Ultrastructural Abnormalities in Cultured ExostosisChondrocytes

Pauline Jackie Duke, PhDDina Montufar-Solis, BS

The University of Texas Health Science Center at Houston,Dental Branch, Department of Orthodontics, Houston,Texas, USA

Richard Haynes, MD

Shriners Hospital for Children¡Houston,Houston, Texas, USA

Jacqueline T. Hecht, PhD

The University of Texas Health Science Center atHouston, Medical School, Department of Pediatrics,Houston, Texas, USA; and Shriners Hospital forChildren¡Houston, Houston, Texas, USA

Hereditary multiple exostoses (HME) is an autosomal dominantdisorder characterized by inappropriate chondrocyte proliferation andbone growth arising at the juxtaepiphyseal region of the long bones.HME is caused by mutations in the EXT 1 and EXT 2 genes, whichhave glycosyltransferase activity. These genes are responsible forsynthesis of heparan sulfate (HS) chains, which are importantsignaling molecules in chondrocyte differentiation. HMEchondrocytes in monolayer culture have been shown by transmissionelectron and deconvolution microscopy to contain enormous bundlesof actin, cross-linked with muscle specific a-actinin. Here additionalultrastructural anomalies in HME chondrocytes are reported,including lobulated nuclei, shortened channels of rER, large numbersof cell processes and podosomes, nontypical junctions, elongated,bulbous-ended mitochondria, and reduced extracellular matrix.Microfilaments are present throughout the cytoplasm,compartmentalizing it, and isolating organelles. The excessmicrofilaments, attributed to increased cell adhesiveness, are likely tointerfere with secretion and cytokinesis, and sterically hinderintracellular organelle differentiation. The observed surfacemodifications and cytoskeletal abnormalities are proposed to play arole in development of the mutant phenotype, via changes in celladhesiveness and=or binding of signals to receptors, which results inloss of the unidirectionality of growth in the epiphyseal plate.

Keywords chondrocyte, EXT, heparan sulfate, hereditary multipleexostosis, microfilaments, ultrastructure

In the chondrocyte disorder hereditary multipleexostoses (HME), cartilage capped tumors, calledosteochondromas or exostoses, extend from the sur-face of the bone into the surrounding soft tissue [1, 2].Growth of exostoses in HME is primarily associatedwith the more rapidly growing end of long bones, andtumors seldom develop at the apophyseal (i.e.,slower-growing) end [3]. The formation of theseexostoses is accompanied by typical skeletaldeformities, e.g., abnormalities of bone joints, bonysynostoses, and limb inequalities. The exostosis is anintegral part of the bone, and consists of a cartilage capoverlying an ossified region, which surrounds a marrowcavity continuous with that of the parent bone. Growthof exostoses usually ceases at the end of puberty,although exceptions are known and warrant medical

examination. Malignant transformation into chondro-sarcoma can occur in early adulthood [4].

HME is an autosomal dominant condition withgreater than 95% penetrance [4], and is caused bymutations in the genes EXT 1 or EXT 2 [5, 6], theproducts of which are type II transmembraneglycoproteins, residing in the endoplasmic reticulum[7]. EXT 1 and EXT 2 have glycosyltransferase activityand are responsible for the modifications of heparansulfate glycosaminoglycans (GAGs), important com-ponents of cell surface proteoglycans involved in cellsignaling [8]. To become active, EXT 1 and EXT 2 forma homo=heterooligomeric complex, which localizes inthe Golgi [9]. This complex forms and localizes toGolgi even in COS-7 cells expressing EXT 1 or EXT 2mutations [10]. Little is known about location andfunction of EXT gene products in chondrocytes.

Exostosis chondrocytes grown in monolayer culturehave a stellate appearance with sticklike forms in thecytoplasm [11]. Transmission electron and deconvo-lution microscopy of these cells identified thesestructures as bands of microfilaments, found byWestern analysis to be actin bundles cross-linked bymuscle-specific a-actinin. In vivo, immunocytochem-istry of chondrocytes in an excised single exostosis

Received 3 July2001;accepted 8 October 2001.

Supported byShriners Hospital for Children grant15955 to JTH.

Address correspondence to Pauline Jackie Duke,PhD,The Universityof TexasHealth Science Center at Houston, Dental Branch, Department of Orthodon-tics,Rm.371,P.O.Box 20068,Houston,TX77225-0068,USA.E-mail: jduke@mail.db.uth.tmc.edu

Ultrastructural Pathology, 26:99±106, 2002

Copyright # 2002 Taylor & Francis

0191-3123 /02 $12.00 ‡ .00

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confirmed that the intracellular fibers were composedof actin bundles.

Many theories regarding the origin and developmentof cartilage capped exostoses (osteochondromas) havebeen proposed [3]. These theories have in common theidea that (1) the exostosis develops from a stem cellpopulation, i.e., a resting zone or perichondrial typecell, and that (2) the cells in this population somehowescape the ordinarily stringent controls placedon directionality of cell division within the growth plate[1, 2]. In this paper, we describe electron microscopystudies showing an altered ultrastructural phenotype incultures of chondrocytes derived from exostosesexcised from one individual with HME, which mayresult from, or contribute to, the signaling defect thatinterferes with the normal differentiation of the cells inthe epiphyseal growth plate. A model of exostosisformation is suggested.

MATERIALS AND METHODSThe exostosis chondrocyte sample was obtained at

surgery from a prepubertal patient with HME afterinformed consent. She had a positive family history ofHME in her sister and father. Mutational analysis wasperformed as described in Bernard et al. [11]. Cos-tochondral cartilage from an individual undergoingsternoplasty served as a source of control chon-drocytes. Both HME and control chondrocytes werereleased from matrix and expanded in monolayer aspreviously described [11]. This study used chon-drocytes from passages 3¡5.

Chondrocytes were fixed overnight with 2%paraformaldehyde in 0.1 M cacodylate buffer, and0.5% glutaraldehyde, pH 7.4, at 4¯C, and prepared forelectron microscopy as previously described [11]. Inbrief, cells were fixed, removed from the plate, pelleted,and embedded in Epon. Ultrathin sections were stainedwith uranyl acetate and lead citrate, and viewed in aJEOL 100CXII microscope at 60 kV. Forty-threesections and 154 photographs were examined.

RESULTS

Mutational AnalysisMutational analysis revealed a germline mutation,

907delG, in the EXT 2 gene. This mutation segregatedwith the HME in the family.

Light MicroscopyHME chondrocytes in subconfluent culture are very

spread and appear larger than costochondral controlchondrocytes (Figure 1). Sticklike structures, pre-viously shown to consist of huge bundles ofmicrofilaments, are visible in the cytoplasm of HMEcells, becoming less visible as the culture reachesconfluency. Cells form large numbers of focaladhesions, which are evident at the light microscopy

level during trypsination. Impaired ability of HME cellsto separate in cultures was observed, and as measuredby differences in passage time, HME chondrocytestake longer to reach confluency than do controlchondrocytes.

Transmission Electron Microscopyof Cultured Cells

Electron microscopy of cultured chondrocytesshowed HME chondrocytes to have several ultra-structural abnormalities compared to controlchondrocytes (Figure 2). Particularly noticeable are thehuge numbers of microfilaments in HME cells.Microfilaments are also present in cells from controlcultures, but in normal amounts.

The microfilaments in HME cells are foundthroughout the cell, appearing as ``rivers’’ ofmicrofilaments between the organelles (Figure 2B).

FIG. 1 Chondrocytes in monolayer culture. …A†Control chondrocytes demonstrate the stellateand spindle shapes of cells in subconfluentcultures. Stress fibers are apparent in somecells. …B† HME chondrocytes appear morespread than controls, have visible actin bands(sticklike structures in their cytoplasm), andhave many cellular processes. A,actin-associated structures; PR, processes.Bar ˆ 100 mm.

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The orientation of the microfilaments generally followsthat seen in the controls, i.e., along the long axis of thecell. However, microfilaments in HME cells often runperpendicular to the long axis of the cell. HMEchondrocytes do not have the extensive RER networkusually associated with chondrocyte differentiation. Incontrast to the normal appearing long channels of RERfilled with an electron-dense material in controlchondrocytes (Figure 2), only short channels of RERare present in HME chondrocytes. In some HMEchannels, the material appears fibrous, and manychannels appear to have ruptured with loss of theircontents into the cytoplasm.

HME chondrocytes have many cell processes (bothshort and long), which are often filled withmicrofilaments (Figures 3A and B); and sometimesattached to other cells via junctional complexes.HME cells were also attached to the substrate bynumerous podo somes, into which microfilaments

insert (Figure 3C). Fewer cell processes and onlyoccasional podosomes are seen in controlchondrocytes.

Nuclei of HME chondrocytes exhibit abnormalnuclear shapes. Though control chondrocytes oftenhave lobed nuclei, HME chondrocytes have lobulatednuclei with abnormal constrictions (Figure 3). Thelobes are connected by short bridges, comprisingmostly nuclear membrane, with some associatedchromatin.

Numerous lysosomes and abnormal mitochondriaare found in HME chondrocytes. These mitochondriaare extremely long, with bulbous regions in whichcisternae are not evident (Figure 4). Controlchondrocytes had fewer lysosomes and did not containsuch mitochondria.

Very little extracellular matrix was associated withHME chondrocytes (Figure 5). Extracellular matrix wasvisible on surfaces of control chondrocytes andbetween cells, with typical collagen fibrils andproteoglycan granules (Figures 1 and 5).

Golgi was observed in both cell types, but morefrequently in control chondrocytes (Figure 5). Largenumbers of pinocytotic vesicles are seen on thesurfaces of both HME and control chondrocytes andboth free ribosomes and groups of ribosomes arepresent in the cytoplasm.

DISCUSSIONIn this study, we demonstrate that HME

chondrocytes grown in monolayer culture exhibit anabnormal ultrastructure, including alterations in cellattachment, cytoskeleton, intracellular differentiation,and extracellular matrix. This altered ultrastructuralphenotype affects cell¡cell and cell¡matrix interac-tions involved in signaling events necessary forchondrocyte differentiation. Interference with signalingactivity has been implicated in a number of chon-drodysplasias [12].

Disruptions in cell surface architecture, including thenature, number, and manner of display of surfacemolecules, has been suggested as a cause ofHME-linked bone tumors [12]. HME cells areabnormally adhesive, forming large numbers of focaladhesions (podosomes in Figure 3C) and largeaggregates of tightly adherent cells when cultured inrotation [13]. The increased adhesiveness of these cellscompared to control chondrocytes indicates that thesurface molecules of HME cells differ from those ofcontrol cells. In the mouse chondrodysplasia brachy-podism (bpH=bpH), the lack of cell surfacedifferentiation, which includes changes in cell surfacecarbohydrates, results in increased and prolongedadhesiveness and ultimately shortened long bones[14¡17]. Thus, an inability to glycosylate surfacemolecules at the appropriate time in chondrocytedifferentiation can lead to abnormally adhesive cells[18]. The increased adhesiveness noted in HME cellsmay also be due to changes in heparan sulfateglycosylation.

FIG. 2 Transmission electron micrographs showingcross sections of cultured chondrocytes. …A†Control chondrocytes show normalmitrochondria …M†, collagen filbrils andproteoglycan granules are seen in the matrix…MX†, and microfilaments are present in normalamounts. …B† HME chondrocytes have manymore microfilaments (arrows), shorter channelsof rough endoplasmic reticulum …ER†, andmore numerous podosomes …P† than controls.N, nucleus. Bar ˆ 0.5 mm.

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Heparan sulfate proteoglycans, found on cellsurfaces and at cell¡matrix interfaces, modulatenumerous extracellular ligands involved in cell¡cellinteractions, growth regulation, and differentiation ofdeveloping tissues, including the differentiationof cartilage [19]. When added to micromass cultures ofembryonic chick limb mesenchyme, heparan sulfatestimulates chondrogenesis as assessed by cartilagenodule formation and growth [20]. Heparan sulfatemay interact with specific cell surface receptors ormodify the extracellular domain to operate as receptorsfor growth factors. Syndecan 3, a heparan sulfateproteoglycan with extracellular, transmembrane, andintracellular domains, is expressed in chondrocytes at ahigh level during the proliferative stage of endo-chondral ossification [21]. Thus, chondrocytes withaltered amounts of heparan sulfate may have alteredbinding of growth factors involved in regulatingchondrocyte proliferation and=or differentiation.Moreover, HME is a disorder that affects chondrocytesand only secondarily affects bone growth.

The signaling defect may also be related to theobserved alterations in the cytoskeleton of HME cells,namely, the large amounts of actin bundles,cross-linked with muscle specific a-actinin. Actin isinvolved in signaling via cell processes, and in signaltransduction via the integrin family of cell surfacereceptors. The binding of integrins to an alteredcytoskeletal complex may result in incorrect intra-cellular signaling affecting gene expression andregulation of chondrocyte differentiation [22]. Also,studies have suggesed that actin organization, rather

FIG. 3 Transmission electron micrographs of HME condrocytes. …A† Cross section of cell showing numerouscell processes …PR†, abnormal nuclear constrictions …arrows†, and large amounts of microfilaments …F†.Bar ˆ 1 m. …B† Higher magnification of a cellular process showing microfilaments …F†. Bar ˆ 0.25 mm.…C† Podosome …P†with associated microfilaments …F†. Bar ˆ 0.5 mm.

FIG. 4 Elongated mitochondrion in EXT chondrocyte.Note degeneration of cisternae at eitherend …arrowheads†. Bar ˆ 0.5 mm.

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than changes in cell shape, may be involved inmodulating the chondrogenic phenotype. Chondro-cytes with microfibrillar actin synthesize chondroitinsulfate proteoglycan (CSPG) and type II collagen, butchondrocytes showing well-defined actin cables lacksuch synthesis [23].

It is not known if the abnormal cell interactionsinduce the excessive cytoskeleton in HME cells, or ifthe abnormal numbers of microfilaments appear first,perhaps participating in organizing of surface mol-ecules into focal adhesions. There are some indicationsthat the former is the case. In pancreatic acinar cells,activation of focal adhesion kinase upregulates mRNAfor a-actin [24]. Syndecan 4 operates via the cyto-sekeleton and is involved in formaiton of focaladhesions [21], and exostotic chondrocytes inmonolayer culture form abnormal numbers of focaladhesions [11].

The unusual orientation of actin bundles, perpen-dicular to the cell’s long axis, contributes tothe compartmentalization by microfilaments of thecytoplasmandisolationof theorganelleswithin theHMEcell (Figure 2). This compartmentalization may interferewith the normal events of intracellular differentiation, inpart, due to a simple matter of volumes. ER cisternaecannot expand, trapped as they are in the cage offilaments, so less matrix molecules are produced,requiring less Golgi for secretion. The paucity oforganized, functioning Golgi correlates with theobserved deficiency of secreted material on to the cell’ssurface. HME chondrocytes do make and secrete matrixin vivo, but the interference of microfilaments withsecretion is indicated by the accumulation of meta-

chromatic vesicles along the actin bundles, represen-tative of impedance of secretory vesicle movement [11].The presence of more lysosomes in HME chondrocytesthan control chondrocytes may result from the rERcontentsbeing released into the cytoplasm, and removalof the defective mitochondria. The elongatedmitochondria seen in HME chondrocytes are typical ofcells from pathological conditions such as in epilepticchildren treated with valproic acid [25].

The bizarre nuclear lobulation seen ultrastructurallymay be secondary to the network of microfilaments,which have been reported to distend the chondrocyte,often pushing aside the nucleus [11]. Microfilamentsare also important in cell division, and the impairedability of HME cells to separate in culture could be dueto physical hindrance. Cytokinesis is most probablyprolonged, since division of the cell’s cytoplasmrequires that the gigantic microfilament bundles besevered, a task made more difficult by the excessivecross-linking seen previously [11]. In vivo, this inabilityto separate leads to the observed nests of cells [1, 2].

While the mechanism by which exostoses in HMEarise is not known, the orderly pattern of the growthplate is disrupted and the normal progression of cellsthrough the phenotypic modulations of the growthplate is altered [1, 2, 13]. Usually, a normalchondrocyte in the resting zone of the growth platereceives a signal that stimulates it to divide, with thedaughter cell flattening onto the top of the underlyingcolumn of the proliferative zone [26]. It is in theproliferative zone that most of the mitoses within theplate take place, mostly confined along the longitudinalplane. Growth in width is thought to occur by

FIG. 5 Electron micrograph of …A† EXT chondrocyte showing a portion of a chromatin bridge …arrow† in the nucleus…N†, Golgi region …G†, numerous podosomes …arrowheads†, cell process …P†, short channels of roughendoplasmic reticulum …ER†, microfilaments …F†, and low amount of matrix in the extracellular space;…B†control chondrocyte showing, normal amounts of matrix in the extracellular space …MX†, normal roughendoplasmic reticulum …ER† and Golgi region …G†. Cell¡substrate interactions are apparent …arrowhead†but organization into focal adhesions …podosomes† does not regularly occur.

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FIG. 6 …A† Diagram of a normal condition of a growth plate showing normal endochondral ossification throughreserve zone …RZ†, proliferative zone …PZ†, and hypertrophy=calcification zone …HCZ†, which results in anormal bone. …B¡E† Diagram of a growth plate with EXT mutation. The shaded cell in EXT respondsabnormally, temporally and spatially. The cell is left behind in the differential process while other cellsrespond normally …C†. When an EXT cell enters the proliferative stage, it doesn’t divide in the correctdirection to continue with the normal clonal expansion of cells along the axis of the bone …D†. Instead, thedelayed secondary endochondral ossification area forms an outgrowth in the bone …E†.

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recruitment of cells from the perichondrium sur-rounding the growth plate. Mitoses on the horizontalplane have also been observed leading to the formationof new columns of cells, which then undergohypertrophy, mineralization, and apoptosis [27].

In HME, with expression of an abnormal EXT gene, achondrocyteora perichondrialprechondrocyte isunableto undergo the normal progression of cell differentiationin the growth plate. We hypothesize that it is the initialfailure of a stem cell (from the reserve zone or theperichondrium) to undergo the phenotypic modulationassociated with the proliferative zone of the growth platethat may be the origin of the tumor itself (Figure 6). Themutated cell, which may undergo additional mutationalsteps [28], looses its ability to establish the signalingevents associated with normal growth plate regulation.Instead, the mutated cell responds abnormally, bothtemporally and spatially.The stemcell is leftbehind in thedifferential process while other cells respond normally.When this cell enters the proliferative stage, it cannotrespond correctly to signaling events ensuring that thenormal clonal expansion of cells occurs in the rightdirection, i.e., along the long axis of the bone. Instead,the cells do not form columns and proliferate in thewrong direction. Growth of the exostosis forming anoutgrowth in the bone occurs by endochondralossification, sothat thecells canatsomepoint respondtosome signaling events, forming columns, becominghypertrophic, secreting type X collagen, mineralizing,and being replaced by bone.

Cells within the secondary growth-plate-like regionformed by this first errant chondrocyte may repeat theprocess producing additional branching growths,resulting in a cauliflowerlike construction such asoccurs in organs like the salivary gland. In the typicalHME lesion, a ``club-shaped thickening of themetaphysis heaped and cleft by innumerable bonyexcrescences,’’ which may become ``cauliflowerlike,’ ’the cartilage cap appears to have numerous growthplates [2]. Each growth plate perhaps arises from aseparate nonresponsive and thus misdirectedchondrocyte.

In summary, the altered ultrastructural phenotypeobserved in HME chondrocytes grown in monolayerculture is evidence of abnormal signaling events takingplace during differentiation of these cells. We arecurrently developing 3-dimensional culture systems forproliferation and differentiation of human chondrocytes[29, 30] which may allow us to determine how thealtered products of the EXT genes affect thechondrocyte’s ability to respond to signals necessaryfor patterning of the growth plate.

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5. Hecht JT, Hogue D, Strong LC, Hansen MF, Blanton SH, WagnerM. Hereditary multiple exostosis and chondrosarcoma: linkage tochromosome 11 and loss of heterozygosity for EXT-linkedmarkers on chromosomes 11 and 8. Am J Hum Genet.1995;56:1125¡1131.

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7. McCormick C, Leduc Y, Martindale D, et al. The putative tumoursuppressor EXT1 alters the expression of cell-surface heparansulfate. Nat Genet. 1998;19:158¡161.

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21. Shimazu A, Nah HD, Kirsch T, et al. Syndecan-3 and the controlof chondrocyte proliferation during endochondral ossification.Exp Cell Res. 1996;229:126¡136.

22. Loeser RF. Chondroycyte integrin expression and function.Biorheology. 2000;37:109¡116.

23. Mallein-Gerin F, Garrone R, van der Rest M. Proteoglycan andcollagen synthesis are correlated with actin organization indedifferentiating chondrocytes. Eur J Cell Biol.1991;56:364¡373.

24. Kiehne K, Herzig KH, Folsch UR. Regulation of the actincytoskeleton by p125 focal adhesion kinase in rat pancreaticacinar cells. Digestion. 1999;60:153¡160.

25. Melegh B, Trombitas K. Valproate treatment induces lipid globuleaccumulation with ultrastructural abnormalities of mitochondriain skeletal muscle. Neuropediatrics. 1997;28:257¡261.

26. Hunziker EB, Schenk RK. Physiological mechanisms adopted bychondrocytes in regulating longitudinal bone growth in rats.J Physiol. 1989;414:55¡71.

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