Molecular and Cellular Endocrinology 228 (2004) 79–102

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. Vincent’s Hospital, 4th Floor, Daly Wing, 35 Victoria Parade, Fitzroy, Vic. 3065, Australia

b St. Vincent’s 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 phosphatecrystals deposited in an organic matrix. Bone has two main functions. It forms a rigid skeleton and has a central role in calcium and phosphatehomeostasis. The major cell types of bone are osteoblasts, osteoclasts and chondrocytes. In the laboratory, primary cultures or cell linesestablished from each of these different cell types provide valuable information about the processes of skeletal development, bone formationand bone resorption, leading ultimately, to the formulation of new forms of treatment for common bone diseases such as osteoporosis.©

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eywords:Cell lines; Primary cell cultures; Bone cell biology

. Introduction

Bone has several major functions. It forms a rigid skeletono provide a framework for the body, support for soft tissues,oints of attachment for skeletal muscles, protection for inter-al organs, housing for bone marrow as well as a central role

n mineral homeostasis, principally of calcium and phosphateons, but also of sodium and magnesium.

Bone is a dynamic tissue that is constantly remodeledhroughout life. During fetal development, most of the skele-on develops from cartilage anlagen which is eventually re-orbed and replaced with bone by a process termed endochon-ral ossification. In contrast, bones which form the calvaria,andible and maxilla are developed from mesenchyme byprocess termed intramembranous ossification. Bone mod-

ling is the process associated with growth and reshaping ofones in childhood and adolescence. In bone modeling, longi-

udinal growth of long bones depends on proliferation and dif-erentiation of cartilage cells at the growth plate while growthn width and thickness is accomplished by formation of bonet the periosteal surface with resorption at the endosteal sur-

and endochondral bone formation cease but remodelibone continues. Remodeling constitutes the lifelong renprocess whereby the mechanical integrity of the skeletpreserved. It implies the continuous removal of bone (bonsorption) followed by synthesis of new bone matrix and ssequent mineralization (bone formation). The maintenof normal, healthy bone requires the coupling of bonemation to bone resorption, with intercellular communicabetween osteoblasts and osteoclasts integral to the acment of a balance between the two processes. Furtherbone remodeling is an integral part of the calcium hoostatic system (Eriksen et al., 1993) that also involves thparathyroid glands, intestinal system and the kidneys.

Many aspects of the processes described above cinvestigated in the laboratory using primarily cell cultuThe major cell types are the bone-forming osteoblasts, bresorbing osteoclasts and cartilage-forming chondrocytthorough understanding of the factors regulating the diffetiation of each of these cell types, the mechanisms by wregulatory factors influence their function, and the mannwhich these cells communicate and interact with each oth

ace. 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).

central to the design of rational therapeutic strategies to treatbone diseases such as osteoporosis. This review will focus oncell lines that are established in the laboratory from these dif-ferent cell types. While much information has been derived

reserv

303-7207/$ – see front matter © 2004 Elsevier Ireland Ltd. All rightsoi:10.1016/j.mce.2003.06.002

ed.

80 V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102

from established cell lines, particularly in osteoblast biology,a substantial amount of work is nonetheless still being carriedout with primary cultures of osteoblasts, chondrocytes and os-teoclasts, and attention will be drawn to these, where relevant.

In bone cell biology, cell cultures are used mainly to ex-amine:

• Regulation of expression of phenotypic characteristics typ-ical of osteoblasts, chondrocytes and osteoclasts.

• Regulation of differentiation of relatively undifferentiatedmesenchymal cells along different lineages, for example,muscle, osteoblasts, chondrocytes and adipocytes.

• Signaling pathways relevant to osteoblast, osteoclast andchondrocyte functions.

• Effects of over-expression and under-expression of partic-ular gene products on cell function.

• In vitro bone formation/mineralization.• Interactions between osteoblasts and osteoclasts, particu-

larly in the regulation of osteoclast formation in vitro.

2. Osteoblasts

2.1. Osteoblast ontogeny

Osteogenic cells arise from pluripotential mesenchymals ntiatei hon-d ewedi n eta ia-t rig-i theo

gen-i illus-t beu er ofr mingg em es-e ld onem nicd ffer-e ar eci-fi lin-e rizedce 1t wnt havea cyticc Fore Shh)a mal

Fig. 1. Origin of cells of the osteoblast and chondrocyte lineages (modifiedfrom Nijweide et al., 1986).

stem cells C3H10T1/2 both in the presence and absence ofBMP-2, while committing these pluripotent cells into the os-teoblastic lineage. Treatment of C3H10T1/2 cells with BMP-7 has also been shown to induce both chondrogenesis andosteogenesis (Gerstenfeld et al., 2002) while treatment withthe potent DNA demethylating agent 5-azacytidine has pre-viously been known to induce differentiation to myoblasts,adipocytes and chondrocytes (Taylor and Jones, 1979).

A variety of cell culture models and other tools suchas the use of monoclonal antibodies have been employedby researchers to track the various stages of osteogenesis.The murine IgM monoclonal antibody STRO-1 recognizesa cell surface antigen expressed by stromal cells in humanbone marrow and is used to identify clonogenic bone mar-row stromal cell progenitors (fibroblast colony-forming units[CFU-F] (Simmons and Torok-Storb, 1991). Gronthos andcolleagues used dual-color fluorescence-activated cell sort-ing to identify cells expressing STRO-1 and ALP in primarycultures of normal human bone cells (NHMC). They showedthat preosteoblastic STRO-1+/ALP− cells did not expressbone-related markers such as bone sialoprotein, osteopontin,and parathyroid hormone receptor and had a reduced abilityto form a mineralized bone matrix over time. The majorityof NHBCs representing fully differentiated osteoblasts, ex-pressed STRO-1−/ALP+ and STRO-1−/ALP− phenotypes,w edi-a ALPN , con-fie re-v aOS-2 wasd mit-t

tem cells. These stem cells have the capacity to differento lineages other than osteoblasts, including those of croblasts, fibroblasts, adipocytes, and myoblasts (revi

n Nijweide et al., 1986; Friedenstein et al., 1987; Aubil., 1995). By analogy with hematopoietic cell different

ion, each of these differentiation lineages is thought to onate from a different committed progenitor, which forsteogenic lineage is called the osteoprogenitor.

Osteodifferentiation progresses via a number of protor and precursor stages to the mature osteoblast, asrated inFig. 1. Pluripotent mesenchymal cell lines cansed to study this process which is regulated by a numbegulatory molecules such as members of the transforrowth factor beta (TGF�) superfamily, including the bonorphogenetic proteins (BMPs). When murine C2C12 mnchymal precursor cells are treated with TGF�1, terminaifferentiation into myotubes is blocked. Treatment with borphogenetic protein 2 (BMP-2) similarly blocks myogeifferentiation of C2C12 cells but induces osteoblast dintiation (Lee et al., 2000). Further evidence implicatingole of the BMP receptors (type IA and IB) in both the spcation and differentiation of osteoblastic and adipocyticages comes from recent studies using a well-charactelonal cell line 2T3, derived from mouse calvariae (Chent al., 1998). In other studies (Spinella-Jaegle et al., 200),

he proteins of the hedgehog (Hh) family which are knoo regulate various aspects of normal limb patterning,lso been shown to influence the osteoblastic and adipoommitment/differentiation of mesenchymal stem cells.xample, recombinant N-terminal sonic hedgehog (N-bolishes adipocytic differentiation of murine mesenchy

hile the STRO-1+/ALP+ subset represented an intermte preosteoblastic stage of development. All STRO-1/HBC subsets expressed the transcription factor cbfa-1rming that they were committed osteogenic cells (Gronthost al., 1999). A survey of human osteosarcoma cell linesealed that STRO-1 was expressed by MG-63 but not S. Among murine cell lines tested, expression of STRO-1etected in the bipotential line BMS-2 but not the com

ed osteoblast precursor MC3T3-E1 (Stewart et al., 1999).

V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102 81

Human fetal bone marrow STRO-1 expressing stromal cellswith weak ALP activity could be induced by dexametha-sone and 1,25-dihydroxyvitamin D3 to increase their expres-sion of ALP activity. Furthermore, when cultured in three-dimensional aggregates, osteogenesis occurred along withthe expression of osteocalcin, a marker of differentiated os-teoblasts (Oyajobi et al., 1999). Thus, a hierarchy of bonecell development in vitro could be identified to facilitate thestudy of bone cell differentiation and function, using STRO-1 expression to identify less well-differentiated cells of theosteoblast lineage.

Important in vitro cell culture models employed to furtherstudy the various stages of osteogenesis, include clonal celllines derived either from bone tumors (ROS 17/2.8 or UMR106; human MG-63 or SaOS-2) or from primary bone cellcultures (MC3T3-E1, UMR 201 and RCJ cell lines). Thesecell lines will be discussed in detail later. MC3T3-E1 andUMR 201 cells have been described as representatives of pre-osteoblastic cells while ROS 17/2.8 as more differentiatedosteoblasts based on the profile of expression of osteoblastphenotypic features. Extensive in vitro analyses of the variouscell lines have provided evidence for considerable flexibilityin the repertoire of osteoblast-associated genes expressed atmultiple differentiation stages as osteoprogenitors mature toform functional osteoblasts.

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Table 1Systemic agents that influence osteoblast function

Agent References

Endocrine hormonesParathyroid hormone (PTH) Parsons (1976), Tam et al. (1982)1,25(OH)2D3 Chen et al. (1983a), Narbaitz et al.

(1983)Growth hormone (GH) Stracke et al. (1984), Barnard et al.

(1991)Glucocorticoid hormones Dietrich et al. (1979), Canalis

(1983)Gonadal steroids (estrogen,

testosterone)Komm et al. (1988)

Insulin Raisz and Kream (1983), Kream etal. (1985)

Retinoids Ng et al. (1985, 1988), Zhou et al.(1991)

Other systemic agentsEpidermal growth factor (EGF) Ng et al. (1983)Transforming growth factor�

(TGF�)Ibbotson et al. (1985)

Prostaglandins (PGs) Raisz and Martin (1984)Parathyroid hormone-related

protein (PTHrP)Yang and Stewart (1996)

Calcitonin gene-related peptide(CGRP)

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 producea specific membrane-bound molecule known as receptor acti-vator of NF�B ligand) due to its ability to induce nuclear fac-tor �B) (Anderson et al., 1997a, 1997b). It is responsible forprogramming osteoclast differentiation and also functions asa 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 var-ious systemic and local (paracrine/autocrine) factors. Theseare summarized inTables 1 and 2.

2.3. Osteoblast cell culture methods

Cell culture techniques combined with basic molecular ge-netic techniques have become highly popular and widespreadtools that are routinely applied to bone cells (Peck et al.,1964). Some of the basic experimental approaches used in os-teoblastic cell culture, including system design, specific cellculture systems and various biochemical and morphologicalassays defining osteoblasts in cell culture, are discussed indepth in the excellent review byMajeska (1996).

In general, most systems currently in use share routineb nce.O yerc ifiedE dw um( (2 /l

.2. Morphology and function

Mature osteoblast cells constitute a lineage of highlyerentiated cells which differ substantially in their propert different stages of development. Thus, depending otage of functional differentiation, they are representedistinctive phenotype, morphology and biosynthetic actictive osteoblasts are plump, cuboidal, mononuclear

ying on the matrix which they have synthesized. Osteocre mature osteoblasts which have become trapped withone matrix and are responsible for its maintenance.

ining cells are flat, elongated cells that cover bone surfhat are undergoing neither bone formation nor resorpMiller and Jee, 1987). Inactive osteoblasts are much ther and more elongated cells, yet they are morphologi

ndistinguishable from the lining cells.Actively synthesizing osteoblasts contain an exten

one oriented secretory organelle apparatus, a largeomplex near the nucleus and a high mitochondrial conll reflecting the capacity of osteoblasts for their prim

unction of protein synthesis (Doty and Schofield, 1976). Os-eoblasts are rich in alkaline phosphatase enzyme (Heath andeynolds, 1990). They synthesize and secrete type I coen, glycoproteins such as osteopontin (rich in sialic a- and O-linked oligosaccharides) and osteocalcin (conlutamic and aspartic acid residues), cytokines and gr

actors into a region of unmineralized matrix (osteoid)ween the cell body and the mineralized matrix. Osteobave receptors and responses to parathyroid hormone (,25-dihydroxyvitamin D3 [1,25(OH)2D3], prostagland

,

asic culture environments and methods of maintenasteoblast cell lines are routinely grown in monola

ulture in standard tissue culture flasks in alpha-modagle’s minimal essential medium (�MEM), supplementeith 10% fetal calf serum (FCS) or fetal bovine ser

FBS), 0.2% sodium bicarbonate, 16 mM HEPESN--hydroxyethylpiperazine-N′-2-ethanesulfonic acid), 1 mg

82 V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102

Table 2Local agents that influence osteoblast function

Agent References

Paracrine factorsParathyroid hormone-related protein (PTHrP) Moseley and Martin (1996)Transforming growth factor� (TGF-�1, -�2, and -�3) Centrella et al. (1987), Hock et al. (1990)Fibroblast growth factors (FGF 1 and 2) Rodan et al. (1987a), Canalis et al. (1988)Insulin-like growth factor (IGF I and II) Schoenle et al. (1982)Platelet-derived growth factor (PDGF) Canalis (1981)Bone morphogenetic proteins (BMPs 2–7) Hammonds et al. (1990), Wozney et al. (1988)Tumor necrosis factors (TNFs� and�) Bertolini et al. (1986), Ng et al. (1989a, 1989b)Interleukin-1 (IL-1) Canalis (1986), Mundy (1993)Interleukin-6 (IL-6) Mundy (1993)Prostaglandin E2 (PGE2) Chyun and Raisz (1984)Calcitonin gene-related peptide (CGRP) Michelangeli et al. (1989)Leukemia inhibitory factor (LIF) Allan et al. (1990)Vasoactive intestinal peptide (VIP) Hohmann et al. (1986)

Autocrine factorsTransforming growth factor� (TGF-�1, -�2, and -�3) Centrella et al. (1987), Hock et al. (1990)Fibroblast growth factors (FGF 1 and 2) Rodan et al. (1987a), Canalis et al. (1988)Insulin-like growth factor (IGF I and II) Schoenle et al. (1982)Platelet-derived growth factor (PDGF) Canalis (1981)

minocycline and 80 mg/l gentamicin. Incubation conditionsare usually maintained at 37◦C in a humidified atmospherewith 5% CO2, although there are some cell lines that requirespecial growth conditions. Culture medium is changed ev-ery 48 h. For routine maintenance, cells are subcultured atconfluence whereby medium is removed and cells detachedfrom the flask by enzymatic digestion. A 10-min incubationat 37◦C in 0.025% trypsin in versene (140 mM NaCl, 2 mMKCl, 1 mM KH2PO4, 8 mM Na2HPO4, 0.05 M EDTA) is usu-ally sufficient for cells to lift off. The cell suspension is pouredinto a sterile 20ml tube and cells are pelleted at 1500 rpm for5 min in a centrifuge. Cells are resuspended in 10 ml of freshmedium, and after separating them by passage through a 21gauge needle on a syringe, the appropriate volume of cellsis 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. Thesecultures are washed twice in warm phosphate-buffered saline(PBS) pH 7.4 and then digested in a solution of collagenaseand dispase for 90 min at 37◦C. Cell suspensions are subse-quently washed twice in growth medium supplemented with5% FCS before being passed through a cell strainer to obtainsingle cell suspensions (Gronthos et al., 1999).

2g

fullyu withe s in-c enti-a d locaf rganc rom

fetal calvaria or subperiosteal fetal long bones, establishedclonal cell lines from cells isolated from bone tumors (typi-cally osteosarcoma), non-transformed cell lines, experimen-tally immortalized cell lines, and bone marrow cultures. Anextensive list of some of the commonly used osteoblast celllines is outlined inStewart et al. (1999).

2.4.1. Primary cultures of bone cells and organ culturesSome of the in vitro models, especially the calvaria-

derived primary cell cultures, undergo changes which mimicosteoblastic differentiation in vivo, thus providing insightsinto the stage-wise regulation of gene expression in osteoblas-tic cells. Observations made from these systems have beenextrapolated to adult bone in vivo to develop concepts of the“osteoblast phenotype”. A summary of the properties associ-ated with this phenotype is provided inTable 3. It must be em-phasized at this point, that depending on the cell system andculture conditions used, temporal aspects of the osteoblastdifferentiation process and ability to ascertain a sequentialexpression of markers varies markedly and is sometimes con-tradictory (Aubin et al., 1993, 1995). Many of the systemsmentioned above have limitations such as the inherent in-stability and the possibility of aberrant behaviour associatedwith many of the clonal cell lines, or in the case of primarycultures, the fact that they contain a heterogeneous mixtureo andca l-t d ratb ulturem stents zedn

e int n oc-

.4. Model systems of osteoblast differentiation andene expression

A variety of cultured cell systems have been successsed to study the pathways of osteoblast differentiationach model system offering unique advantages towardreasing our understanding of bone development, differtion, gene expression and responses to hormones an

actors. Commonly employed model systems include oultures, primary cultures of osteoblastic cells derived f

l

f osteoblastic cells at different stages of differentiationells of other lineages including fibroblastic cells (Aubin etl., 1993; Liu et al., 1994). Nevertheless, differentiating cu

ures from both the extensively studied rat calvaria anone marrow stromal systems and various other bone codels when plated appropriately, go through a consi

eries of differentiating events culminating in mineraliodule formation.

In calvarial-derived cultures grown to post-confluenche presence of serum and ascorbic acid, cell proliferatio

V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102 83

Table 3Osteoblast phenotypic characteristics (modified from Martin et al., 1993)

Proteins Receptors and/or responses

Alkaline phosphatase (ALP) Parathyroid hormone (PTH)Type I collagen (COL I) Parathyroid hormone-related protein (PTHrP)Osteocalcin ProstanoidsOsteopontin (OP) 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3)Osteonectin RetinoidsBone proteoglycan I (biglycan) Epidermal growth factor (EGF)Bone proteoglycan II (decorin) Tumor necrosis factor� (TNF�)Thrombospondin Tumor necrosis factor� (TNF�)Fibronectin (FN) Interleukin-1 (IL-1)Vitronectin (VN) Interleukin-6 (IL-6)Bone morphogenetic proteins (BMPs) Transforming growth factor� (TGF�)Transforming growth factor� (TGF�) Bone morphogenetic proteins (BMPs)Fibroblast growth factors (FGFs) Transforming growth factor� (TGF�)Insulin-like growth factor I (IGF I and II) GlucocorticoidsInterleukin-1 (IL-1) Insulin-like growth factor I (IGF I)Interleukin-6 (IL-6) Insulin-like growth factor II (IGF II)Interleukin-11 (IL-11) InsulinCiliary neurotrophic factor (CNTF) InhibinTumor necrosis factor� (TNF�) ActivinLeukemia inhibitory factor (LIF) Estrogen receptors� and� (ER�; ER�)Colony-stimulating factors (e.g. CSF-1) Leukemia inhibitory factor (LIF)Prostanoids Atrial naturetic peptide (ATP)Noggin Calcitonin gene-related peptide (CGRP)Receptor activator of NF�B ligand Calcium sensing receptor (CSR)(RANKL) Vasoactive intestinal peptide (VIP)Osteoprotegerin (OPG) Vascular endothelial growth factor receptorsOsteoclast inhibitory lectin (OCIL) (VEGFR)Notch 2 Fibroblast growth factor receptor-2 (FGFR2)Jagged 2 Growth hormone (GH)Vascular endothelial growth factors Connective tissue growth factor-like(VEGF) (CTGF-L)Phosphate-regulating gene with homologies to endopeptidases

on the X chromosome (Phex)Phosphate-regulating gene with homologies to endopeptidaseson the X chromosome (Phex)

Amylin Intercellular adhesion molecule (ICAM)Oncostatin M (OSM) Soluble low density lipoprotein receptor-related protein (SLRPs)Cardiotrophin-1 (CT-1)Homeobox (Hox) TGF-� inducible early gene (TIEG)Matrix metalloproteinase protein-13 Catenins(MMP-13) STRO-1Cyclo-oxygenase 2Cadherins

curs in scattered foci, which develop into multilayered struc-tures called “nodules” (Bellows and Aubin, 1989). Thesenodules consist of a top layer of osteoblast-like cells whichstain intensely for alkaline phosphatase, sitting underneathan osteoid layer containing collagen fibrils (Bellows et al.,1987; Bhargava et al., 1988).

The process of bone nodule formation as studied in ratcalvaria populations has been subdivided into three devel-opmental stages: proliferation, extracellular matrix develop-ment and maturation, and matrix mineralization. Character-istic changes in genes associated with proliferative and cellcycling activity and those associated with specific osteoblastactivities are observed throughout all stages (reviewed inAubin et al., 1993, 1995; Lian and Stein, 1995; Stein andLian, 1993). In the first phase, active proliferation is reflectedby mitotic activity with expression of genes associated withcell cycling (e.g., histone) and growth (e.g., proto-oncogenes

c-myc, c-fos, andc-jun) (McCabe et al., 1995). Several othergenes associated with formation of the extracellular matrix(type I collagen, fibronectin, and TGF�) are also actively ex-pressed (Aronow et al., 1990; Owen et al., 1991) and thengradually down-regulated with collagen mRNA being main-tained at a low basal level during subsequent stages of os-teoblast differentiation.

Immediately after the down-regulation of proliferation,proteins associated with the osteoblast phenotype are de-tected such as alkaline phosphatase. With progression intothe mineralization stage, all cells become positive foralkaline phosphatase. Other osteoblast-related genes such asbone sialoprotein (BSP) (Nagata et al., 1991), osteopontin(OP), and osteocalcin (Owen et al., 1990) are induced fol-lowing the onset of mineralization. OP is expressed duringthe period of active proliferation (at 25% of maximal levels),decreases post-proliferatively, and then is induced again at the

84 V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102

onset of mineralization achieving peak levels of expression.Consistent with high levels of osteopontin expression laterin the osteoblast developmental sequence are the calciumbinding properties of this acidic glycoprotein containingO-phosphoserine (Glimcher, 1989). The Vitamin K-dependentprotein, osteocalcin (Lian and Friedman, 1978), in contrastto OP, is mainly expressed post-proliferatively with the onsetof nodule formation. Late expression of osteocalcin in theosteoblast development sequence, suggests that it is a markerof the mature osteoblast, which is consistent with a possiblerole for the synthesis and binding of osteocalcin to mineralin the coupling of bone formation to resorption.

A similar temporal pattern of gene expression reflect-ing stages of progressive bone formation has been observedin cultured normal diploid osteoblasts derived from chick(Gerstenfeld et al., 1987; Shalhoub et al., 1989; Aronow etal., 1990), bovine (Ibaraki et al., 1992), and in cell lines ofhuman (Keeting et al., 1992) and mouse (Quarles et al., 1992)origin. Furthermore, it has also been possible to discern cellsof the osteoblast lineage at different stages of differentiationin situ and note the pattern of expression of osteoblast-relatedmarkers 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, theo d re-fl ines.F ) aree ationp heP enti-ae

blastc eren-t re-s erem fromf t-likec ta .,1

a-t rma-t f theb sitiv-i nee fi-b dif-f h-n

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U ring

the late 1970s by the Martin and Rodan laboratories. UMR106 is a cell line derived from a transplantable rat32P-inducedmalignant osteogenic sarcoma (Martin et al., 1976; Partridgeet al., 1983). The cell line has been extensively characterizedwith properties of enrichment in ALP activity, type I collagenproduction, adenylate cyclase responsiveness to PTH, PGE2,ability to mineralize in vivo, prostaglandin production, col-lagenase production and receptors for 1,25(OH)2D3, EGFand PTH (Forrest et al., 1985; Mitchell et al., 1990; Ng etal., 1983; Partridge et al., 1980, 1983, 1987). The twosubclones reported for UMR 106 cells are UMR 106-01 and UMR 106-06 (Forrest et al., 1985) with the onlyknown substantial difference between the two clones be-ing the expression of calcitonin receptors in the UMR106-06.

The family of clonal cell lines designated ROS (ROS 17/2and its subclone ROS 17/2.8) were derived from a sponta-neous tumor in an ACI rat that had been propagated by serialsubcutaneous transplantation (Majeska et al., 1980). Thesecells exhibit adenylate cyclase activity in response to PTHas well as a high ALP activity which is regulated by PTH(Majeska and Rodan, 1982a) and 1,25(OH)2D3 (Majeskaand Rodan, 1982b). The subclone ROS 17/2.8 constitutivelyexpresses osteocalcin mRNA (Price and Baukol, 1980) andproduces calcified matrix when implanted in diffusion cham-ba steo-p 88b

ano4 taI arilyu bonem n tofi

newh selyr comac rest-i ieticc gu-l osar-c -t SFa lasticc MG-6 L72c cans itorsi pri-m dif-f oree ,2

steoblast differentiation process is both determined anected by the expression of certain hormones and cytokor instance, the bone morphogenetic proteins (BMPsxpressed in early osteoblasts and in the late mineralizhase (Harris et al., 1994a, 1994b), and the expression of tTH receptor appears to correlate with increasing differtion (reviewed inAubin et al., 1995; Suda et al., 1996;Bost al., 1996).

To date several bone organ cultures and primary osteoultures have been widely used to study osteoblast diffiation (Owen et al., 1990) as well as the osteoblasticponse to growth factors (Centrella et al., 1987; Guentht al., 1988), and hormones (Wong and Cohn, 1975). Theajor bone culture systems use long bones or calvaria

etal/neonatal rats and mice. Populations of osteoblasells can be isolated by using enzymatic digestion (Peck el., 1964) or by mechanical methods (Ecarot-Charrier et al983).

Although the available models provide important informion on the fundamental processes involved in bone foion, it is important to note that the source and the age oone, the medium, and culture system, all affect the sen

ty of bones to hormones (Stern and Krieger, 1983; Soskolit al., 1986). In addition, the variable proportion ofroblasts and osteoblast cells at different stages of

erentiation provides a further limitation to this tecique.

.4.2. Osteosarcoma cell linesThe most widely used osteosarcoma cell lines are

MR 106 and the ROS 17/2 which were established du

ers (Shteyer et al., 1986). The cells respond to TGF� withn increase in ALP, type I collagen, osteonectin, and oontin mRNA (Noda and Rodan, 1987; Noda et al., 19)ut with a decrease in mRNA for osteocalcin (Noda, 1989).

Other models of osteoblastic cells derived from humsteosarcomas include the SaOS (Rodan et al., 1987b), OHS-(Fournier and Price, 1991), TE-85, MG-63 (Francheschi el., 1985, 1988), KPDXM and TPXM (Bruland et al., 1988).

nterestingly, some of these cell lines have been primsed as models to study the control of expression ofatrix proteins, including integrin-mediated cell adhesio

bronectin (Dedhar et al., 1987; Rodan et al., 1994).Rochet and colleagues have recently characterized a

uman osteosarcoma cell line, CAL72, which is more cloelated to normal osteoblasts than any of the osteosarells previously described, and could also provide an inteng tool to study the role of osteoblastic cells in hematopoell growth and differentiation. The cell line exhibits a sinar cytokine expression profile compared to other osteoma cell lines (Rochet et al., 1999). CAL72 cells constituively express mRNA coding for IL-6, GM-CSF, and G-Cnd thus appear to be closer to human primary osteobells than the well-described osteosarcoma cell lines3 and SaOS-2. In contrast to MG-63 or SaOS-2, CAells do not inhibit hematopoietic colony formation andustain the limited expansion of hematopoietic progenn a way similar to that described for normal human

ary osteoblasts. In addition, CAL72 cells induce theerentiation of promyelocytic cells into macrophages mfficiently than other osteosarcoma cell lines (Rochet et al.003).

V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102 85

2.4.3. Non-transformed cell linesThe UMR 201 cell line is a clonal non-transformed cell

line derived from neonatal rat calvaria. The cell line was es-tablished byNg et al. (1988)as a non-immortalized cell linewith a limited lifespan (12 passages in culture) and pheno-typic features suggestive of pre-osteoblasts. They have un-detectable alkaline phosphatase activity and mRNA for ALPwhich is significantly induced by the differentiating agent,retinoic acid (Ng et al., 1988, 1989a). TNF�, dexametha-sone and 1,25(OH)2D3 also modulate retinoic acid (RA)-induced ALP activity and mRNA for ALP in these cells (Nget al., 1989a, 1989b). This cell line therefore, provides auseful model to study the osteoblastic differentiation fromprogenitor cell populations and also the regulation of os-teoblast differentiation by systemic hormones and localgrowth factors. Furthermore, in subsequent studies the samegroup established immortalized subclones of UMR 201 us-ing SV40 large T antigen (see section below) hence, al-lowing careful comparison of phenotypic characteristicsbetween the parent and derived cell lines (Zhou et al.,1991).

MC3T3-E1 is a clonal non-transformed cell line estab-lished from newborn mouse calvaria (Kodama et al., 1981;Sudo et al., 1983). This cell line has also been shownto increase cyclic AMP production in response to PTH( yw apa-b atrixv n bemi -E1c rtiesm ,1 T3-E tiala ays.C xtra-c in-i , 30,a rre-l lonet r ande blastr evelso cel-l tedi ilityt iche rma-t isb enee

7/7,C lonaln latedf GF

Table 4Properties of the CRP clonal cell lines

Clone ALP (activity) Response toPTH PGE2

OsteocalcinmRNA

CRP 4/7 Absent Absent AbsentCRP 7/7 Present Absent AbsentCRP 7/4 Absent Present PresentCRP 10/3 Present Absent PresentCRP 10/30 Present Present Present

and EGF (Guenther et al., 1989). The various properties ofthese clonal cell lines are shown inTable 4.

2.4.4. Experimentally immortalized cell linesThe immortalization of cells by transfection with a re-

combinant retrovirus containing the cDNA for SV40 large Tantigen has been used to establish immortalized osteoblasticcell lines (Jat and Sharp, 1986). RCT-1 and RCT-3 cell lineswere derived from isolated and fractionated embryonic ratcalvarial cells. RCT-1 cells were established from the earlydigest population and expressed osteoblastic traits [ALP, pro-�1(I) collagen, PTH-responsive adenylate cyclase] after in-duction by retinoic acid. RCT-3 cells on the other hand, wereestablished from the more osteoblastic late digest populationand were found to constitutively express osteoblastic markersexcept osteocalcin (Heath et al., 1989).

KS-4 is a clonal cell line which was isolated from mousecalvaria by transfection with the c-Ha-ras-1 gene. The cellsdisplay low ALP activity at confluence, low type I collagenproduction and low cAMP accumulation in response to PTH.The cells also display low mRNA levels for pro-�1(I) colla-gen, osteonectin and bone proteoglycan I but not osteocalcin.Importantly, KS-4 cells have the ability to stimulate osteo-clast formation on co-culture with spleen cells (Yamashita etal., 1990a, 1990b).

celll ationie OB( db , de-r allT ell-d selya Theyea -d os-t unc-t ot ther thec

de-r riage( is-s ure-

Kumegawa et al., 1984) and exhibits a high ALP activithich is regulated by PTH, PGE2, 1,25(OH)2D3 and is cle of collagen synthesis. In addition, these cells form mesicles which are deposited on collagen fibrils and caineralized in vitro (Sudo et al., 1983). Although originally

solated as a clonal cell line, different variants of MC3T3ells have been isolated with different phenotypic propeost likely as a result of prolonged passaging (Leis et al.997). Wang and colleagues isolated 10 subclonal MC31 cell lines exhibiting high or low mineralization potenfter growth in ascorbic acid-containing medium for 10 dlones 4, 8, 11, 14, and 26 formed a well-mineralized eellular matrix after incubation for 2 days in medium conta

ng 3.0 mM inorganic phosphate, whilst clones 17, 20, 24nd 35 failed to form any detectable mineral. A good co

ation was observed between the ability of a given subco activate the osteoblast-specific osteocalcin promotendogenous expression of osteocalcin and other osteoelated mRNAs. Essentially, subclones expressing high lf osteoblast marker mRNAs formed a mineralized extra

ular matrix in culture and were osteogenic when implannto mice. In addition, all subclones, regardless of their abo differentiate, expressed high levels of cbfa-1 mRNA, whncodes a transcription factor necessary for osteoblast fo

ion (Otto et al., 1997), implying that the presence of cbfa-1y itself insufficient for induction of osteoblast-specific gxpression (Wang et al., 1999).

The cell lines represented by CRP 4/7, CRP 7/4, CRPRP 10/3 and CRP 10/30 also belong to the group of con-transformed osteoblastic cells. These cells were iso

rom neonatal calvarial bone cells in the presence of T�

-

Other model systems of experimentally immortalizedines used to study certain stages of osteoblast differentinclude the adult human osteoblast-like (hOB) cells (Keetingt al., 1992) and the human fetal osteoblast cell line hFHarris et al., 1995). The hOB cell line was immortalizey transfecting normal adult human osteoblast-like cellsived from a 68-year-old woman, with the large and sm

antigens of the SV40 virus. The cells represent a wifferentiated, steroid-responsive clonal cell line that clopproximates the phenotype of the mature osteoblast.xpress mRNA for�(I)-procollagen, osteopontin, TGF�,nd interleukin-1 beta (IL-1�), while treatment with 1,25ihydroxyvitamin D3 results in increased expression of

eocalcin and alkaline phosphatase mRNA and protein. Fional estrogen and androgen receptors are present but neceptor for PTH. When�-glycerophosphate is added toultures, the cells produce a matrix that mineralizes.

The human fetal osteoblast cell line (hFOB) wasived from biopsies obtained from a spontaneous miscarHarris et al., 1995). Primary cultures isolated from fetal tue were immortalized by transfection with a temperat

86 V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102

sensitive mutant (tsA58) of SV40 large T antigen. hFOB 1.19,the highest alkaline phosphatase-expressing clone, increasedalkaline phosphatase activity and osteocalcin secretion in adose dependent manner following 1,25-dihydroxyvitamin D3treatment. Differentiated hFOB cells showed high levels ofosteopontin, osteonectin, BSP, and type I collagen expres-sion. Treatment of hFOB cells with parathyroid hormone(1–34) resulted in increased cAMP levels. In addition, uponreaching confluence, hFOB cultures formed mineralized nod-ules.

2.5. A model of metabolic bone disease

Type I collagen is the most abundant and ubiquitouslydistributed of the collagen family of proteins. It is a het-erotrimer comprising two alpha1 (I) chains and one alpha2 (I)chain, which are encoded by the unlinked loci COL1A1 andCOL1A2, respectively. Mutations at these loci result primar-ily in the connective tissue disorders osteogenesis imperfecta(OI) and Ehlers–Danlos syndrome. Osteogenesis imperfectais a heterogenous genetic disorder associated with increasedfractures (Rowe, 2002). In its most severe form multiple frac-tures occur in utero and the disorder is lethal. In milder forms(type I) there may be an increase in fractures in childhoodwhich then cease after puberty, with a subsequent increase inwy sis ofO eend ,1 uta-to ationw f an rinem v1 on-f ta l-l llst ireda llagenm lum( andB omO1 fer-e ntoo neticp

im-p f os-t rmali entlyb enew oly-m tion

factor Sp1 in the first intron of COLIA1, and has been foundto be associated with bone mass and osteoporotic fracture inseveral Caucasian populations (Ralston, 1999).

2.6. Osteocytes

Osteocytes are terminally differentiated cells of the os-teoblast lineage that have become embedded in mineralizedmatrix. Individual osteocytes communicate with each otherand with cells on the bone surface such as lining osteoblastcells, through long intercellular processes. Their location andmorphology renders them particularly well suited to transferinformation between cells within bone. For example, whenthe skeleton is undergoing mechanical stress, osteocytes areideally located to sense pressure changes in bone, whichcould result in specific chemical messages being relayed tothe surface cells to respond either by formation or resorp-tion (Lanyon, 1993; Turner et al., 1994; Weinbaum et al.,1994; Klein-Nulend et al., 1995). It has also been hypothe-sized that osteocytes may have the capacity to regulate cal-cium homeostasis (Rubinacci et al., 1998). Unfortunately,their peculiar location within bone makes them the most in-accessible type of osteoblast to obtain in culture for in vitrostudy.

Bonewald and colleagues have established several im-m ter-i enicm alcinp hol-o t ofc 4)w witho nsive,c , os-t pro-d alinep last-s pe Ic -p cre-t ur-fa diums pti-m driticp

O-A byt hlye eveni acid( llsa stager

thesep in.

omen after the menopause (Paterson et al., 1984). It is be-ond the scope of this review to discuss the molecular baI. A comprehensive listing of the mutations that have biscovered within human type I collagen genes (Dalgleish997), is maintained in the osteogenesis imperfecta m

ion database (http://www.le.ac.uk/genetics/collagen). Theim/oim mouse which arose from a spontaneous mutithin the COL1A2 gene resulting in the production oon-functional alpha2 (I) chain, is a widely used muodel of OI (Chipman et al., 1993). The heterozygous Mo3 mouse in which one of the two COLIA1 genes is n

unctional, is the only murine model of type I OI (Bonadio el., 1990). Apart from defective formation of the type I co

agen triple helix, the ability of OI fibroblasts or bone ceo produce collagen and proliferate in vitro is also impand that is probably a consequence of the retained procoolecules within the distended rough endoplasmic reticu

Lamande et al., 1995; Fitzgerald et al., 1999; Lamandeateman, 1999). In vitro studies of osteoblasts derived frI humans (Fedarko et al., 1996) or oim mouse (Balk et al.,997) show diminished markers of osteoblastic difntiation. However the cells can still differentiate isteoblasts under the influence of bone morphogeroteins.

Knowledge obtained from the study of osteogenesiserfecta has also provided clues about the genetics o

eoporosis. Although the collagen protein chains are non most osteoporotic patients, a polymorphism has receen identified in a regulatory region of the COLIA1 ghich is more common in osteoporotic patients. This porphism is located at a binding site for the transcrip

ortalized cell lines in culture with phenotypic characstics of osteocytes. Bone cells were derived from transg

ice over-expressing T-antigen driven by the osteocromoter. They chose cells expressing a dendritic morpgy as the initial criterion for selection and establishmenlonal cell lines. MLO-Y4 (murine long bone osteocyte Yas one of the immortalized clonal lines establishedsteocyte-like characteristics. These cells produce exteomplex dendritic processes, are positive for T-antigeneopontin, neural antigen CD44 and connexin 43. Theyuce large amounts of osteocalcin, have low levels of alkhosphatase activity, lack detectable mRNA for osteobpecific factor 2, and produce very small amounts of tyollagen (Kato et al., 1997). The MLO-Y4 cells also suport osteoclast formation and activation through the se

ion of M-CSF and expression of RANKL on their sace and their dendritic processes (Zhao et al., 2002). Cellsre grown on collagen-coated surfaces in culture meupplemented with 5% FBS and 5% calf serum for oal growth and maintenance of the osteophyte denhenotype.

Four other immortalized osteocyte-like cell lines (ML5, MLO-A2, MLO-D1 and MLO-D6) were established

his group. Out of these, MLO-A5 cells were shown to higxpress BSP and mineralize spontaneously in culture

n the absence of beta-glycerophosphate and ascorbicKato et al., 2001). The authors claim that the MLO-A5 cere representative of the post-osteoblast, preosteocyteesponsible for triggering mineralization of osteoid.

To date, there is no published data on the responses ofresumptive osteocyte-like cell lines to mechanical stra

V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102 87

3. Osteoclasts

3.1. Osteoclast ontogeny

Multinucleate osteoclasts are responsible for bone re-sorption. Their chief functional characteristic is the abil-ity to pump acid into specialized resorption pits to dissolvebone mineral as well as to provide an optimum environmentfor the enzymatic degradation of demineralized extracellu-lar bone matrix. Osteoclasts are derived from hematopoieticstem cells that differentiate along the monocyte/macrophagelineage (Martin et al., 1989; Suda et al., 1992). Directcontact of mononuclear hematopoietic precursors with os-teoblast/stomal cells expressing the membrane protein Re-ceptor Activator of NF-kappa B Ligand (RANKL) is neces-sary before they can differentiate into osteoclast precursorsand proceed to fuse into mature, multinucleate osteoclasts(Suda et al., 1995; Lacey et al., 1998). This is depicted dia-grammatically inFig. 2.

3.2. Phenotypic characteristics of osteoclasts

The mature osteoclast is a functionally polarized cell thatattaches via its apical pole to the mineralized bone matrixby forming a tight ring-like zone of adhesion, the sealingzone. This attachment involves the specific interaction be-t rins)a fam-i ainsi do-m edi-a

F ltinucle matopo act with of osteof tiate fir P positiva ntually

The space contained inside this ring of attachment and be-tween the osteoclast and the bone matrix constitutes the bone-resorbing compartment. The cell membrane of the apicalpole is invaginated to form a ruffled border. Osteoclasts areactively engaged in the synthesis and secretion of severalclasses of enzymes formed in the Golgi region and vectoriallytransported to the apical pole through their association withmannose-6-phosphate receptors. At their destination, the en-zymes bound to mannose-6-phosphate receptors fuse withthe ruffled border apical membrane and their contents dis-charged into the bone-resorbing compartment (Baron et al.,1993).

Acidification of the extracellular bone-resorbing compart-ment is one of the most important features of osteoclastaction. The osteoclast is highly enriched in carbonic anhy-drase (Gay and Mueller, 1974). Carbonic anhydrase gener-ates protons and bicarbonate from carbon dioxide and wa-ter, providing the cells with protons to be extruded acrossthe cell membrane into the bone-resorbing compartment byproton pumps (H+ ATPases) located in the ruffle borderapical membrane. Regulation of H+ transport at the api-cal surface of the osteoclast, which is tightly linked to theregulation of intracellular pH and membrane potential, ismostly accomplished by ion exchangers, pumps and channelspresent in the basolateral membrane of the cell (Baron et al.,1

velyr highn

cter-i

ween adhesion molecules in the cell membrane (integnd some bone matrix proteins. The integrins are a

ly of transmembrane proteins whose cytoplasmic domnteract with the cytoskeleton while their extracellular

ains bind to bone matrix proteins, enabling them to mte cell–substratum and cell–cell interactions (Hynes, 1987).

ig. 2. Diagrammatic representation of the formation of mature, musteoclast progenitors present in bone marrow come into direct cont

actors such as PTH, PGE2, IL-11 or 1,25(OH)2D3 (1). They differennd calcitonin receptor (CTR) positive mononucleate cells (3) that eve

ated osteoclasts from mononuclear hematopoietic progenitors. Heoieticosteoblast or stromal cells expressing RANKL under the influencelytic

stly into TRAP positive mononuclear cells (2) before becoming TRAefuse to form multinucleate, functional mature osteoclasts (4).

993).The activity of mature osteoclast is directly and negati

egulated by calcitonin, for which the cell expresses aumber of receptors (Nicholson et al., 1986).

A summary of the main osteoclast phenotypic charastics is provided inTable 5.

88 V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102

Table 5Phenotypic characteristics of osteoclasts

Lysosomal enzymesTartrate-resistant acid phosphatase�-GlycerophosphateArylsulfatase�-GlucuronidaseCysteine-proteinases (cathepsin B, C, K, L)

Non-lysosomal enzymesCollagenaseStromelysinTissue plasminogen activatorLysozyme

Matrix proteinsOsteopontinBone sialoproteinTGF�

ReceptorsRANK (membrane)Calcitonin (membrane)Vitronectin (�v�3) (membrane)Integrins with�1 subunitsMannose-6-phosphate (intracellular)

Proton pumpVacuolar H+ ATPase

Ion transportCalcium channelsPotassium channelsChloride channelsCalcium ATPaseNa, K-ATPaseNa+/H+ antiporterBicarbonate/chloride exchanger

Membrane associated proteinsCarbonic anhydrasec-src

3.3. In vitro methods to study osteoclast formation andfunction

Unlike osteoblasts, osteoclasts are difficult to study in vitrobecause they are relatively scarce, terminally differentiated,adherent to mineralized surfaces and fragile. Methods havebeen developed to isolate these cells in vitro or to induce theirformation in bone marrow cultures. The major criteria gener-ally used to identify osteoclasts are multinuclearity, positivestaining for tartrate resistant acid phosphatase (TRAP), ex-pression of calcitonin receptors and the ability to resorb calci-fied matrices (Takahashi et al., 1988a; Hattersley and Cham-bers, 1989; Shinar et al., 1990). TRAP staining and someof the other criteria used for identification, such as cathep-sin K, vitronectin receptor, are not specific for osteoclasts,being also expressed by macrophages (Table 6). However,these markers are often useful to identify osteoclasts whenmacrophage expression of these markers can be effectivelyexcluded. Indeed, the simplest method of estimating osteo-

clast number in in vitro assays is a count of TRAP positive orvitronectin receptor positive multinucleated cells. However,prolonged culture (more than 7 days) frequently results incalcitonin receptor negative, TRAP positive multinucleatedmacrophages, and that is a common pitfall. Conversely, theabsence of these markers indicates the absence of osteoclasts.

Mature, multinucleated functional osteoclasts are ob-tained either directly from bone as the primary source, or elsethey are secondarily generated in vitro from hematopoieticprogenitors obtained from a source of hematopoietic cells ormacrophages such as bone marrow, spleen, human peripheralblood mononuclear cells and human umbilical cord blood.

3.3.1. Primary sources of osteoclasts3.3.1.1. Mechanical disaggregation of osteoclasts.Matureosteoclasts can be isolated by mechanical disaggregationfrom the long bones of neonatal rats, rabbits or chicks. Thismethod involves curetting the long bones with a scalpel bladeto release bone fragments into the surrounding medium. Thefragments are triturated into a suspension with a wide-borepipette before plating onto glass coverslips for 15–30 minto allow the large, highly adherent osteoclasts to adhere tothe glass surface, before washing vigorously with mediumto remove non-adherent and other contaminating cell types(Chambers and Magnus, 1982). A longer settling time in-c er ofn teo-c ationo ctorsof udiedw ta con-t t maya steo-c lturet ulateot thel

3 ),a lasmso ten-s ablen t thes uceo ecur-s eo-c lyu clasts( aP , andt tionm ll sus-p 0%

reases the yield of osteoclasts, but also the numbon-osteoclastic contaminating cells. Relatively ‘pure’ oslasts have been obtained this way to enable the identificf calcitonin receptors and effects of bone-resorbing fan cytoplasmic spreading (Nicholson et al., 1986). The ef-

ects of osteotropic factors on bone resorption can be sthen the osteoclasts are placed on bone slices (Chambers el., 1985). The main disadvantages of this assay are the

aminating osteoblasts in the osteoclast preparation thaffect the experimental outcomes and the sensitivity of olasts to the pH of the assay medium, especially when cuimes exceed 24 h. An acid pH has been shown to stimsteoclast bone resorption (Arnett and Dempster, 1990), po-

entially accounting for some of the conflicting reports initerature.

.3.1.2. Giant cell tumors of bone.Giant cell tumors (GCTlso known as osteoclastomas, are rare primary neopf the skeleton. They are locally destructive, causing exive osteolysis. GCT contain within the tumor mass, variumbers of large, multinucleated cells. It is believed thatromal cells of GCT are the tumor cells, and they indsteoclastic bone resorption by recruiting osteoclast prors, promoting their differentiation into functional ostlasts (James et al., 1996). Until recently, this was the onseful source of human osteoclasts. To obtain the osteoGoldring et al., 1987), the giant cell tumor is dissected inetri dish under sterile conditions using a scalpel blade

hen enzymatically digested for cell culture in a digesixture made up from collagenase and dispase. The ceension is diluted in alpha-modified MEM containing 1

V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102 89

Table 6Identification of osteoclast markers

Marker Specificity/use Comment

Bone resorption Definitive Requires live, active cells; time dependentCalcitonin receptors Specific (hematopoietic lineage) Difficult without live cellsMultinuclearity Typical but not essential Indicates terminal differentiationTRAP Useful marker; easy to perform Indicative in vitro, but also expressed by activated macrophagesCathepsin K Useful marker Also expressed by macrophages/tumor cellsVitronectin receptor Occasionally useful Also expressed by macrophagesActin ring Indicates active osteoclast (on calcified substrate)

FBS, and filtered through a 40�m cell strainer. Cells arethen either cryopreserved or cultured in medium (Atkins etal., 2001).

3.3.2. Secondary sources of osteoclasts. In vitrogeneration3.3.2.1. Bone marrow cultures.Bone marrow consists of amixed cell population that is rich in hematopoietic but rel-atively poor in osteoblastic stromal cells. They are usefulfor examining the process of osteoclast differentiation andformation in culture under the influence of bone-resorbingagents. Murine bone marrow cultures have been the mostwidely studied (Takahashi et al., 1988a, 1988c: see methodbelow).Takahashi et al. (1988b)reported that treatment with1,25(OH)2D3 or human PTH (1–34) resulted in an increase inthe number of TRAP positive multinucleated cells that satisfythe major criteria for osteoclasts. Subsequently, a similar in-crease was observed with prostaglandins, PTHrP (1–34) andIL-1 (Akatsu et al., 1989a, 1989b, 1991). Two other importantobservations were made. Firstly, time course studies showedthat the appearance of TRAP positive mononucleated cellsprecede that of TRAP positive multinucleated cells, imply-ing that TRAP positive mononucleated cells are precursorsof the multinucleated cells. Secondly, osteotropic hormonessuch as 1,25(OH)2D3 and PTH induce the differentiationo TR-n ors( i-l 7a mi orsf enti ent.T ce ofm te thei

3 ells.A ast ieticp ands tionc -v ntiale ayer;

(ii) a source of hematopoietic cells such as murine spleen orbone marrow cells; and (iii) hormonal stimulation.

(i) Preparation of primary osteoblastPrimary osteoblasts are usually obtained from the cal-varia of newborn C57BL/6J mice. Calvaria are removedfrom the newborn mice under sterile conditions andtransferred to a sterile 30 ml tube containing 6 ml di-gest fluid made up immediately before use. The calvar-ial digest fluid is made from 30 mg collagenase type IIand 60 mg dispase dissolved in 30 ml PBS and filteredthrough a 0.2�m Acrodisc® 32 Supor®. The tube con-taining calvaria is shaken in a 37◦C water bath for 5 min,allowed to settle and the supernatant discarded by pipet-ting. Six millilitres of fresh digest fluid is added to thetube and incubated at 37◦C for 10 min. The cell sus-pension is collected in a separate sterile 30 ml tube. Thedigest is repeated a further three times and the cell sus-pensions pooled in the 30 ml tube. This is centrifugedat 2000 rpm for 5 min and the supernatant discarded.The cell pellet of primary osteoblasts is re-suspended in10 ml�MEM + 10% FBS and used immediately. Alter-natively, osteoblasts can be grown in culture for a fewdays to increase their numbers. Cells are seeded at adensity of 5× 106 cells in 10 ml of medium in Petridishes and incubated at 37◦C in a humidified incuba-

arefrom

by

ne3-

fromD3.

eatlyromalthelinest-

(dultwith

f immature precursors, characterized by TRAP and Cegativity, into mature TRAP and CTR-positive precursTakahashi et al., 1988b) (Fig. 2). Osteoclasts have also simarly been obtained using rabbit (Fuller and Chambers, 198)nd feline (Ibbotson et al., 1984) bone marrow. This syste

s limited by the inability to identify osteoclastic precursrom the mixed cell population and the difficulties inhern studying osteoclast activation as opposed to recruitmhe dependence of osteoclast formation on the presenesenchymal stromal cells or osteoblasts also complica

nterpretation of results.

.3.2.2. Co-cultures of osteoblastic and hematopoietic cn important finding from murine bone marrow cultures w

he demonstration that it was necessary for hematoporecursors to come into direct contact with osteoblaststromal cells before osteoclast differentiation and formaould occur (Takahashi et al., 1988b). This led to the deelopment of co-culture systems comprising three esselements: (i) stromal cells or osteoblasts as a feeder l

tor with 5% CO2. The adherent calvarial osteoblastsconfluent in 2–3 days and can be used up to 1 weektheir initial preparation. They are dispersed for usestandard trypsinization methods.

(ii) Stromal and osteoblast-like cell linesUdagawa et al. (1989)demonstrated that two bomarrow-derived pre-adipocytic cell lines, MC3TG2/PA6 and ST2, can support osteoclast formationmurine spleen cells in the presence of 1,25(OH)2The simultaneous addition of dexamethasone grenhanced osteoclast numbers. Other osteoblast/stcell lines shown to support osteoclast formation inco-culture system include the rat osteoblast-like cellUMR 106 (Quinn et al., 1994), murine stromal cell linetsJ2 and 10 (Chambers et al., 1993), murine osteoblaslike cell lines KS-4 (Yamashita et al., 1990a, 1990b) andKUSA/O (Umezawa et al., 1992).

iii) Preparation of bone marrow cellsLong bones (femur and tibia) are obtained from amale mice (4–8 weeks old). Each bone is flushed

90 V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102

PBS (5 ml per bone) from a syringe and samples fromall bones are pooled. Bone marrow cells are plated (106

cells per well in 1 ml) into the wells of a 16 mm diame-ter culture dish, together with osteoblasts (typically 2×105 cells/dish). The cultures are then maintained for ap-proximately 8 days in�MEM supplemented with 10%FBS at 37◦C in a humidified atmosphere of 5% CO2with fresh medium and treatments added every 3 days.Osteoclast formation begins typically at day 4 and in-creases thereafter.

(iv) Preparation of spleen cellsSpleen cells are obtained from either newborn mice (forexample, mice used in the osteoblast calvarial prepara-tion), or adult mice. The spleens are removed and col-lected in a sterile wire sieve over a Petri dish half-filledwith medium. Using the plunger from a disposable 10 mlsyringe, the spleens are gently crushed through the sieveand the sieve rinsed over the Petri dish with medium.The disaggregated spleen cells are transferred to a freshsterile tube and centrifuged at 2000 rpm for 5 min. Thepelleted cells are then re-suspended in�MEM + 10%FBS in similar numbers to bone marrow cells describedabove.

The cell mixture of hematopoietic cells and stroma/osteoblasts must be stimulated by osteolytic factors, the mostp nMpi tal too

3u

ands ren-t teints in-u wheni nu-c a,1 ae r-a ck inop fac-t1 98;

TI al cells

C e marrrow ma

S NKL/TC oclast tors

Hofbauer et al., 1999), at the same time, reducing the pro-duction of the secreted RANKL decoy receptor, osteoprote-gerin. RANKL also enhances the activity of mature osteo-clasts (Burgess et al., 1998) and inhibits osteoclast apoptosis(Fuller et al., 1998; Udagawa et al., 1999). Several factors in-cluding IL-1, M-CSF and TNF� (Kobayashi et al., 2000) alsohave this ability. Recombinant protein corresponding to theextracellular domain of RANKL stimulates the formation ofactive, bone-resorbing osteoclasts from hematopoietic cellsin the presence of M-CSF. RANKL plus M-CSF are suf-ficient to cause osteoclast formation from human or mousehematopoietic precursors, even in the absence of osteoblasticstromal cells (Quinn et al., 1998). This is a major advance inthe development of in vitro methods to study osteoclast dif-ferentiation and formation, enabling investigators to obtainosteoclasts in culture and in sufficient numbers without theconfounding presence of other cell types such as osteoblastsor stromal cells. In vitro models of mouse osteoclast forma-tion using hematopoietic cells as a source of precursors, andstimulated by soluble factors M-CSF, RANKL, TNF� or IL-1is summarized inTable 7andFig. 3.

3.3.3.1. Bone marrow and bone marrow macrophages(BMM). Quinn et al. (2002)reported a method of obtain-ing highly enriched osteoclast-lineage cell populations usingM n ofb rent,p her-e re-c pho-c /6Jm ormn e ande stingt gen-is tiveo es ce of2 heB d forf

3 e-c ive tof esis.R ca-p with

otent being a mixture of 10 nM 1,25(OH)2D3 and 100rostaglandin E2. The addition of dexamethasone (10−8 M)

ncreases the yield of osteoclasts, but may be detrimensteoclast morphology.

.3.3. New methods for osteoclast generation in vitro,sing RANKL, M-CSF, TNF� and IL-1

The membrane factor expressed by osteoblaststromal cells that was essential for osteoclast diffeiation was identified as the membrane-bound proermed receptor activator of NF�B ligand (RANKL). Ittimulates the differentiation and formation of multcleated osteoclasts from mononuclear precursors

t binds to its receptor, RANK, expressed on monolear hematopoietic precursors (Anderson et al., 1997997b; Wong et al., 1997a, 1997b; Lacey et al., 1998; Yasudt al., 1998; Hsu et al., 1999). RANK-deficient mice are chacterized by profound osteopetrosis resulting from a blosteoclast differentiation (Dougall et al., 1999). RANKL ex-ression in osteoblasts is stimulated by bone-resorbing

ors such as PTH, PGE2, 1,25(OH)2D3, interleukin 1� (IL-�) and IL-11 (Horwood et al., 1998; Yasuda et al., 19

able 7n vitro models of mouse osteoclast formation in the absence of strom

ell types Spleen cells, bone marrow or bloodmonocytes

Bonmar

timulus RANKL/TNF + M-CSF RAells present Hematopoietic cells, lymphocytes

(B, T, NK cells), stromal cellsOsteitors

ow macrophages or bonecrophage precursors

RAW 264.7

NF + M-CSF RANKL/TNFand macrophage progen- Osteoclast and macrophage progeni

-CSF stimulated bone marrow cells. M-CSF stimulatioone marrow cells results in large numbers of non-adheroliferating macrophage precursors that rapidly form adnt bone marrow macrophages (BMM). BMM and their pursors can be isolated free from mesenchymal and lymytic cells. BMM precursors derived from CBA or C57BLouse bone marrow, when cocultured with ST2 cells, fumerous mononuclear osteoclasts which resorb bonxpress TRAP and calcitonin (CTR) receptors, suggehat they are a highly enriched source of osteoclast protors. Recombinant RANKL/M-CSF, together with TGF�,timulate BMM precursors to form almost pure CTR-posisteoclasts after 7 days. Typically, 105 bone marrow cells areeded per 10 mm diameter culture dish in the presen5 ng/ml M-CSF and 50–100 ng/ml RANKL for 7 days. TMMs generated with this method can be cryopreserve

uture use.

.3.3.2. Osteoclast cell lines.At least two osteoclast prursor cell lines have been established as an alternatresh hematopoietic cells in the study of osteoclastogenAW264.7 is a mouse osteoclast-like myeloma cell lineable of differentiating into osteoclasts when treated

V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102 91

Fig. 3. This diagram is a representation of the differentiation pathway from mononucleate hematopoietic progenitors to functional, mature, multinucleateosteoclasts. It illustrates the points along this pathway that are acted upon by soluble factors M-CSF, RANKL and IL-1. These factors would normally besecreted or expressed by osteoblasts or stromal cells.

RANKL (Hsu et al., 1999). Unlike murine spleen cells, bonemarrow cells or BMMs, RAW264.7 cells do not require M-CSF to differentiate into osteoclasts. Furthermore, they can-not be co-cultured with stromal cells or osteoblasts. Thereason for this is not known. C7 is an immortalized mousemacrophage-like cell line that can be used for a similar pur-pose (Yasuda et al., 1998).

3.3.3.3. Human osteoclasts.The formation of human osteo-clasts in coculture has proved to be a challenge because ofa lack of a suitable human osteoblastic stromal cell line touse in a coculture system.Fujikawa et al. (1996)isolated hu-man monocytes [CD14, CD11a, CD11b, HLA-DR positive,TRAP, CTR, vitronectin receptor (VNR) negative] and cocul-tured these cells for up to 21 days with either osteoblast-likeUMR 106 (rat) or ST2 (mouse) stromal cells in the presenceof 1,25(OH)2D3, dexamethasone and human M-CSF (rat M-CSF is inactive on human cells). Numerous TRAP, VNR andCTR-positive multinucleated cells, capable of extensive la-cunar bone resorption, formed in these cocultures. This workwas extended to include human hematopoietic marrow cells,blood monocytes and peritoneal macrophages, all of whichwere capable of differentiating into mature functional os-teoclasts (Quinn et al., 1998b). Matsuzaki et al. (1999)es-tablished subclones of the human osteosarcoma cell line,S , ands riph-e lturew ncedb wass u-m redi s-

teoclasts. However, PBMC are heterogeneous, consisting ofsubsets of monocytes, lymphocytes and other blood cells.Nicholson et al. (2000)showed that a highly purified popu-lation of osteoclast-forming PBMC can be obtained by se-lecting for the expression of CD14, a marker that is stronglyexpressed in monocytes, the putative osteoclast precursor inperipheral blood.

A novel and exciting new method of obtaining osteo-clast progenitors from human umbilical cord blood was re-cently described (Hodge et al., 2002). A mononuclear cellfraction containing monocytes and lymphocytes, isolatedfrom human umbilical cord blood by Ficoll–Paque densitygradient centrifugation, is cultured in semi-solid medium,and incubated at 37◦C in a humidified atmosphere of 5%CO2–air for 7–14 days. Pooled colonies identified as CFU-GM are harvested and transferred into 96-well plates con-taining dentine slices in the presence of RANKL and hu-man M-CSF for a further 6 days. Cultures are then fixedin 1% formalin and reacted for TRAP activity. The forma-tion of bone-resorbing multinucleated osteoclasts is assessedby transmission light microscopy and quantified using com-puter image analysis. Using human umbilical cord bloodmononuclear cells (CBMC) as a source of osteoclast pro-genitors, it was shown that clonal expansion of CFU-GMprogenitors markedly increases osteoclastogenic potential,b riort sis,d ion.T ingh f dif-f tageo uree

aOS-2, expressing the human PTH/PTHrP receptorhowed that mouse bone marrow cells or human peral blood mononuclear cells formed osteoclasts in cocuhen treated with PTH. The response was greatly enhay adding dexamethasone, but no osteoclast formationeen with the addition of 1,25(OH)2D3, PGE2 or IL-6. Han peripheral blood mononuclear cells (PBMC) cultu

n vitro with soluble RANKL and human M-CSF, form o

ut exposure of pooled colonies to GM-CSF or IL-3 po RANKL stimulus completely inhibits osteoclastogeneirecting cells instead towards dendritic cell differentiathis may prove to be a very useful method for obtainuman osteoclast progenitors to study the regulation o

erentiation along different cell lineages. A further advanf this method is the ability to cryopreserve CBMC for futxperiments.

92 V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102

4. Chondrocytes

Cartilage is a specialized form of connective tissue thatpossesses a firm pliable matrix, which endows it with theresilience that allows the tissue to bear mechanical stresseswithout distortion. Articular cartilage, smooth surfaced andresilient, provides a shock-absorbing sliding area for joints tofacilitate movement of bone, while cartilage is also essentialfor the embryonic development and, thereafter, growth oflong bones.

Cartilage consists of chondrocytes and an extensive extra-cellular matrix. The characteristics of cartilage stem mainlyfrom the nature and predominance of ground substance inthe extracellular matrix. Glycoproteins, containing a highproportion of sulfated polysaccharides, make up the groundsubstance and account for the solid, yet flexible properties ofcartilage. The functional differences between cartilage andbone relate principally to the different nature and proportionof the ground substance and fibrous elements of the extracel-lular matrix. Nonetheless, chondrocytes and osteoblasts sharea common origin from primitive mesenchymal cells (Marksand Hermey, 1996).

Hyaline cartilage is the most prevalent type of cartilage.During embryonic development, it forms the cartilage tem-plate of many of the developing bones until replaced by bonei s, thee hysisi hint cretes ion,a l ofp en-t leton(

anyd ogly-c omet istedi useg notpY

4

r thei x-p sys-tC har-a s ofc wella tap den-s in

Table 8A summary of the phenotypic characteristics of chondrocytes

CollagensType I collagenType II collagenType VI collagenType IX collagenType X collagenType X1 collagen

Proteoglycans and other proteinsAggrecanLink proteinBiglycanFibronectinOsteopontinCartilage oligomeric matrix proteinMatrix gla proteinChondromodulin-ICalmodulinFibromodulinCartilage homeoprotein IPerlecanTropomodulinOsteonectin

ReceptorsGrowth hormoneTGF-betaBMPPTHrPIGF-1Retinoic acidFibroblast growth factor receptors 1 and 3Thyroid hormones

medium supplemented with serum and by passage (Heringet al., 1994). Growth of chondrocytes under conditions thatsupport a rounded morphology also facilitates maintenanceof the differentiated chondrocytic phenotype (Bonaventureet al., 1994; Hauselmann et al., 1994; Binette et al., 1998).Stewart et al. (2000)studied the phenotypic stability of ar-ticular chondrocytes in vitro and demonstrated that the in-fluence of BMP-2 and serum on expression of chondrocyte-specific matrix proteins [procollagen type I and II, aggrecanand (V + C)− fibronectin] varies depending on cellular mor-phology, and/or cytoskeletal organization when chondrocytesare grown as monolayer, aggregate, pellet or explant cultures.

Despite these limitations, some measure of success hasbeen achieved with autologous chondrocyte transplantationto repair cartilage defects. Focal chondral or osteochondraldefects, usually the result of trauma, have a poor capacity forrepair and predispose patients to osteoarthritis. In a surveyof one thousand consecutive knee arthroscopies, chondral orosteochondral lesions were found in 61% of the patients, withfocal chondral or osteochondral defects accounting for 19%with a mean defect area of 2.1 cm2 (Hjelle et al., 2002). Au-tologous chondrocyte transplantation (ACT) was first usedin humans in 1987 and the first pilot study was published in1994 (Brittberg et al., 1994) in 23 patients with deep cartilagedefects in the knee. Cartilage slices were obtained througha pper

n the process of endochondral ossification. In long bonepiphyseal growth plate between the epiphysis and diap

s responsible for the longitudinal growth of bone. Withe growth plate, chondrocytes undergo a series of distages of differentiation, namely, proliferation, maturatnd hypertrophy. The strict spatial and temporal controroliferation and differentiation of chondrogenic cells is c

ral to the coordinated development of the vertebrate skeErlebacher et al., 1995).

Chondrocytes are unique cells, in that they have mifferentiated markers such as large cartilage-type proteans (aggrecan) and collagen types II, IX, X, and XI. Sypical phenotypic characteristics of chondrocytes are ln Table 8. A comprehensive list of several hundred morowth cartilage-derived gene products, including manyreviously reported, was recently published (Okihana andamada, 1999).

.1. Propagation of chondrocytes in culture

Various cell culture models have been developed fonvestigation of chondrocyte biology in vitro, including elant models, several forms of three-dimensional culture

ems, and monolayer cultures (Adolphe and Benya, 1992).hondrocytes grown in monolayer culture undergo a ccteristic process of dedifferentiation, marked by a losollagen type II and aggrecan core protein expression ass the induction of collagen type I expression (Takigawa el., 1987; Hering et al., 1994; Lefebvre et al., 1994). Thishenomenon is influenced to some extent by seedingity (Ronziere et al., 1997) and is accelerated by growth

n arthroscope from a minor-load-bearing area on the u

V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102 93

medial femoral condyle of the damaged knee, placed inchilled sterile normal saline and processed within 2–5 h. Thecartilage specimens were minced, washed in culture mediumcontaining Ham’s F12 medium supplemented with HEPESbuffer, antibiotics and ascorbic acid and digested for 16 h inculture medium containing clostridial collagenase and de-oxyribonuclease I. The cells were then filtered through ny-lon mesh, resuspended in culture medium supplemented with15% of the patient’s serum (autologous serum), and grownin culture flasks for 14–21 days before being used to fillthe cartilage defect. A long-term follow up of a larger se-ries of 61 patients treated for isolated cartilage defects onthe femoral condyle or patella was recently reported. After 2years, 50 patients had good or excellent results and 51 of 61had good or excellent results at 5–11 years later (Peterson etal., 2002). Matrix-induced autologous chondrocyte implan-tation (MACI) is a refinement of ACI. The technique involvesgrowing chondrocytes on a Matricel membrane prior to trans-plantation. Matricel membranes are biocompatible, porcinein origin and composed of type I/type III collagen, avoid-ing type II-, XI- and V-induced osteoarthritis. They have asmooth and a coarse surface. Chondrocytes are seeded ontothe coarse surface to form multilayers in culture, while thesmooth surface of the Matricel membrane closely mimics thesurface of articular cartilage (Zheng and Wood, 2003).

ro-c nalc ytes,a

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ain-lture

ighde-seandho-on-rkerdsytesrate

dingther

( mb-tia--hy-alka

esis.( ng

ing

a culture model of chondrocytes derived from epiphy-ses of long bones of fetal calves. Chondrocytes were in-duced to differentiate by treatment with 5-azacytidine, apotent DNA demethylating agent shown to induce dif-ferentiation of the pluripotent mesenchymal cell line,C3H10T1/2, to myoblasts, adipocytes and chondro-cytes (Taylor and Jones, 1979). After treatment with 5-azacytidine (aza-C) for 48 h, cultures maintained withoutaza-C and harvested at selected time points, were shownto undergo a sequence of differentiation steps leadingto hypertrophic chondrocytes, mimicking events occur-ring during endochondral ossification in vivo. Cells inthis culture model of endochondral ossification were syn-chronized, allowing the study of individual stages of thedifferentiation pathway.

4.1.2. Chondrocyte cell linesChondrocyte cell lines are used as an alternative to primary

chondrocytes because they represent a renewable source ofmaterial.

4.1.2.1. Normal clonal chondrocyte cell lines.CFK2 is anestablished chondrocytic cell line derived from fetal rat cal-variae. Extended culture results in the appearance of gly-cosaminoglycans and type II collagen in the cell layer ina if-f one-s tioni owthf soneap ns-f ns lls inw car-t andI d.

-t car-t itro.T cloneR tiatei thep

4e tingc thatc denti hicc

asc ytesw ingt cul-t re as

In vitro methods currently employed to study chondytes in vitro, range from primary chondrocytes, to clohondrocyte cell lines established from normal chondrocnd transformed chondrocyte cell lines.

.1.1. Primary chondrocytesa) Most studies have used chick chondrocytes. Thes

be isolated from whole chick embryo sterna and mtained for extended periods of time in suspension custabilized with agarose (Bruckner et al., 1989). The cellsremain viable without FBS only when seeded at hdensities. They do not proliferate at a high rate, butposit extracellular matrix with fibrils resembling thoof authentic embryonic cartilage in their appearancecollagen composition. The cells exhibit many morplogical and biochemical characteristics of resting chdrocytes and they do not produce collagen X, a mafor hypertrophic chondrocytes. Addition of FBS leato profound changes in the phenotype of chondrocseeded at low density. They form colonies at a highand assume properties of hypertrophic cells, incluthe synthesis of collagen X, thus resembling adult rathan embryonic cartilage.

b) A culture system using chondrocytes derived from eryonic chick vertebrae was shown to undergo differention in vitro (Gerstenfeld and Landis, 1991). Supplementation with ascorbic acid led to the expression of thepertrophic phenotype as assessed by an increase inline phosphatase activity and collagen type X synth

c) Cheung et al. (2001)described a method of studyimammalian chondrocyte differentiation in vitro us

-

ssociation with the formation of focal nodes of cells. Derentiated CFK2 cells show enhanced parathyroid hormtimulated adenylate cyclase activity and their proliferas stimulated by regulatory factors such as epidermal gractor, parathyroid hormone, and inhibited by dexamethas well as retinoic acid (Bernier and Goltzman, 1993). Ex-ression of matrix proteins was inhibited in CFK2 cells tra

ected with PTHrP (Henderson et al., 1996). Thus CFK2 caerve as a non-transformed model of rat chondrocytic cehich both induction and regulation of the expression of

ilaginous matrix components by factors such as PTHrPndian Hedgehog (Deckelbaum et al., 2002) can be observe

Grigoriadis et al. (1996)established a family of nonransformed, clonal rat chondrogenic cell lines in whichilage differentiation and metabolism can be studied in vhese were established from a parental chondroblastCJ3.1C5 that was originally demonstrated to differen

nto three-dimensional cartilage nodules when grown inresence of 15% FCS (Grigoriadis et al., 1989).

.1.2.2. Transformed chondrocyte cell lines.ATDC5 is anmbryonal carcinoma cell line isolated from a differentiaulture of AT805 teratocarcinoma (Atsumi et al., 1990),an be induced to undergo a reproducible, time-depen

n vitro progression from early precursors to hypertrophondrocytes (Shukunami et al., 1997, 1998).

IRC is an immortalized rat chondrocyte cell line that wreated by transformation of primary rat costal chondrocith a recombinant murine retrovirus (NIH/J-2) carry

he v-myc and v-raf oncogenes. It grows in suspensionure as multicellular aggregates and in monolayer cultu

94 V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102

polygonal cells, which accumulate an alcian-blue stainablematrix. IRC cells synthesize typical cartilage proteins, aggre-can 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 cellline, N1511, from rib cartilage of a p53-null mouse. BMP-2and insulin treatment induces full differentiation toward hy-pertrophic chondrocytes, whereas treatment with PTH anddexamethasone slows and limits differentiation. Recovery ofp53 expression in N1511 cells by transient transfection in-hibits proliferation, suggesting that cell proliferation can beregulated with p53 in this cell line. These results would in-dicate that N1511 is the only cell line with known geneticmutation, which undergoes multiple steps of chondrocytedifferentiation towards hypertrophy, and may also be usedto study the function of p53.

HCS-2/8 is an immortalized clonal cell line derived from awell-differentiated human chondrosarcoma (Takigawa et al.,1989), with phenotypic features resembling normal chondro-cytes. The cells synthesize aggrecan, integrins, collagen typesII, IX and XI, show the same responses to growth factors asnormal chondrocytes and maintain their cartilage phenotypeover more than 3 years in culture (Takigawa et al., 1997).

5

ro-c euro-t ctiono hina inatet roidg

singr gicalc Re-d lt ina tion.P ctlyo on-i tiono is of1 sti-n se ofc in-c re-s al-i ls of1 n an izedc enalc l cal-c

ls oft rcal-

cemia but in man, no essential function has yet been foundfor calcitonin. Steady-state plasma calcium shows little orno change with either complete absence or a large excess ofcalcitonin (Parfitt, 1993).

5.1. Parathyroid cells in culture

Work in the laboratory has generally been performed ondispersed bovine parathyroid cells. Fresh bovine parathyroidglands, transported in cold Hank’s solution, are washed in70% ethanol, dissected free of surrounding fat tissue andfinely minced in Hank’s solution. They are transferred totissue culture flasks, 8–10 glands in 15 ml, and dispersedby shaking with 1.25 mg/ml collagenase type I suspendedin Eagle basal medium containing 15% FCS. The cells arefiltered through 200 mm cell dissociation sieve and 40 mmnylon mesh. Cell suspension is washed by centrifugation inHank’s solution and dispersed in M-199 medium containing1.25 nM Ca2+ and 15% newborn calf serum. Cells are platedin 24 multiplate dish at a density of 1× 106 cells per welland cultured at 37◦C in 5% CO2 for 24 h to allow attachment(Moallem et al., 1995).

The extracellular calcium sensing receptor was clonedfrom bovine parathyroid cells (Brown et al., 1993), and thesecells have been very useful in determining the role of cal-cium fluxes in the regulation of parathyroid hormone secre-t

5

(C)c C)i Celll pro-v de-v cellst Theh tM -c an-a f thetf ex-p ecep-t Met-e omo-g therp o eta nH 7i O

6

udyo uce

. Calcium homeostasis

Calcium is an essential ion for many physiological pesses such as cell motility, muscle contraction and nransmitter release. In mammals, these processes funptimally when extracellular calcium is maintained witnormal range by regulatory mechanisms that coord

he metabolic activities of the kidneys, intestine, parathylands, and bone.

Parathyroid cells express a cell surface calcium-seneceptor that recognizes and responds to physiolohanges in extracellular ionized calcium concentration.uctions in serum calcium of the order of 1–2% resuprompt increase in parathyroid hormone (PTH) secreTH acts directly on the kidneys and skeleton and indiren the intestine to normalize any fall in extracellular i

zed calcium. In the kidney, PTH stimulates re-absorpf calcium in the distal tubules and increases synthes,25(OH)2D3. 1,25-Dihydroxyvitamin D increases inteal absorption of calcium and also stimulates the releaalcium from bone by stimulating bone resorption. Thereased flux of calcium ions into the extracellular fluidtores circulating levels of calcium toward normal. Normzation of serum calcium as well as the increased leve,25-dihydroxyvitamin D inhibits further PTH synthesis iegative feedback loop. An increase in extracellular ionalcium inhibits PTH secretion, resulting in increased ralcium excretion and reduction in net release of skeletaium as well as intestinal absorption of calcium.

In several species, calcitonin secretion by the C-celhe thyroid is part of the homeostatic response to hype

ion (Chang et al., 2001).

.2. C-cells of the thyroid

Calcitonin is a secretory product of the parafollicularells of the thyroid, and medullary thyroid carcinoma (MTs a neuroendocrine tumor of the parafollicular cells.ines established from human and animal MTC tumorside a useful system to analyze genes involved in theelopment of this neoplasia, as well as a source of Co determine the regulation of calcitonin production.MTC cell line, TT cells (Leong et al., 1981) and the raTC line, 6–23 cells (Zeytinoglu et al., 1980) can be pur

hased from the American Type Culture Collection (Mssas, VA). TT cells display an impaired expression o

umor suppressor gene p53 (Velasco et al., 1997). Apartrom calcitonin and the calcitonin receptor, TT cellsress carcino-embryonic antigen, somatostatin and its r

ors, neurotensin, gastrin-releasing peptide, Leu- andnkephalin, parathyroid hormone-releasing peptide, chrranin A, synaptophysin, 1,25(OH)2D3 receptor and oeptides (Frendo et al., 1994; Zabel et al., 1995; Velascl., 1997; Zatelli et al., 2001). TT cells are routinely grown iam’s F12K medium supplemented with 10% FBS at 3◦C

n a humidified atmosphere containing 95% air and 5% C2.

. Discussion

Although cell culture has proved invaluable in the stf bone biology, in vitro model systems cannot reprod

V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102 95

the complex three-dimensional architecture of bone that isrequired for the proper expression of the functional capabilityof the cells that make up its microenvironment. Nonetheless,despite the limitations of the various systems described inthis review, significant and important contributions havebeen made to our understanding of the normal processesleading to bone formation, remodeling and resorption aswell as how these processes can be deranged to result inmetabolic bone diseases.

The term osteoblast describes a lineage of cells that dif-fer substantially in their properties at different stages of de-velopment. Although many ‘characteristic’ properties of os-teoblasts have been described, it does not follow that all cellsof the lineage possess each of these features. At differentstages of differentiation, and at different sites in bone carry-ing out specific functions, osteoblasts are likely to expressonly a proportion of the features associated with the pheno-type. The challenge of identifying the cellular pathways andthe factors that regulate progression from osteoprogenitors tomature osteoblasts is facilitated by the ability to isolate andanalyse in culture, osteoblasts at various stages of differen-tiation, and in particular, clonal cell lines from bone or bonetumors. These are used as models of osteoblasts representingdifferent stages of differentiation, enabling investigators todefine the heterogeneity of bone cell populations with morep on-t os-t es ofm capa-b ;M lyu owea hichs iffer-e res.H ro-m a1-p rotein( nd a2 -kbp RNAt ctedG blastl e thatw ot-e olates tionf icef ;K

use-f imu-l last-l thee l.,1

cells (Umezawa et al., 1992). Cell lines established frompluripotent mesenchymal cells provide valuable informa-tion on the factors and mechanisms regulating differentiationalong the osteoblast, chondrocyte, adipocyte and myocytelineages.

In the case of osteoclasts, considerable progress has beenmade in the past twenty years in the development of methodsto study their function and formation in vitro. Early studiesusing relatively crude methods to disaggregate osteoclastsfrom bone were cumbersome, succeeding in obtaining onlysmall numbers of osteoclasts that could not be separated fromother cell types such as stromal cells and osteoblasts, andresults were difficult to reproduce. Investigators were facedwith the challenging task of obtaining sufficient numbers ofrelatively ‘pure’ preparations of osteoclasts in culture. Thiswas clearly not attainable using bone as a primary source offunctional, mature osteoclasts. Knowledge that osteoclastsare derived from hematopoietic stem cells that differentiatealong the monocyte/macrophage lineage, and the realizationin the late eighties, that direct contact between osteoclastprogenitors and stromal cells/osteoblasts is required for os-teoclast differentiation, led to the widely-used coculture sys-tem to study the regulation of osteoclast differentiation frommononucleate progenitors to mature, functional multinucle-ate osteoclasts. It became feasible to generate functional os-t h theo n os-t cadel in-t coyr thesed regu-l titu-t ticc ingf rna-t tisedb oriesh lture.U rowo sideR notk th-w

lowo ouse aryc heng m-p owna angeo lter-n

aryc thep one

recision. In contrast, primary calvarial cells probably cain osteoblasts at all stages of differentiation, includingeoprogenitors that proliferate before undergoing a seriaturational steps to become differentiated osteoblastsle of forming mineralized nodules in culture (Aubin, 1998alavalk et al., 1999). Primary calvarial cultures are widesed to study the progression of differentiation in vitro. Rnd colleagues recently described an elegant method in wubpopulations of osteoblasts at different stages of dntiation can be isolated from primary osteoblast cultuaving identified fragments of the rat type I (Col1a1) poter that show preferential expression in different Col1roducing tissues, they generated green fluorescent pGFP)-expressing transgenic mice containing a 3.6- a.3-kb rat type I collagen promoter fragment. The 3.6romoter directed strong expression of GFP messenger

o preosteoblastic cells, while the 2.3-kb promoter direFP mRNA expression to a cell that is late in the osteo

ineage, extending into mature osteocytes. They concludith further refinement of this method, using other promrs and color isomers of GFP, it should be possible to isubpopulations of cells at different stages of differentiarom primary cultures derived from these transgenic mor molecular and cellular analysis (Bogdanovic et al., 1994alajzic et al., 2002).Established osteoblast-like cell lines are particularly

ul models to study signaling pathways in response to station by osteotropic factors. The great majority of osteobike cell lines however, do not mineralize in culture, withxception of MC3T3-E1 (Kodama et al., 1981; Sudo et a983), 2T3 (Ghosh-Choudhury et al., 1996) and KUSA-O

eoclasts in culture in far greater numbers, even thougsteoclasts could not be separated from the companio

eoblasts/stromal cells for separate analysis. Almost a deater, the pivotal role of RANKL in osteoclastogenesis, itseraction with its cognate receptor RANK as well as the deeceptor osteoprotegerin were revealed. Not only wereiscoveries major advances in the understanding of the

ation of osteoclast formation, it made possible the subsion of soluble RANKL and M-CSF for stromal/osteoblasells, thus considerably simplifying the method for obtainunctional osteoclasts in vitro. The use of cell lines as alteive sources of osteoclast progenitors is not widely pracecause of the lack of suitable cell lines. Some laboratave used RAW264.7 cells to generate osteoclasts in cunlike hematopoietic progenitors derived from bone marr spleen, RAW264.7 cells do not require M-CSF alongANKL. The mechanism underlying this difference isnown, but it may imply a difference in the signaling paay leading to osteoclastogenesis.Chondrocytes are unique in their ability to exist in a

xygen tension environment, isolated within a voluminxtracellular matrix devoid of a vascular supply. Primhondrocytes retain their phenotypic characteristics wrown in the form of a three-dimensional multicellular colex, but undergo a process of dedifferentiation when grs monolayer cultures with serum supplementation. A rf established chondrocytic cell lines are available as aatives.

Work carried out on established cell lines and primell cultures have provided much valuable insight intohenotypic characteristics of cells belonging to the b

96 V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102

microenvironment, and the factors governing their develop-ment and function. Although it is not possible to grow bone inthe laboratory, much knowledge has been generated about theprocesses leading to bone formation and resorption and thefactors that regulate the fine balance essential for the main-tenance of the integrity of the skeleton. Targeted gene dele-tions have allowed the development of animal models withaltered skeletal phenotypes, but the mechanisms leading tosuch alterations remain largely unexplored in the laboratoryusing cell lines established from these models. The refine-ment of methods for selection and screening of rare homol-ogous events in mammalian cells by the introduction of se-lectable markers in the transfected piece of DNA (Mansour etal., 1988) and the isolation of pluripotent murine embryonicstem (ES) cells (Evans and Kaufman, 1981; Martin, 1981)that can be grown and manipulated in tissue culture havebroadened the scope of gene targeting approaches. Finally,the transfection of osteoblastic cell lines with putative con-structs has enabled the investigation of tissue-specific regu-lation of expression of the gene of interest at the cellular andmolecular level.

Acknowledgements

This work is supported by Program Grant 003211 from theN lia).T tionoa

R

A ytes:he,Ann

A i, A.,and

iner.

A ata,clast-

iner.

A ose-TH)-atedures.

A lf,s-

bitory

A C.,man,

its390,

Anderson, D.M., Maraskovsky, E., Billingsley, W.L., Dougall, W.C.,Tometsko, M.E., Roux, E.R., Teepe, M.C., DuBose, R.F., Cosman,D., Galibert, L., 1997b. A homologue of the TNF receptor and itsligand enhance T-cell growth and dendritic-cell function. Nature 390,175–179.

Arnett, T.R., Dempster, D.W., 1990. Protons and osteoclasts. J. BoneMiner. Res. 5, 1099–1103.

Aronow, M.A., Gerstenfeld, L.C., Owen, T.A., Tassinari, M.S., Stein,G.S., Lian, J.B., 1990. Factors that promote progressive developmentof the osteoblast phenotype in cultured fetal rat calvaria cells. J. Cell.Physiol. 143, 213–221.

Atkins, G.J., Bouralexis, S., Haynes, D.R., Graves, S.E., Geary, S.M.,Evdokiou, A., Zannettino, A.C.W., Hay, S., Findlay, D.M., 2001. Os-teoprotegerin inhibits osteoclast formation and bone resorbing activityin giant cell tumors of bone. Bone 28, 370–377.

Aubin, J.E., 1998. Advances in the osteoblast lineage. Biochem. CellBiol. 76, 899–910.

Aubin, J.E., Liu, F., Malaval, L., Gupta, A.K., 1995. Osteoblast and chon-droblast differentiation. Bone 17 (Suppl. 2), S77–S83.

Aubin, J.E., Turksen, K., Heersche, J.N.M., 1993. Osteoblastic cell lin-eage. In: Noda, M. (Ed.), Cellular and Molecular Biology of Bone.Academic Press, San Diego, pp. 1–45.

Balk, M.L., Bray, J., Day, C., Epperly, M., Greenberger, J., Evans, C.H.,Niyibizi, C., 1997. Bone 21, 7–15.

Barnard, R., Ng, K.W., Martin, T.J., Waters, M.J., 1991. Growth hormone(GH) receptors in clonal osteoblast-like cells mediate a mitogenicresponse to growth hormone. Endocrinology 128, 1459–1464.

Baron, R., Chakraborty, M., Chatterjee, D., Horne, W., Lomri, A., Raves-loot, J.-H., 1993. Biology of the osteoclast. In: Mundy, G.R., Martin,T.J. (Eds.), Physiology and Pharmacology of Bone, vol. 107. Springer-

B gen-

B tionsated

B f theone

B .R.,tion

.B ural

arial

B her-tilagecular

B , O.,.C.,of the385–

B n, J.,an,of theSci.

B non,ecific

ed in

B .M.,rrela-alcif.

ational Health and Medical Research Council (Austrahe help provided by Dr. Julian Quinn with the preparaf the manuscript,Figs. 2 and 3andTable 7, is gratefullycknowledged.

eferences

dolphe, M., Benya, P., 1992. Different types of cultured chondrocthe in vitro approach to the study of biological regulation. In: AdolpM. (Ed.), Biological Regulation of the Chondrocytes. CRC Press,Arbor, MI, pp. 105–139.

katsu, T., Takahashi, N., Udagawa, N., Imamura, K., YamaguchSato, K., Nagata, N., Suda, T., 1991. Role of prostaglandinsinterleukin-1-induced bone resorption in mice in vitro. J. Bone MRes. 6, 183–190.

katsu, T., Takahashi, N., Debari, K., Morita, I., Murota, S., NagN., Takatani, O., Suda, T., 1989a. Prostaglandins promote osteolike cell formation by a mechanism involving cyclic adenosine 3′,5′-monophosphate in mouse bone marrow cell cultures. J. Bone MRes. 4, 29–35.

katsu, T., Takahashi, N., Udagawa, N., Sato, K., Nagata, N., Mley, J.M., Martin, T.J., Suda, T., 1989b. Parathyroid hormone (Prelated protein is a potent stimulator of osteoclast-like multinuclecell formation to the same extent as PTH in mouse marrow cultEndocrinology 125, 20–27.

llan, E.H., Hilton, D.J., Brown, M.A., Evely, R.S., Yumita, S., MetcaD., Gough, N.M., Ng, K.W., Nicola, N.A., Martin, T.J., 1990. Oteoblasts display receptors for and responses to leukemia-inhifactor. J. Cell. Physiol. 145, 110–119.

nderson, D.M., Maraskovsky, E., Billingsley, W.L., Dougall, W.Tometsko, M.E., Roux, E.R., Teepe, M.C., DuBose, R.F., CosD., Galibert, L., 1997a. A homologue of the TNF receptor andligand enhance T-cell growth and dendritic-cell function. Nature175–179.

Verlag, New York (pp. 111–147).ellows, C., Aubin, J., 1989. Determination of numbers of osteopro

itors present in fetal rat calvaria in vitro. Dev. Biol. 133, 8–13.ellows, C., Aubin, J., Heersche, J., 1987. Physiological concentra

of glucocorticoids stimulate formation of bone nodules from isolrat calvaria cells. Endocrinology 121, 1985–1992.

ernier 1st, S.M., Goltzman, D., 1993. Regulation of expression ochondrocyte phenotype in a skeletal cell line (CFK2) in vitro. J. BMiner. Res. 8, 475–484.

ertolini, D.R., Nedwin, G.E., Bringman, T.S., Smith, D.D., Mundy, G1986. Stimulation of bone resorption and inhibition of bone formain vitro by human tumor necrosis factors. Nature 319, 516–518

hargava, U., Bar-Lev, M., Bellows, C., Aubin, J., 1988. Ultrastructanalysis of bone nodules formed in vitro by isolated fetal rat calvcells. Bone 9, 155–163.

inette, F., McQuaid, D.P., Haudenschild, D.R., Yaeger, P.C., McPson, J.M., Tubo, R., 1998. Expression of a stable articular carphenotype without evidence of hypertrophy by adult human artichondrocytes in vitro. J. Orthop. Res. 16, 207–216.

ogdanovic, Z., Bedalov, A., Krebsbach, P.H., Pavlin, D., Woody, CoClark, S.H., Thomas, H.F., Rowe, D.W., Kream, B.E., Lichtler, A1994. Upstream regulatory elements necessary for expressionrat COL1A1 promoter in transgenic mice. J. Bone Miner. Res. 9,392.

onadio, J., Saunders, T.L., Tsai, E., Goldstein, S.A., Morris-WimaBrinkley, L., Dolan, D.F., Altschuler, R.A., Hawkins Jr., J.E., BatemJ.F., Mascara, T., Jaenisch, R., 1990. Transgenic mouse modelmild dominant form of osteogeneis imperfecta. Proc. Natl. Acad.U.S.A. 87, 7145–7149.

onaventure, J., Kadhom, N., Cohen-Solal, L., Ng, K.H., BourguigJ., Lasselin, C., Freisinger, P., 1994. Reexpression of cartilage-spgenes by dedifferentiated human articular chondrocytes culturalginate beads. Exp. Cell. Res. 212, 97–104.

os, M.P., Van Der Meer, J.M., Feyen, J.H.M., Hermann-Erlee, M.P1996. Expression of the parathyroid hormone receptor and cotion with other osteoblastic parameters in fetal rat osteoblasts. CTissue Int. 58, 95–100.

V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102 97

Brittberg, M., Lindahl, A., Nilsson, A., Ohlsson, C., Isaksson, O., Peter-son, L., 1994. Treatment of deep cartilage defects in the knee with au-tologous chondrocyte transplantation. N. Engl. J. Med. 331, 889–895.

Brown, E.M., Gamba, G., Riccardi, D., Lombardi, M., Butters, R., Kifor,O., Sun, A., Hediger, M.A., Lytton, J., Hebert, S.C., 1993. Cloningand characterization of an extracellular Ca(2+)-sensing receptor frombovine parathyroid. Nature 366, 575–580.

Bruckner, P., Horler, I., Mendler, M., Houze, Y., Winterhalter, K.H., Eich-Bender, S.G., Spycher, M.A., 1989. Induction and prevention of chon-drocyte hypertrophy in culture. J. Cell. Biol. 109, 2537–2545.

Bruland, O.S., Fodstad, O., Stenwig, A.E., Pihl, A., 1988. Expression andcharacteristics of a novel human osteosarcoma-associated cell surfaceantigen. Cancer Res. 48, 5302–5309.

Canalis, E., 1981. Effect of platelet-derived growth factor on DNA andprotein synthesis in cultured rat calvaria. Metabolism 30, 970–975.

Canalis, E., 1983. Effect of glucocorticoids on type I collagen synthesis,alkaline phosphatase activity and DNA content in cultured rat calvaria.Endocrinology 112, 931–939.

Canalis, E., 1986. Interleukin-1 has independent effects on DNA andcollagen synthesis in cultures of rat calvariae. Endocrinology 118,74–81.

Canalis, E., Centrella, M., McCarthy, T., 1988. Effects of basic fibroblastgrowth factor on bone formation in vitro. J. Clin. Invest. 81, 1572–1577.

Centrella, M., McCarthy, T.L., Canalis, E., 1987. Transforming growthfactor � is a bifunctional regulator of replication and collagen syn-thesis in osteoblast-enriched cell cultures from fetal rat bone. J. Biol.Chem. 262, 2869–2874.

Chambers, T.J., Magnus, C.J., 1982. Calcitonin alters behaviour of iso-lated osteoclasts. J. Pathol. 136, 27–39.

C . Theboneones.

C 1993.from90,

C , A.,sitiveysiol.

C A.J.,onenti-oblast

C min258,

C .J.P.,ovellian

C arksJ.R.,

muta-Natl.

C by

D . Nu-

D .C.,and115,

Dedhar, S., Argraves, W.S., Suzuki, S., Ruoslahti, E., Pierschbacher, M.D.,1987. Human osteosarcoma cells resistant to detachment by an arg-gly-asp containing peptide overproduce the fibronectin receptor. J.Cell. Biol. 105, 1175–1182.

Dietrich, J.W., Canalis, E.M., Maine, D.M., Raisz, L.G., 1979. Effectsof glucocorticoids on fetal rat bone collagen synthesis in vitro. En-docrinology 104, 715–721.

Doty, S.B., Schofield, B.H., 1976. Enzyme histochemistry of bone andcartilage cells. Prog. Histochem. Cytochem. 8, 1–38.

Dougall, W.C., Glaccum, M., Charrier, K., Rohrbach, K., Brasel, K., DeSmedt, T., Daro, E., Smith, J., Tometsko, M.E., Maliszewski, C.R.,Armstrong, A., Shen, V., Bain, S., Cosman, D., Anderson, D., Mor-rissey, P.J., Peschon, J.J., Schuh, J., 1999. RANK is essential forosteoclast and lymph node development. Genes Dev. 13, 2412–2424.

Ecarot-Charrier, B., Glorieux, F.H., van der Rest, M., Pereira, G., 1983.Osteoblasts isolated from mouse calvaria initiate matrix mineraliza-tion. J. Cell. Biol. 96, 639–643.

Eriksen, E.F., Vesterby, A., Kassem, M., Melsen, F., Mosekilde, L., 1993.Bone remodelling and bone structure. In: Mundy, G.R., Martin, T.J.(Eds.), Physiology and Pharmacology of Bone, vol. 107. Springer-Verlag, New York (pp. 67–101).

Erlebacher, A., Filvaroff, E.H., Gitelman, S.E., Derynck, R., 1995. Cell80, 371–378.

Evans, M.J., Kaufman, M.H., 1981. Establishment in culture of pluripo-tential cells from mouse embryos. Nature 292, 154–156.

Fedarko, N.S., Sponseller, P.D., Shapiro, J.R., 1996. Long-term extracel-lular matrix metabolism by cultured human osteogenesis imperfectaosteoblasts. J. Bone Miner. Res. 11, 800–805.

Fitzgerald, J., Lamande, S.R., Bateman, J.F., 1999. Proteasomal degrada-tion of unassembled mutant type I collagen pro-alpha1 (I) chains. J.

F .A.,of anroid

F eosar-

F Dman

F ype Ir-

F N.,lci-oma

F mar-n in

F .A.,frac-

F res of452.

F CEbone

G local-434.

G pres-ken

G G.L.,that

stem

hambers, T.J., McSheehy, P.M.J., Thompson, B.M., Fuller, K., 1985effect of calcium regulating hormones and prostaglandins onresorption by osteoclasts disaggregated from neonatal rabbit bEndocrinology 116, 234–239.

hambers, T.J., Owens, J.M., Hattersley, G., Jat, P.S., Fuller, K.,Generation of osteoclast-inductive and osteoclastogenic cell linestheH-2KbtsA58 transgenic mouse. Proc. Natl. Acad. Sci. U.S.A.5578–5582.

hang, W., Pratt, S.A., Chen, T.-H., Tu, C.-L., Mikala, G., SchwartzShoback, D., 2001. Parathyroid cells express dihydropyridine-sencation currents and L-type calcium channel subunits. Am. J. PhEndocrinol. Metab. 281, E180–E189.

hen, D., Ji, X., Harris, M.A., Feng, J.Q., Karsenty, G., Celeste,Rosen, V., Mundy, G.R., Harris, S.E., 1998. Differential roles for bmorphogenetic protein (BMP) receptor type IB and IA in differeation and specification of mesenchymal precursor cells to osteand adipocyte lineages. J. Cell. Biol. 142, 295–305.

hen, T.L., Cone, C.M., Morey-Hilton, E., 1983a. 1,25-DihydroxyvitaD3 receptors in cultured rat osteoblast-like cells. J. Biol. Chem.4350–4355.

heung, J.O.P., Hillarby, M.C., Ayad, S., Hoyland, J.A., Jones, CDenton, J., Thomas, J.T., Wallis, G.A., Grant, M.E., 2001. A ncell culture model of chondrocyte differentiation during mammaendochondral ossification. J. Bone Miner. Res. 16, 309–318.

hipman, S.D., Sweet, H.O., McBride Jr., D.J., Davisson, M.T., MJr., S.C., Shuldiner, A.R., Wenstrup, R.J., Rowe, D.W., Shapiro,1993. Defective pro alpha 2(I) collagen synthesis in a recessivetion in mice: a model of human osteogenesis imperfecta. Proc.Acad. Sci. U.S.A. 90, 1701–1705.

hyun, Y.S., Raisz, L.G., 1984. Stimulation of bone formationprostaglandin E2. Prostaglandins 27, 97–103.

algleish, R., 1997. The human type I collagen mutation databasecleic Acids Res. 25, 181–187.

eckelbaum, R.A., Chan, G., Miao, D., Gotzman, D., Karaplis, A2002. Ihh enhances differentiation of CFK-2 chondrocytic cellsantagonizes PTHrP-mediated activation of PKA. J. Cell. Sci.3015– 3025.

Biol. Chem. 274, 27392–27398.orrest, S.M., Ng, K.W., Findlay, D.M., Michelangeli, V.P., Livesey, S

Partridge, N.C., Zajac, J.D., Martin, T.J., 1985. Characterizationosteoblast-like clonal cell line which responds to both parathyhormone and calcitonin. Calcif. Tissue Int. 37, 51–56.

ournier, B., Price, P., 1991. Characterization of a new human ostcoma cell line OHS-4. J. Cell. Biol. 114, 577–583.

rancheschi, R.T., James, W.M., Zerlauth, G., 1985. 1,25-Dihydroxy3-specific regulation of growth, morphology and fibronectin in a huosteosarcoma cell line. J. Cell. Physiol. 123, 401–409.

rancheschi, R.T., Romano, P.R., Park, K.Y., 1988. Regulation of tcollagen synthesis by 1,25-dihydroxyvitamin D3 in human osteosacoma cells. J. Biol. Chem. 263, 18938–18945.

rendo, J.L., Pichaud, F., Mourroux, R.D., Bouizar, A., Segond,Moukhtar, M.S., Julienne, A., 1994. An isoform of the human catonin receptor is expressed in TT cells and in medullary carcinof the thyroid. FEBS Lett. 342, 214–216.

riedenstein, A.J., Chailakhyan, R.K., Gerasimov, U.V., 1987. Bonerow osteogenic stem cells: in vitro cultivation and transplantatiodiffusion chambers. Cell Tissue Kinet. 20, 263–272.

ujikawa, Y., Quinn, J.M., Sabokbar, A., McGee, J.O., Athanasou, N1996. The human osteoclast precursor circulates in the monocytetion. Endocrinology 137, 4058–4060.

uller, K., Chambers, T.J., 1987. Generation of osteoclasts in culturabbit bone marrow and spleen cells. J. Cell. Physiol. 132, 441–

uller, K., Wong, B., Fox, S., Choi, Y., Chambers, T.J., 1998. TRANis necessary and sufficient for osteoblast-mediated activation ofresorption in osteoclasts. J. Exp. Med. 188, 997–1001.

ay, C.V., Mueller, W.J., 1974. Carbonic anhydrase and osteoclasts:ization by labelled inhibitor autoradiography. Science 183, 432–

erstenfeld, L.C., Chipman, S.D., Glowacki, J., Lian, J.B., 1987. Exsion of differentiated function by mineralizing cultures of chicosteoblasts. Dev. Biol. 122, 49–60.

erstenfeld, L.C., Cruceta, J., Shea, C.M., Sampath, K., Barnes,Einhorn, T.A., 2002. Chondrocytes provide morphogenic signalsselectively induce osteogenic differentiation of mesenchymalcells. J. Bone Miner. Res. 17, 221–230.

98 V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102

Gerstenfeld, L.C., Landis, W.J., 1991. Gene expression and extracellularmatrix ultrastructure of a mineralising chondrocyte cell culture system.J. Cell. Biol. 112, 501–513.

Ghosh-Choudhury, N., Windle, J.J., Koop, B.A., Harris, M.A., Guerrero,D.L., Wozney, J.M., Mundy, G.R., Harris, S.E., 1996. Immortalizedmurine osteoblasts derived from BMP 2-T-antigen expressing trans-genic mice. Endocrinology 137, 331–339.

Glimcher, M.J., 1989. Mechanism of calcification: role of collagen fibrilsand collagen–phosphoprotein complexes in vitro and in vivo. Anat.Rec. 224, 139–153.

Goldring, S.R., Roelke, M.S., Petrison, K., Bhan, A.K., 1987. Humangiant cell tumors of the bone: identification and characterization ofcell types. J. Clin. Invest. 79, 481–491.

Grigoriadis, A.E., Aubin, J.E., Heersche, J.N., 1989. Effects of dexam-ethasone and Vitamin D3 on cartilage differentiation in a clonal chon-drogenic cell population. Endocrinology 125, 2103–2110.

Grigoriadis, A.E., Heersche, J.N., Aubin, J.E., 1996. Analysis of chon-droprogenitor frequency and cartilage differentiation in a novel fam-ily of clonal chondrogenic rat cell lines. Differentiation 60, 299–307.

Gronthos, S., Zannettino, A.C., Graves, S.E., Ohta, S., Hay, S.J., Sim-mons, P.J., 1999. Differential cell surface expression of the STRO-1and alkaline phosphatase antigens on discrete developmental stages inprimary cultures of human bone cells. J. Bone Miner. Res. 14, 47–56.

Guenther, H.L., Cecchini, M.G., Elford, P.R., Fleisch, H., 1988. Effectsof transforming growth factor type beta upon bone cell populationsgrown either in monolayer of semisolid medium. J. Bone Miner. Res.3, 269–278.

Guenther, H.L., Hofstetter, W., Stutzer, A., Nuhlbauer, R., Fleisch, H.,e de-

ingntly

H Lee,Thethe

brid

H mentl os-

H eng,ffectsandontin,tures3.

H ndy,engerner.

H nctionistantiation.

H , J.A.,ypice in

H tech-iques

H arialandrinol-

Henderson, J.E., He, B., Goltzman, D., Karaplis, A.C., 1996. Constitutiveexpression of parathyroid hormone-related peptide (PTHrP) stimulatesgrowth and inhibits differentiation of CFK2 chondrocytes. J. Cell.Physiol. 169, 33–41.

Hering, T., Kollar, J., Huynh, T.D., Varelas, J.B., Sandell, L.J., 1994. Mod-ulation of extracellular matrix gene expression in bone high-densitychondrocyte cultures by ascorbic acid and enzymatic resuspension.Arch. Biochem. Biophys. 314, 90–98.

Hjelle, K., Solheim, E., Strand, T., Muri, R., Brittberg, M., 2002. Ar-ticular cartilage defects in 1000 knee arthroscopies. Arthroscopy 18,730–740.

Hock, J.M., Canalis, E., Centrella, M., 1990. Transforming growth factorbeta (TGF-beta-1) stimulates bone matrix apposition and bone cellreplication in cultured fetal rat calvariae. Endocrinology 126, 421–426.

Hodge, J.M., Kirkland, M.A., Aitken, C.J., Myers, D.E., Nicholson, G.C.,2002. Osteoclastic potential of human cord blood mononuclear cellsand derived CFU-GM: effects of M-CSF, GM-CSF and IL-3. J. BoneMiner. Res. 12 (Suppl. 1), S455.

Hofbauer, L.C., Lacey, D.L., Dunstan, C.R., Spelsberg, T.C., Riggs, B.L.,Khosla, S., 1999. Interleukin-1beta and tumor necrosis factor-alpha,but not interleukin-6, stimulate osteoprotegerin ligand gene expressionin human osteoblastic cells. Bone 25, 255–259.

Hohmann, E.L., Elde, R.P., Rysavy, J.A., Einzig, S., Gebhard, R.L., 1986.Innervation of periosteum and bone by sympathetic vasoactive intesti-nal peptide-containing nerve fibers. Science 232, 868–871.

Horton Jr., W.E., Cleveland, J., Rapp, U., Nemuth, G., Bolander, M.,Doege, K., Yamada, Y., Hassell, J.R., 1988. An established rat cellline expressing chondrocyte properties. Exp. Cell Res. 178, 457–468.

Horwood, N.J., Elliott, J., Martin, T.J., Gillespie, M.T., 1998. Osteotropicagents regulate the expression of osteoclast differentiation factor

139,

H ms,Z.,S.,actorandSci.

H ll 48,

I ma-. J.

I daro,by

1009.I 984.

theirCell.

J nnor,Pu-sors.

J and59,

K H.,res-erent.

K an-1511.

K .F.,-A5

1989. Evidence for heterogeneity of the osteoblastic phenotyptermined with clonal rat bone cells established from transformgrowth factor-�-induced cell colonies grown anchorage independein semisolid medium. Endocrinology 125, 2092–2102.

ammonds, R.G., Schwall, R., Dudley, A., Berkemeier, L., Lai, C.,J., Cunningham, N., Reddi, A.H., Wood, W.I., Mason, A.J., 1990.bone inducing activity of recombinant BMP-2A and BMP-2B andpurification and characterization of mature BMP-2B from a hyBMP-2A/2B precursor. Mol. Endocrinol. 4, 149–155.

arris, S.A., Enger, R.J., Riggs, B.L., Spelsberg, T.C., 1995. Developand characterization of a conditionally immortalized human fetateoblastic cell line. J. Bone Miner. Res. 10, 178–186.

arris, S.E., Bonewald, L.F., Harris, M.A., Sabatini, M., Dallas, S., FJ.Q., Ghosh-Choudhury, N., Wozney, J., Mundy, G.R., 1994a. Eof transforming growth factor beta on bone nodule formationexpression of bone morphogenetic protein 2, osteocalcin, osteopalkaline phosphatase, and type I collagen mRNA in long-term culof fetal rat calvarial osteoblasts. J. Bone Miner. Res. 9, 855–86

arris, S.E., Sabatini, M., Harris, M.A., Feng, J.Q., Wozney, J., MuG.R., 1994b. Expression of bone morphogenetic protein messRNA in prolonged cultures of fetal rat calvarial cells. J. Bone MiRes. 9, 389–394.

attersley, G., Chambers, T.J., 1989. Generation of osteoclastic fuin mouse bone marrow cultures: multinuclearity and tartrate-resacid phosphatase are unreliable markers for osteoclast differentEndocrinology 125, 1606–1612.

auselmann, H.J., Fernandes, R.J., Mok, S.S., Schnid, T.M., BlockAydelotte, M.B., Kuettner, K.E., Thonar, E.J.-M.A., 1994. Phenotstability of bovine articular chondrocytes after long-term culturalginate beads. J. Cell. Sci. 107, 17–27.

eath, J.K., Reynolds, J.J., 1990. Bone cell physiology and in vitroniques in its investigation. In: Stevenson, J.C. (Ed.), New Technin Metabolic Bone Disease. Wright Press, London, pp. 21–39.

eath, J.K., Rodan, S.B., Yoon, K., Rodan, G.A., 1989. Rat calvcell lines immortalized with SV-40 large T antigen: constitutiveretinoic acid-inducible expression of osteoblastic features. Endocogy 124, 3060–3068.

and osteoprotegerin in osteoblastic stromal cells. Endocrinology4743–4746.

su, H., Lacey, D.L., Dunstan, C.R., Solovyev, I., Colombero, A., TimE., Tan, H.L., Elliott, G., Kelley, M.J., Sarosi, I., Wang, L., Xia, X.Elliott, R., Chiu, L., Black, T., Scully, S., Capparelli, C., Morony,Shimamoto, G., Bass, M.B., Boyle, W.J., 1999. Tumor necrosis freceptor family member RANK mediates osteoclast differentiationactivation induced by osteoprotegerin ligand. Proc. Natl. Acad.U.S.A. 96, 3540–3545.

ynes, R.O., 1987. Integrins: a family of cell-surface receptors. Ce549–554.

baraki, K., Termine, J.D., Whitson, W.S., Young, M.F., 1992. Bonetrix mRNA expression in differentiating fetal bovine osteoblastsBone Miner. Res. 7, 743–754.

bbotson, K.J., Twardzik, D.R., D’Souza, S.M., Hargreaves, W.R., ToG.J., Mundy, G.R., 1985. Stimulation of bone resorption in vitrosynthetic transforming growth factor alpha. Science 228, 1007–

bbotson, K.J., Roodman, G.D., McManus, L.M., Mundy, G.R., 1Identification and characterization of osteoclast-like cells andprogenitors in cultures of feline marrow mononuclear cells. J.Biol. 99, 471–480.

ames, I.E., Dodds, R.A., Lee-Rykaczewski, E., Eichman, C.F., CoJ.R., Hart, T.K., Maleeff, B.E., Lackman, R.D., Gowen, M., 1996.rification and characterization of fully functional osteoclast precurJ. Bone Miner. Res. 11, 1608–1618.

at, P., Sharp, P.A., 1986. Large T antigens of simian virus 40polyomavirus efficiently establish primary fibroblasts. J. Virol.746–750.

alajzic, I., Kalajzic, Z., Kaliterna, M., Gronowicz, G., Clark, S.Lichtler, A.C., Rowe, D., 2002. Use of type I collagen green fluocent protein transgenes to identify subpopulations of cells at diffstages of the osteoblast lineage. J. Bone Miner. Res. 17, 15–25

amiya, N., Jikko, A., Kimata, K., Damsky, C., Shimizu, K., Watabe, H., 2002. Establishment of a novel chondrocytic cell line Nderived from p53-null mice. J. Bone Miner. Res. 17, 1832–1842

ato, Y., Boskey, A., Spevak, L., Dallas, M., Hori, M., Bonewald, L2001. Establishment of an osteoid preosteocyte-like cell MLO

V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102 99

that spontaneously mineralises in culture. J. Bone Miner. Res. 16,1622–1633.

Kato, Y., Windle, J.J., Koop, B.A., Mundy, G.R., Bonewald, L.F., 1997.Establishment of an osteocyte-like cell line, MLO-Y4. J. Bone Miner.Res. 12, 2014–2023.

Keeting, P.E., Scott, R.E., Colvard, D.S., Anderson, M.A., Oursler, M.J.,Spelsberg, T.C., Riggs, B.L., 1992. Development and characterizationof a rapidly proliferating, well-differentiated cell line derived fromnormal adult human osteoblast-like cells transfected with SV40 largeT antigen. J. Bone Miner. Res. 7, 127–136.

Klein-Nulend, J., van der Plas, A., Semeins, C.M., Ajubi, N.E., Frangos,J.A., Nijweide, P.J., Burger, E.H., 1995. Sensitivity of osteocytes tobiomechanical stress in vitro. FASEB J. 9, 441–445.

Kobayashi, K., Takahashi, N., Jimi, E., Udagawa, N., Takami, M., Kotake,S., Nakagawa, N., Kinosaki, M., Yamaguchi, K., Shima, N., Yasuda,H., Morinaga, T., Higashio, K., Martin, T.J., Suda, T., 2000. Tumornecrosis factor alpha stimulates osteoclast differentiation by a mech-anism independent of the ODF/RANKL–RANK interaction. J. Exp.Med. 191, 275–286.

Kodama, H., Amagai, Y., Sudo, H., Kasai, S., Yamamoto, S., 1981. Estab-lishment of a clonal osteogenic cell line from newborn mouse calvaria.Jpn. J. Oral Biol. 23, 899–901.

Komm, B.S., Terpening, C.M., Benz, D.J., Greame, E.A., O’Malley, B.W.,Haussler, M.R., 1988. Estrogen binding receptor mRNA, and biologicresponse in osteoblast-like osteosarcoma cells. Science 241, 81–84.

Kream, B.E., Smith, M.D., Canalis, E., Raisz, L.G., 1985. Characteriza-tion of the effect of insulin on collagen synthesis in fetal rat bone.Endocrinology 116, 296–302.

Kumegawa, J., Ikeda, E., Tanaka, S., Haneji, T., Yora, T., Sakahishi,Y., Minami, N., Hiramatsu, M., 1984. The effects of prostaglandin

cliclastic

L ess,n,

uf-y, J.,lates

L embly:rones.

L Cole,lum-rom

1 (I)ly. J.

L and

L ey,nx2bonemad5mes-92.

L ehon-trix

L , H.,t-like

E2. 12,

L cellAn-

dreoli, M., Monaco, F. (Eds.), Advances in thyroid neoplasia.. FieldEducational, Rome, pp. 95–105.

Lian, J.B., Friedman, P.A., 1978. The Vitamin K-dependent synthesis ofgamma-carboxyglutamic acid by bone microsomes. J. Biol. Chem.253, 6623–6626.

Lian, J.B., Stein, G.S., 1995. Development of the osteoblast phenotype:molecular mechanisms mediating osteoblast growth and differentia-tion. Iowa Orthop. J. 15, 118–140.

Liu, F., Malaval, L., Gupta, A.K., Aubin, J.E., 1994. Simultaneous de-tection of multiple bone-related mRNAs and protein expression dur-ing osteoblast differentiation: polymerase chain reaction and immuno-cytochemical studies at the single cell level. Dev. Biol. 166, 220–234.

Lyons, K.M., Pelton, R.W., Hogan, B.L.M., 1989. Patterns of expressionof murine Vgr-1 and BMP-2a RNA suggest that transforming growthfactor-�-like genes coordinately regulate aspects of embryonic devel-opment. Genes Dev. 1, 1657–1668.

Majeska, R.J., 1996. Culture of osteoblastic cells. In: Bilezikian, J.P.,Raisz, L.G., Rodan, G.A. (Eds.), Principles of Bone Biology. Aca-demic Press, San Diego, pp. 1229–1237.

Majeska, R.J., Rodan, G.A., 1982a. Alkaline phosphatase inhibition byparathyroid hormone and isoproterenol in a clonal rat osteosarcomacell line. Possible mediation by cyclic AMP. Calcif. Tissue Int. 34,59–66.

Majeska, R.J., Rodan, G.A., 1982b. The effect of 1,25(OH)2D3 on al-kaline phosphatase in osteoblastic osteosarcoma cells. J. Biol. Chem.257, 3362–3365.

Majeska, R.J., Rodan, S.B., Rodan, G.A., 1980. Parathyroid hormone-responsive clonal cell lines from rat osteosarcoma. Endocrinology 107,1494–1502.

M teo-ell.

M f theneral336,

M nt ofiples

M usestem

M lan-eny-in the

M ne.nd

M inol.

M awa,M.,rmedOS-

lated

M ctiveration

M tin,MPRes.

M type?

E2, parathyroid hormone, 1,25-dihydroxycholecalciferol, and cynucleotide analogs on alkaline phosphatase activity in osteobcells. Calcif. Tissue Int. 36, 72–76.

acey, D.L., Timms, E., Tan, H.L., Kelley, M.J., Dunstan, C.R., BurgT., Elliott, R., Colombero, A., Elliott, G., Scully, S., Hsu, H., SullivaJ., Hawkins, N., Davy, E., Capparelli, C., Eli, A., Qian, Y.X., Kaman, S., Sarosi, I., Shalhoub, V., Senaldi, G., Guo, J., DelaneBoyle, W.J., 1998. Osteoprotegerin ligand is a cytokine that reguosteoclast differentiation and activation. Cell 93, 165–176.

amande, S.R., Bateman, J.F., 1999. Procollagen folding and assthe role of endoplasmic reticulum enzymes and molecular chapeSemin. Cell Dev. Biol. 10, 455–464.

amande, S.R., Chessler, S.D., Golub, S.B., Byers, P.H., Chan, D.,W.G., Sillence, D.O., Bateman, J.F., 1995. Endoplasmic reticumediated quality control of type I collagen production by cells fosteogenesis imperfecta patients with mutations in the pro alphachain carboxy-terminal propeptide which impair subunit assembBiol. Chem. 270, 8642–8649.

anyon, L.E., 1993. Osteocytes, strain detection, bone modellingremodelling. Calcif. Tissue Int. 53 (Suppl. 1), S102–S106.

ee, K.S., Kim, H.J., Li, Q.L., Chi, X.Z., Ueta, C., Komori, T., WoznJ.M., Kim, E.G., Choi, J.Y., Ryoo, H.M., Bae, S.C., 2000. Ruis a common target of transforming growth factor beta1 andmorphogenetic protein 2, and cooperation between Runx2 and Sinduces osteoblast-specific gene expression in the pluripotentenchymal precursor cell line C2C12. Mol. Cell. Biol. 20, 8783–87

efebvre, V., Garafalo, S., Zhou, G., Metsaranta, M., Vucurio, E., DCrombrugghe, B., 1994. Characterization of primary cultures of cdrocytes from type II collagen/�-galactosidase transgenic mice. MaBiol. 14, 329–335.

eis, H.J., Hulla, W., Gruber, R., Huber, E., Zach, D., GleispachWindischhofer, W., 1997. Phenotypic heterogeneity of osteoblasMC3T3-E1 cells: changes of bradykinin-induced prostaglandinproduction during osteoblast maturation. J. Bone Miner. Res541–551.

eong, S.S., Horoszewicz, J.S., Shimaoka, K., et al., 1981. A newline for study of human medullary carcinoma. In: Robbins, J.,

alavalk, L., Liu, F., Roche, P., Aubin, J.E., 1999. Kinetics of osprogenitor proliferation and osteoblast differentiation in vitro. J. CBiochem. 7, 616–627.

ansour, S.L., Thomas, K.R., Capecchi, M.R., 1988. Disruption oproto-oncogene int-2 in mouse embryo-derived stem cells: a gestrategy for targeting mutations to non-selectable genes. Nature348–352.

arks, S.C., Hermey, D.C., 1996. The structure and developomebone. In: Bilezikian, J.P., Raisz, L.G., Rodan, G.A. (Eds.), Princof Bone Biology. Academic Press, San Diego, pp. 3–14.

artin, G.R., 1981. Isolation of a pluripotent cell line from early moembryos cultured in medium conditioned by teratocarcinomacells. Proc. Natl. Acad. Sci. U.S.A. 78, 7634–7638.

artin, T.J., Ingleton, P.M., Underwood, J.C.E., Melick, R.A., Michegeli, V.P., Hunt, N.H., 1976. Parathyroid hormone responsive adlate cyclase in an induced transplantable osteogenic sarcomarat. Nature 260, 436–438.

artin, T.J., Ng, K.W., Nicholson, G.C., 1988. Cell biology of boIn: Sheppard, M.C. (Ed.), Bailliere’s Clinical Endocrinology aMetabolism, vol. 2. Bailliere Tindall, London (pp. 1–29).

artin, T.J., Ng, K.W., Suda, T., 1989. Bone cell physiology. EndocrMetab. Clin. North Am. 18, 833–858.

atsuzaki, K., Katayama, K., Takahashi, Y., Nakamura, I., UdagN., Tsurukai, T., Nishinakamura, R., Toyama, Y., Yabe, Y., Hori,Takahashi, N., Suda, T., 1999. Human osteoclast-like cells are fofrom peripheral blood mononuclear cells in a coculture with Sa2 cells transfected with the parathyroid hormone (PTH)/PTH-reprotein receptor gene. Endocrinology 140, 925–932.

cCabe, L.R., Kockx, M., Lian, J., Stein, J., Stein, G., 1995. Seleexpression of fos- and jun-related genes during osteoblast prolifeand differentiation. Exp. Cell Res. 218, 255–262.

ichelangeli, V.P., Fletcher, A.E., Allan, E.H., Nicholson, G.C., MarT.J., 1989. Effects of calcitonin gene-related peptide on cyclic Aformation in chicken, rat, and mouse bone cells. J. Bone Miner.4, 269–272.

iller, S.C., Jee, W.S.S., 1987. The bone lining cell: a distinct phenoCalcif. Tissue Int. 41, 1–5.

100 V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102

Mitchell, J., Rouleau, M.F., Goltzman, D., 1990. Biochemical and mor-phological characterization of parathyroid hormone receptor bind-ing to the rat osteosarcoma cell line UMR-106. Endocrinology 126,2327–2335.

Moallem, E., Silver, J., Naveh-Many, T., 1995. Regulation of parathyroidhormone messenger RNA levels by protein kinase A and C in bovineparathyroid cells. J. Bone Miner. Res. 10, 447–452.

Moseley, J.M., Martin, T.J., 1996. Parathyroid hormone-related protein:physiological actions. In: Bilezikian, J.P., Raisz, L.G., Rodan, G.A.(Eds.), Principles of Bone Biology. Academic Press, San Diego, pp.363–376.

Mundy, G.R., 1993. Cytokines of bone. In: Mundy, G.R., Martin, T.J.(Eds.), Physiology and Pharmacology of Bone, vol. 107. Springer-Verlag, Berlin (pp. 185–214).

Nagata, T., Bellows, C.G., Kasugai, S., Butler, W.T., Sodek, J., 1991.Biosynthesis of bone proteins [SPP-1 (secreted phosphoprotein-1, os-teopontin), BSP (bone sialoprotein) and SPARC (osteonectin)] in as-sociation with mineralized-tissue formation by fetal-rat calvarial cellsin culture. J. Biochem. 274, 513–520.

Narbaitz, R., Stumpf, W., Sar, M., Huang, S., DeLuca, H.F.,1983. Autoradiographic demonstration of target cells for 1,25-dihydroxycholecalciferol in bones from fetal rats. Calcif. Tissue Int.35, 177–182.

Ng, K.W., Gummer, P.R., Michelangeli, V.P., Bateman, J.F., Mascara, T.,Cole, W.G., Martin, T.J., 1988. Regulation of alkaline phosphataseexpression in a neonatal rat clonal calvarial cell strain by retinoicacid. J. Bone Miner. Res. 3, 53–61.

Ng, K.W., Hudson, P.J., Power, B.E., Manji, S.S., Gummer, P.R., Martin,T.J., 1989a. Retinoic acid and tumor necrosis factor-� act in concert tocontrol the level of alkaline phosphatase mRNA. J. Mol. Endocrinol.

N 85.truc-.

N ingntrol

N wthnd in

N ay,E.,eralkap-

N Mar-steo-

N rolif-66,

N n byol-

N Fotype-ysiol.

N 88.osar-3,

N .M.,(os-

06,

Okihana, H., Yamada, K., 1999. Preparation of a cDNA library and pre-liminary assessment of 1400 genes from mouse growth cartilage. J.Bone Miner. Res. 14, 304–310.

Otto, F., Thornell, A.P., Crompton, T., Denzel, A., Gilmour, K.C.,Rosewell, I.R., Owen, M.J., 1997. Cbfa1, a candidate gene for clei-docranial dysplasia syndrome, is essential for osteoblast differentiationand bone development. Cell 89, 765–771.

Owen, T.A., Aronow, M.S., Barone, L.M., Bettencourt, B., Stein, G.S.,Lian, J.B., 1991. Pleiotropic effects of Vitamin D on osteoblast geneexpression are related to the proliferative and differentiated state ofthe bone cell phenotype: dependency upon basal levels of gene ex-pression, duration of exposure, and bone matrix competency in normalrat osteoblast cultures. Endocrinology 128, 1496–1504.

Owen, T.A., Aronow, M., Shalhoub, V., Barone, L.M., Wilming, L., Tassi-nari, M.S., Kennedy, M.B., Pockwinse, S., Lian, J.B., Stein, G.S.,1990. Progressive development of the osteoblast phenotype in vitro:reciprocal relationships in expression of genes associated with os-teoblast proliferation and differentiation during formation of the boneextracellular matrix. J. Cell. Physiol. 143, 420–430.

Oxford, J.T., Doege, K.J., Horton Jr., W.E., Morris, N.P., 1994. Character-ization of type II and type XI collagen synthesis by an immortalizedrat chondrocyte cell line (IRC) having a low level of type II collagenmRNA expression. Exp. Cell. Res. 213, 28–36.

Oyajobi, B.O., Lomri, A., Hott, M., Marie, P.J., 1999. Isolation and char-acterization of human clonogenic osteoblast progenitors immunose-lected from fetal bone marrow stroma using STRO-1 monoclonal an-tibody. J. Bone Miner. Res. 14, 351–361.

Parfitt, R., 1993. Calcium homeostasis. In: Mundy, G.R., Martin, T.J.(Eds.), Physiology and Pharmacology of Bone, vol. 107. Springer-Verlag, New York (pp. 1–66).

P urne,mic

P .J.,onal308–

P lms,

cells.

P r, C.,duc-

os-

P mper-

P l and1477.

P A.,and

P -cells.

Q 992.-E1one

Q .A.,pop-

Q ionatingon in

3, 57–64.g, K.W., Livesey, S.A., Collier, F., Gummer, P.R., Martin, T.J., 19

Effect of retinoids on the growth, ultrastructure, and cytoskeletal stures of malignant rat osteoblasts. Cancer Res. 45, 5106–5113

g, K.W., Manji, S.S., Young, M.F., Findlay, D.M., 1989b. Opposinfluences of glucocorticoid and retinoic acid on transcriptional coin preosteoblasts. Mol. Endocrinol. 3, 2079–2085.

g, K.W., Partridge, N.C., Niall, M., Martin, T.J., 1983. Epidermal grofactor receptors in clonal lines of a rat osteogenic sarcoma aosteoblast-rich rat bone cells. Calcif. Tissue Int. 35, 293–303.

icholson, G.C., Malakellis, M., Collier, F.M., Cameron, P.U., HollowW.R., Gough, T.J., Gregorio-King, C., Kirkland, M.A., Myers, D.2000. Induction of osteoclast from CD14-positive human periphblood mononuclear cells by receptor activator of nuclear factorpaB ligand (RANKL). Clin. Sci. 99, 133–140.

icholson, G.C., Moseley, J.M., Sexton, P.M., Mendelsohn, F.A.O.,tin, T.J., 1986. Abundant calcitonin receptors in isolated rat oclasts. J. Clin. Invest. 78, 355–360.

ijweide, P.J., Burger, E.H., Feyen, J.M., 1986. Cells of bone: peration, differentiation and hormonal regulation. Physiol. Rev.855–886.

oda, M., 1989. Transcriptional regulation of osteocalcin productiotransforming growth factor-� in rat osteoblast-like cells. Endocrinogy 124, 612–617.

oda, M., Rodan, G.A., 1987. Type� transforming growth factor (TG�) regulation of alkaline phosphatase expression and other phenrelated mRNAs in osteoblastic rat osteosarcoma cells. J. Cell. Ph133, 426–437.

oda, M., Yoon, K., Prince, C.W., Butler, W.T., Rodan, G.A., 19Transcriptional regulation of osteopontin production in rat ostecoma cells by type� transforming growth factor. J. Biol. Chem. 2613916–13921.

omura, S., Wills, A.J., Edwards, D.R., Heath, J.K., Hogan, B.L1988. Developmental expression of 2ar (osteopontin) and SPARCteonectin) RNA as revealed by in situ hybridization. J. Cell. Biol. 1441–450.

arsons, J.A., 1976. Parathyroid physiology and the skeleton. In: BoG.H. (Ed.), The Biochemistry and Physiology of Bone. AcadePress, New York, pp. 159–214.

artridge, N.C., Alcorn, D., Michelangeli, V.P., Ryan, G.B., Martin, T1983. Morphological and biochemical characterization of four closteogenic sarcoma cell lines of rat origin. Cancer Res. 43, 44314.

artridge, N.C., Frampton, R.J., Eisman, J.A., Michelangeli, V.P., EE., Bradley, T.R., Martin, T.J., 1980. Receptors for 1,25(OH)2-VitaminD3 enriched in cloned osteoblast-like rat osteogenic sarcomaFEBS Lett. 115, 139–142.

artridge, N.C., Jeffrey, J.J., Ehlich, L.S., Teitelbaum, S.L., FliszaWelgus, H.G., Kahn, A.J., 1987. Hormonal regulation of the protion of collagenase and a collagenase inhibitor activity by ratteogenic sarcoma cells. Endocrinology 12, 1956–1962.

aterson, C.R., McAllion, S., Stellman, J.L., 1984. Osteogenesis ifecta after the menopause. N. Engl. J. Med. 310, 1694–1696.

eck, W.A., Birge, S.J., Fedak, S.A., 1964. Bone cells: biochemicabiological studies after enzymatic isolation. Science 146, 1476–

eterson, L., Brittberg, M., Kiviranta, I., Akerlund, E.L., Lindahl,2002. Autologous chondrocyte transplantation. Biomechanicslong-term durability. Am. J. Sports Med. 30, 2–12.

rice, P.A., Baukol, S.A., 1980. 1,25-Dihydroxyvitamin D3 increases synthesis of the Vitamin K-dependent bone protein by osteosarcomaJ. Biol. Chem. 255, 11660–11663.

uarles, L.D., Yohay, D.A., Lever, L.W., Caton, R., Wenstrup, R.J., 1Distinct proliferative and differentiated stages of murine MC3T3cells in culture: an in vitro model of osteoblast development. J. BMiner. Res. 7, 683–692.

uinn, J.M.W., Whitty, G.A., Byrne, R.J., Gillespie, M.T., Hamilton, J2002. The generation of highly enriched osteoclast-lineage cellulations. Bone 30, 164–170.

uinn, J.M., Elliott, J., Gillespie, M.T., Martin, T.J., 1998. A combinatof osteoclast differentiation factor and macrophage-colony stimulfactor is sufficient for both human and mouse osteoclast formativitro. Endocrinology 139, 4424–4427.

V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102 101

Quinn, J.M., McGee, J.O., Athanasou, N.A., 1994. Cellular and hor-monal factors influencing monocyte differentiation in osteoclasticbone-resorbing cells. Endocrinology 134, 2416–2423.

Quinn, J.M., Neale, S., Fujikawa, Y., McGee, J.O., Athanasou, N.A.,1998b. Human osteoclast formation from blood monocytes, peritonealmacrophages, and bone marrow cells. Calcif. Tissue Int. 62, 527–531.

Raisz, L.G., Kream, B.E., 1983. Regulation of bone formation. N. Engl.J. Med. 309, 29–35.

Raisz, L.G., Martin, T.J., 1984. Prostaglandins in bone and mineralmetabolism. J. Bone Miner. Res. 2, 286–311.

Ralston, S.H., 1999. The genetics of osteoporosis. Bone 1, 85–86.Reid, I.R., Cornish, J., 1996. Amylin and CGRP. In: Bilezikian, J.P.,

Raisz, L.G., Rodan, G.A. (Eds.), Principles of Bone Biology. Aca-demic Press, San Diego, pp. 495–505.

Rochet, N., Dubousset, J., Mazeau, C., Zanghellini, E., Farges, M.F.,de Novion, H.S., Chompret, A., Delpech, B., Cattan, N., Frenay, M.,Gioanni, J., 1999. Establishment, characterization and partial cytokineexpression profile of a new human osteosarcoma cell line (CAL 72).Int. J. Cancer 82, 282–285.

Rochet, N., Leroy, P., Far, D.F., Ollier, L., Loubat, A., Rossi, B., 2003.CAL72: a human osteosarcoma cell line with unique effects onhematopoietic cells. Eur. J. Haematol. 70, 43–52.

Rodan, S.B., Imai, Y., Thiede, M.A., Wesolowski, G., Thompson, D.,Bar-Shavit, Z., Shull, S., Mann, K., Rodan, G.A., 1987b. Characteri-zation of a human osteosarcoma cell line (SaOS-2) with osteoblasticproperties. Cancer Res. 47, 4961–4966.

Rodan, S.B., Majeska, R.J., Rodan, G.A., 1994. Osteosarcoma cells asmodels for osteoblasts. In: Novak, J.F., MacMaster, J.H. (Eds.), Fron-tiers of Osteosarcoma Research. Holgrefe and Huber, Seattle, pp.193–203.

R owthwth

R age,ed

hritis

R aisz,mic

R .,stemone.

S f the

S -liketure

S 989.dur-n of

S ieticbone

S wicz,byInt.

S Y.,ma-es.

S ntialnetic

protein-2 in chondrogenic cell line ATDC5. Exp. Cell. Res. 241, 1–11.

Simmons, P.J., Torok-Storb, B., 1991. Identification of stromal cell precur-sors in human bone marrow by a novel monoclonal antibody, STRO-1.Blood 78, 55–62.

Soskoline, W., Schwartz, Z., Ornoy, A., 1986. The development of fetalmice long bones in vitro: an assay of bone modeling. Bone 7, 41–48.

Spinella-Jaegle, S., Rawadi, G., Kawai, S., Gallea, S., Faucheu, C., Mollat,P., Courtois, B., Bergaud, B., Ramez, V., Blanchet, A.M., Adelmant,G., Baron, R., Roman-Roman, S., 2001. Sonic hedgehog increasesthe commitment of pluripotent mesenchymal cells into the osteoblas-tic lineage and abolishes adipocytic differentiation. J. Cell. Sci. 114,2085–2094.

Stein, G.S., Lian, J.B., 1993. Molecular mechanisms mediating prolifera-tion/differentiation interrelationships during progressive developmentof the osteoblast phenotype. Endocrine Rev. 14, 424–442.

Stern, P., Krieger, N., 1983. Comparison of fetal limb bones andneonatal mouse calvaria: effects of parathyroid hormone and 1,25-dihydroxyvitamin D3. Calcif. Tissue Int. 35, 172–176.

Stewart, K., Walsh, S., Screen, J., Jefferiss, C.M., Chainey, J., Jordan,G.R., Beresford, J.N., 1999. Further characterization of cells express-ing STRO-1 in cultures of adult human bone marrow stromal cells.J. Bone Miner. Res. 14, 1345–1356.

Stewart, M.C., Saunder, K.M., Burton-Wurster, N., Macleod, J.N., 2000.Phenotypic stability of articular chondrocytes in vitro: the effects ofculture models, bone morphogenetic protein 2, and serum supplemen-tation. J. Bone Miner. Res. 15, 166–174.

Stracke, H., Schultz, A., Moeller, D., Rossol, S., Schatz, H., 1984. Effectof growth hormone on osteoblasts and demonstration of somatomedinC/IGF-1 in bone organ culture. Acta Endocrinol. 107, 16–24.

S clast

S clasts.),ress,

S 3. Incell198.

T eley,cep-ted123,

T ose-lved

T .D.,ation

arrow

T , J.,II

arkercep-

ll line,

T ki,lture

449–

T uki,an

s. 49,

T rathy-of its

odan, S.B., Wesolowski, G., Thomas, K., Rodan, G.A., 1987a. Grstimulation of rat calvaria osteoblastic cells by acidic fibroblast grofactor. Endocrinology 121, 1917–1923.

onziere, M.-C., Farjanel, J., Freyria, A.-M., Hartmann, D.J., HerbD., 1997. Analysis of types I, II, III, IX and XI collagens synthesizby fetal bovine chondrocytes in high-density culture. OsteoartCartilage 5, 205–214.

owe, D.W., 2002. Osteogenesis imperefecta. In: Bilezikian, J.P., RG.L., Rodan, G.A. (Eds.), Principles of Bone Biology. AcadePress, San Diego, pp. 1177–1193.

ubinacci, A., Villa, I., Dondi Benelli, F., Borgo, E., Ferretti, MPalumbo, C., Marotti, G., 1998. Osteocyte-bone lining cell syat the origin of steady ionic current in damaged amphibian bCalcif. Tissue Int. 63, 331–339.

andberg, M., Autio-Harmainen, H., Vuorio, E., 1988. Localization oexpression of types I, III, and IV collagen, TGF-�1 andc-fos genesin developing human calvarial bones. Dev. Biol. 130, 324–334.

choenle, E., Zapf, J., Hubel, R.E., Froesch, E.R., 1982. Insulingrowth factor I stimulates growth in hypophysectomized rats. Na296, 252–253.

halhoub, V., Gerstenfeld, L.C., Collart, D., Lian, J.B., Stein, G.S., 1Downregulation of cell growth and cell cycle regulated genesing chick osteoblast differentiation with the reciprocal expressiohistone gene variants. Biochemistry 28, 5318–5322.

hinar, D.M., Sato, M., Rodan, G.A., 1990. The effect of hemopogrowth factors on the generation of osteoclast-like cell in mousemarrow cultures. Endocrinology 126, 1728–1735.

hteyer, A., Gazit, D., Passi-even, L., Rab, I., Majeska, R.J., GronoG., Lurie, A., Rodan, G.A., 1986. Formation of calcifying matrixosteosarcoma cells in diffusion chambers in vivo. Calcif. Tissue39, 49–54.

hukunami, C., Ishizeki, K., Atsumi, T., Ohta, Y., Suzuki, F., Hiraki,1997. Cellular hypertrophy and calcification of embryonal carcinoderived chondrogenic cell line ATDC5 in vitro. J. Bone Miner. R12, 1174–1188.

hukunami, C., Ohta, Y., Sakuda, M., Hiraki, Y., 1998. Sequeprogression of the differentiation program by bone morphoge

uda, T., Takahashi, N., Martin, T.J., 1992. Modulation of osteodifferentiation. Endocrine Rev. 13, 66–80.

uda, T., Takahashi, N., Martin, T.J., 1995. Modulation of osteodifferentiation: update 1995. In: Bikle, D.D., Negro-vilar, A. (EdEndocrine Reviews, Monographs, vol. 4. The Endocrine Society PBethesda (pp. 266–270).

udo, H., Kodama, H.A., Amagai, Y., Yamamoto, S., Kasai, S., 198vitro differentiation and calcification in a new clonal osteogenicline derived from newborn mouse calvaria. J. Cell. Biol. 96, 191–

akahashi, N., Akatsu, T., Sasaki, T., Nicholson, G.C., MosJ.M., Martin, T.J., Suda, T., 1988a. Induction of calcitonin retors by 1,25-dihydroxyvitamin D3 in osteoclast-like multinucleacells formed from mouse bone marrow cells. Endocrinology1504–1510.

akahashi, N., Akatsu, T., Udagawa, N., Sasaki, T., Yamaguchi, Y., Mley, J.M., Martin, T.J., Suda, T., 1988c. Osteoblastic cells are invoin osteoclast formation. Endocrinology 123, 2600–2602.

akahashi, N., Yamana, H., Yoshiki, S., Roodman, G.D., Mundy, GJones, S.J., Boyde, A., Suda, T., 1988b. Osteoclast-like cell formand its regulation by osteotropic hormones in mouse bone mcultures. Endocrinology 122, 1373–1382.

akigawa, M., Okawa, T., Pan, H., Aoki, C., Takahashi, K., ZueSuzuki, F., Kinoshita, A., 1997. Insulin-like growth factors I andare autocrine factors in stimulating proteoglycan synthesis, a mof differentiated chondrocytes, acting through their respective retors on a clonal human chondrosarcoma-derived chondrocyte ceHCS-2/8. Endocrinology 138, 4390–4400.

akigawa, M., Shirai, E., Fukuo, K., Tajima, K., Mori, Y., SuzuF., 1987. Chondrocytes dedifferentiated by serial monolayer cuform cartilage nodules to nude mice. J. Bone Miner. Res. 2,462.

akigawa, M., Tajima, K., Pan, H.O., Enomoto, M., Kinoshita, A., SuzF., Takano, Y., Mori, Y., 1989. Establishment of a clonal humchondrosarcoma cell line with cartilage phenotypes. Cancer Re3996–4002.

am, C.S., Heersche, J.N.M., Murray, T.M., Parsons, J.A., 1982. Paroid hormone stimulates the bone apposition rate independently

102 V. Kartsogiannis, K.W. Ng / Molecular and Cellular Endocrinology 228 (2004) 79–102

resorptive action. Differential effects of intermittent and continuousadministration. Endocrinology 110, 506–512.

Taylor, S.M., Jones, P.A., 1979. Multiple new phenotypes induced in10T1/2 and 3T3 cells treated with 5-azacytidine. Cell 17, 771–779.

Turner, C.H., Forwood, M.R., Otter, M.W., 1994. Mechanotransductionin bone: do bone cells act as sensors of fluid flow? J. Biomech. 27,339–360.

Udagawa, N., Takahashi, N., Akatsu, T., Sasaki, T., Yamaguchi, A., Ko-dama, H., Martin, T.J., Suda, T., 1989. The bone marrow-derived stro-mal cell lines MC3T3-G2/PA6 and ST2 support osteoclast-like celldifferentiation in cocultures with mouse spleen cells. Endocrinology125, 1805–1813.

Udagawa, N., Takahashi, N., Jimi, E., Matsuzaki, K., Tsurukai, T., Itoh,K., Nakagawa, N., Yasuda, H., Goto, M., Tsuda, E., Higashio, K.,Gillespie, M.T., Martin, T.J., Suda, T., 1999. Osteoblasts/stromal cellsstimulate osteoclast activation through expression of osteoclast differ-entiation factor/RANKL but not macrophage colony-stimulating fac-tor: receptor activator of NF-kappa B ligand. Bone 25, 517–523.

Umezawa, A., Maruyama, T., Segawa, K., Shadduck, R.K., Waheed, A.,Hata, J., 1992. Multipotent marrow stromal cell line is able to inducehematopoiesis in vivo. J. Cell. Physiol. 151, 197–205.

Velasco, J.A., Medina, D.L., Romero, J., Mato, M.E., Santisteban, P.,1997. Introduction of p53 induces cell-cycle arrest in p53-deficienthuman medullar thyroid carcinoma cells. Int. J. Cancer 73, 449–455.

Wang, D., Christensen, K., Chawla, K., Xiao, G., Krebsbach, P.H.,Franceschi, R.T., 1999. Isolation and characterization of MC3T3-E1preosteoblast subclones with distinct in vitro and in vivo differentia-tion/mineralization potential. J. Bone Miner. Res. 14, 893–903.

Weinbaum, S., Cowin, S.C., Zeng, Y., 1994. A model for the excitation ofosteocytes by mechanical loading-induced bone fluid shear stresses.

W lineing rat31–

W .M.,tedmi-ctor.

W M.,.Y.,

osisin T

W andiges-aces.

Yamashita, T., Asano, K., Takahashi, N., Akatsu, T., Udagawa, N., Sasaki,T., Martin, T.J., Suda, T., 1990a. Cloning of an osteoblastic cell lineinvolved in the formation of osteoclast-like cells. J. Cell. Physiol. 145,587–595.

Yamashita, T., Asano, K., Takahashi, N., Akatsu, T., Udagawa, T., Sasaki,T., Martin, T.J., Suda, T., 1990b. Cloning of an osteoblastic cell lineinvolved in the formation of osteoclast-like cells. J. Cell. Physiol. 145,587–595.

Yang, K.H., Stewart, A.F., 1996. Parathyroid hormone-related protein:the gene, its mRNA species, and protein products. In: Bilezikian,J.P., Raisz, L.G., Rodan, G.A. (Eds.), Principles of Bone Biology.Academic Press, San Diego, pp. 347–362.

Yasuda, H., Shima, N., Nakagawa, N., Yamaguchi, K., Kinosaki, M.,Mochizuki, S., Tomoyasu, A., Yano, K., Goto, M., Murakami, A.,Tsuda, E., Morinaga, T., Higashio, K., Udagawa, N., Takahashi,N., Suda, T., 1998. Osteoclast differentiation factors is a ligand forosteoprotegerin/osteoclastogenesis-inhibitory factor and is identical toTRANCE/RANKL. Proc. Natl. Acad. Sci. U.S.A. 95, 3597–3602.

Yoon, K., Buenaga, R.F., Rodan, G.A., 1987. Tissue specificity and de-velopmental expression of rat osteopontin. Biochem. Biophys. Res.Commun. 148, 1129–1136.

Zabel, M., Seidel, J., Kaczmarek, A., Surdyk-Zasasa, J., Grzeszkowiak,J., Corny, A., 1995. Immunocytochemical and immuno-ultrastructuralstudy of calcitonin gene expression in cultured medullary carcinomacells. Histochemistry 102, 323–327.

Zatelli, M.C., Tagliati, F., Taylor, J.E., Rossi, R., Culler, M.D., degliUberti, E.R., 2001. Somatostatin receptor subtypes 2 and 5 differ-entially affect proliferation in vitro of the human medullary thy-roid carcinoma cell line TT. J. Clin. Endocrinol. Metab. 86, 2161–2169.

Z Es-omaure.

Z L.F.,and

Z cellu-ousLon-

Z .W.,blasts. 9,

Z T.J.,nant,es. 6,

J. Biomech. 27, 339–360.einreb, M., Shinar, D., Rodan, G.A., 1990. Different pattern of alka

phosphatase, osteopontin, and osteocalcin expression in developbone visualized by in situ hybridization. J. Bone Miner. Res. 5, 8842.

ong, B.R., Josien, R., Lee, S.Y., Sauter, B., Li, H.L., Steinman, RChoi, Y., 1997a. TRANCE (tumor necrosis factor [TNF]-relaactivation-induced cytokine), a new TNF family member predonantly expressed in T cells, is a dendritic cell-specific survival faJ. Exp. Med. 186, 2075–2080.

ong, B.R., Rho, J., Arron, J., Robinson, E., Orlinick, J., Chao,Kalachikov, S., Cayani, E., Bartlett III, F.S., Frankel, W.N., Lee, SChoi, Y., 1997b. TRANCE is a novel ligand of the tumor necrfactor receptor family that activates c-June N-terminal kinasecells. J. Biol. Chem. 272, 25190–25194.

ong, F.L., Cohn, D.V., 1975. Target cells in bone for parathormonecalcitonin are different. Enrichment for each type by sequential dtion of mouse calvaria and selective adhesion to polymeric surfProc. Natl. Acad. Sci. U.S.A. 72, 3167–3171.

eytinoglu, F.N., Gagel, R.F., Wolfe, H.J., Tashjian Jr., A.H., 1980.tablishment of a calcitonin-producing rat medullary thyroid carcincell line. I. Morphological studies of the tumor and cells in cultEndocrinology 107, 509–515.

hao, S., Kato, Y., Zhang, Y., Harris, S., Ahuja, S.S., Bonewald,2002. MLO-Y4 osteocyte-like cells support osteoclast formationactivation. J. Bone Miner. Res. 17, 2068–2079.

heng, M.-H., Wood, D., 2003. The basic science of matrices andlar repair. In: Bentley, G. (Ed.), Current Developments in AutologChondrocyte Transplantation. Royal Society of Medicine Press,don, UK, pp. 49–54.

hou, H., Choong, P., McCarthy, R., Chou, S.T., Martin, T.J., Ng, K1994. In situ hybridization to show sequential expression of osteogene markers during bone formation in vivo. J. Bone Miner. Re1489–1499.

hou, H., Hammonds Jr., R.G., Findlay, D.M., Fuller, P.J., Martin,Ng, K.W., 1991. Retinoic acid-induced gene expression in malignon-transformed and immortalized osteoblasts. J. Bone Miner. R767–775.