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  Molecular and Cellular Endocrinology 228 (2004) 79–102

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

  Vicky Kartsogiannis b, Kong Wah Ng a,

  a Department of Endocrinology and Diabetes, St. Vincent’sHospital, 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 tissueconsistingof a mineralphase of hydroxyapatiteand amorphouscalciumphosphatecrystals deposited in an organic matrix. Bone has two mainfunctions. It forms a rigid skeleton and has a central role incalcium and phosphatehomeostasis. The major cell types of bone areosteoblasts, osteoclasts and chondrocytes. In the laboratory,primary cultures or cell linesestablished from each of thesedifferent cell types provide valuable information about theprocesses of skeletal development, bone formationand boneresorption, leading ultimately, to the formulation of new forms oftreatment for common bone diseases such as osteoporosis.© 2004Elsevier Ireland Ltd. All rights reserved.

  Keywords: Cell lines; Primary cell cultures; Bone cellbiology

  1. Introduction

  Bone has severalmajor functions. It forms a rigid skeletontoprovide a framework for the body, support for soft tissues,pointsofattachment forskeletalmuscles, protection for inter-nal organs,housing for bone marrow as well as a central rolein mineralhomeostasis,principally of calcium andphosphateions, but also ofsodium and magnesium.

  Bone is a dynamic tissue that is constantly remodeledthroughoutlife. During fetal development, most of the skele-ton develops fromcartilage anlagen which is eventually re-sorbedand replacedwithbone bya process termedendochon-dral ossication. In contrast, boneswhich form the calvaria,mandible and maxilla are developed frommesenchyme bya process termed intramembranous ossication. Bonemod-

  eling is the process associated with growth and reshaping ofbonesin childhoodandadolescence. In bonemodeling, longi-tudinalgrowthof long bonesdepends onproliferation anddif-ferentiationofcartilagecells at the growthplate while growthin width andthickness is accomplished by formation of boneat the periostealsurface with resorption at the endosteal sur-face. In adults, afterthe epiphyses close, growth in length

  Corresponding author. Tel.: +61 3 9288 3568; fax: +61 3 92883590. E-mail address: [emailprotected] (K.W. Ng).

  and endochondral bone formation cease but remodeling of bonecontinues. Remodeling constitutes the lifelong renewalprocesswhereby the mechanical integrity of the skeleton ispreserved. Itimplies thecontinuousremovalofbone (bonere-sorption) followed bysynthesis of new bone matrix and sub-sequent mineralization (boneformation). The maintenanceof normal, healthy bone requires thecoupling of bone for-mation to bone resorption, with intercellularcommunicationbetween osteoblasts and osteoclasts integral to theachieve-ment of a balance between the two processes.Furthermore,bone remodeling is an integral part of the calciumhome-ostatic system ( Eriksen et al., 1993 ) that also involvestheparathyroid glands, intestinal system and the kidneys.

  Many aspects of the processes described above can beinvestigatedin 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 thedifferen-tiation of each of these cell types, the mechanisms bywhichregulatory factors inuence their function, and the mannerinwhich thesecellscommunicateandinteractwitheach other, iscentralto the design of rational therapeutic strategies to treatbonediseases such as osteoporosis. This review will focus oncell linesthat are established in the laboratory from these dif-ferent celltypes. While much information has been derived

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

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  from established cell lines, particularly in osteoblastbiology,a substantial amountof work is nonetheless still beingcarriedoutwithprimary culturesof osteoblasts,chondrocytesandos-teoclasts, andattentionwill be drawn tothese,whererelevant.

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

  • Regulationof expressionof phenotypiccharacteristics typ-icalof osteoblasts, chondrocytes and osteoclasts.

  • Regulation of differentiation of relativelyundifferentiatedmesenchymal cells along different lineages, forexample,muscle, osteoblasts, chondrocytes and adipocytes.

  • Signaling pathways relevant to osteoblast, osteoclastandchondrocyte functions.

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

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

  larly in the regulation of osteoclast formation in vitro.

  2. Osteoblasts

  2.1. Osteoblast ontogeny

  Osteogenic cells arise from pluripotential mesenchymalstemcells. These stem cells have the capacity to differentiateintolineages 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 analogywith hematopoietic cell differentia-tion, each of thesedifferentiation lineages is thought to orig-

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

  Osteodifferentiation progresses via a number of progen-itor andprecursor stages to the mature osteoblast, as illus-trated in Fig.1. Pluripotent mesenchymal cell lines can beused to study thisprocess which is regulated by a number of regulatory molecules suchas members of the transforminggrowth factor beta (TGF )superfamily, including the bonemorphogenetic proteins (BMPs). Whenmurine C2C12 mes-enchymal precursor cells are treated with TGF 1,terminaldifferentiationintomyotubesisblocked.Treatmentwithbonemorphogenetic protein 2(BMP-2) similarlyblocks myogenicdifferentiation of C2C12 cells butinduces osteoblast differ-entiation ( Lee et al., 2000 ). Furtherevidence implicating arole of the BMP receptors (type IA and IB) inboth the speci-cation and differentiation of osteoblasticandadipocytic lin-eages comes from recent studies using awell-characterizedclonal cell line 2T3, derived from mousecalvariae ( Chenet al., 1998). In other studies ( Spinella-Jaegleet al., 2001 ),the proteins of the hedgehog (Hh) family which areknownto regulate various aspects of normal limb patterning,havealso been shown to inuence the osteoblastic andadipocyticcommitment/differentiation of mesenchymal stem cells.Forexample, recombinant N-terminal sonic hedgehog (N-Shh)abolishesadipocytic differentiation of murine mesenchymal

  Fig. 1. Origin of cells of the osteoblast and chondrocytelineages (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-teoblasticlineage. Treatment of C3H10T1/2cells with BMP-7 has also been shownto induce both chondrogenesis andosteogenesis ( Gerstenfeld et al.,2002 ) while treatment withthe potent DNA demethylating agent5-azacytidine has pre-viously been known to induce differentiationto 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 employedbyresearchers to track the various stages of osteogenesis.The murineIgM monoclonal antibody STRO-1 recognizesa cell surface antigenexpressed by stromal cells in humanbone marrow and is used toidentify clonogenic bone mar-row stromal cell progenitors (broblastcolony-forming units[CFU-F] ( Simmons and Torok-Storb, 1991 ).Gronthos andcolleagues used dual-color uorescence-activated cellsort-ing to identify cells expressing STRO-1 and ALP inprimarycultures of normal human bone cells (NHMC). They showedthatpreosteoblastic STRO-1+/ALP − cells did not expressbone-relatedmarkers such as bone sialoprotein, osteopontin,and parathyroidhormone receptor and had a reduced abilityto form a mineralizedbone matrix over time. The majorityof NHBCs representing fullydifferentiated osteoblasts, ex-pressed STRO-1 − /ALP+ and STRO-1 −/ALP− phenotypes,while the STRO-1+/ALP+ subset represented anintermedi-ate preosteoblastic stage of development. AllSTRO-1/ALPNHBCsubsets expressed the transcription factor cbfa-1,con-rming that they were committed osteogenic cells ( Gronthosetal., 1999). A survey of human osteosarcoma cell lines re-vealedthat STRO-1 was expressed by MG-63 but not SaOS-2. Among murinecelllines tested, expressionof STRO-1wasdetected in the bipotentialline 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 Martinet al., 1993)

  Proteins Receptors and/or responses

  Alkaline phosphatase (ALP) Parathyroid hormone (PTH)Type Icollagen (COL I) Parathyroid hormone-related protein(PTHrP)Osteocalcin ProstanoidsOsteopontin (OP)1,25-Dihydroxyvitamin D3 (1,25(OH)2D3)Osteonectin RetinoidsBoneproteoglycan I (biglycan) Epidermal growth factor (EGF)Boneproteoglycan II (decorin) Tumor necrosis factor (TNF)Thrombospondin Tumor necrosis factor (TNF )Fibronectin (FN)Interleukin-1 (IL-1)Vitronectin (VN) Interleukin-6 (IL-6)Bonemorphogenetic 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 (IGFII)Interleukin-11 (IL-11) InsulinCiliary neurotrophic factor (CNTF)InhibinTumor necrosis factor (TNF ) ActivinLeukemia inhibitoryfactor (LIF) Estrogen receptors and (ER ; ER )Colony-stimulatingfactors (e.g. CSF-1) Leukemia inhibitory factor (LIF)ProstanoidsAtrial naturetic peptide (ATP)Noggin Calcitonin gene-relatedpeptide (CGRP)Receptor activator of NF B ligand Calcium sensingreceptor (CSR)(RANKL) Vasoactive intestinal peptide(VIP)Osteoprotegerin (OPG) Vascular endothelial growth factorreceptorsOsteoclast inhibitory lectin (OCIL) (VEGFR)Notch 2Fibroblast growth factor receptor-2 (FGFR2)Jagged 2 Growth hormone(GH)Vascular endothelial growth factors Connective tissue growthfactor-like(VEGF) (CTGF-L)Phosphate-regulating gene with hom*ologiesto endopeptidases

  on the X chromosome (Phex)Phosphate-regulating gene withhom*ologies 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 earlygene (TIEG)Matrix metalloproteinase protein-13 Catenins(MMP-13)STRO-1Cyclo-oxygenase 2Cadherins

  curs in scattered foci, which develop into multilayeredstruc-tures called “nodules” ( Bellows and Aubin, 1989 ).Thesenodules consist of a top layer of osteoblast-like cellswhichstain intensely for alkaline phosphatase, sitting underneathanosteoid layer containing collagen brils ( Bellows et al.,1987;Bhargava et al., 1988 ).

  The process of bone nodule formation as studied in ratcalvariapopulations has been subdivided into three devel-opmental stages:proliferation, extracellular matrix develop-ment and maturation,and matrix mineralization. Character-istic changes in genesassociated with proliferative and cellcycling activity and thoseassociated with specic osteoblastactivities are observed throughoutall stages (reviewed inAubin et al., 1993, 1995; Lian and Stein,1995; Stein andLian, 1993 ). In the rst phase, active proliferationis reectedby mitotic activity with expression of genes associatedwithcell cycling (e.g., histone) and growth (e.g.,proto-oncogenes

  c-myc , c-fos , and c-jun ) (McCabe et al., 1995 ). Severalothergenes associated with formation of the extracellularmatrix(type I collagen, bronectin, and TGF ) are also activelyex-pressed ( Aronow et al., 1990; Owen et al., 1991 ) andthengradually down-regulated with collagen mRNA being main-tainedat a low basal level during subsequent stages of os-teoblastdifferentiation.

  Immediately after the down-regulation of proliferation,proteinsassociated with the osteoblast phenotype are de-tected such asalkaline phosphatase. With progression intothe mineralizationstage, all cells become positive foralkaline phosphatase. Otherosteoblast-related genes such asbone sialoprotein (BSP) ( Nagata etal., 1991 ), osteopontin(OP), and osteocalcin ( Owen et al., 1990 )are induced fol-lowing the onset of mineralization. OP is expressedduringthe period of active proliferation (at 25% of maximallevels),decreasespost-proliferatively, and thenis induced againatthe

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  onset of mineralization achieving peak levels ofexpression.Consistent with high levels of osteopontin expressionlaterin the osteoblast developmental sequence are thecalciumbinding properties of this acidic glycoprotein containingO-phosphoserine ( Glimcher, 1989 ). The Vitamin K-dependentprotein,osteocalcin ( Lian and Friedman, 1978 ), in contrast

  to OP, is mainly expressed post-proliferatively with the onsetofnodule formation. Late expression of osteocalcin in theosteoblastdevelopment sequence, suggests that it is a markerof the matureosteoblast, which is consistent with a possiblerole for thesynthesis and binding of osteocalcin to mineralin the coupling ofbone formation to resorption.

  A similar temporal pattern of gene expression reect-ing stagesof progressive bone formation has been observedin cultured normaldiploid osteoblasts derived from chick (Gerstenfeld et al., 1987;Shalhoub et al., 1989; Aronow etal., 1990 ), bovine (Ibaraki etal., 1992 ), and in cell lines of human ( Keeting et al., 1992 )and mouse ( Quarles et al., 1992 )origin. Furthermore, it has alsobeen possible to discern cellsof the osteoblast lineage atdifferent stages of differentiationin situ andnote the pattern ofexpression of osteoblast-relatedmarkers in relation to theirlocation in bone ( Yoonet al., 1987;Nomuraet al., 1988; Sandbergetal., 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 andre-ected by the expression of certain hormones and cytokines.Forinstance, the bone morphogenetic proteins (BMPs) areexpressed inearly osteoblasts and in the late mineralizationphase (Harris etal., 1994a, 1994b ), and the expression of thePTH receptor appearsto correlate with increasing differenti-

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

  To date severalbone organcultures andprimary osteoblastcultureshave been widely used to study osteoblast differen-tiation (Owen etal., 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 bonesor calvaria fromfetal/neonatal rats and mice. Populations ofosteoblast-likecells can be isolated by using enzymatic digestion (Peck etal., 1964 ) or by mechanical methods ( Ecarot-Charrier etal.,1983).

  Although the available models provide important informa-tion onthe fundamental processes involved in bone forma-tion, it isimportant to note that the source and the age of thebone, themedium, and culture system, all affect the sensitiv-ity of bones tohormones ( Stern and Krieger, 1983; Soskolineet al., 1986). Inaddition, the variable proportion of -broblasts and osteoblastcells at different stages of dif-ferentiation provides a furtherlimitation to this tech-nique.

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

  See Also

  Health Keet on LinkedIn: How Does Ionized Alkaline Water Work In Our Body?Unveiling the Chemistry of Radiolaria: Exploring Elemental Insights and Environmental Significance - Bioengineering HyperbookUS Patent for Humanized anti-S100A9 antibody and uses thereof Patent (Patent # 11,359,010 issued June 14, 2022)Verwandeln Sie Leitungswasser mit dem VEVOR-Wasserionisierer in alkalisches und saures Wasser

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

  the late 1970s by the Martin and Rodan laboratories. UMR106is acell line derived from a transplantable rat 32P-inducedmalignantosteogenic sarcoma ( Martin et al., 1976; Partridgeet al., 1983).The cell line has been extensively characterizedwith properties ofenrichment in ALP activity, type I collagenproduction, adenylatecyclase 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 forUMR 106 cells are UMR 106-01 and UMR 106-06 ( Forrest et al., 1985) with the onlyknown substantial difference between the two clonesbe-ing the expression of calcitonin receptors in the UMR106-06.

  The family of clonal cell lines designated ROS (ROS 17/2and itssubclone ROS 17/2.8) were derived from a sponta-neous tumor in anACI rat that had been propagated by serialsubcutaneoustransplantation ( Majeska et al., 1980 ). Thesecells exhibitadenylate cyclase activity in response to PTHas well as a high ALPactivity which is regulated by PTH(Majeska and Rodan, 1982a ) and1,25(OH)2D3 ( Majeskaand Rodan, 1982b ). The subclone ROS 17/2.8constitutivelyexpresses 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 increasein ALP, type I collagen, osteonectin, and osteo-pontin mRNA ( Nodaand Rodan, 1987; Noda et al., 1988 )but with a decrease in mRNA forosteocalcin ( Noda, 1989 ).

  Other models of osteoblastic cells derived fromhumanosteosarcomas 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 primarilyusedas models to study the control of expression of bonematrixproteins, including integrin-mediated cell adhesion tobronectin (Dedhar et al., 1987; Rodan et al., 1994 ).

  Rochet and colleagues have recently characterized a newhumanosteosarcomacell line, CAL72,which is more closelyrelated to normalosteoblasts than any of the osteosarcomacells previously described,andcould also provide an interest-ing tool to study therole ofosteoblasticcells in hematopoieticcell growth and differentiation.The cell line exhibits a singu-lar cytokine expression prolecompared 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 primaryosteoblasticcells than the well-described osteosarcoma cell linesMG-63 and SaOS-2. In contrast to MG-63 or SaOS-2, CAL72cells do notinhibit hematopoietic colony formation and cansustain the limitedexpansion of hematopoietic progenitorsin a way similar to thatdescribed for normal human pri-mary osteoblasts. In addition, CAL72cells induce the dif-ferentiation of promyelocytic cells intomacrophages 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 aclonal non-transformed cell

  line derived from neonatal rat calvaria. The cell line wases-tablished by Ng et al. (1988) as a non-immortalized celllinewith a limited lifespan (12 passages in culture) andpheno-typic features suggestive of pre-osteoblasts. They haveun-

  detectable alkaline phosphatase activity and mRNA for ALPwhichis signicantly induced by the differentiating agent,retinoic acid (Ng et al., 1988, 1989a ). TNF , dexametha-sone and 1,25(OH)2D3 alsomodulate retinoic acid (RA)-induced ALP activity and mRNA for ALPin these cells ( Nget al., 1989a, 1989b ). This cell linetherefore, provides auseful model to study the osteoblasticdifferentiation fromprogenitor cell populations and also theregulation of os-teoblast differentiation by systemic hormones andlocalgrowth factors. Furthermore, in subsequent studies thesamegroup established immortalized subclones of UMR 201 us-ing SV40large T antigen (see section below) hence, al-lowing carefulcomparison of phenotypic characteristicsbetween the parent andderived cell lines ( Zhou et al.,1991).

  MC3T3-E1 is a clonal non-transformed cell line estab-lished fromnewborn mouse calvaria ( Kodama et al., 1981;Sudo et al., 1983 ).This cell line has also been shownto increase cyclic AMP productionin response to PTH(Kumegawa et al., 1984 ) and exhibits a high ALPactivitywhich is regulated by PTH, PGE2, 1,25(OH)2D3 andis capa-bleof collagen synthesis. In addition, these cells form matrixvesicleswhich are deposited on collagen brils and can bemineralized invitro ( Sudo et al., 1983 ). Although originallyisolated as aclonal cell line, different variants of MC3T3-E1

  cells have been isolated with different phenotypicpropertiesmost likely as a result of prolonged passaging ( Leis etal.,1997). Wang and colleagues isolated 10 subclonal MC3T3-E1 celllines exhibiting high or low mineralization potentialafter growthin ascorbic acid-containingmedium for 10 days.Clones 4, 8, 11, 14,and 26 formed a well-mineralized extra-cellular matrixafterincubation for 2 days in mediumcontain-ing3.0mM inorganicphosphate, whilst clones17,20,24,30,and 35 failed to form anydetectable mineral. A good corre-lation was observed between theability of a given subcloneto activate the osteoblast-specicosteocalcin promoter andendogenous expression of osteocalcin andother osteoblast-related mRNAs.Essentially, subclonesexpressinghighlevelsof osteoblast marker mRNAs formed a mineralizedextracel-lular matrix in culture and were osteogenic whenimplantedinto mice. In addition,allsubclones, regardless of theirabilityto differentiate,expressed highlevelsof cbfa-1 mRNA,whichencodes a transcription factor necessaryfor osteoblastforma-tion (Otto et al., 1997 ), implying that thepresence ofcbfa-1 isby itself insufcient for induction of osteoblast-specicgeneexpression ( Wang et al., 1999 ).

  The cell lines represented by CRP 4/7, CRP 7/4, CRP 7/7,CRP 10/3and CRP 10/30 also belong to the group of clonalnon-transformedosteoblastic cells. These cells were isolatedfrom neonatalcalvarial 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 7/4Absent Present PresentCRP 10/3 Present Absent PresentCRP 10/30Present Present Present

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

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

  combinant retrovirus containing the cDNA for SV40 large Tantigenhas been used to establish immortalized osteoblasticcell lines (Jat and Sharp, 1986 ). RCT-1 and RCT-3 cell lineswere derived fromisolated and fractionated embryonic ratcalvarial cells. RCT-1 cellswere established from the earlydigest populationand expressedosteoblastic traits [ALP, pro-

  1(I) collagen, PTH-responsive adenylate cyclase] afterin-duction by retinoic acid. RCT-3 cells on the other hand,wereestablished from the more osteoblastic late digestpopulationandwere foundto constitutivelyexpressosteoblasticmarkersexcept osteocalcin ( Heath et al., 1989 ).

  KS-4 is a clonal cell line which was isolated from mousecalvariaby transfection with the c-Ha-ras-1 gene. The cellsdisplay low ALPactivity at conuence, low type I collagenproduction and low cAMPaccumulation in response to PTH.The cells also display low mRNAlevels for pro- 1(I) colla-

  gen, osteonectin and bone proteoglycan I but notosteocalcin.Importantly, KS-4 cells have the ability to stimulateosteo-clast formation on co-culture with spleen cells ( Yamash*taetal., 1990a, 1990b ).

  Other model systems of experimentally immortalized celllinesused to study certain stages of osteoblast differentiationincludethe 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 adulthuman osteoblast-like cells, de-rived from a 68-year-old woman,with the large and smallT antigens of the SV40 virus. The cellsrepresent a well-differentiated, steroid-responsive clonal cellline that closelyapproximates the phenotype of the matureosteoblast. Theyexpress mRNA for (I)-procollagen, osteopontin, TGF,and interleukin-1 beta (IL-1 ), while treatment with1,25-dihydroxyvitamin D3 results in increased expression ofos-teocalcinand alkaline phosphatase mRNA andprotein. Func-tionalestrogen andandrogen receptors are present but not thereceptor forPTH. When -glycerophosphate is added to thecultures, the cellsproduce a matrix that mineralizes.

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

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  sensitivemutant (tsA58) of SV40 large T antigen.hFOB 1.19,thehighest alkaline phosphatase-expressing clone, increasedalkalinephosphatase activity and osteocalcin secretion in adosedependentmanner following1,25-dihydroxyvitaminD3treatment. DifferentiatedhFOB cells showed high levels of osteopontin, osteonectin, BSP, andtype 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 ubiquitouslydistributedof the collagen family of proteins. It is a het-erotrimercomprisingtwoalpha1 (I) chainsandonealpha2 (I)chain, which are encoded by theunlinked loci COL1A1 andCOL1A2, respectively. Mutations at theseloci result primar-ily in the connective tissue disordersosteogenesis imperfecta(OI) and Ehlers–Danlos syndrome.Osteogenesis imperfectais a heterogenous genetic disorderassociated with increasedfractures ( Rowe, 2002 ). In its mostsevere form multiple frac-tures occur in utero and the disorder islethal. In milder forms(type I) there may be an increase infractures in childhoodwhich then cease after puberty, with asubsequent increase inwomen after the menopause ( Paterson et al.,1984 ). It is be-yond thescope of this review to discussthemolecularbasis of OI. A comprehensive listing of the mutationsthat have beendiscovered within human type I collagen genes (Dalgleish,1997), is maintained in the osteogenesis imperfectamuta-tion database ( http://www.le.ac.uk/genetics/collagen ).The

  oim/oim mouse which arose from a spontaneous mutationwithin theCOL1A2 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 COLIA1genes is non-functional, is the only murine model of type I OI (Bonadio etal., 1990 ). Apart from defective formation of the type Icol-lagen triple helix, the ability of OI broblasts or bone cellstoproduce collagen and proliferate in vitro is also impairedandthatisprobablya consequenceof theretainedprocollagenmolecules within thedistended roughendoplasmic reticulum(Lamande et al., 1995;Fitzgerald et al., 1999; Lamande andBateman, 1999 ). In vitrostudies of osteoblasts derived fromOI humans ( Fedarko et al., 1996) or oim mouse ( Balk et al.,1997) show diminished markers ofosteoblastic differ-entiation. However the cells can stilldifferentiate intoosteoblasts under the inuence of bonemorphogeneticproteins.

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

  factor Sp1 in the rst intron of COLIA1, and has been foundto beassociated with bone mass and osteoporotic fracture inseveralCaucasian populations ( Ralston, 1999 ).

  2.6. Osteocytes

  Osteocytes are terminally differentiated cells of theos-teoblast lineage that have become embedded in mineralizedmatrix.Individual osteocytes communicate with each otherand with cells onthe bone surface such as lining osteoblastcells, through longintercellular processes. Their location andmorphology renders themparticularly well suited to transferinformation between cellswithin bone. For example, whenthe skeleton is undergoing mechanicalstress, osteocytes areideally located to sense pressure changes inbone, whichcould result in specic chemical messages being relayedtothe 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 thatosteocytes may have the capacity to regulate cal-cium homeostasis (Rubinacci et al., 1998 ). Unfortunately,their peculiar locationwithin bone makes them the most in-accessible type of osteoblast toobtain in culture for in vitrostudy.

  Bonewald and colleagues have established several im-mortalizedcell lines in culture with phenotypic character-istics ofosteocytes. Bone cells were derived from transgenicmiceover-expressing T-antigen driven by the osteocalcinpromoter. Theychose cells expressing a dendritic morphol-ogy as the initialcriterion for selection and establishment of clonal cell lines.MLO-Y4 (murine long bone osteocyte Y4)

  was one of the immortalized clonal lines establishedwithosteocyte-like characteristics. These cells produceextensive,complex dendritic processes, are positive for T-antigen,os-teopontin, neural antigen CD44 and connexin 43. They pro-ducelarge amounts of osteocalcin,have low levels of alkalinephosphataseactivity, lack detectable mRNA for osteoblast-specic factor 2, andproduce very small amounts of type Icollagen ( Kato et al., 1997 ).The MLO-Y4 cells also sup-port osteoclast formation and activationthrough the secre-tion of M-CSF and expression of RANKL on theirsur-face and their dendritic processes ( Zhao et al., 2002 ).Cellsare grown on collagen-coated surfaces in culturemediumsupplemented with 5% FBS and 5% calf serum for opti-malgrowth 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 ofthese, MLO-A5 cells were shown to highlyexpress BSP and mineralizespontaneously in culture evenin the absence ofbeta-glycerophosphate and ascorbic acid(Kato et al., 2001 ). Theauthors claim that the MLO-A5 cellsare representative of thepost-osteoblast, preosteocyte stageresponsible for triggeringmineralization of osteoid.

  To date, there isnopublisheddataon theresponses ofthesepresumptive osteocyte-like cell lines to mechanicalstrain.

  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 acidinto specialized resorption pits to dissolvebone mineral as well asto provide an optimum environmentfor the enzymatic degradation ofdemineralized extracellu-lar bone matrix. Osteoclasts are derivedfrom hematopoieticstem cells that differentiate along themonocyte/macrophagelineage ( Martin et al., 1989; Suda et al., 1992). Directcontact of mononuclear hematopoietic precursors withos-teoblast/stomal cells expressing the membrane protein Re-ceptorActivator of NF-kappa B Ligand (RANKL) is neces-sary before theycan differentiate into osteoclast precursorsand proceed to fuseinto mature, multinucleate osteoclasts(Suda et al., 1995; Lacey etal., 1998 ). This is depicted dia-grammatically in Fig. 2.

  3.2. Phenotypic characteristics of osteoclasts

  The mature osteoclast is a functionally polarized cellthatattaches via its apical pole to the mineralized bone matrixbyforming a tight ring-like zone of adhesion, the sealingzone. Thisattachment involves the specic interaction be-tween adhesionmolecules in the cell membrane (integrins)and some bone matrixproteins. The integrins are a fam-ily of transmembrane proteinswhose cytoplasmic domainsinteract with the cytoskeleton while theirextracellular do-mains bind to bone matrix proteins, enabling themto medi-ate cell–substratum and cell–cell interactions ( Hynes,1987 ).

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

  The space contained inside this ring of attachment and be-tweentheosteoclast andthebone matrixconstitutes thebone-resorbingcompartment. The cell membrane of the apicalpole is invagin*ted toform a rufed border. Osteoclasts areactively engaged in thesynthesis and secretion of severalclasses of enzymes formedintheGolgi regionandvectorially

  transported to the apical pole through their associationwithmannose-6-phosphate receptors. At their destination, theen-zymes bound to mannose-6-phosphate receptors fuse withthe rufedborder apical membrane and their contents dis-charged into thebone-resorbing compartment ( Baron et al.,1993).

  Acidication of the extracellular bone-resorbingcompart-ment isone of the most important features of osteoclastaction. Theosteoclast is highly enriched in carbonic anhy-drase (Gay andMueller, 1974 ). Carbonic anhydrase gener-ates protons andbicarbonate from carbon dioxide and wa-ter, providing the cellswith protons to be extruded acrossthe cell membrane into thebone-resorbing compartment byproton pumps (H + ATPases) located inthe rufe borderapical membrane. Regulation of H + transport at theapi-cal surface of the osteoclast, which is tightly linked totheregulation of intracellular pH and membrane potential, ismostlyaccomplishedby ion exchangers, pumpsand channelspresent in thebasolateral membrane of the cell ( Baron et al.,1993).

  Theactivityof mature osteoclast is directly andnegativelyregulated by calcitonin, for which the cell expresses ahighnumber of receptors ( Nicholson et al., 1986 ).

  A summary of the main osteoclast phenotypic character-istics isprovided 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 plasminogenactivatorLysozyme

  Matrix proteinsOsteopontinBone sialoproteinTGF

  ReceptorsRANK (membrane)

  Calcitonin (membrane)Vitronectin ( v 3) (membrane)Integrins with1 subunitsMannose-6-phosphate (intracellular)

  Proton pumpVacuolar H + ATPase

  Ion transportCalcium channelsPotassium channelsChloridechannelsCalcium ATPaseNa, K-ATPaseNa+ /H+ antiporter

  Bicarbonate/chloride exchanger

  Membrane associated proteinsCarbonic anhydrasec-src

  3.3. In vitro methods to study osteoclast formation andfunction

  Unlike osteoblasts,osteoclasts aredifcult to studyinvitrobecause they are relatively scarce, terminallydifferentiated,adherent to mineralized surfaces and fragile.Methods havebeen developed to isolate these cells in vitro or toinduce theirformation in bone marrow cultures. The major criteriagener-ally used to identify osteoclasts are multinuclearity,positivestaining for tartrate resistant acid phosphatase (TRAP),ex-pression of calcitonin receptorsandtheability to resorbcalci-edmatrices ( Takahashi et al., 1988a; Hattersley and Cham-bers, 1989;Shinar et al., 1990 ). TRAP staining and someof the other criteriaused 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 identifyosteoclasts whenmacrophage expression of these markers can beeffectivelyexcluded. Indeed, the simplest method of estimatingosteo-

  clast number in in vitro assays is a count of TRAP positiveorvitronectin receptor positive multinucleated cells.However,prolonged culture (more than 7 days) frequently resultsincalcitonin receptor negative, TRAP positivemultinucleatedmacrophages, and that is a common pitfall.Conversely, theabsence of these markers indicates theabsence ofosteoclasts.

  Mature, multinucleated functional osteoclasts are ob-tainedeither directly from bone as the primary source, or elsethey aresecondarily generated in vitro from hematopoieticprogenitorsobtained from a source of hematopoietic cells ormacrophages such asbone marrow, spleen, human peripheralblood mononuclear cells andhuman umbilical cord blood.

  3.3.1. Primary sources of osteoclasts3.3.1.1. Mechanicaldisaggregation of osteoclasts. Matureosteoclasts can be isolated bymechanical disaggregationfrom the long bones of neonatal rats,rabbits or chicks. Thismethod involves curetting the long boneswith a scalpel bladeto release bone fragments into the surroundingmedium. Thefragments are triturated into a suspension with awide-borepipette before plating onto glass coverslips for 15–30minto allow the large, highly adherent osteoclasts to adhere totheglass surface, before washing vigorously with mediumto removenon-adherent and other contaminating cell types(Chambers andMagnus, 1982 ). A longer settling time in-creases the yield ofosteoclasts, but also the number of non-osteoclasticcontaminatingcells. Relatively‘pure’osteo-clastshave been obtainedthis way to enable the identicationof calcitonin receptors andeffects of bone-resorbing factorson cytoplasmic spreading (Nicholson et al., 1986 ). The ef-fects of osteotropic factors onbone resorption can be studied

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

  3.3.1.2. Giant cell tumors of bone. Giant cell tumors(GCT),alsoknown as osteoclastomas, are rare primary neoplasmsof the skeleton.They are locally destructive, causing exten-sive osteolysis. GCTcontain within the tumor mass, variablenumbers of large,multinucleated cells. It is believed that thestromal cells of GCTare the tumor cells, and they induceosteoclastic bone resorption byrecruiting osteoclast precur-sors, promoting their differentiationinto functional osteo-clasts (James et al., 1996 ). Until recently,this was the onlyuseful source of human osteoclasts. To obtain theosteoclasts(Goldring et al., 1987 ), the giant cell tumor isdissected in aPetri dish under sterile conditions using a scalpelblade, andthen enzymatically digested for cell culture in adigestionmixture made up from collagenase and dispase. The cellsus-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; timedependentCalcitonin receptors Specic (hematopoietic lineage)Difcult without live cellsMultinuclearity Typical but not essentialIndicates terminal differentiationTRAP Useful marker; easy toperform Indicative in vitro, but also expressed by activatedmacrophagesCathepsin K Useful marker Also expressed bymacrophages/tumor cellsVitronectin receptor Occasionally usefulAlso expressed by macrophagesActin ring Indicates active osteoclast(on calcied substrate)

  FBS, and ltered through a 40 m cell strainer. Cells aretheneither cryopreserved or cultured in medium ( Atkins etal., 2001).

  3.3.2. Secondary sources of osteoclasts. Invitrogeneration3.3.2.1. Bone marrow cultures. Bone marrow consistsof amixed cell population that is rich in hematopoietic butrel-

  atively poor in osteoblastic stromal cells. They are usefulforexamining the process of osteoclast differentiation andformation inculture under the inuence of bone-resorbingagents. Murine bonemarrow cultures have been the mostwidely studied ( Takahashi etal., 1988a, 1988c : see methodbelow). Takahashi et al. (1988b)reported that treatment with1,25(OH)2D3or humanPTH(1–34) resultedinan increase inthenumberof TRAP positivemultinucleated cells thatsatisfythe major criteria for osteoclasts. Subsequently, a similarin-crease was observed with prostaglandins, PTHrP (1–34) andIL-1(Akatsuet al., 1989a,1989b, 1991 ). Two otherimportantobservationswere made. Firstly, time course studies showed

  that the appearance of TRAP positive mononucleated cellsprecedethat of TRAP positive multinucleated cells, imply-ing that TRAPpositive mononucleated cells are precursorsof the multinucleatedcells. Secondly, osteotropic hormonessuch as 1,25(OH)2D3 and PTHinduce the differentiationof immature precursors, characterized byTRAP and CTR-negativity, into mature TRAP and CTR-positiveprecursors(Takahashi et al., 1988b ) (Fig. 2). Osteoclasts havealso simi-larly been obtained using rabbit ( Fuller and Chambers,1987 )and feline ( Ibbotson et al., 1984 ) bone marrow. Thissystemis limited by the inability to identify osteoclasticprecursorsfrom the mixed cell population and the difcultiesinherentin studying osteoclast activation as opposed torecruitment.The dependence of osteoclast formation on the presenceof mesenchymal stromal cells or osteoblasts also complicatetheinterpretation of results.

  3.3.2.2. Co-cultures of osteoblastic and hematopoietic cells.Animportant nding from murine bone marrow cultures wasthedemonstration that it was necessary for hematopoieticprecursors tocome into direct contact with osteoblasts andstromal cells beforeosteoclast differentiation and formationcould occur ( Takahashi etal., 1988b ). This led to the de-velopment of co-culture systemscomprising three essentialelements: (i) stromal cells orosteoblasts as a feeder layer;

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

  (i) Preparation of primary osteoblastPrimary osteoblasts areusually obtained from the cal-varia of newborn C57BL/6J mice.Calvaria are removedfrom the newborn mice under sterile conditionsandtransferred to a sterile 30 ml tube containing 6 ml di-gest uidmade up immediately before use. The calvar-ial digest uid is madefrom 30 mg collagenase type IIand 60 mg dispase dissolved in 30 mlPBS and lteredthrough a 0.2 m Acrodisc ® 32 Supor ®. The tubecon-taining calvaria is shakenin a 37 ◦ C water bath for 5min,allowed to settle and the supernatant discarded by pipet-ting.Six millilitres of fresh digest uid is added to thetube andincubated at 37 ◦ C for 10 min. The cell sus-pension is collectedin a separate sterile 30 ml tube. Thedigest is repeated a furtherthree times and the cell sus-pensions pooled in the 30 ml tube.This is centrifugedat 2000 rpm for 5 min and the supernatantdiscarded.The cell pellet of primary osteoblasts is re-suspendedin

  10ml MEM + 10% FBS and used immediately. Alter-natively,osteoblasts can be grown in culture for a fewdays to increase theirnumbers. Cells are seeded at adensity of 5 × 106 cells in 10 ml ofmedium in Petridishes and incubated at 37 ◦ C in a humidiedincuba-tor with 5% CO 2 . The adherent calvarial osteoblastsareconuent in 2–3 daysand can beused up to 1 week fromtheir initialpreparation. They are dispersed for use bystandard trypsinizationmethods.

  (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 frommurinespleen cells in the presence of 1,25(OH)2D3.The simultaneousaddition of dexamethasone greatlyenhanced osteoclast numbers. Otherosteoblast/stromalcell lines shown to support osteoclast formationin theco-culture systeminclude the ratosteoblast-likecell lineUMR106 ( Quinn et al., 1994 ), murine stromal cell linestsJ2 and 10(Chambers et al., 1993 ), murine osteoblast-like cell lines KS-4 (Yamash*ta et al., 1990a, 1990b ) andKUSA/O ( Umezawa et al., 1992).

  (iii) Preparation of bone marrow cellsLong bones (femur andtibia) are obtained from adultmale mice (4–8 weeks old). Each boneis ushed with

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

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

  3.3.3.3. Humanosteoclasts. The formation of human osteo-clastsin coculture has proved to be a challenge because of a lack of asuitable human osteoblastic stromal cell line touse in a coculturesystem. 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 witheither osteoblast-likeUMR 106 (rat) or ST2 (mouse) stromal cells inthe presenceof 1,25(OH)2D3, dexamethasone and human M-CSF (ratM-CSF is inactive on human cells). Numerous TRAP, VNRandCTR-positive multinucleated cells, capable of extensive la-cunarbone resorption, formed in these cocultures. This work was extendedto include human hematopoietic marrow cells,blood monocytes andperitoneal macrophages, all of whichwere capable of differentiatinginto mature functional os-teoclasts ( Quinn et al., 1998b ).Matsuzaki et al. (1999) es-tablished subclones of the humanosteosarcoma cell line,SaOS-2, expressing the human PTH/PTHrPreceptor, andshowed that mouse bone marrow cells or humanperiph-eral blood mononuclear cells formed osteoclasts incoculturewhen treated with PTH. The response was greatly enhancedbyadding dexamethasone, but no osteoclast formation wasseen with theaddition of 1,25(OH)2D3, PGE2 or IL-6. Hu-man peripheral bloodmononuclear cells (PBMC) culturedin vitro with soluble RANKL andhuman M-CSF, form os-

  teoclasts. However, PBMC are heterogeneous, consisting ofsubsets of monocytes, lymphocytes and other blood cells.Nicholsonet al. (2000) showed that a highly puried popu-lation ofosteoclast-forming PBMC can be obtained by se-lecting for theexpression of CD14, a marker that is stronglyexpressed inmonocytes, the putative osteoclast precursor inperipheralblood.

  A novel and exciting new method of obtaining osteo-

  clast progenitors from human umbilical cord blood was re-centlydescribed ( Hodge et al., 2002 ). A mononuclear cellfractioncontaining monocytes and lymphocytes, isolatedfrom human umbilicalcord blood by Ficoll–Paque densitygradient centrifugation, iscultured in semi-solid medium,and incubated at 37 ◦ C in a humidiedatmosphere of 5%CO2–air for 7–14 days. Pooled colonies identied asCFU-GM are harvested and transferred into 96-well platescon-taining dentine slices in the presence of RANKL and hu-manM-CSF for a further 6 days. Cultures are then xedin 1% formalin andreacted for TRAP activity. The forma-tion of bone-resorbingmultinucleated osteoclasts is assessedby transmission lightmicroscopy and quantied using com-puter image analysis. Using humanumbilical cord bloodmononuclear cells (CBMC) as a source ofosteoclast pro-genitors, it was shown that clonal expansion ofCFU-GMprogenitors markedly increases osteoclastogenic potential,butexposure of pooled colonies to GM-CSF or IL-3 priorto RANKLstimulus completely inhibits osteoclastogenesis,directing cellsinstead towards dendritic cell differentiation.This may prove to bea very useful method for obtaininghuman osteoclast progenitors tostudy the regulation of dif-ferentiation along different celllineages.A further advantageof this method is the ability tocryopreserve CBMC for futureexperiments.

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  4. Chondrocytes

  Cartilage is a specialized form of connective tissuethatpossesses a rm pliable matrix, which endows it withtheresilience that allows the tissue to bear mechanicalstresseswithout distortion. Articular cartilage, smooth surfacedand

  resilient, provides a shock-absorbing sliding area for jointstofacilitate movement of bone, while cartilage is also essentialforthe embryonic development and, thereafter, growth of longbones.

  See Also

  Сe înseamnă ALKALINE DRINKING în Română - Română Traducere

  Cartilage consists of chondrocytes and an extensiveextra-cellular matrix. The characteristics of cartilage stemmainlyfrom the nature and predominance of ground substance intheextracellular matrix. Glycoproteins, containing a highproportion ofsulfated polysaccharides, make up the groundsubstance and accountfor the solid, yet exible properties of cartilage. The functionaldifferences between cartilage andbone relate principally to thedifferent nature and proportionof the ground substance and brouselements of the extracel-lularmatrix.Nonetheless,chondrocytesandosteoblasts sharea common origin fromprimitive mesenchymal cells ( Marksand Hermey, 1996 ).

  Hyaline cartilage is the most prevalent type of cartilage.Duringembryonic development, it forms the cartilage tem-plate of many ofthe developing bones until replaced by bonein theprocess ofendochondralossication. In long bones, theepiphyseal growth platebetween the epiphysis and diaphysisis responsible for thelongitudinal growth of bone. Withinthe growth plate, chondrocytesundergo a series of discretestages of differentiation, namely,proliferation, maturation,and hypertrophy. The strict spatial andtemporal control of

  proliferation and differentiation of chondrogenic cells iscen-tral to the coordinated development of the vertebrateskeleton(Erlebacher et al., 1995 ).

  Chondrocytes are unique cells, in that they havemanydifferentiatedmarkers suchas large cartilage-typeproteogly-cans (aggrecan) and collagen types II, IX, X, and XI.Sometypical phenotypic characteristics of chondrocytes are listedinTable 8 . A comprehensive list of several hundred mousegrowthcartilage-derived gene products, including many notpreviouslyreported, was recently published ( Okihana andYamada, 1999 ).

  4.1. Propagation of chondrocytes in culture

  Various cell culture models have been developed fortheinvestigation of chondrocyte biology in vitro, includingex-plant models, several forms of three-dimensional culturesys-tems, and monolayer cultures ( Adolphe and Benya, 1992).Chondrocytes grown in monolayer culture undergo a char-acteristicprocess of dedifferentiation, marked by a loss of collagen type IIand aggrecan core protein expression as wellas the induction ofcollagen type I expression ( Takigawa etal., 1987; Hering et al.,1994; Lefebvre et al., 199 4). Thisphenomenon is inuenced to someextent by seeding den-sity (Ronzi ère et al., 1997 ) and isaccelerated by growth in

  Table 8A summary of the phenotypic characteristics ofchondrocytes

  CollagensType I collagenType II collagenType VI collagenType IXcollagen

  Type X collagenType X1 collagen

  Proteoglycans and other proteinsAggrecanLinkproteinBiglycanFibronectinOsteopontinCartilage oligomeric matrixproteinMatrix glaproteinChondromodulin-ICalmodulinFibromodulinCartilage homeoproteinIPerlecanTropomodulinOsteonectin

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

  medium supplemented with serum and by passage ( Hering

  et al., 1994). Growth of chondrocytes under conditionsthatsupport a rounded morphology also facilitates maintenanceof thedifferentiated chondrocytic phenotype ( Bonaventureet al., 1994; Häuselmann et al., 1994; Binette et al., 1998 ).Stewart et al.(2000) studied the phenotypic stability of ar-ticular chondrocytesin vitro and demonstrated that the in-uence of BMP-2 and serum onexpression of chondrocyte-specic matrix proteins [procollagen typeI and II, aggrecanand (V + C)− bronectin] varies depending oncellular mor-phology, and/orcytoskeletalorganizationwhenchondrocytesare grown as monolayer,aggregate, pellet or explant cultures.

  Despite these limitations, some measure of success hasbeenachieved with autologous chondrocyte transplantationto repaircartilage defects. Focal chondral or osteochondraldefects, usuallythe result of trauma, have a poor capacity forrepair and predisposepatients to osteoarthritis. In a surveyof one thousand consecutiveknee arthroscopies, chondral orosteochondral lesions were found in61% of the patients,withfocal chondral or osteochondral defectsaccounting for 19%with a mean defect area of 2.1 cm 2 (Hjelle etal., 2002 ). Au-tologous chondrocyte transplantation (ACT) was rstusedin humans in 1987 and the rst pilot study was published in1994( Brittberg et al., 1994 ) in 23 patients with deepcartilagedefects in the knee. Cartilage slices were obtainedthroughan arthroscope from a minor-load-bearing area on theupper

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  polygonal cells, which accumulate an alcian-bluestainablematrix. IRCcells synthesize typical cartilage proteins,aggre-can and link protein, but show reduced collagen IIexpression(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 towardhy-pertrophic chondrocytes, whereas treatment with PTHanddexamethasone slows and limits differentiation. Recovery of p53expression in N1511 cells by transient transfection in-hibitsproliferation, suggesting that cell proliferation can beregulatedwith p53 in this cell line. These results would in-dicate thatN1511 is the only cell line with known geneticmutation, whichundergoes multiple steps of chondrocytedifferentiation towardshypertrophy, and may also be usedto study the function of p53.

  HCS-2/8 is an immortalizedclonalcell line derived fromawell-differentiated human chondrosarcoma ( Takigawa et al.,1989),with phenotypic features resembling normalchondro-cytes.Thecellssynthesize aggrecan, integrins, collagen typesII, IX andXI, show the same responses to growth factors asnormal chondrocytesand maintain their cartilage phenotypeover more than 3 years inculture ( Takigawa et al., 1997 ).

  5. Calcium homeostasis

  Calcium is an essential ion for many physiological pro-cessessuch as cell motility, muscle contraction and neuro-transmitterrelease. In mammals, these processes functionoptimally whenextracellular calcium is maintained within

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

  Parathyroid cells express a cell surface calcium-sensingreceptorthat recognizes and responds to physiologicalchanges inextracellular ionized calcium concentration. Re-ductions in serumcalcium of the order of 1–2% result ina prompt increase inparathyroid hormone (PTH) secretion.PTH acts directly on thekidneys and skeleton and indirectlyon the intestine to normalizeany fall in extracellular ion-ized calcium. In the kidney, PTHstimulates re-absorptionof calcium in the distal tubules andincreases synthesis of 1,25(OH)2D3. 1,25-Dihydroxyvitamin Dincreases intesti-nal absorption of calcium and also stimulates therelease of calcium from bone by stimulating bone resorption. Thein-creased ux of calcium ions into the extracellular uid re-storescirculating levels of calcium toward normal. Normal-ization ofserum calcium as well as the increased levels of1,25-dihydroxyvitamin D inhibits further PTH synthesis in anegativefeedback loop. An increase in extracellular ionizedcalcium inhibitsPTH secretion, resulting in increased renalcalcium excretion andreduction in net release of skeletal cal-cium as well as intestinalabsorption of calcium.

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

  cemia but in man, no essential function has yet been foundforcalcitonin. Steady-state plasma calcium shows little orno changewith 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 ondispersedbovine 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’ssolution. They are transferred totissue culture asks, 8–10 glandsin 15 ml, and dispersedby shaking with 1.25 mg/ml collagenase typeI suspendedin Eagle basal medium containing 15% FCS. The cellsareltered through 200 mm cell dissociation sieve and 40 mmnylonmesh. Cell suspension is washed by centrifugation inHank’s solutionand 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 wellandcultured at 37 ◦ Cin5%CO 2 for 24h to allow attachment(Moallem etal., 1995 ).

  The extracellular calcium sensing receptor was clonedfrom bovineparathyroid cells ( Brown et al., 1993 ), and thesecells have beenvery useful in determining the role of cal-cium uxes in theregulation 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)cellsof the thyroid, and medullary thyroid carcinoma (MTC)is aneuroendocrine tumor of the parafollicular cells. Celllinesestablished from human and animal MTC tumors pro-vide a usefulsystem to analyze genes involved in the de-velopment of thisneoplasia, as well as a source of C cellsto determine theregulation of calcitonin production. ThehMTC cell line, TT cells (Leong et al., 1981 ) and the ratMTC line, 6–23 cells ( Zeytinogluet al., 1980 ) can be pur-chased from the American Type CultureCollection (Man-assas, VA). TT cells display an impaired expressionof thetumor suppressor gene p53 ( Velasco et al., 1997 ). Apartfromcalcitonin and the calcitonin receptor, TT cells ex-presscarcino-embryonic antigen, somatostatin and its recep-

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

  6. Discussion

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

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  the complex three-dimensional architecture of bone thatisrequired for theproperexpression of the functional capabilityofthe cells that make up its microenvironment. Nonetheless,despitethe limitations of the various systems described inthis review,signicant and important contributions havebeen made to ourunderstanding of the normal processes

  leading to bone formation, remodeling and resorption aswell ashow these processes can be deranged to result inmetabolic bonediseases.

  The term osteoblast describes a lineage of cells that dif-fersubstantially in their properties at different stages ofde-velopment. Although many ‘characteristic’ properties ofos-teoblasts have been described, it does not follow that allcellsof the lineage possess each of these features. Atdifferentstages of differentiation, and at different sites in bonecarry-ing out specic functions, osteoblasts are likely toexpressonly a proportion of the features associated with thepheno-type. The challenge of identifying the cellular pathwaysandthe factors that regulate progression from osteoprogenitorstomature osteoblasts is facilitated by the ability to isolateandanalyse in culture, osteoblasts at various stages ofdifferen-tiation, and in particular, clonal cell lines from bone orbonetumors. These are used as models of osteoblastsrepresentingdifferent stages of differentiation, enablinginvestigators todene the heterogeneity of bone cell populationswith moreprecision. In contrast, primary calvarial cells probablycon-tain osteoblasts at all stages of differentiation, includingos-teoprogenitors that proliferate before undergoing a series ofmaturational steps to become differentiatedosteoblasts capa-ble offorming mineralized nodules in culture ( Aubin, 1998;Malavalk etal., 1999 ). Primary calvarial cultures are widely

  used to study theprogression of differentiation in vitro.Roweandcolleaguesrecently describedan elegant methodinwhichsubpopulations of osteoblasts at different stages ofdiffer-entiation can be isolated from primary osteoblastcultures.Having identied fragments of the rat type I (Col1a1)pro-moter that show preferential expression in differentCol1a1-producing tissues, they generated green uorescentprotein(GFP)-expressing transgenic mice containing a 3.6- anda2.3-kb rat type I collagen promoter fragment. The 3.6-kbpromoterdirected strong expression of GFP messenger RNAto preosteoblasticcells, while the 2.3-kb promoter directedGFP mRNA expression to acell that is late in the osteoblastlineage,extendinginto matureosteocytes. They conclude thatwith further renement of this method,using other promot-ers and color isomers of GFP, it should bepossible to isolatesubpopulations of cells at different stages ofdifferentiationfrom primary cultures derived from these transgenicmicefor molecular and cellular analysis ( Bogdanovic et al.,1994;Kalajzic et al., 2002 ).

  Established osteoblast-like cell lines are particularly use-fulmodels to study signaling pathways in response to stimu-lation byosteotropic factors.Thegreatmajorityof osteoblast-like cell lineshowever, do not mineralize in culture, with theexception ofMC3T3-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 establishedfrompluripotent mesenchymal cells provide valuable informa-tion onthe factors and mechanisms regulating differentiationalong theosteoblast, chondrocyte, adipocyte and myocytelineages.

  In the case of osteoclasts, considerable progress has been

  made in the past twenty years in the development of methodstostudy their function and formation in vitro. Early studiesusingrelatively crude methods to disaggregate osteoclastsfrom bone werecumbersome, succeeding in obtaining onlysmall numbers ofosteoclasts that could notbe separatedfromother cell types such asstromal cells and osteoblasts, andresults were difcult toreproduce. Investigators were facedwith the challenging task ofobtaining sufcient numbers of relatively ‘pure’ preparations ofosteoclasts in culture. Thiswas clearly not attainable using boneas a primary source of functional, mature osteoclasts. Knowledgethat osteoclastsare derived from hematopoietic stem cells thatdifferentiatealong the monocyte/macrophage lineage, and therealizationin the late eighties, that direct contact betweenosteoclastprogenitors and stromal cells/osteoblasts is required foros-teoclast differentiation, led to the widely-used coculturesys-tem to study the regulation of osteoclast differentiationfrommononucleate progenitors to mature, functional multinucle-ateosteoclasts. It became feasible to generate functional os-teoclastsin culture in far greater numbers, even though theosteoclasts couldnot be separated from the companion os-teoblasts/stromal cells forseparate analysis.Almost a decadelater, the pivotal role of RANKLin osteoclastogenesis, its in-teractionwith itscognate receptorRANK aswell as thedecoyreceptor osteoprotegerin were revealed. Notonly were these

  discoveries major advances in the understanding of theregu-lation of osteoclast formation, it made possible thesubstitu-tion of soluble RANKL and M-CSF forstromal/osteoblasticcells, thus considerably simplifying the methodfor obtainingfunctional osteoclasts in vitro.Theuseof cell lines asalterna-tive sources of osteoclast progenitors is not widelypractisedbecause of the lack of suitable cell lines. Somelaboratorieshave used RAW264.7 cells to generate osteoclasts inculture.Unlike hematopoietic progenitors derived from bone marroworspleen, RAW264.7 cells do not require M-CSF alongsideRANKL. Themechanism underlying this difference is notknown, but it may implya difference in the signaling path-way leading toosteoclastogenesis.

  Chondrocytes are unique in their ability to exist in a lowoxygentension environment, isolated within a voluminousextracellularmatrix devoid of a vascular supply. Primarychondrocytes retaintheir phenotypic characteristics whengrown in the form of athree-dimensional multicellular com-plex, but undergo a process ofdedifferentiation when grownas monolayer cultures with serumsupplementation. A rangeof established chondrocytic cell lines areavailable as alter-natives.

  Work carried out on established cell lines and primarycellcultures have provided much valuable insight into thephenotypiccharacteristics of cells belonging to the bone

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