin gene-related peptide
(CGRP)
References
Parsons (1976), Tam et al. (1982)
Chen et al. (1983a), Narbaitz et al.
(1983)
Stracke et al. (1984), Barnard et al.
(1991)
Dietrich et al. (1979), Canalis
(1983)
Komm et al. (1988)
Raisz and Kream (1983), Kream et
al. (1985)
Ng et al. (1985, 1988), Zhou et al.
(1991)
Ng et al. (1983)
Ibbotson et al. (1985)
Raisz and Martin (1984)
Yang and Stewart (1996)
Reid and Cornish (1996)
(PGs), epidermal and transforming growth factors (EGFs,

TGFs) and tumor necrosis factors (TNFs) (Martin et al., 1988;
Heath and Reynolds, 1990). In addition, osteoblasts produce
a specific membrane-bound molecule known as receptor activator of NFB ligand) due to its ability to induce nuclear factor B) (Anderson et al., 1997a, 1997b). It is responsible for
programming osteoclast differentiation and also functions as
a dendritic cell survival factor (Anderson et al., 1997a, 1997b;
Wong et al., 1997a, 1997b; Lacey et al., 1998; Yasuda et al.,
1998).
Osteoblast function is under the regulatory control of various systemic and local (paracrine/autocrine) factors. These
are summarized in Tables 1 and 2.
2.3. Osteoblast cell culture methods
Cell culture techniques combined with basic molecular genetic techniques have become highly popular and widespread
tools that are routinely applied to bone cells (Peck et al.,
1964). Some of the basic experimental approaches used in osteoblastic cell culture, including system design, specific cell
culture systems and various biochemical and morphological
assays defining osteoblasts in cell culture, are discussed in
depth in the excellent review by Majeska (1996).
In general, most systems currently in use share routine
basic culture environments and methods of maintenance.
Osteoblast cell lines are routinely grown in monolayer
culture in standard tissue culture flasks in alpha-modified
Eagles minimal essential medium (MEM), supplemented
with 10% fetal calf serum (FCS) or fetal bovine serum
(FBS), 0.2% sodium bicarbonate, 16 mM HEPES (N2-hydroxyethylpiperazine-N -2-ethanesulfonic acid), 1 mg/l
82
Table 2
Local agents that influence osteoblast function
Agent
References
Paracrine factors
Parathyroid hormone-related protein (PTHrP)
Transforming growth factor (TGF-1, -2, and -3)
Fibroblast growth factors (FGF 1 and 2)
Insulin-like growth factor (IGF I and II)
Platelet-derived growth factor (PDGF)
Bone morphogenetic proteins (BMPs 27)
Tumor necrosis factors (TNFs and )
Interleukin-1 (IL-1)
Prostaglandin E2 (PGE2)
Calcitonin gene-related peptide (CGRP)
Leukemia inhibitory factor (LIF)
Vasoactive intestinal peptide (VIP)
Moseley and Martin (1996)

Centrella et al. (1987), Hock et al. (1990)
Rodan et al. (1987a), Canalis et al. (1988)
Schoenle et al. (1982)
Canalis (1981)
Hammonds et al. (1990), Wozney et al. (1988)
Bertolini et al. (1986), Ng et al. (1989a, 1989b)
Canalis (1986), Mundy (1993)
Mundy (1993)
Chyun and Raisz (1984)
Michelangeli et al. (1989)
Allan et al. (1990)
Hohmann et al. (1986)
Autocrine factors
Transforming growth factor (TGF-1, -2, and -3)
Fibroblast growth factors (FGF 1 and 2)
Insulin-like growth factor (IGF I and II)
Platelet-derived growth factor (PDGF)
Centrella et al. (1987), Hock et al. (1990)

Rodan et al. (1987a), Canalis et al. (1988)
Schoenle et al. (1982)
Canalis (1981)
minocycline and 80 mg/l gentamicin. Incubation conditions

are usually maintained at 37 C in a humidified atmosphere
with 5% CO2, although there are some cell lines that require
special growth conditions. Culture medium is changed every 48 h. For routine maintenance, cells are subcultured at
confluence whereby medium is removed and cells detached
from the flask by enzymatic digestion. A 10-min incubation
at 37 C in 0.025% trypsin in versene (140 mM NaCl, 2 mM
KCl, 1 mM KH2 PO4 , 8 mM Na2 HPO4 , 0.05 M EDTA) is usually sufficient for cells to lift off. The cell suspension is poured
into a sterile 20ml tube and cells are pelleted at 1500 rpm for
5 min in a centrifuge. Cells are resuspended in 10 ml of fresh
medium, and after separating them by passage through a 21
gauge needle on a syringe, the appropriate volume of cells
is pipetted into a fresh flask containing medium. Routinely,
cells are usually passaged weekly at 1:20.
When working with confluent primary bone cell cultures,
a more rigid enzymatic digestion step may be required. These
cultures are washed twice in warm phosphate-buffered saline
(PBS) pH 7.4 and then digested in a solution of collagenase
and dispase for 90 min at 37 C. Cell suspensions are subsequently washed twice in growth medium supplemented with
5% FCS before being passed through a cell strainer to obtain
single cell suspensions (Gronthos et al., 1999).
2.4. Model systems of osteoblast differentiation and
gene expression
A variety of cultured cell systems have been successfully
used to study the pathways of osteoblast differentiation with
each model system offering unique advantages towards increasing our understanding of bone development, differentiation, gene expression and responses to hormones and local
factors. Commonly employed model systems include organ
cultures, primary cultures of osteoblastic cells derived from
fetal calvaria or subperiosteal fetal long bones, established

clonal cell lines from cells isolated from bone tumors (typically osteosarcoma), non-transformed cell lines, experimentally immortalized cell lines, and bone marrow cultures. An
extensive list of some of the commonly used osteoblast cell
lines is outlined in Stewart et al. (1999).
2.4.1. Primary cultures of bone cells and organ cultures
Some of the in vitro models, especially the calvariaderived primary cell cultures, undergo changes which mimic
osteoblastic differentiation in vivo, thus providing insights
into the stage-wise regulation of gene expression in osteoblastic cells. Observations made from these systems have been
extrapolated to adult bone in vivo to develop concepts of the
osteoblast phenotype. A summary of the properties associated with this phenotype is provided in Table 3. It must be emphasized at this point, that depending on the cell system and
culture conditions used, temporal aspects of the osteoblast
differentiation process and ability to ascertain a sequential
expression of markers varies markedly and is sometimes contradictory (Aubin et al., 1993, 1995). Many of the systems
mentioned above have limitations such as the inherent instability and the possibility of aberrant behaviour associated
with many of the clonal cell lines, or in the case of primary
cultures, the fact that they contain a heterogeneous mixture
of osteoblastic cells at different stages of differentiation and
cells of other lineages including fibroblastic cells (Aubin et
al., 1993; Liu et al., 1994). Nevertheless, differentiating cultures from both the extensively studied rat calvaria and rat
bone marrow stromal systems and various other bone culture
models when plated appropriately, go through a consistent
series of differentiating events culminating in mineralized
nodule formation.
In calvarial-derived cultures grown to post-confluence in
the presence of serum and ascorbic acid, cell proliferation oc-
83
Table 3
Osteoblast phenotypic characteristics (modified from Martin et al., 1993)
Proteins
Receptors and/or responses
Alkaline phosphatase (ALP)

Type I collagen (COL I)
Osteocalcin
Osteopontin (OP)
Osteonectin
Bone proteoglycan I (biglycan)
Bone proteoglycan II (decorin)
Thrombospondin
Fibronectin (FN)
Vitronectin (VN)
Bone morphogenetic proteins (BMPs)
Transforming growth factor (TGF)
Fibroblast growth factors (FGFs)
Insulin-like growth factor I (IGF I and II)
Ciliary neurotrophic factor (CNTF)
Tumor necrosis factor (TNF)
Colony-stimulating factors (e.g. CSF-1)
Prostanoids
Noggin
Receptor activator of NFB ligand
(RANKL)
Osteoprotegerin (OPG)
Osteoclast inhibitory lectin (OCIL)
Notch 2
Jagged 2
Vascular endothelial growth factors
(VEGF)
Phosphate-regulating gene with homologies to endopeptidases
on the X chromosome (Phex)
Amylin
Oncostatin M (OSM)
Cardiotrophin-1 (CT-1)
Homeobox (Hox)
Matrix metalloproteinase protein-13
(MMP-13)
Cyclo-oxygenase 2
Cadherins
Parathyroid hormone (PTH)

Parathyroid hormone-related protein (PTHrP)
Prostanoids
1,25-Dihydroxyvitamin D3 (1,25(OH)2D3)
Retinoids
Epidermal growth factor (EGF)
Bone morphogenetic proteins (BMPs)
Glucocorticoids
Insulin-like growth factor I (IGF I)
Insulin-like growth factor II (IGF II)
Insulin
Inhibin
Activin
Estrogen receptors and (ER; ER)
Atrial naturetic peptide (ATP)
Calcitonin gene-related peptide (CGRP)
Calcium sensing receptor (CSR)
Vasoactive intestinal peptide (VIP)
Vascular endothelial growth factor receptors
(VEGFR)
Fibroblast growth factor receptor-2 (FGFR2)
Growth hormone (GH)
Connective tissue growth factor-like
(CTGF-L)
Phosphate-regulating gene with homologies to endopeptidases
on the X chromosome (Phex)
Intercellular adhesion molecule (ICAM)
Soluble low density lipoprotein receptor-related protein (SLRPs)
curs in scattered foci, which develop into multilayered structures called nodules (Bellows and Aubin, 1989). These
nodules consist of a top layer of osteoblast-like cells which
stain intensely for alkaline phosphatase, sitting underneath
an osteoid layer containing collagen fibrils (Bellows et al.,
1987; Bhargava et al., 1988).
The process of bone nodule formation as studied in rat
calvaria populations has been subdivided into three developmental stages: proliferation, extracellular matrix development and maturation, and matrix mineralization. Characteristic changes in genes associated with proliferative and cell
cycling activity and those associated with specific osteoblast
activities are observed throughout all stages (reviewed in
Aubin et al., 1993, 1995; Lian and Stein, 1995; Stein and
Lian, 1993). In the first phase, active proliferation is reflected
by mitotic activity with expression of genes associated with
cell cycling (e.g., histone) and growth (e.g., proto-oncogenes
TGF- inducible early gene (TIEG)

Catenins
STRO-1
c-myc, c-fos, and c-jun) (McCabe et al., 1995). Several other

genes associated with formation of the extracellular matrix
(type I collagen, fibronectin, and TGF) are also actively expressed (Aronow et al., 1990; Owen et al., 1991) and then
gradually down-regulated with collagen mRNA being maintained at a low basal level during subsequent stages of osteoblast differentiation.
Immediately after the down-regulation of proliferation,
proteins associated with the osteoblast phenotype are detected such as alkaline phosphatase. With progression into
the mineralization stage, all cells become positive for
alkaline phosphatase. Other osteoblast-related genes such as
bone sialoprotein (BSP) (Nagata et al., 1991), osteopontin
(OP), and osteocalcin (Owen et al., 1990) are induced following the onset of mineralization. OP is expressed during
the period of active proliferation (at 25% of maximal levels),
decreases post-proliferatively, and then is induced again at the
84
onset of mineralization achieving peak levels of expression.

Consistent with high levels of osteopontin expression later
in the osteoblast developmental sequence are the calcium
binding properties of this acidic glycoprotein containing Ophosphoserine (Glimcher, 1989). The Vitamin K-dependent
protein, osteocalcin (Lian and Friedman, 1978), in contrast
to OP, is mainly expressed post-proliferatively with the onset
of nodule formation. Late expression of osteocalcin in the
osteoblast development sequence, suggests that it is a marker
of the mature osteoblast, which is consistent with a possible
role for the synthesis and binding of osteocalcin to mineral
in the coupling of bone formation to resorption.
A similar temporal pattern of gene expression reflecting stages of progressive bone formation has been observed
in cultured normal diploid osteoblasts derived from chick
(Gerstenfeld et al., 1987; Shalhoub et al., 1989; Aronow et
al., 1990), bovine (Ibaraki et al., 1992), and in cell lines of
human (Keeting et al., 1992) and mouse (Quarles et al., 1992)
origin. Furthermore, it has also been possible to discern cells
of the osteoblast lineage at different stages of differentiation
in situ and note the pattern of expression of osteoblast-related
markers in relation to their location in bone (Yoon et al., 1987;
Nomura et al., 1988; Sandberg et al., 1988; Lyons et al., 1989;
Weinreb et al., 1990; Zhou et al., 1994).
In addition to the expression of matrix components, the
osteoblast differentiation process is both determined and reflected by the expression of certain hormones and cytokines.
For instance, the bone morphogenetic proteins (BMPs) are
expressed in early osteoblasts and in the late mineralization
phase (Harris et al., 1994a, 1994b), and the expression of the
PTH receptor appears to correlate with increasing differentiation (reviewed in Aubin et al., 1995; Suda et al., 1996; Bos
et al., 1996).
To date several bone organ cultures and primary osteoblast
cultures have been widely used to study osteoblast differentiation (Owen et al., 1990) as well as the osteoblastic response to growth factors (Centrella et al., 1987; Guenther
et al., 1988), and hormones (Wong and Cohn, 1975). The
major bone culture systems use long bones or calvaria from
fetal/neonatal rats and mice. Populations of osteoblast-like
cells can be isolated by using enzymatic digestion (Peck et
al., 1964) or by mechanical methods (Ecarot-Charrier et al.,
1983).
Although the available models provide important information on the fundamental processes involved in bone formation, it is important to note that the source and the age of the
bone, the medium, and culture system, all affect the sensitivity of bones to hormones (Stern and Krieger, 1983; Soskoline
et al., 1986). In addition, the variable proportion of fibroblasts and osteoblast cells at different stages of differentiation provides a further limitation to this technique.
2.4.2. Osteosarcoma cell lines
The most widely used osteosarcoma cell lines are the
UMR 106 and the ROS 17/2 which were established during
the late 1970s by the Martin and Rodan laboratories. UMR

106 is a cell line derived from a transplantable rat 32 P-induced
malignant osteogenic sarcoma (Martin et al., 1976; Partridge
et al., 1983). The cell line has been extensively characterized
with properties of enrichment in ALP activity, type I collagen
production, adenylate cyclase responsiveness to PTH, PGE2,
ability to mineralize in vivo, prostaglandin production, collagenase production and receptors for 1,25(OH)2D3, EGF
and PTH (Forrest et al., 1985; Mitchell et al., 1990; Ng et
al., 1983; Partridge et al., 1980, 1983, 1987). The two
subclones reported for UMR 106 cells are UMR 10601 and UMR 106-06 (Forrest et al., 1985) with the only
known substantial difference between the two clones being the expression of calcitonin receptors in the UMR
106-06.
The family of clonal cell lines designated ROS (ROS 17/2
and its subclone ROS 17/2.8) were derived from a spontaneous tumor in an ACI rat that had been propagated by serial
subcutaneous transplantation (Majeska et al., 1980). These
cells exhibit adenylate cyclase activity in response to PTH
as well as a high ALP activity which is regulated by PTH
(Majeska and Rodan, 1982a) and 1,25(OH)2D3 (Majeska
and Rodan, 1982b). The subclone ROS 17/2.8 constitutively
expresses osteocalcin mRNA (Price and Baukol, 1980) and
produces calcified matrix when implanted in diffusion chambers (Shteyer et al., 1986). The cells respond to TGF with
an increase in ALP, type I collagen, osteonectin, and osteopontin mRNA (Noda and Rodan, 1987; Noda et al., 1988)
but with a decrease in mRNA for osteocalcin (Noda, 1989).
Other models of osteoblastic cells derived from human
osteosarcomas include the SaOS (Rodan et al., 1987b), OHS4 (Fournier and Price, 1991), TE-85, MG-63 (Francheschi et
al., 1985, 1988), KPDXM and TPXM (Bruland et al., 1988).
Interestingly, some of these cell lines have been primarily
used as models to study the control of expression of bone
matrix proteins, including integrin-mediated cell adhesion to
fibronectin (Dedhar et al., 1987; Rodan et al., 1994).
Rochet and colleagues have recently characterized a new
human osteosarcoma cell line, CAL72, which is more closely
related to normal osteoblasts than any of the osteosarcoma
cells previously described, and could also provide an interesting tool to study the role of osteoblastic cells in hematopoietic
cell growth and differentiation. The cell line exhibits a singular cytokine expression profile compared to other osteosarcoma cell lines (Rochet et al., 1999). CAL72 cells constitutively express mRNA coding for IL-6, GM-CSF, and G-CSF
and thus appear to be closer to human primary osteoblastic
cells than the well-described osteosarcoma cell lines MG63 and SaOS-2. In contrast to MG-63 or SaOS-2, CAL72
cells do not inhibit hematopoietic colony formation and can
sustain the limited expansion of hematopoietic progenitors
in a way similar to that described for normal human primary osteoblasts. In addition, CAL72 cells induce the differentiation of promyelocytic cells into macrophages more
efficiently than other osteosarcoma cell lines (Rochet et al.,
2003).
2.4.3. Non-transformed cell lines

The UMR 201 cell line is a clonal non-transformed cell
line derived from neonatal rat calvaria. The cell line was established by Ng et al. (1988) as a non-immortalized cell line
with a limited lifespan (12 passages in culture) and phenotypic features suggestive of pre-osteoblasts. They have undetectable alkaline phosphatase activity and mRNA for ALP
which is significantly induced by the differentiating agent,
retinoic acid (Ng et al., 1988, 1989a). TNF, dexamethasone and 1,25(OH)2D3 also modulate retinoic acid (RA)induced ALP activity and mRNA for ALP in these cells (Ng
et al., 1989a, 1989b). This cell line therefore, provides a
useful model to study the osteoblastic differentiation from
progenitor cell populations and also the regulation of osteoblast differentiation by systemic hormones and local
growth factors. Furthermore, in subsequent studies the same
group established immortalized subclones of UMR 201 using SV40 large T antigen (see section below) hence, allowing careful comparison of phenotypic characteristics
between the parent and derived cell lines (Zhou et al.,
1991).
MC3T3-E1 is a clonal non-transformed cell line established from newborn mouse calvaria (Kodama et al., 1981;
Sudo et al., 1983). This cell line has also been shown
to increase cyclic AMP production in response to PTH
(Kumegawa et al., 1984) and exhibits a high ALP activity
which is regulated by PTH, PGE2, 1,25(OH)2D3 and is capable of collagen synthesis. In addition, these cells form matrix
vesicles which are deposited on collagen fibrils and can be
mineralized in vitro (Sudo et al., 1983). Although originally
isolated as a clonal cell line, different variants of MC3T3-E1
cells have been isolated with different phenotypic properties
most likely as a result of prolonged passaging (Leis et al.,
1997). Wang and colleagues isolated 10 subclonal MC3T3E1 cell lines exhibiting high or low mineralization potential
after growth in ascorbic acid-containing medium for 10 days.
Clones 4, 8, 11, 14, and 26 formed a well-mineralized extracellular matrix after incubation for 2 days in medium containing 3.0 mM inorganic phosphate, whilst clones 17, 20, 24, 30,
and 35 failed to form any detectable mineral. A good correlation was observed between the ability of a given subclone
to activate the osteoblast-specific osteocalcin promoter and
endogenous expression of osteocalcin and other osteoblastrelated mRNAs. Essentially, subclones expressing high levels
of osteoblast marker mRNAs formed a mineralized extracellular matrix in culture and were osteogenic when implanted
into mice. In addition, all subclones, regardless of their ability
to differentiate, expressed high levels of cbfa-1 mRNA, which
encodes a transcription factor necessary for osteoblast formation (Otto et al., 1997), implying that the presence of cbfa-1 is
by itself insufficient for induction of osteoblast-specific gene
expression (Wang et al., 1999).
The cell lines represented by CRP 4/7, CRP 7/4, CRP 7/7,
CRP 10/3 and CRP 10/30 also belong to the group of clonal
non-transformed osteoblastic cells. These cells were isolated
from neonatal calvarial bone cells in the presence of TGF
85
Table 4
Properties of the CRP clonal cell lines
Clone
ALP (activity)
Response to
PTH PGE2
Osteocalcin
mRNA
CRP 4/7
CRP 7/7
CRP 7/4
CRP 10/3
CRP 10/30
Absent
Present
Absent
Present
Present
Absent
Absent
Present
Absent
Present
Absent
Absent
Present
Present
Present
and EGF (Guenther et al., 1989). The various properties of

these clonal cell lines are shown in Table 4.
2.4.4. Experimentally immortalized cell lines
The immortalization of cells by transfection with a recombinant retrovirus containing the cDNA for SV40 large T
antigen has been used to establish immortalized osteoblastic
cell lines (Jat and Sharp, 1986). RCT-1 and RCT-3 cell lines
were derived from isolated and fractionated embryonic rat
calvarial cells. RCT-1 cells were established from the early
digest population and expressed osteoblastic traits [ALP, pro1(I) collagen, PTH-responsive adenylate cyclase] after induction by retinoic acid. RCT-3 cells on the other hand, were
established from the more osteoblastic late digest population
and were found to constitutively express osteoblastic markers
except osteocalcin (Heath et al., 1989).
KS-4 is a clonal cell line which was isolated from mouse
calvaria by transfection with the c-Ha-ras-1 gene. The cells
display low ALP activity at confluence, low type I collagen
production and low cAMP accumulation in response to PTH.
The cells also display low mRNA levels for pro-1(I) collagen, osteonectin and bone proteoglycan I but not osteocalcin.
Importantly, KS-4 cells have the ability to stimulate osteoclast formation on co-culture with spleen cells (Yamashita et
al., 1990a, 1990b).
Other model systems of experimentally immortalized cell
lines used to study certain stages of osteoblast differentiation
include the adult human osteoblast-like (hOB) cells (Keeting
et al., 1992) and the human fetal osteoblast cell line hFOB
(Harris et al., 1995). The hOB cell line was immortalized
by transfecting normal adult human osteoblast-like cells, derived from a 68-year-old woman, with the large and small
T antigens of the SV40 virus. The cells represent a welldifferentiated, steroid-responsive clonal cell line that closely
approximates the phenotype of the mature osteoblast. They
express mRNA for (I)-procollagen, osteopontin, TGF,
and interleukin-1 beta (IL-1), while treatment with 1,25dihydroxyvitamin D3 results in increased expression of osteocalcin and alkaline phosphatase mRNA and protein. Functional estrogen and androgen receptors are present but not the
receptor for PTH. When -glycerophosphate is added to the
cultures, the cells produce a matrix that mineralizes.
The human fetal osteoblast cell line (hFOB) was derived from biopsies obtained from a spontaneous miscarriage
(Harris et al., 1995). Primary cultures isolated from fetal tissue were immortalized by transfection with a temperature-
86
sensitive mutant (tsA58) of SV40 large T antigen. hFOB 1.19,

the highest alkaline phosphatase-expressing clone, increased
alkaline phosphatase activity and osteocalcin secretion in a
dose dependent manner following 1,25-dihydroxyvitamin D3
treatment. Differentiated hFOB cells showed high levels of
osteopontin, osteonectin, BSP, and type I collagen expression. Treatment of hFOB cells with parathyroid hormone
(134) resulted in increased cAMP levels. In addition, upon
reaching confluence, hFOB cultures formed mineralized nodules.
2.5. A model of metabolic bone disease
Type I collagen is the most abundant and ubiquitously
distributed of the collagen family of proteins. It is a heterotrimer comprising two alpha1 (I) chains and one alpha2 (I)
chain, which are encoded by the unlinked loci COL1A1 and
COL1A2, respectively. Mutations at these loci result primarily in the connective tissue disorders osteogenesis imperfecta
(OI) and EhlersDanlos syndrome. Osteogenesis imperfecta
is a heterogenous genetic disorder associated with increased
fractures (Rowe, 2002). In its most severe form multiple fractures occur in utero and the disorder is lethal. In milder forms
(type I) there may be an increase in fractures in childhood
which then cease after puberty, with a subsequent increase in
women after the menopause (Paterson et al., 1984). It is beyond the scope of this review to discuss the molecular basis of
OI. A comprehensive listing of the mutations that have been
discovered within human type I collagen genes (Dalgleish,
1997), is maintained in the osteogenesis imperfecta mutation database (http://www.le.ac.uk/genetics/collagen). The
oim/oim mouse which arose from a spontaneous mutation
within the COL1A2 gene resulting in the production of a
non-functional alpha2 (I) chain, is a widely used murine
model of OI (Chipman et al., 1993). The heterozygous Mov
13 mouse in which one of the two COLIA1 genes is nonfunctional, is the only murine model of type I OI (Bonadio et
al., 1990). Apart from defective formation of the type I collagen triple helix, the ability of OI fibroblasts or bone cells
to produce collagen and proliferate in vitro is also impaired
and that is probably a consequence of the retained procollagen
molecules within the distended rough endoplasmic reticulum
(Lamande et al., 1995; Fitzgerald et al., 1999; Lamande and
Bateman, 1999). In vitro studies of osteoblasts derived from
OI humans (Fedarko et al., 1996) or oim mouse (Balk et al.,
1997) show diminished markers of osteoblastic differentiation. However the cells can still differentiate into
osteoblasts under the influence of bone morphogenetic
proteins.
Knowledge obtained from the study of osteogenesis imperfecta has also provided clues about the genetics of osteoporosis. Although the collagen protein chains are normal
in most osteoporotic patients, a polymorphism has recently
been identified in a regulatory region of the COLIA1 gene
which is more common in osteoporotic patients. This polymorphism is located at a binding site for the transcription
factor Sp1 in the first intron of COLIA1, and has been found
to be associated with bone mass and osteoporotic fracture in
several Caucasian populations (Ralston, 1999).
2.6. Osteocytes
Osteocytes are terminally differentiated cells of the osteoblast lineage that have become embedded in mineralized
matrix. Individual osteocytes communicate with each other
and with cells on the bone surface such as lining osteoblast
cells, through long intercellular processes. Their location and
morphology renders them particularly well suited to transfer
information between cells within bone. For example, when
the skeleton is undergoing mechanical stress, osteocytes are
ideally located to sense pressure changes in bone, which
could result in specific chemical messages being relayed to
the surface cells to respond either by formation or resorption (Lanyon, 1993; Turner et al., 1994; Weinbaum et al.,
1994; Klein-Nulend et al., 1995). It has also been hypothesized that osteocytes may have the capacity to regulate calcium homeostasis (Rubinacci et al., 1998). Unfortunately,
their peculiar location within bone makes them the most inaccessible type of osteoblast to obtain in culture for in vitro
study.
Bonewald and colleagues have established several immortalized cell lines in culture with phenotypic characteristics of osteocytes. Bone cells were derived from transgenic
mice over-expressing T-antigen driven by the osteocalcin
promoter. They chose cells expressing a dendritic morphology as the initial criterion for selection and establishment of
clonal cell lines. MLO-Y4 (murine long bone osteocyte Y4)
was one of the immortalized clonal lines established with
osteocyte-like characteristics. These cells produce extensive,
complex dendritic processes, are positive for T-antigen, osteopontin, neural antigen CD44 and connexin 43. They produce large amounts of osteocalcin, have low levels of alkaline
phosphatase activity, lack detectable mRNA for osteoblastspecific factor 2, and produce very small amounts of type I
collagen (Kato et al., 1997). The MLO-Y4 cells also support osteoclast formation and activation through the secretion of M-CSF and expression of RANKL on their surface and their dendritic processes (Zhao et al., 2002). Cells
are grown on collagen-coated surfaces in culture medium
supplemented with 5% FBS and 5% calf serum for optimal growth and maintenance of the osteophyte dendritic
phenotype.
Four other immortalized osteocyte-like cell lines (MLOA5, MLO-A2, MLO-D1 and MLO-D6) were established by
this group. Out of these, MLO-A5 cells were shown to highly
express BSP and mineralize spontaneously in culture even
in the absence of beta-glycerophosphate and ascorbic acid
(Kato et al., 2001). The authors claim that the MLO-A5 cells
are representative of the post-osteoblast, preosteocyte stage
responsible for triggering mineralization of osteoid.
To date, there is no published data on the responses of these
presumptive osteocyte-like cell lines to mechanical strain.
3. Osteoclasts
3.1. Osteoclast ontogeny
Multinucleate osteoclasts are responsible for bone resorption. Their chief functional characteristic is the ability to pump acid into specialized resorption pits to dissolve
bone mineral as well as to provide an optimum environment
for the enzymatic degradation of demineralized extracellular bone matrix. Osteoclasts are derived from hematopoietic
stem cells that differentiate along the monocyte/macrophage
lineage (Martin et al., 1989; Suda et al., 1992). Direct
contact of mononuclear hematopoietic precursors with osteoblast/stomal cells expressing the membrane protein Receptor Activator of NF-kappa B Ligand (RANKL) is necessary before they can differentiate into osteoclast precursors
and proceed to fuse into mature, multinucleate osteoclasts
(Suda et al., 1995; Lacey et al., 1998). This is depicted diagrammatically in Fig. 2.
3.2. Phenotypic characteristics of osteoclasts
The mature osteoclast is a functionally polarized cell that
attaches via its apical pole to the mineralized bone matrix
by forming a tight ring-like zone of adhesion, the sealing
zone. This attachment involves the specific interaction between adhesion molecules in the cell membrane (integrins)
and some bone matrix proteins. The integrins are a family of transmembrane proteins whose cytoplasmic domains
interact with the cytoskeleton while their extracellular domains bind to bone matrix proteins, enabling them to mediate cellsubstratum and cellcell interactions (Hynes, 1987).
87
The space contained inside this ring of attachment and between the osteoclast and the bone matrix constitutes the boneresorbing compartment. The cell membrane of the apical
pole is invaginated to form a ruffled border. Osteoclasts are
actively engaged in the synthesis and secretion of several
classes of enzymes formed in the Golgi region and vectorially
transported to the apical pole through their association with
mannose-6-phosphate receptors. At their destination, the enzymes bound to mannose-6-phosphate receptors fuse with
the ruffled border apical membrane and their contents discharged into the bone-resorbing compartment (Baron et al.,
1993).
Acidification of the extracellular bone-resorbing compartment is one of the most important features of osteoclast
action. The osteoclast is highly enriched in carbonic anhydrase (Gay and Mueller, 1974). Carbonic anhydrase generates protons and bicarbonate from carbon dioxide and water, providing the cells with protons to be extruded across
the cell membrane into the bone-resorbing compartment by
proton pumps (H+ ATPases) located in the ruffle border
apical membrane. Regulation of H+ transport at the apical surface of the osteoclast, which is tightly linked to the
regulation of intracellular pH and membrane potential, is
mostly accomplished by ion exchangers, pumps and channels
present in the basolateral membrane of the cell (Baron et al.,
1993).
The activity of mature osteoclast is directly and negatively
regulated by calcitonin, for which the cell expresses a high
number of receptors (Nicholson et al., 1986).
A summary of the main osteoclast phenotypic characteristics is provided in Table 5.
Fig. 2. Diagrammatic representation of the formation of mature, multinucleated osteoclasts from mononuclear hematopoietic progenitors. Hematopoietic
osteoclast progenitors present in bone marrow come into direct contact with osteoblast or stromal cells expressing RANKL under the influence of osteolytic
factors such as PTH, PGE2, IL-11 or 1,25(OH)2D3 (1). They differentiate firstly into TRAP positive mononuclear cells (2) before becoming TRAP positive
and calcitonin receptor (CTR) positive mononucleate cells (3) that eventually fuse to form multinucleate, functional mature osteoclasts (4).
88
Table 5
Phenotypic characteristics of osteoclasts
Lysosomal enzymes
Tartrate-resistant acid phosphatase
-Glycerophosphate
Arylsulfatase
-Glucuronidase
Cysteine-proteinases (cathepsin B, C, K, L)
Non-lysosomal enzymes
Collagenase
Stromelysin
Tissue plasminogen activator
Lysozyme
Matrix proteins
Osteopontin
Bone sialoprotein
TGF
Receptors
RANK (membrane)
Calcitonin (membrane)
Vitronectin (v3) (membrane)
Integrins with 1 subunits
Mannose-6-phosphate (intracellular)
Proton pump
Vacuolar H+ ATPase
Ion transport
Calcium channels
Potassium channels
Chloride channels
Calcium ATPase
Na, K-ATPase
Na+ /H+ antiporter
Bicarbonate/chloride exchanger
Membrane associated proteins
Carbonic anhydrase
c-src
3.3. In vitro methods to study osteoclast formation and

function
Unlike osteoblasts, osteoclasts are difficult to study in vitro
because they are relatively scarce, terminally differentiated,
adherent to mineralized surfaces and fragile. Methods have
been developed to isolate these cells in vitro or to induce their
formation in bone marrow cultures. The major criteria generally used to identify osteoclasts are multinuclearity, positive
staining for tartrate resistant acid phosphatase (TRAP), expression of calcitonin receptors and the ability to resorb calcified matrices (Takahashi et al., 1988a; Hattersley and Chambers, 1989; Shinar et al., 1990). TRAP staining and some
of the other criteria used for identification, such as cathepsin K, vitronectin receptor, are not specific for osteoclasts,
being also expressed by macrophages (Table 6). However,
these markers are often useful to identify osteoclasts when
macrophage expression of these markers can be effectively
excluded. Indeed, the simplest method of estimating osteo-
clast number in in vitro assays is a count of TRAP positive or

vitronectin receptor positive multinucleated cells. However,
prolonged culture (more than 7 days) frequently results in
calcitonin receptor negative, TRAP positive multinucleated
macrophages, and that is a common pitfall. Conversely, the
absence of these markers indicates the absence of osteoclasts.
Mature, multinucleated functional osteoclasts are obtained either directly from bone as the primary source, or else
they are secondarily generated in vitro from hematopoietic
progenitors obtained from a source of hematopoietic cells or
macrophages such as bone marrow, spleen, human peripheral
blood mononuclear cells and human umbilical cord blood.
3.3.1. Primary sources of osteoclasts
3.3.1.1. Mechanical disaggregation of osteoclasts. Mature
osteoclasts can be isolated by mechanical disaggregation
from the long bones of neonatal rats, rabbits or chicks. This
method involves curetting the long bones with a scalpel blade
to release bone fragments into the surrounding medium. The
fragments are triturated into a suspension with a wide-bore
pipette before plating onto glass coverslips for 1530 min
to allow the large, highly adherent osteoclasts to adhere to
the glass surface, before washing vigorously with medium
to remove non-adherent and other contaminating cell types
(Chambers and Magnus, 1982). A longer settling time increases the yield of osteoclasts, but also the number of
non-osteoclastic contaminating cells. Relatively pure osteoclasts have been obtained this way to enable the identification
of calcitonin receptors and effects of bone-resorbing factors
on cytoplasmic spreading (Nicholson et al., 1986). The effects of osteotropic factors on bone resorption can be studied
when the osteoclasts are placed on bone slices (Chambers et
al., 1985). The main disadvantages of this assay are the contaminating osteoblasts in the osteoclast preparation that may
affect the experimental outcomes and the sensitivity of osteoclasts to the pH of the assay medium, especially when culture
times exceed 24 h. An acid pH has been shown to stimulate
osteoclast bone resorption (Arnett and Dempster, 1990), potentially accounting for some of the conflicting reports in the
literature.
3.3.1.2. Giant cell tumors of bone. Giant cell tumors (GCT),
also known as osteoclastomas, are rare primary neoplasms
of the skeleton. They are locally destructive, causing extensive osteolysis. GCT contain within the tumor mass, variable
numbers of large, multinucleated cells. It is believed that the
stromal cells of GCT are the tumor cells, and they induce
osteoclastic bone resorption by recruiting osteoclast precursors, promoting their differentiation into functional osteoclasts (James et al., 1996). Until recently, this was the only
useful source of human osteoclasts. To obtain the osteoclasts
(Goldring et al., 1987), the giant cell tumor is dissected in a
Petri dish under sterile conditions using a scalpel blade, and
then enzymatically digested for cell culture in a digestion
mixture made up from collagenase and dispase. The cell suspension is diluted in alpha-modified MEM containing 10%
89
Table 6
Identification of osteoclast markers
Marker
Specificity/use
Comment
Bone resorption
Calcitonin receptors
Multinuclearity
TRAP
Cathepsin K
Vitronectin receptor
Actin ring
Definitive
Specific (hematopoietic lineage)
Typical but not essential
Useful marker; easy to perform
Useful marker
Occasionally useful
Indicates active osteoclast (on calcified substrate)
Requires live, active cells; time dependent

Difficult without live cells
Indicates terminal differentiation
Indicative in vitro, but also expressed by activated macrophages
Also expressed by macrophages/tumor cells
Also expressed by macrophages
FBS, and filtered through a 40 m cell strainer. Cells are

then either cryopreserved or cultured in medium (Atkins et
al., 2001).
3.3.2. Secondary sources of osteoclasts. In vitro
generation
3.3.2.1. Bone marrow cultures. Bone marrow consists of a
mixed cell population that is rich in hematopoietic but relatively poor in osteoblastic stromal cells. They are useful
for examining the process of osteoclast differentiation and
formation in culture under the influence of bone-resorbing
agents. Murine bone marrow cultures have been the most
widely studied (Takahashi et al., 1988a, 1988c: see method
below). Takahashi et al. (1988b) reported that treatment with
1,25(OH)2D3 or human PTH (134) resulted in an increase in
the number of TRAP positive multinucleated cells that satisfy
the major criteria for osteoclasts. Subsequently, a similar increase was observed with prostaglandins, PTHrP (134) and
IL-1 (Akatsu et al., 1989a, 1989b, 1991). Two other important
observations were made. Firstly, time course studies showed
that the appearance of TRAP positive mononucleated cells
precede that of TRAP positive multinucleated cells, implying that TRAP positive mononucleated cells are precursors
of the multinucleated cells. Secondly, osteotropic hormones
such as 1,25(OH)2D3 and PTH induce the differentiation
of immature precursors, characterized by TRAP and CTRnegativity, into mature TRAP and CTR-positive precursors
(Takahashi et al., 1988b) (Fig. 2). Osteoclasts have also similarly been obtained using rabbit (Fuller and Chambers, 1987)
and feline (Ibbotson et al., 1984) bone marrow. This system
is limited by the inability to identify osteoclastic precursors
from the mixed cell population and the difficulties inherent
in studying osteoclast activation as opposed to recruitment.
The dependence of osteoclast formation on the presence of
mesenchymal stromal cells or osteoblasts also complicate the
interpretation of results.
3.3.2.2. Co-cultures of osteoblastic and hematopoietic cells.
An important finding from murine bone marrow cultures was
the demonstration that it was necessary for hematopoietic
precursors to come into direct contact with osteoblasts and
stromal cells before osteoclast differentiation and formation
could occur (Takahashi et al., 1988b). This led to the development of co-culture systems comprising three essential
elements: (i) stromal cells or osteoblasts as a feeder layer;
(ii) a source of hematopoietic cells such as murine spleen or

bone marrow cells; and (iii) hormonal stimulation.
(i) Preparation of primary osteoblast
Primary osteoblasts are usually obtained from the calvaria of newborn C57BL/6J mice. Calvaria are removed
from the newborn mice under sterile conditions and
transferred to a sterile 30 ml tube containing 6 ml digest fluid made up immediately before use. The calvarial digest fluid is made from 30 mg collagenase type II
and 60 mg dispase dissolved in 30 ml PBS and filtered
through a 0.2 m Acrodisc 32 Supor . The tube containing calvaria is shaken in a 37 C water bath for 5 min,
allowed to settle and the supernatant discarded by pipetting. Six millilitres of fresh digest fluid is added to the
tube and incubated at 37 C for 10 min. The cell suspension is collected in a separate sterile 30 ml tube. The
digest is repeated a further three times and the cell suspensions pooled in the 30 ml tube. This is centrifuged
at 2000 rpm for 5 min and the supernatant discarded.
The cell pellet of primary osteoblasts is re-suspended in
10 ml MEM + 10% FBS and used immediately. Alternatively, osteoblasts can be grown in culture for a few
days to increase their numbers. Cells are seeded at a
density of 5 106 cells in 10 ml of medium in Petri
dishes and incubated at 37 C in a humidified incubator with 5% CO2 . The adherent calvarial osteoblasts are
confluent in 23 days and can be used up to 1 week from
their initial preparation. They are dispersed for use by
standard trypsinization methods.
(ii) Stromal and osteoblast-like cell lines
Udagawa et al. (1989) demonstrated that two bone
marrow-derived pre-adipocytic cell lines, MC3T3G2/PA6 and ST2, can support osteoclast formation from
murine spleen cells in the presence of 1,25(OH)2D3.
The simultaneous addition of dexamethasone greatly
enhanced osteoclast numbers. Other osteoblast/stromal
cell lines shown to support osteoclast formation in the
co-culture system include the rat osteoblast-like cell line
UMR 106 (Quinn et al., 1994), murine stromal cell lines
tsJ2 and 10 (Chambers et al., 1993), murine osteoblastlike cell lines KS-4 (Yamashita et al., 1990a, 1990b) and
KUSA/O (Umezawa et al., 1992).
(iii) Preparation of bone marrow cells
Long bones (femur and tibia) are obtained from adult
male mice (48 weeks old). Each bone is flushed with
90
PBS (5 ml per bone) from a syringe and samples from

all bones are pooled. Bone marrow cells are plated (106
cells per well in 1 ml) into the wells of a 16 mm diameter culture dish, together with osteoblasts (typically 2
105 cells/dish). The cultures are then maintained for approximately 8 days in MEM supplemented with 10%
FBS at 37 C in a humidified atmosphere of 5% CO2
with fresh medium and treatments added every 3 days.
Osteoclast formation begins typically at day 4 and increases thereafter.
(iv) Preparation of spleen cells
Spleen cells are obtained from either newborn mice (for
example, mice used in the osteoblast calvarial preparation), or adult mice. The spleens are removed and collected in a sterile wire sieve over a Petri dish half-filled
with medium. Using the plunger from a disposable 10 ml
syringe, the spleens are gently crushed through the sieve
and the sieve rinsed over the Petri dish with medium.
The disaggregated spleen cells are transferred to a fresh
sterile tube and centrifuged at 2000 rpm for 5 min. The
pelleted cells are then re-suspended in MEM + 10%
FBS in similar numbers to bone marrow cells described
above.
The cell mixture of hematopoietic cells and stroma/
osteoblasts must be stimulated by osteolytic factors, the most
potent being a mixture of 10 nM 1,25(OH)2D3 and 100 nM
prostaglandin E2. The addition of dexamethasone (108 M)
increases the yield of osteoclasts, but may be detrimental to
osteoclast morphology.
3.3.3. New methods for osteoclast generation in vitro,
using RANKL, M-CSF, TNF and IL-1
The membrane factor expressed by osteoblasts and
stromal cells that was essential for osteoclast differentiation was identified as the membrane-bound protein
termed receptor activator of NFB ligand (RANKL). It
stimulates the differentiation and formation of multinucleated osteoclasts from mononuclear precursors when
it binds to its receptor, RANK, expressed on mononuclear hematopoietic precursors (Anderson et al., 1997a,
1997b; Wong et al., 1997a, 1997b; Lacey et al., 1998; Yasuda
et al., 1998; Hsu et al., 1999). RANK-deficient mice are characterized by profound osteopetrosis resulting from a block in
osteoclast differentiation (Dougall et al., 1999). RANKL expression in osteoblasts is stimulated by bone-resorbing factors such as PTH, PGE2, 1,25(OH)2D3, interleukin 1 (IL1) and IL-11 (Horwood et al., 1998; Yasuda et al., 1998;
Hofbauer et al., 1999), at the same time, reducing the production of the secreted RANKL decoy receptor, osteoprotegerin. RANKL also enhances the activity of mature osteoclasts (Burgess et al., 1998) and inhibits osteoclast apoptosis
(Fuller et al., 1998; Udagawa et al., 1999). Several factors including IL-1, M-CSF and TNF (Kobayashi et al., 2000) also
have this ability. Recombinant protein corresponding to the
extracellular domain of RANKL stimulates the formation of
active, bone-resorbing osteoclasts from hematopoietic cells
in the presence of M-CSF. RANKL plus M-CSF are sufficient to cause osteoclast formation from human or mouse
hematopoietic precursors, even in the absence of osteoblastic
stromal cells (Quinn et al., 1998). This is a major advance in
the development of in vitro methods to study osteoclast differentiation and formation, enabling investigators to obtain
osteoclasts in culture and in sufficient numbers without the
confounding presence of other cell types such as osteoblasts
or stromal cells. In vitro models of mouse osteoclast formation using hematopoietic cells as a source of precursors, and
stimulated by soluble factors M-CSF, RANKL, TNF or IL-1
is summarized in Table 7 and Fig. 3.
3.3.3.1. Bone marrow and bone marrow macrophages
(BMM). Quinn et al. (2002) reported a method of obtaining highly enriched osteoclast-lineage cell populations using
M-CSF stimulated bone marrow cells. M-CSF stimulation of
bone marrow cells results in large numbers of non-adherent,
proliferating macrophage precursors that rapidly form adherent bone marrow macrophages (BMM). BMM and their precursors can be isolated free from mesenchymal and lymphocytic cells. BMM precursors derived from CBA or C57BL/6J
mouse bone marrow, when cocultured with ST2 cells, form
numerous mononuclear osteoclasts which resorb bone and
express TRAP and calcitonin (CTR) receptors, suggesting
that they are a highly enriched source of osteoclast progenitors. Recombinant RANKL/M-CSF, together with TGF,
stimulate BMM precursors to form almost pure CTR-positive
osteoclasts after 7 days. Typically, 105 bone marrow cells are
seeded per 10 mm diameter culture dish in the presence of
25 ng/ml M-CSF and 50100 ng/ml RANKL for 7 days. The
BMMs generated with this method can be cryopreserved for
future use.
3.3.3.2. Osteoclast cell lines. At least two osteoclast precursor cell lines have been established as an alternative to
fresh hematopoietic cells in the study of osteoclastogenesis.
RAW264.7 is a mouse osteoclast-like myeloma cell line capable of differentiating into osteoclasts when treated with
Table 7
In vitro models of mouse osteoclast formation in the absence of stromal cells
Cell types
Stimulus
Cells present
Spleen cells, bone marrow or blood

monocytes
RANKL/TNF + M-CSF
Hematopoietic cells, lymphocytes
(B, T, NK cells), stromal cells
Bone marrow macrophages or bone

marrow macrophage precursors
RANKL/TNF + M-CSF
Osteoclast and macrophage progenitors
RAW 264.7
RANKL/TNF
Osteoclast and macrophage progenitors
91
Fig. 3. This diagram is a representation of the differentiation pathway from mononucleate hematopoietic progenitors to functional, mature, multinucleate
osteoclasts. It illustrates the points along this pathway that are acted upon by soluble factors M-CSF, RANKL and IL-1. These factors would normally be
secreted or expressed by osteoblasts or stromal cells.
RANKL (Hsu et al., 1999). Unlike murine spleen cells, bone

marrow cells or BMMs, RAW264.7 cells do not require MCSF to differentiate into osteoclasts. Furthermore, they cannot be co-cultured with stromal cells or osteoblasts. The
reason for this is not known. C7 is an immortalized mouse
macrophage-like cell line that can be used for a similar purpose (Yasuda et al., 1998).
3.3.3.3. Human osteoclasts. The formation of human osteoclasts in coculture has proved to be a challenge because of
a lack of a suitable human osteoblastic stromal cell line to
use in a coculture system. Fujikawa et al. (1996) isolated human monocytes [CD14, CD11a, CD11b, HLA-DR positive,
TRAP, CTR, vitronectin receptor (VNR) negative] and cocultured these cells for up to 21 days with either osteoblast-like
UMR 106 (rat) or ST2 (mouse) stromal cells in the presence
of 1,25(OH)2D3, dexamethasone and human M-CSF (rat MCSF is inactive on human cells). Numerous TRAP, VNR and
CTR-positive multinucleated cells, capable of extensive lacunar bone resorption, formed in these cocultures. This work
was extended to include human hematopoietic marrow cells,
blood monocytes and peritoneal macrophages, all of which
were capable of differentiating into mature functional osteoclasts (Quinn et al., 1998b). Matsuzaki et al. (1999) established subclones of the human osteosarcoma cell line,
SaOS-2, expressing the human PTH/PTHrP receptor, and
showed that mouse bone marrow cells or human peripheral blood mononuclear cells formed osteoclasts in coculture
when treated with PTH. The response was greatly enhanced
by adding dexamethasone, but no osteoclast formation was
seen with the addition of 1,25(OH)2D3, PGE2 or IL-6. Human peripheral blood mononuclear cells (PBMC) cultured
in vitro with soluble RANKL and human M-CSF, form os-
teoclasts. However, PBMC are heterogeneous, consisting of

subsets of monocytes, lymphocytes and other blood cells.
Nicholson et al. (2000) showed that a highly purified population of osteoclast-forming PBMC can be obtained by selecting for the expression of CD14, a marker that is strongly
expressed in monocytes, the putative osteoclast precursor in
peripheral blood.
A novel and exciting new method of obtaining osteoclast progenitors from human umbilical cord blood was recently described (Hodge et al., 2002). A mononuclear cell
fraction containing monocytes and lymphocytes, isolated
from human umbilical cord blood by FicollPaque density
gradient centrifugation, is cultured in semi-solid medium,
and incubated at 37 C in a humidified atmosphere of 5%
CO2 air for 714 days. Pooled colonies identified as CFUGM are harvested and transferred into 96-well plates containing dentine slices in the presence of RANKL and human M-CSF for a further 6 days. Cultures are then fixed
in 1% formalin and reacted for TRAP activity. The formation of bone-resorbing multinucleated osteoclasts is assessed
by transmission light microscopy and quantified using computer image analysis. Using human umbilical cord blood
mononuclear cells (CBMC) as a source of osteoclast progenitors, it was shown that clonal expansion of CFU-GM
progenitors markedly increases osteoclastogenic potential,
but exposure of pooled colonies to GM-CSF or IL-3 prior
to RANKL stimulus completely inhibits osteoclastogenesis,
directing cells instead towards dendritic cell differentiation.
This may prove to be a very useful method for obtaining
human osteoclast progenitors to study the regulation of differentiation along different cell lineages. A further advantage
of this method is the ability to cryopreserve CBMC for future
experiments.
92
4. Chondrocytes
Table 8
A summary of the phenotypic characteristics of chondrocytes
Cartilage is a specialized form of connective tissue that

possesses a firm pliable matrix, which endows it with the
resilience that allows the tissue to bear mechanical stresses
without distortion. Articular cartilage, smooth surfaced and
resilient, provides a shock-absorbing sliding area for joints to
facilitate movement of bone, while cartilage is also essential
for the embryonic development and, thereafter, growth of
long bones.
Cartilage consists of chondrocytes and an extensive extracellular matrix. The characteristics of cartilage stem mainly
from the nature and predominance of ground substance in
the extracellular matrix. Glycoproteins, containing a high
proportion of sulfated polysaccharides, make up the ground
substance and account for the solid, yet flexible properties of
cartilage. The functional differences between cartilage and
bone relate principally to the different nature and proportion
of the ground substance and fibrous elements of the extracellular matrix. Nonetheless, chondrocytes and osteoblasts share
a common origin from primitive mesenchymal cells (Marks
and Hermey, 1996).
Hyaline cartilage is the most prevalent type of cartilage.
During embryonic development, it forms the cartilage template of many of the developing bones until replaced by bone
in the process of endochondral ossification. In long bones, the
epiphyseal growth plate between the epiphysis and diaphysis
is responsible for the longitudinal growth of bone. Within
the growth plate, chondrocytes undergo a series of discrete
stages of differentiation, namely, proliferation, maturation,
and hypertrophy. The strict spatial and temporal control of
proliferation and differentiation of chondrogenic cells is central to the coordinated development of the vertebrate skeleton
(Erlebacher et al., 1995).
Chondrocytes are unique cells, in that they have many
differentiated markers such as large cartilage-type proteoglycans (aggrecan) and collagen types II, IX, X, and XI. Some
typical phenotypic characteristics of chondrocytes are listed
in Table 8. A comprehensive list of several hundred mouse
growth cartilage-derived gene products, including many not
previously reported, was recently published (Okihana and
Yamada, 1999).
Collagens
Type I collagen
Type II collagen
Type VI collagen
Type IX collagen
Type X collagen
Type X1 collagen
4.1. Propagation of chondrocytes in culture

Various cell culture models have been developed for the
investigation of chondrocyte biology in vitro, including explant models, several forms of three-dimensional culture systems, and monolayer cultures (Adolphe and Benya, 1992).
Chondrocytes grown in monolayer culture undergo a characteristic process of dedifferentiation, marked by a loss of
collagen type II and aggrecan core protein expression as well
as the induction of collagen type I expression (Takigawa et
al., 1987; Hering et al., 1994; Lefebvre et al., 1994). This
phenomenon is influenced to some extent by seeding density (Ronzi`ere et al., 1997) and is accelerated by growth in
Proteoglycans and other proteins

Aggrecan
Link protein
Biglycan
Fibronectin
Osteopontin
Cartilage oligomeric matrix protein
Matrix gla protein
Chondromodulin-I
Calmodulin
Fibromodulin
Cartilage homeoprotein I
Perlecan
Tropomodulin
Osteonectin
Receptors
Growth hormone
TGF-beta
BMP
PTHrP
IGF-1
Retinoic acid
Fibroblast growth factor receptors 1 and 3
Thyroid hormones
medium supplemented with serum and by passage (Hering

et al., 1994). Growth of chondrocytes under conditions that
support a rounded morphology also facilitates maintenance
of the differentiated chondrocytic phenotype (Bonaventure
et al., 1994; Hauselmann et al., 1994; Binette et al., 1998).
Stewart et al. (2000) studied the phenotypic stability of articular chondrocytes in vitro and demonstrated that the influence of BMP-2 and serum on expression of chondrocytespecific matrix proteins [procollagen type I and II, aggrecan
and (V + C) fibronectin] varies depending on cellular morphology, and/or cytoskeletal organization when chondrocytes
are grown as monolayer, aggregate, pellet or explant cultures.
Despite these limitations, some measure of success has
been achieved with autologous chondrocyte transplantation
to repair cartilage defects. Focal chondral or osteochondral
defects, usually the result of trauma, have a poor capacity for
repair and predispose patients to osteoarthritis. In a survey
of one thousand consecutive knee arthroscopies, chondral or
osteochondral lesions were found in 61% of the patients, with
focal chondral or osteochondral defects accounting for 19%
with a mean defect area of 2.1 cm2 (Hjelle et al., 2002). Autologous chondrocyte transplantation (ACT) was first used
in humans in 1987 and the first pilot study was published in
1994 (Brittberg et al., 1994) in 23 patients with deep cartilage
defects in the knee. Cartilage slices were obtained through
an arthroscope from a minor-load-bearing area on the upper
medial femoral condyle of the damaged knee, placed in

chilled sterile normal saline and processed within 25 h. The
cartilage specimens were minced, washed in culture medium
containing Hams F12 medium supplemented with HEPES
buffer, antibiotics and ascorbic acid and digested for 16 h in
culture medium containing clostridial collagenase and deoxyribonuclease I. The cells were then filtered through nylon mesh, resuspended in culture medium supplemented with
15% of the patients serum (autologous serum), and grown
in culture flasks for 1421 days before being used to fill
the cartilage defect. A long-term follow up of a larger series of 61 patients treated for isolated cartilage defects on
the femoral condyle or patella was recently reported. After 2
years, 50 patients had good or excellent results and 51 of 61
had good or excellent results at 511 years later (Peterson et
al., 2002). Matrix-induced autologous chondrocyte implantation (MACI) is a refinement of ACI. The technique involves
growing chondrocytes on a Matricel membrane prior to transplantation. Matricel membranes are biocompatible, porcine
in origin and composed of type I/type III collagen, avoiding type II-, XI- and V-induced osteoarthritis. They have a
smooth and a coarse surface. Chondrocytes are seeded onto
the coarse surface to form multilayers in culture, while the
smooth surface of the Matricel membrane closely mimics the
surface of articular cartilage (Zheng and Wood, 2003).
In vitro methods currently employed to study chondrocytes in vitro, range from primary chondrocytes, to clonal
chondrocyte cell lines established from normal chondrocytes,
and transformed chondrocyte cell lines.
4.1.1. Primary chondrocytes
(a) Most studies have used chick chondrocytes. These can
be isolated from whole chick embryo sterna and maintained for extended periods of time in suspension culture
stabilized with agarose (Bruckner et al., 1989). The cells
remain viable without FBS only when seeded at high
densities. They do not proliferate at a high rate, but deposit extracellular matrix with fibrils resembling those
of authentic embryonic cartilage in their appearance and
collagen composition. The cells exhibit many morphological and biochemical characteristics of resting chondrocytes and they do not produce collagen X, a marker
for hypertrophic chondrocytes. Addition of FBS leads
to profound changes in the phenotype of chondrocytes
seeded at low density. They form colonies at a high rate
and assume properties of hypertrophic cells, including
the synthesis of collagen X, thus resembling adult rather
than embryonic cartilage.
(b) A culture system using chondrocytes derived from embryonic chick vertebrae was shown to undergo differentiation in vitro (Gerstenfeld and Landis, 1991). Supplementation with ascorbic acid led to the expression of the hypertrophic phenotype as assessed by an increase in alkaline phosphatase activity and collagen type X synthesis.
(c) Cheung et al. (2001) described a method of studying
mammalian chondrocyte differentiation in vitro using
93
a culture model of chondrocytes derived from epiphyses of long bones of fetal calves. Chondrocytes were induced to differentiate by treatment with 5-azacytidine, a
potent DNA demethylating agent shown to induce differentiation of the pluripotent mesenchymal cell line,
C3H10T1/2, to myoblasts, adipocytes and chondrocytes (Taylor and Jones, 1979). After treatment with 5azacytidine (aza-C) for 48 h, cultures maintained without
aza-C and harvested at selected time points, were shown
to undergo a sequence of differentiation steps leading
to hypertrophic chondrocytes, mimicking events occurring during endochondral ossification in vivo. Cells in
this culture model of endochondral ossification were synchronized, allowing the study of individual stages of the
differentiation pathway.
4.1.2. Chondrocyte cell lines
Chondrocyte cell lines are used as an alternative to primary
chondrocytes because they represent a renewable source of
material.
4.1.2.1. Normal clonal chondrocyte cell lines. CFK2 is an
established chondrocytic cell line derived from fetal rat calvariae. Extended culture results in the appearance of glycosaminoglycans and type II collagen in the cell layer in
association with the formation of focal nodes of cells. Differentiated CFK2 cells show enhanced parathyroid hormonestimulated adenylate cyclase activity and their proliferation
is stimulated by regulatory factors such as epidermal growth
factor, parathyroid hormone, and inhibited by dexamethasone
as well as retinoic acid (Bernier and Goltzman, 1993). Expression of matrix proteins was inhibited in CFK2 cells transfected with PTHrP (Henderson et al., 1996). Thus CFK2 can
serve as a non-transformed model of rat chondrocytic cells in
which both induction and regulation of the expression of cartilaginous matrix components by factors such as PTHrP and
Indian Hedgehog (Deckelbaum et al., 2002) can be observed.
Grigoriadis et al. (1996) established a family of nontransformed, clonal rat chondrogenic cell lines in which cartilage differentiation and metabolism can be studied in vitro.
These were established from a parental chondroblast clone
RCJ3.1C5 that was originally demonstrated to differentiate
into three-dimensional cartilage nodules when grown in the
presence of 15% FCS (Grigoriadis et al., 1989).
4.1.2.2. Transformed chondrocyte cell lines. ATDC5 is an
embryonal carcinoma cell line isolated from a differentiating
culture of AT805 teratocarcinoma (Atsumi et al., 1990), that
can be induced to undergo a reproducible, time-dependent
in vitro progression from early precursors to hypertrophic
chondrocytes (Shukunami et al., 1997, 1998).
IRC is an immortalized rat chondrocyte cell line that was
created by transformation of primary rat costal chondrocytes
with a recombinant murine retrovirus (NIH/J-2) carrying
the v-myc and v-raf oncogenes. It grows in suspension culture as multicellular aggregates and in monolayer culture as
94
polygonal cells, which accumulate an alcian-blue stainable

matrix. IRC cells synthesize typical cartilage proteins, aggrecan and link protein, but show reduced collagen II expression
(Oxford et al., 1994; Horton et al., 1988).
Kamiya et al. (2002) established a clonal chondrocytic cell
line, N1511, from rib cartilage of a p53-null mouse. BMP-2
and insulin treatment induces full differentiation toward hypertrophic chondrocytes, whereas treatment with PTH and
dexamethasone slows and limits differentiation. Recovery of
p53 expression in N1511 cells by transient transfection inhibits proliferation, suggesting that cell proliferation can be
regulated with p53 in this cell line. These results would indicate that N1511 is the only cell line with known genetic
mutation, which undergoes multiple steps of chondrocyte
differentiation towards hypertrophy, and may also be used
to study the function of p53.
HCS-2/8 is an immortalized clonal cell line derived from a
well-differentiated human chondrosarcoma (Takigawa et al.,
1989), with phenotypic features resembling normal chondrocytes. The cells synthesize aggrecan, integrins, collagen types
II, IX and XI, show the same responses to growth factors as
normal chondrocytes and maintain their cartilage phenotype
over more than 3 years in culture (Takigawa et al., 1997).
5. Calcium homeostasis
Calcium is an essential ion for many physiological processes such as cell motility, muscle contraction and neurotransmitter release. In mammals, these processes function
optimally when extracellular calcium is maintained within
a normal range by regulatory mechanisms that coordinate
the metabolic activities of the kidneys, intestine, parathyroid
glands, and bone.
Parathyroid cells express a cell surface calcium-sensing
receptor that recognizes and responds to physiological
changes in extracellular ionized calcium concentration. Reductions in serum calcium of the order of 12% result in
a prompt increase in parathyroid hormone (PTH) secretion.
PTH acts directly on the kidneys and skeleton and indirectly
on the intestine to normalize any fall in extracellular ionized calcium. In the kidney, PTH stimulates re-absorption
of calcium in the distal tubules and increases synthesis of
1,25(OH)2D3. 1,25-Dihydroxyvitamin D increases intestinal absorption of calcium and also stimulates the release of
calcium from bone by stimulating bone resorption. The increased flux of calcium ions into the extracellular fluid restores circulating levels of calcium toward normal. Normalization of serum calcium as well as the increased levels of
1,25-dihydroxyvitamin D inhibits further PTH synthesis in a
negative feedback loop. An increase in extracellular ionized
calcium inhibits PTH secretion, resulting in increased renal
calcium excretion and reduction in net release of skeletal calcium as well as intestinal absorption of calcium.
In several species, calcitonin secretion by the C-cells of
the thyroid is part of the homeostatic response to hypercal-
cemia but in man, no essential function has yet been found

for calcitonin. Steady-state plasma calcium shows little or
no change with either complete absence or a large excess of
calcitonin (Parfitt, 1993).
5.1. Parathyroid cells in culture
Work in the laboratory has generally been performed on
dispersed bovine parathyroid cells. Fresh bovine parathyroid
glands, transported in cold Hanks solution, are washed in
70% ethanol, dissected free of surrounding fat tissue and
finely minced in Hanks solution. They are transferred to
tissue culture flasks, 810 glands in 15 ml, and dispersed
by shaking with 1.25 mg/ml collagenase type I suspended
in Eagle basal medium containing 15% FCS. The cells are
filtered through 200 mm cell dissociation sieve and 40 mm
nylon mesh. Cell suspension is washed by centrifugation in
Hanks solution and dispersed in M-199 medium containing
1.25 nM Ca2+ and 15% newborn calf serum. Cells are plated
in 24 multiplate dish at a density of 1 106 cells per well
and cultured at 37 C in 5% CO2 for 24 h to allow attachment
(Moallem et al., 1995).
The extracellular calcium sensing receptor was cloned
from bovine parathyroid cells (Brown et al., 1993), and these
cells have been very useful in determining the role of calcium fluxes in the regulation of parathyroid hormone secretion (Chang et al., 2001).
5.2. C-cells of the thyroid
Calcitonin is a secretory product of the parafollicular (C)
cells of the thyroid, and medullary thyroid carcinoma (MTC)
is a neuroendocrine tumor of the parafollicular cells. Cell
lines established from human and animal MTC tumors provide a useful system to analyze genes involved in the development of this neoplasia, as well as a source of C cells
to determine the regulation of calcitonin production. The
hMTC cell line, TT cells (Leong et al., 1981) and the rat
MTC line, 623 cells (Zeytinoglu et al., 1980) can be purchased from the American Type Culture Collection (Manassas, VA). TT cells display an impaired expression of the
tumor suppressor gene p53 (Velasco et al., 1997). Apart
from calcitonin and the calcitonin receptor, TT cells express carcino-embryonic antigen, somatostatin and its receptors, neurotensin, gastrin-releasing peptide, Leu- and Metenkephalin, parathyroid hormone-releasing peptide, chromogranin A, synaptophysin, 1,25(OH)2D3 receptor and other
peptides (Frendo et al., 1994; Zabel et al., 1995; Velasco et
al., 1997; Zatelli et al., 2001). TT cells are routinely grown in
Hams F12K medium supplemented with 10% FBS at 37 C
in a humidified atmosphere containing 95% air and 5% CO2 .
6. Discussion
Although cell culture has proved invaluable in the study
of bone biology, in vitro model systems cannot reproduce
the complex three-dimensional architecture of bone that is

required for the proper expression of the functional capability
of the cells that make up its microenvironment. Nonetheless,
despite the limitations of the various systems described in
this review, significant and important contributions have
been made to our understanding of the normal processes
leading to bone formation, remodeling and resorption as
well as how these processes can be deranged to result in
metabolic bone diseases.
The term osteoblast describes a lineage of cells that differ substantially in their properties at different stages of development. Although many characteristic properties of osteoblasts have been described, it does not follow that all cells
of the lineage possess each of these features. At different
stages of differentiation, and at different sites in bone carrying out specific functions, osteoblasts are likely to express
only a proportion of the features associated with the phenotype. The challenge of identifying the cellular pathways and
the factors that regulate progression from osteoprogenitors to
mature osteoblasts is facilitated by the ability to isolate and
analyse in culture, osteoblasts at various stages of differentiation, and in particular, clonal cell lines from bone or bone
tumors. These are used as models of osteoblasts representing
different stages of differentiation, enabling investigators to
define the heterogeneity of bone cell populations with more
precision. In contrast, primary calvarial cells probably contain osteoblasts at all stages of differentiation, including osteoprogenitors that proliferate before undergoing a series of
maturational steps to become differentiated osteoblasts capable of forming mineralized nodules in culture (Aubin, 1998;
Malavalk et al., 1999). Primary calvarial cultures are widely
used to study the progression of differentiation in vitro. Rowe
and colleagues recently described an elegant method in which
subpopulations of osteoblasts at different stages of differentiation can be isolated from primary osteoblast cultures.
Having identified fragments of the rat type I (Col1a1) promoter that show preferential expression in different Col1a1producing tissues, they generated green fluorescent protein
(GFP)-expressing transgenic mice containing a 3.6- and a
2.3-kb rat type I collagen promoter fragment. The 3.6-kb
promoter directed strong expression of GFP messenger RNA
to preosteoblastic cells, while the 2.3-kb promoter directed
GFP mRNA expression to a cell that is late in the osteoblast
lineage, extending into mature osteocytes. They conclude that
with further refinement of this method, using other promoters and color isomers of GFP, it should be possible to isolate
subpopulations of cells at different stages of differentiation
from primary cultures derived from these transgenic mice
for molecular and cellular analysis (Bogdanovic et al., 1994;
Kalajzic et al., 2002).
Established osteoblast-like cell lines are particularly useful models to study signaling pathways in response to stimulation by osteotropic factors. The great majority of osteoblastlike cell lines however, do not mineralize in culture, with the
exception of MC3T3-E1 (Kodama et al., 1981; Sudo et al.,
1983), 2T3 (Ghosh-Choudhury et al., 1996) and KUSA-O
95
cells (Umezawa et al., 1992). Cell lines established from

pluripotent mesenchymal cells provide valuable information on the factors and mechanisms regulating differentiation
along the osteoblast, chondrocyte, adipocyte and myocyte
lineages.
In the case of osteoclasts, considerable progress has been
made in the past twenty years in the development of methods
to study their function and formation in vitro. Early studies
using relatively crude methods to disaggregate osteoclasts
from bone were cumbersome, succeeding in obtaining only
small numbers of osteoclasts that could not be separated from
other cell types such as stromal cells and osteoblasts, and
results were difficult to reproduce. Investigators were faced
with the challenging task of obtaining sufficient numbers of
relatively pure preparations of osteoclasts in culture. This
was clearly not attainable using bone as a primary source of
functional, mature osteoclasts. Knowledge that osteoclasts
are derived from hematopoietic stem cells that differentiate
along the monocyte/macrophage lineage, and the realization
in the late eighties, that direct contact between osteoclast
progenitors and stromal cells/osteoblasts is required for osteoclast differentiation, led to the widely-used coculture system to study the regulation of osteoclast differentiation from
mononucleate progenitors to mature, functional multinucleate osteoclasts. It became feasible to generate functional osteoclasts in culture in far greater numbers, even though the
osteoclasts could not be separated from the companion osteoblasts/stromal cells for separate analysis. Almost a decade
later, the pivotal role of RANKL in osteoclastogenesis, its interaction with its cognate receptor RANK as well as the decoy
receptor osteoprotegerin were revealed. Not only were these
discoveries major advances in the understanding of the regulation of osteoclast formation, it made possible the substitution of soluble RANKL and M-CSF for stromal/osteoblastic
cells, thus considerably simplifying the method for obtaining
functional osteoclasts in vitro. The use of cell lines as alternative sources of osteoclast progenitors is not widely practised
because of the lack of suitable cell lines. Some laboratories
have used RAW264.7 cells to generate osteoclasts in culture.
Unlike hematopoietic progenitors derived from bone marrow
or spleen, RAW264.7 cells do not require M-CSF alongside
RANKL. The mechanism underlying this difference is not
known, but it may imply a difference in the signaling pathway leading to osteoclastogenesis.
Chondrocytes are unique in their ability to exist in a low
oxygen tension environment, isolated within a voluminous
extracellular matrix devoid of a vascular supply. Primary
chondrocytes retain their phenotypic characteristics when
grown in the form of a three-dimensional multicellular complex, but undergo a process of dedifferentiation when grown
as monolayer cultures with serum supplementation. A range
of established chondrocytic cell lines are available as alternatives.
Work carried out on established cell lines and primary
cell cultures have provided much valuable insight into the
phenotypic characteristics of cells belonging to the bone
96
microenvironment, and the factors governing their development and function. Although it is not possible to grow bone in
the laboratory, much knowledge has been generated about the
processes leading to bone formation and resorption and the
factors that regulate the fine balance essential for the maintenance of the integrity of the skeleton. Targeted gene deletions have allowed the development of animal models with
altered skeletal phenotypes, but the mechanisms leading to
such alterations remain largely unexplored in the laboratory
using cell lines established from these models. The refinement of methods for selection and screening of rare homologous events in mammalian cells by the introduction of selectable markers in the transfected piece of DNA (Mansour et
al., 1988) and the isolation of pluripotent murine embryonic
stem (ES) cells (Evans and Kaufman, 1981; Martin, 1981)
that can be grown and manipulated in tissue culture have
broadened the scope of gene targeting approaches. Finally,
the transfection of osteoblastic cell lines with putative constructs has enabled the investigation of tissue-specific regulation of expression of the gene of interest at the cellular and
molecular level.
Acknowledgements
This work is supported by Program Grant 003211 from the
National Health and Medical Research Council (Australia).
The help provided by Dr. Julian Quinn with the preparation
of the manuscript, Figs. 2 and 3 and Table 7, is gratefully
acknowledged.
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Molecular and Cellular Endocrinology 228 (2004) 79102

and endochondral bone formation cease but remodeling of

V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79102

from established cell lines, particularly in osteoblast biology,

Fig. 1. Origin of cells of the osteoblast and chondrocyte lineages (modified

stem cells C3H10T1/2 both in the presence and absence of

V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79102

Human fetal bone marrow STRO-1 expressing stromal cells

(PGs), epidermal and transforming growth factors (EGFs,

V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79102

Moseley and Martin (1996)

Centrella et al. (1987), Hock et al. (1990)

minocycline and 80 mg/l gentamicin. Incubation conditions

fetal calvaria or subperiosteal fetal long bones, established

V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79102

Receptors and/or responses

Alkaline phosphatase (ALP)

Parathyroid hormone (PTH)

TGF- inducible early gene (TIEG)

c-myc, c-fos, and c-jun) (McCabe et al., 1995). Several other

V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79102

onset of mineralization achieving peak levels of expression.

the late 1970s by the Martin and Rodan laboratories. UMR

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2.4.3. Non-transformed cell lines

and EGF (Guenther et al., 1989). The various properties of

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sensitive mutant (tsA58) of SV40 large T antigen. hFOB 1.19,

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V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79102

3.3. In vitro methods to study osteoclast formation and

clast number in in vitro assays is a count of TRAP positive or

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Requires live, active cells; time dependent

FBS, and filtered through a 40 m cell strainer. Cells are

(ii) a source of hematopoietic cells such as murine spleen or

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PBS (5 ml per bone) from a syringe and samples from

Spleen cells, bone marrow or blood

Bone marrow macrophages or bone

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RANKL (Hsu et al., 1999). Unlike murine spleen cells, bone

teoclasts. However, PBMC are heterogeneous, consisting of

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Cartilage is a specialized form of connective tissue that

4.1. Propagation of chondrocytes in culture

Proteoglycans and other proteins

medium supplemented with serum and by passage (Hering

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medial femoral condyle of the damaged knee, placed in

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polygonal cells, which accumulate an alcian-blue stainable

cemia but in man, no essential function has yet been found

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the complex three-dimensional architecture of bone that is

cells (Umezawa et al., 1992). Cell lines established from

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Anderson, D.M., Maraskovsky, E., Billingsley, W.L., Dougall, W.C.,

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V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79102

Gerstenfeld, L.C., Landis, W.J., 1991. Gene expression and extracellular

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