cell lines and primary cell cultures in the study of bone cell biology.pdf

8/9/2019 Cell lines and primary cell cultures in the study of bone cell biology.pdf

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Molecular and Cellular Endocrinology 228 (2004) 79–102

Cell lines and primary cell cultures in the study of bone cell biology

Vicky Kartsogiannis b, Kong Wah Ng a,

a Department of Endocrinology and Diabetes, St. Vincent’s Hospital, 4th Floor, Daly Wing, 35 Victoria Parade, Fitzroy, Vic. 3065, Australiab St. Vincent’s Institute 9 Princes Street, Fitzroy, Vic. 3065, Australia

Received 8 April 2003; accepted 12 June 2003

Abstract

Boneis a metabolicallyactive and highlyorganized tissue consistingof a mineralphase of hydroxyapatiteand amorphouscalcium 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.© 2004 Elsevier Ireland Ltd. All rights reserved.

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

1. Introduction

Bone has severalmajor functions. It forms a rigid skeletonto provide a framework for the body, support for soft tissues,pointsof attachment forskeletalmuscles, protection for inter-nal organs, housing for bone marrow as well as a central rolein mineral homeostasis,principally of calcium andphosphateions, but also of sodium and magnesium.

Bone is a dynamic tissue that is constantly remodeledthroughout life. During fetal development, most of the skele-ton develops from cartilage anlagen which is eventually re-sorbedand replacedwith bone bya process termedendochon-dral ossication. In contrast, bones which form the calvaria,mandible and maxilla are developed from mesenchyme bya process termed intramembranous ossication. Bone mod-

eling is the process associated with growth and reshaping of bonesin childhoodandadolescence. In bonemodeling, longi-tudinal growthof long bonesdepends onproliferation anddif-ferentiationof cartilagecells at the growthplate while growthin width and thickness is accomplished by formation of boneat the periosteal surface with resorption at the endosteal sur-face. In adults, after the epiphyses close, growth in length

Corresponding author. Tel.: +61 3 9288 3568; fax: +61 3 9288 3590. E-mail address: [email protected] (K.W. Ng).

and endochondral bone formation cease but remodeling of bone continues. Remodeling constitutes the lifelong renewalprocess whereby the mechanical integrity of the skeleton ispreserved. It implies thecontinuousremovalofbone (bonere-sorption) followed by synthesis of new bone matrix and sub-sequent mineralization (bone formation). The maintenanceof normal, healthy bone requires the coupling of bone for-mation to bone resorption, with intercellular communicationbetween osteoblasts and osteoclasts integral to the achieve-ment of a balance between the two processes. Furthermore,bone remodeling is an integral part of the calcium home-ostatic system ( Eriksen et al., 1993 ) that also involves theparathyroid glands, intestinal system and the kidneys.

Many aspects of the processes described above can beinvestigated in the laboratory using primarily cell culture.

The major cell types are the bone-forming osteoblasts, bone-resorbing osteoclasts and cartilage-forming chondrocytes. Athorough understanding of the factors regulating the differen-tiation of each of these cell types, the mechanisms by whichregulatory factors inuence their function, and the manner inwhich thesecellscommunicateandinteractwitheach other, iscentral 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

0303-7207/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.mce.2003.06.002


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

from established cell lines, particularly in osteoblast biology,a substantial amountof work is nonetheless still being carriedoutwithprimary culturesof osteoblasts, chondrocytesandos-teoclasts, andattentionwill be drawn to these,whererelevant.

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

• Regulationof expressionof phenotypiccharacteristics 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 mesenchymalstem cells. These stem cells have the capacity to differentiateinto lineages other than osteoblasts, including those of chon-droblasts, broblasts, adipocytes, and myoblasts (reviewedin Nijweide et al., 1986; Friedenstein et al., 1987; Aubin etal., 1995 ). By analogy with hematopoietic cell differentia-tion, each of these differentiation lineages is thought to orig-

inate from a different committed progenitor, which for theosteogenic lineage is called the osteoprogenitor.

Osteodifferentiation progresses via a number of progen-itor and precursor stages to the mature osteoblast, as illus-trated in Fig. 1. Pluripotent mesenchymal cell lines can beused to study this process which is regulated by a number of regulatory molecules such as members of the transforminggrowth factor beta (TGF ) superfamily, including the bonemorphogenetic proteins (BMPs). When murine C2C12 mes-enchymal precursor cells are treated with TGF 1, terminaldifferentiation intomyotubesisblocked.Treatmentwithbonemorphogenetic protein 2 (BMP-2) similarlyblocks myogenicdifferentiation of C2C12 cells but induces osteoblast differ-entiation ( Lee et al., 2000 ). Further evidence implicating arole of the BMP receptors (type IA and IB) in both the speci-cation and differentiation of osteoblastic andadipocytic lin-eages comes from recent studies using a well-characterizedclonal cell line 2T3, derived from mouse calvariae ( Chenet al., 1998). In other studies ( Spinella-Jaegle et al., 2001 ),the proteins of the hedgehog (Hh) family which are knownto regulate various aspects of normal limb patterning, havealso been shown to inuence the osteoblastic and adipocyticcommitment/differentiation of mesenchymal stem cells. Forexample, recombinant N-terminal sonic hedgehog (N-Shh)abolishes adipocytic differentiation of murine mesenchymal

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

stem cells C3H10T1/2 both in the presence and absence of BMP-2, while committing these pluripotent cells into the os-teoblastic lineage. Treatment of C3H10T1/2cells 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 such

as 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 (broblast colony-forming units[CFU-F] ( Simmons and Torok-Storb, 1991 ). Gronthos andcolleagues used dual-color uorescence-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,while the STRO-1+/ALP+ subset represented an intermedi-ate preosteoblastic stage of development. All STRO-1/ALPNHBCsubsets expressed the transcription factor cbfa-1, con-rming that they were committed osteogenic cells ( Gronthoset al., 1999). A survey of human osteosarcoma cell lines re-vealed that STRO-1 was expressed by MG-63 but not SaOS-2. Among murinecell lines tested, expressionof STRO-1wasdetected in the bipotential line BMS-2 but not the commit-ted osteoblast precursor MC3T3-E1 ( Stewart et al., 1999 ).


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Table 3Osteoblast phenotypic characteristics (modied 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 brils ( 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 specic osteoblastactivities are observed throughout all stages (reviewed inAubin et al., 1993, 1995; Lian and Stein, 1995; Stein andLian, 1993 ). In the rst phase, active proliferation is reectedby mitotic activity with expression of genes associated withcell cycling (e.g., histone) and growth (e.g., proto-oncogenes

c-myc , c-fos , and c-jun ) (McCabe et al., 1995 ). Several othergenes associated with formation of the extracellular matrix(type I collagen, bronectin, 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),decreasespost-proliferatively, and thenis induced againat the


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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 containing O-phosphoserine ( Glimcher, 1989 ). The Vitamin K-dependentprotein, osteocalcin ( Lian and Friedman, 1978 ), in contrast

to 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 reect-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 of human ( 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 andnote the pattern of expression of osteoblast-relatedmarkers in relation to their location in bone ( Yoonet al., 1987;Nomuraet al., 1988; Sandberget al., 1988; Lyons et al., 1989;Weinreb et al., 1990; Zhou et al., 1994 ).

In addition to the expression of matrix components, theosteoblast differentiation process is both determined and re-ected by the expression of certain hormones and cytokines.For instance, the bone morphogenetic proteins (BMPs) areexpressed in early osteoblasts and in the late mineralizationphase (Harris et al., 1994a, 1994b ), and the expression of thePTH receptor appears to correlate with increasing differenti-

ation (reviewed in Aubin et al., 1995 ; Suda et al., 1996; Boset al., 1996 ).

To date severalbone organcultures andprimary osteoblastcultures have been widely used to study osteoblast differen-tiation (Owen et al., 1990 ) as well as the osteoblastic re-sponse to growth factors ( Centrella et al., 1987; Guentheret al., 1988 ), and hormones ( Wong and Cohn, 1975 ). Themajor bone culture systems use long bones or calvaria fromfetal/neonatal rats and mice. Populations of osteoblast-likecells can be isolated by using enzymatic digestion ( Peck etal., 1964 ) or by mechanical methods ( Ecarot-Charrier et al.,1983).

Although the available models provide important informa-tion on the fundamental processes involved in bone forma-tion, it is important to note that the source and the age of thebone, the medium, and culture system, all affect the sensitiv-ity of bones to hormones ( Stern and Krieger, 1983; Soskolineet al., 1986). In addition, the variable proportion of -broblasts and osteoblast cells at different stages of dif-ferentiation provides a further limitation to this tech-nique.

2.4.2. Osteosarcoma cell linesThe most widely used osteosarcoma cell lines are the

UMR 106 and the ROS 17/2 which were established during

the late 1970s by the Martin and Rodan laboratories. UMR106is a cell line derived from a transplantable rat 32P-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 e tal., 1983; Partridge et al., 1980, 1983, 1 987). 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 calcied matrix when implanted in diffusion cham-bers (Shteyer et al., 1986 ). The cells respond to TGF withan increase in ALP, type I collagen, osteonectin, and osteo-pontin mRNA ( Noda and Rodan, 1987; Noda et al., 1988 )but with a decrease in mRNA for osteocalcin ( Noda, 1989 ).

Other models of osteoblastic cells derived from humanosteosarcomas include theSaOS ( Rodan et al., 1987b ), OHS-

4 (Fournier and Price, 1991 ), TE-85, MG-63 ( Francheschi etal., 1985, 1988 ), KPDXM and TPXM ( Bruland et al., 1988 ).Interestingly, some of these cell lines have been primarilyused as models to study the control of expression of bonematrix proteins, including integrin-mediated cell adhesion tobronectin ( Dedhar et al., 1987; Rodan et al., 1994 ).

Rochet and colleagues have recently characterized a newhuman osteosarcomacell line, CAL72,which is more closelyrelated to normal osteoblasts than any of the osteosarcomacells previously described, andcould also provide an interest-ing tool to study therole of osteoblasticcells in hematopoieticcell growth and differentiation. The cell line exhibits a singu-lar cytokine expression prole compared to other osteosar-coma cell lines ( Rochet et al., 1999 ). CAL72 cells constitu-tively express mRNA coding for IL-6, GM-CSF, and G-CSFand thus appear to be closer to human primary osteoblasticcells than the well-described osteosarcoma cell lines MG-63 and SaOS-2. In contrast to MG-63 or SaOS-2, CAL72cells do not inhibit hematopoietic colony formation and cansustain the limited expansion of hematopoietic progenitorsin a way similar to that described for normal human pri-mary osteoblasts. In addition, CAL72 cells induce the dif-ferentiation of promyelocytic cells into macrophages moreefciently than other osteosarcoma cell lines ( Rochet et al.,2003).


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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 by Ng 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 signicantly 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(Kumegawa et al., 1984 ) and exhibits a high ALP activitywhich is regulated by PTH, PGE2, 1,25(OH)2D3 andis capa-ble of collagen synthesis. In addition, these cells form matrixvesicles which are deposited on collagen brils and can bemineralized in vitro ( Sudo et al., 1983 ). Although originallyisolated as a clonal cell line, different variants of MC3T3-E1

cells have been isolated with different phenotypic propertiesmost likely as a result of prolonged passaging ( Leis et al.,1997). Wang and colleagues isolated 10 subclonal MC3T3-E1 cell lines exhibiting high or low mineralization potentialafter growth in ascorbic acid-containingmedium for 10 days.Clones 4, 8, 11, 14, and 26 formed a well-mineralized extra-cellular matrixafter incubation for 2 days in mediumcontain-ing3.0mM inorganic phosphate, whilst clones17,20,24,30,and 35 failed to form any detectable mineral. A good corre-lation was observed between the ability of a given subcloneto activate the osteoblast-specic osteocalcin promoter andendogenous expression of osteocalcin and other osteoblast-related mRNAs.Essentially, subclonesexpressing highlevelsof osteoblast marker mRNAs formed a mineralized extracel-lular matrix in culture and were osteogenic when implantedinto mice. In addition,allsubclones, regardless of their abilityto differentiate,expressed highlevelsof cbfa-1 mRNA, whichencodes a transcription factor necessaryfor osteoblast forma-tion (Otto et al., 1997 ), implying that thepresence of cbfa-1 isby itself insufcient for induction of osteoblast-specic geneexpression ( Wang et al., 1999 ).

The cell lines represented by CRP 4/7, CRP 7/4, CRP 7/7,CRP 10/3 and CRP 10/30 also belong to the group of clonalnon-transformed osteoblastic cells. These cells were isolatedfrom neonatal calvarial bone cells in the presence of TGF

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 of these clonal cell lines are shown in Table 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 populationand 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 populationandwere foundto constitutivelyexpressosteoblastic 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 conuence, 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 ).

Other model systems of experimentally immortalized celllines used to study certain stages of osteoblast differentiationinclude the adult human osteoblast-like (hOB) cells ( Keetinget al., 1992) and the human fetal osteoblast cell line hFOB(Harris et al., 1995 ). The hOB cell line was immortalizedby transfecting normal adult human osteoblast-like cells, de-rived from a 68-year-old woman, with the large and smallT antigens of the SV40 virus. The cells represent a well-differentiated, steroid-responsive clonal cell line that closelyapproximates the phenotype of the mature osteoblast. Theyexpress mRNA for (I)-procollagen, osteopontin, TGF ,and interleukin-1 beta (IL-1 ), while treatment with 1,25-dihydroxyvitamin D3 results in increased expression of os-teocalcinand alkaline phosphatase mRNA andprotein. Func-tional estrogen andandrogen receptors are present but not thereceptor for PTH. When -glycerophosphate is added to thecultures, the cells produce a matrix that mineralizes.

The human fetal osteoblast cell line (hFOB) was de-rived from biopsies obtained from a spontaneous miscarriage(Harris et al., 1995 ). Primary cultures isolated from fetal tis-sue were immortalized by transfection with a temperature-


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sensitivemutant (tsA58) of SV40 large T antigen.hFOB 1.19,the highest alkaline phosphatase-expressing clone, increasedalkaline phosphatase activity and osteocalcin secretion in adosedependent manner following1,25-dihydroxyvitaminD3treatment. Differentiated hFOB cells showed high levels of osteopontin, osteonectin, BSP, and type I collagen expres-

sion. Treatment of hFOB cells with parathyroid hormone(1–34) resulted in increased cAMP levels. In addition, uponreachingconuence, hFOBcultures formed mineralizednod-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-erotrimercomprising twoalpha1 (I) chainsandonealpha2 (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 inwomen after the menopause ( Paterson et al., 1984 ). It is be-yond thescope of this review to discuss themolecularbasis of OI. A comprehensive listing of the mutations that have beendiscovered within human type I collagen genes ( Dalgleish,1997), is maintained in the osteogenesis imperfecta muta-tion database ( http://www.le.ac.uk/genetics/collagen ). The

oim/oim mouse which arose from a spontaneous mutationwithin the COL1A2 gene resulting in the production of anon-functional alpha2 (I) chain, is a widely used murinemodel of OI ( Chipman et al., 1993 ). The heterozygous Mov13 mouse in which one of the two COLIA1 genes is non-functional, is the only murine model of type I OI ( Bonadio etal., 1990 ). Apart from defective formation of the type I col-lagen triple helix, the ability of OI broblasts or bone cellsto produce collagen and proliferate in vitro is also impairedandthatis probablya consequenceof theretainedprocollagenmolecules within the distended roughendoplasmic reticulum(Lamande et al., 1995; Fitzgerald et al., 1999; Lamande andBateman, 1999 ). In vitro studies of osteoblasts derived fromOI humans ( Fedarko et al., 1996 ) or oim mouse ( Balk et al.,1997) show diminished markers of osteoblastic differ-entiation. However the cells can still differentiate intoosteoblasts under the inuence of bone morphogeneticproteins.

Knowledge obtained from the study of osteogenesis im-perfecta has also provided clues about the genetics of os-teoporosis. Although the collagen protein chains are normalin most osteoporotic patients, a polymorphism has recentlybeen identied in a regulatory region of the COLIA1 genewhich is more common in osteoporotic patients. This poly-morphism is located at a binding site for the transcription

factor Sp1 in the rst 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 specic 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-mortalized cell lines in culture with phenotypic character-istics of osteocytes. Bone cells were derived from transgenicmice over-expressing T-antigen driven by the osteocalcinpromoter. They chose cells expressing a dendritic morphol-ogy as the initial criterion for selection and establishment of clonal cell lines. MLO-Y4 (murine long bone osteocyte Y4)

was one of the immortalized clonal lines established withosteocyte-like characteristics. These cells produce extensive,complex dendritic processes, are positive for T-antigen, os-teopontin, neural antigen CD44 and connexin 43. They pro-duce large amounts of osteocalcin,have low levels of alkalinephosphatase activity, lack detectable mRNA for osteoblast-specic factor 2, and produce very small amounts of type Icollagen ( Kato et al., 1997 ). The MLO-Y4 cells also sup-port osteoclast formation and activation through the secre-tion of M-CSF and expression of RANKL on their sur-face and their dendritic processes ( Zhao et al., 2002 ). Cellsare grown on collagen-coated surfaces in culture mediumsupplemented with 5% FBS and 5% calf serum for opti-mal growth and maintenance of the osteophyte dendriticphenotype.

Four other immortalized osteocyte-like cell lines (MLO-A5, MLO-A2, MLO-D1 and MLO-D6) were established bythis group. Out of these, MLO-A5 cells were shown to highlyexpress BSP and mineralize spontaneously in culture evenin the absence of beta-glycerophosphate and ascorbic acid(Kato et al., 2001 ). The authors claim that the MLO-A5 cellsare representative of the post-osteoblast, preosteocyte stageresponsible for triggering mineralization of osteoid.

To date, there isnopublisheddataon theresponses of thesepresumptive osteocyte-like cell lines to mechanical strain.

http://www.le.ac.uk/genetics/collagenhttp://www.le.ac.uk/genetics/collagen


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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., 1 992). 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 in Fig. 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 specic interaction be-tween adhesion molecules in the cell membrane (integrins)and some bone matrix proteins. The integrins are a fam-ily of transmembrane proteins whose cytoplasmic domainsinteract with the cytoskeleton while their extracellular do-mains bind to bone matrix proteins, enabling them to medi-ate cell–substratum and cell–cell interactions ( Hynes, 1987 ).

Fig. 2. Diagrammatic representation of the formation of mature, multinucleated osteoclasts from mononuclear hematopoietic progenitors. Hematopoieticosteoclast progenitors present in bone marrow come into direct contact with osteoblast or stromal cells expressing RANKL under the inuence of osteolyticfactors such as PTH, PGE2, IL-11 or 1,25(OH)2D3 (1). They differentiate rstly into TRAP positive mononuclear cells (2) before becoming TRAP positiveand calcitonin receptor (CTR) positive mononucleate cells (3) that eventually fuse to form multinucleate, functional mature osteoclasts (4).

The space contained inside this ring of attachment and be-tween theosteoclast andthebone matrixconstitutes thebone-resorbing compartment. The cell membrane of the apicalpole is invaginated to form a rufed border. Osteoclasts areactively engaged in the synthesis and secretion of severalclasses of enzymes formedin theGolgi regionandvectorially

transported 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 rufed border apical membrane and their contents dis-charged into the bone-resorbing compartment ( Baron et al.,1993).

Acidication of the extracellular bone-resorbingcompart-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 rufe 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 accomplishedby ion exchangers, pumpsand channelspresent in the basolateral membrane of the cell ( Baron et al.,1993).

Theactivityof mature osteoclast is directly and negativelyregulated by calcitonin, for which the cell expresses a highnumber of receptors ( Nicholson et al., 1986 ).

A summary of the main osteoclast phenotypic character-istics is provided in Table 5 .


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Table 5Phenotypic characteristics of osteoclasts

Lysosomal enzymesTartrate-resistant acid phosphatase

-GlycerophosphateArylsulfatase

-Glucuronidase

Cysteine-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+ antiporter

Bicarbonate/chloride exchanger

Membrane associated proteinsCarbonic anhydrasec-src

3.3. In vitro methods to study osteoclast formation and function

Unlike osteoblasts,osteoclasts aredifcult to studyin 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 receptorsandtheability to resorbcalci-ed matrices ( Takahashi et al., 1988a; Hattersley and Cham-bers, 1989; Shinar et al., 1990 ). TRAP staining and someof the other criteria used for identication, such as cathep-sin K, vitronectin receptor, are not specic 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 theabsence 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-creases the yield of osteoclasts, but also the number of non-osteoclastic contaminatingcells. Relatively‘pure’osteo-clastshave been obtained this way to enable the identicationof calcitonin receptors and effects of bone-resorbing factorson cytoplasmic spreading ( Nicholson et al., 1986 ). The ef-fects of osteotropic factors on bone resorption can be studied

when the osteoclasts are placed on bone slices ( Chambers etal., 1985). The main disadvantages of this assay are the con-taminating osteoblasts in the osteoclast preparation that mayaffect the experimental outcomes and the sensitivityof osteo-clasts to the pH of the assay medium, especially when culturetimes exceed 24 h. An acid pH has been shown to stimulateosteoclast bone resorption ( Arnett and Dempster, 1990 ), po-tentially accounting for some of the conicting reports in theliterature.

3.3.1.2. Giant cell tumors of bone. Giant cell tumors(GCT),also known as osteoclastomas, are rare primary neoplasmsof the skeleton. They are locally destructive, causing exten-sive osteolysis. GCT contain within the tumor mass, variablenumbers of large, multinucleated cells. It is believed that thestromal cells of GCT are the tumor cells, and they induceosteoclastic bone resorption by recruiting osteoclast precur-sors, promoting their differentiation into functional osteo-clasts (James et al., 1996 ). Until recently, this was the onlyuseful source of human osteoclasts. To obtain the osteoclasts(Goldring et al., 1987 ), the giant cell tumor is dissected in aPetri dish under sterile conditions using a scalpel blade, andthen enzymatically digested for cell culture in a digestionmixture made up from collagenase and dispase. The cell sus-pension is diluted in alpha-modied MEM containing 10%


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Table 6Identication of osteoclast markers

Marker Specicity/use Comment

Bone resorption Denitive Requires live, active cells; time dependentCalcitonin receptors Specic (hematopoietic lineage) Difcult 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 calcied substrate)

FBS, and ltered 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 inuence 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)2D3or humanPTH(1–34) resulted inan increase inthenumberof TRAP positivemultinucleated cells that satisfythe major criteria for osteoclasts. Subsequently, a similar in-crease was observed with prostaglandins, PTHrP (1–34) andIL-1 (Akatsuet al., 1989a,1989b, 1991 ). Two otherimportantobservations were made. Firstly, time course studies showed

that 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 differentiationof immature precursors, characterized by TRAP and CTR-negativity, into mature TRAP and CTR-positive precursors(Takahashi et al., 1988b ) (Fig. 2). Osteoclasts have also simi-larly been obtained using rabbit ( Fuller and Chambers, 1987 )and feline ( Ibbotson et al., 1984 ) bone marrow. This systemis limited by the inability to identify osteoclastic precursorsfrom the mixed cell population and the difculties inherentin studying osteoclast activation as opposed to recruitment.The dependence of osteoclast formation on the presence of mesenchymal stromal cells or osteoblasts also complicate theinterpretation of results.

3.3.2.2. Co-cultures of osteoblastic and hematopoietic cells.An important nding from murine bone marrow cultures wasthe demonstration that it was necessary for hematopoieticprecursors to come into direct contact with osteoblasts andstromal cells before osteoclast differentiation and formationcould occur ( Takahashi et al., 1988b ). This led to the de-velopment of co-culture systems comprising three essentialelements: (i) stromal cells or osteoblasts as a feeder layer;

(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 uid made up immediately before use. The calvar-ial digest uid is made from 30 mg collagenase type IIand 60 mg dispase dissolved in 30 ml PBS and lteredthrough a 0.2 m Acrodisc ® 32 Supor ®. The tube con-taining calvaria is shakenin a 37 ◦ C water bath for 5 min,allowed to settle and the supernatant discarded by pipet-ting. Six millilitres of fresh digest uid 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 in

10ml 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 humidied incuba-tor with 5% CO 2 . The adherent calvarial osteoblasts areconuent in 2–3 daysand can beused up to 1 week fromtheir initial preparation. They are dispersed for use bystandard trypsinization methods.

(ii) Stromal and osteoblast-like cell linesUdagawa et al. (1989) demonstrated that two bonemarrow-derived pre-adipocytic cell lines, MC3T3-G2/PA6 andST2, cansupport osteoclast formation frommurine spleen cells in the presence of 1,25(OH)2D3.The simultaneous addition of dexamethasone greatlyenhanced osteoclast numbers. Other osteoblast/stromalcell lines shown to support osteoclast formation in theco-culture systeminclude the ratosteoblast-likecell lineUMR 106 ( Quinn et al., 1994 ), murine stromal cell linestsJ2 and 10 (Chambers et al., 1993 ), murine osteoblast-like 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 adultmale mice (4–8 weeks old). Each bone is ushed with


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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. Humanosteoclasts. The formation of human osteo-clasts in coculture has proved to be a challenge because of a 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, vitronectinreceptor (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 work was 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,SaOS-2, expressing the human PTH/PTHrP receptor, andshowed that mouse bone marrow cells or human periph-eral blood mononuclear cells formed osteoclasts in coculturewhen treated with PTH. The response was greatly enhancedby adding dexamethasone, but no osteoclast formation wasseen with the addition of 1,25(OH)2D3, PGE2 or IL-6. Hu-man peripheral blood mononuclear cells (PBMC) culturedin vitro with soluble RANKL and human M-CSF, form os-

teoclasts. However, PBMC are heterogeneous, consisting of subsets of monocytes, lymphocytes and other blood cells.Nicholson et al. (2000) showed that a highly puried 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 humidied atmosphere of 5%CO2–air for 7–14 days. Pooled colonies identied 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 xedin 1% formalin and reacted for TRAP activity. The forma-tion of bone-resorbing multinucleated osteoclasts is assessedby transmission light microscopy and quantied 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,but exposure of pooled colonies to GM-CSF or IL-3 priorto RANKL stimulus completely inhibits osteoclastogenesis,directing cells instead towards dendritic cell differentiation.This may prove to be a very useful method for obtaininghuman osteoclast progenitors to study the regulation of dif-ferentiation along different cell lineages.A further advantageof this method is the ability to cryopreserve CBMC for futureexperiments.


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4. Chondrocytes

Cartilage is a specialized form of connective tissue thatpossesses a rm pliable matrix, which endows it with theresilience that allows the tissue to bear mechanical stresseswithout distortion. Articular cartilage, smooth surfaced and

resilient, provides a shock-absorbing sliding area for joints tofacilitate movement of bone, while cartilage is also essentialfor the embryonic development and, thereafter, growth of long 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 exible properties of cartilage. The functional differences between cartilage andbone relate principally to the different nature and proportionof the ground substance and brous elements of the extracel-lularmatrix. Nonetheless,chondrocytesandosteoblasts 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 bonein theprocess of endochondralossication. In long bones, theepiphyseal growth plate between the epiphysis and diaphysisis responsible for the longitudinal growth of bone. Withinthe growth plate, chondrocytes undergo a series of discretestages of differentiation, namely, proliferation, maturation,and hypertrophy. The strict spatial and temporal control of

proliferation and differentiation of chondrogenic cells is cen-tral to the coordinated development of the vertebrate skeleton(Erlebacher et al., 1995 ).

Chondrocytes are unique cells, in that they have manydifferentiatedmarkers suchas large cartilage-type proteogly-cans (aggrecan) and collagen types II, IX, X, and XI. Sometypical phenotypic characteristics of chondrocytes are listedin Table 8 . A comprehensive list of several hundred mousegrowth cartilage-derived gene products, including many notpreviously reported, was recently published ( Okihana andYamada, 1999 ).

4.1. Propagation of chondrocytes in culture

Various cell culture models have been developed for theinvestigation of chondrocyte biology in vitro, including ex-plant models, several forms of three-dimensional culture sys-tems, and monolayer cultures ( Adolphe and Benya, 1992 ).Chondrocytes grown in monolayer culture undergo a char-acteristic process of dedifferentiation, marked by a loss of collagen type II and aggrecan core protein expression as wellas the induction of collagen type I expression ( Takigawa etal., 1987; Hering et al., 1994; Lefebvre et al., 199 4). Thisphenomenon is inuenced to some extent by seeding den-sity (Ronzi ère et al., 1997 ) and is accelerated by growth in

Table 8A summary of the phenotypic characteristics of chondrocytes

CollagensType I collagenType II collagenType VI collagenType IX collagen

Type 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 ( Hering

et al., 1994). Growth of chondrocytes under conditions thatsupport a rounded morphology also facilitates maintenanceof the differentiated chondrocytic phenotype ( Bonaventureet al., 1994; H äuselmann 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-uence of BMP-2 and serum on expression of chondrocyte-specic matrix proteins [procollagen type I and II, aggrecanand (V + C)− bronectin] varies depending on cellular mor-phology, and/or cytoskeletalorganizationwhenchondrocytesare 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 cm 2 (Hjelle et al., 2002 ). Au-tologous chondrocyte transplantation (ACT) was rst usedin humans in 1987 and the rst pilot study was published in1994 ( Brittberg et al., 1994 ) in 23 patients with deep cartilagedefects in the knee. Cartilage slices were obtained throughan arthroscope from a minor-load-bearing area on the upper


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polygonal cells, which accumulate an alcian-blue stainablematrix. IRCcells synthesize typical cartilage proteins, aggre-can and link protein, but show reduced collagen II expression(Oxford et al., 1994; Horton et al., 1988 ).

Kamiyaet al. (2002) establisheda clonal chondrocytic cellline, N1511, from rib cartilage of a p53-null mouse. BMP-2

and insulin treatment induces full differentiation toward hy-pertrophic chondrocytes, whereas treatment with PTH anddexamethasone slows and limits differentiation. Recovery of p53 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 immortalizedclonalcell line derived from awell-differentiated human chondrosarcoma ( Takigawa et al.,1989), with phenotypic features resembling normalchondro-cytes. Thecellssynthesize 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. Calcium homeostasis

Calcium is an essential ion for many physiological pro-cesses such as cell motility, muscle contraction and neuro-transmitter release. In mammals, these processes functionoptimally when extracellular calcium is maintained within

a normal range by regulatory mechanisms that coordinatethe metabolic activities of the kidneys, intestine, parathyroidglands, and bone.

Parathyroid cells express a cell surface calcium-sensingreceptor that recognizes and responds to physiologicalchanges in extracellular ionized calcium concentration. Re-ductions in serum calcium of the order of 1–2% result ina prompt increase in parathyroid hormone (PTH) secretion.PTH acts directly on the kidneys and skeleton and indirectlyon the intestine to normalize any fall in extracellular ion-ized calcium. In the kidney, PTH stimulates re-absorptionof calcium in the distal tubules and increases synthesis of 1,25(OH)2D3. 1,25-Dihydroxyvitamin D increases intesti-nal absorption of calcium and also stimulates the release of calcium from bone by stimulating bone resorption. The in-creased ux of calcium ions into the extracellular uid re-stores circulating levels of calcium toward normal. Normal-ization of serum calcium as well as the increased levels of 1,25-dihydroxyvitamin D inhibits further PTH synthesis in anegative feedback loop. An increase in extracellular ionizedcalcium inhibits PTH secretion, resulting in increased renalcalcium excretion and reduction in net release of skeletal cal-cium as well as intestinal absorption of calcium.

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

cemia but in man, no essential function has yet been foundfor calcitonin. Steady-state plasma calcium shows little orno change with either complete absence or a large excess of calcitonin ( Partt, 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 andnely minced in Hank’s solution. They are transferred totissue culture asks, 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 areltered 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 Ca 2+ and 15% newborn calf serum. Cells are plated

in 24 multiplate dish at a density of 1 × 106 cells per welland cultured at 37 ◦ Cin5%CO 2 for 24h 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 uxes in the regulation of parathyroid hormone secre-tion (Chang et al., 2001 ).

5.2. C-cells of the thyroid

Calcitonin is a secretory product of the parafollicular (C)cells of the thyroid, and medullary thyroid carcinoma (MTC)is a neuroendocrine tumor of the parafollicular cells. Celllines established from human and animal MTC tumors pro-vide a useful system to analyze genes involved in the de-velopment of this neoplasia, as well as a source of C cellsto determine the regulation of calcitonin production. ThehMTC cell line, TT cells ( Leong et al., 1981 ) and the ratMTC line, 6–23 cells ( Zeytinoglu et al., 1980 ) can be pur-chased from the American Type Culture Collection (Man-assas, VA). TT cells display an impaired expression of thetumor suppressor gene p53 ( Velasco et al., 1997 ). Apartfrom calcitonin and the calcitonin receptor, TT cells ex-press carcino-embryonic antigen, somatostatin and its recep-

tors, neurotensin, gastrin-releasing peptide, Leu- and Met-enkephalin, parathyroid hormone-releasing peptide, chromo-granin A, synaptophysin, 1,25(OH)2D3 receptor and otherpeptides ( Frendo et al., 1994; Zabel et al., 1995; Velasco etal., 1997; Zatelli et al., 2001 ). TT cells are routinely grown inHam’s F12K medium supplemented with 10% FBS at 37 ◦ Cin a humidied atmosphere containing 95% air and 5% CO 2 .

6. Discussion

Although cell culture has proved invaluable in the studyof bone biology, in vitro model systems cannot reproduce


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the complex three-dimensional architecture of bone that isrequired for theproperexpression of the functional capabilityof the cells that make up its microenvironment. Nonetheless,despite the limitations of the various systems described inthis review, signicant and important contributions havebeen made to our understanding of the normal processes

leading 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 specic 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 todene the heterogeneity of bone cell populations with moreprecision. In contrast, primary calvarial cells probably con-tain osteoblasts at all stages of differentiation, including os-teoprogenitors that proliferate before undergoing a series of maturational steps to become differentiatedosteoblasts capa-ble of forming mineralized nodules in culture ( Aubin, 1998;Malavalk et al., 1999 ). Primary calvarial cultures are widely

used to study theprogression of differentiation in vitro. Roweandcolleaguesrecently describedan elegant methodin whichsubpopulations of osteoblasts at different stages of differ-entiation can be isolated from primary osteoblast cultures.Having identied fragments of the rat type I (Col1a1) pro-moter that show preferential expression in different Col1a1-producing tissues, they generated green uorescent protein(GFP)-expressing transgenic mice containing a 3.6- and a2.3-kb rat type I collagen promoter fragment. The 3.6-kbpromoter directed strong expression of GFP messenger RNAto preosteoblastic cells, while the 2.3-kb promoter directedGFP mRNA expression to a cell that is late in the osteoblastlineage,extendinginto mature osteocytes. They conclude thatwith further renement of this method, using other promot-ers and color isomers of GFP, it should be possible to isolatesubpopulations of cells at different stages of differentiationfrom primary cultures derived from these transgenic micefor molecular and cellular analysis ( Bogdanovic et al., 1994;Kalajzic et al., 2002 ).

Established osteoblast-like cell lines are particularly use-ful models to study signaling pathways in response to stimu-lation by osteotropic factors.Thegreatmajorityof osteoblast-like cell lines however, do not mineralize in culture, with theexception of MC3T3-E1 ( Kodama et al., 1981; Sudo et al.,1983), 2T3 (Ghosh-Choudhury et al., 1996 ) and KUSA-O

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 been

made 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 notbe separatedfromother cell types such as stromal cells and osteoblasts, andresults were difcult to reproduce. Investigators were facedwith the challenging task of obtaining sufcient numbers of relatively ‘pure’ preparations of osteoclasts in culture. Thiswas clearly not attainable using bone as a primary source of functional, 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-teoclasts in culture in far greater numbers, even though theosteoclasts could not be separated from the companion os-teoblasts/stromal cells for separate analysis.Almost a decadelater, the pivotal role of RANKL in osteoclastogenesis, its in-teractionwith itscognate receptor RANK aswell as thedecoyreceptor osteoprotegerin were revealed. Not only were these

discoveries major advances in the understanding of the regu-lation of osteoclast formation, it made possible the substitu-tion of soluble RANKL and M-CSF for stromal/osteoblasticcells, thus considerably simplifying the method for obtainingfunctional osteoclasts in vitro.Theuseof cell lines as alterna-tive sources of osteoclast progenitors is not widely practisedbecause of the lack of suitable cell lines. Some laboratorieshave used RAW264.7 cells to generate osteoclasts in culture.Unlike hematopoietic progenitors derived from bone marrowor spleen, RAW264.7 cells do not require M-CSF alongsideRANKL. The mechanism underlying this difference is notknown, but it may imply a difference in the signaling path-way leading to osteoclastogenesis.

Chondrocytes are unique in their ability to exist in a lowoxygen tension environment, isolated within a voluminousextracellular matrix devoid of a vascular supply. Primarychondrocytes retain their phenotypic characteristics whengrown in the form of a three-dimensional multicellular com-plex, but undergo a process of dedifferentiation when grownas monolayer cultures with serum supplementation. A rangeof established chondrocytic cell lines are available as alter-natives.

Work carried out on established cell lines and primarycell cultures have provided much valuable insight into thephenotypic characteristics of cells belonging to the bone


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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.

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