Phenotypic switching of in vitro mandibular condylar cartilage during matrix mineralization

Anatomical Science International

(2002)

77

, 237–246

Blackwell Science, Ltd

Original Article

Phenotypic switching of in vitro mandibular condylar cartilage during matrix mineralization

Hiroyuki

Inoue

,

1

Yuji

Hiraki

,

1

Tokio

Nawa

2

and Kiyoto

Ishizeki

2

1 Department of Molecular Interaction and Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto and

2 Department of Oral Anatomy, School of Dentistry, Iwate Medical University, Morioka, Japan

Abstract

In order to analyze the phenotypic conversion of chondrocytes, mandibular condyles of mice and rabbitswere cultured under cell and organ culture systems, and then examined by a combination of morphologicaland biochemical procedures. In organ culture, mandibular condylar cartilage (MCC) obtained from newbornmice began to mineralize from the central zone and then progressively widened towards the peripheralzone. Electron microscopic observations showed that with the increasing duration of the organ culture,chondrocytes at the central zone converted into spindle-shaped osteoblastic cells accompanying theformation of the bone type of thick-banded collagen fibrils. To obtain a better understanding of thechondrocytic conversion, immunolocalizations for type I and type X collagens and osteocalcin (OC) wereexamined in mouse MCC cells in cell culture. Type X collagen and OC were expressed almost simultaneouslyat the late stage of culture, and type I collagen was detected along the calcified nodues after the productionof these proteins. Northern blot analysis in cell cultures of rabbit MCC indicated that type II collagen andalkaline phosphatase (ALPase) messenger ribonucleic acids (mRNAs) were highly expressed at day 7, butsubsequently decreased. In contrast, mRNA for type I collagen was expressed at a low level on day 7 andpeaked on day 12. The present results suggest that, morphologically and biochemically, cellular modificationin MCC cells under culture conditions occurs at a cellular morphological level and also at marker-gene-expression level.

Key words:

bone-marker genes, cell culture, chondrocyte, mandibular condyle, mineralization.

Introduction

Mandibular condylar cartilage (MCC) is secondarycartilage and functions as an important growthcenter of the mandible by an endochondral type ofbone formation during embryonic morphogenesis.The MCC is composed of four distinct zones offibroblast-like cells covering the articular surface,chondroprogenitor cells, chondroblasts, and themature chondrocyte zone that includes hypertro-phic cells (Silbermann

et al

., 1983; Weiss

et al

., 1986;Lewinson

et al

., 1991). In general, it has beenaccepted that during endochondral bone formation,chondrocytes reaching cellular hypertrophy degen-erate and ultimately die at the osseous-cartilagejunction (Jee, 1988). However, several studies havesuggested that some hypertrophic chondrocytesare released into marrow spaces (Yoshioka & Yagi,1980, 1988) and might be actively involved in bone

formation (Holtrop, 1972; Silbermann & Frommer, 1972,1974; Galotto

et al

., 1994). Ben-Ami

et al

. (1993) havereported that during the culture of mandibular con-dyles, the chondroprogenitor cells undergo osteogenicdifferentiation and form new bone. Biochemical findingssuggested that under certain culture conditions notonly cells in the progenitor zone in the MCC but alsothose in the maturation zone start to express genestypifying osteoblast differentiation (Celeste

et al

.,1986; Schmidt

et al

., 1986; Yoon

et al

., 1988; Lian

et al

.,1989). These two zones have also shown markedincreases in expressions of messenger ribonucleicacids (mRNAs) for bone-type proteins such as collagentype I, osteonectin, alkaline phosphatase (ALPase),osteopontin and osteocalcin (OC) during organ cultureof MCC isolated from newborn mice (Strauss

et al

.,1990). Therefore, it is noteworthy that cells in MCCundergo the expression of genes characteristic ofosteogenic differentiation.

The chondrocytes in the growth plate of longbones

in vivo

are reported, morpholologically andbiochemically, to convert into bone-forming cellsincluding osteoblasts (Holtrop, 1972; Silbermann &Frommer, 1974; Galotto

et al

., 1994), but that suchphenotypic switching occurs less frequently than that

Correspondence: Kiyoto Ishizeki, Department of Oral Anatomy, School of Dentistry, Iwate Medical University, Morioka 020-8505, Japan. Email: [email protected]

Received 23 January 2002; accepted 5 June 2002.

ASI_031.fm Page 237 Tuesday, October 29, 2002 3:57 PM

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et al.

in MCC. However, it is difficult to confirm at a cellularlevel the conversion into osteoblastic cells

in vivo

.Although MCC in culture is a powerful model forstudying cellular conversion, only a few studies usinga cell-culture system have been reported (Takigawa

et al

., 1984; Engel

et al

., 1990). Here, we investivatedosteoblastic conversion of MCC cells in cell as wellas organ culture. Cellular phenotypes were char-acterized by morphological and biochemical ana-lyses. The aim of this study was to determine whetherMCC in organ- and cell-culture systems recapitulatephenomena for conversion into osteoblastic cells.Hence, we investigated, using morphological andbiochemical combinations, the characteristics ofMCC under culture conditions.

Materials and methods

Dissection of the MCC

Newborn mice (ddY strain) and 2–3-week-old maleNew Zealand rabbits were killed with an overdoseof carbon dioxide according to the protocols forresearch projects approved by the New Energy andIndustrial Technology Development Organization(NEDO) of Kyoto University and conforming to theprovisions of the Declaration of Helsinki in 1995(as revised in Edinburgh 2000). Both mandibularcondyles with part of the ramus were removedaseptically from the temporomandibular joints. TheMCC was dissected under a microscope, andspecial care was taken to eliminate any remainingfragments of attached muscles, fibrous articular layersconnected to the periosteum, and any underlyingbone. The apical progenitor zone of mouse MCC waseliminated prior to starting the organ culture becauseof the possibility that osteoprogenitor cells werecontained in this zone.

Organ culture of mouse MCC

The MCC isolated from 1-day-old mice was grownby a modified Trowell-type culture system accordingto Ishizeki

et al

. (1996a). In brief, explants wereplaced on a membrane filter (Chemotaxicell; Curabo,Osaka, Japan) at the medium–gas interface andwere cultured for 4 weeks in a-modified Eagle’smedium (

α

-MEM; Irvine Scientific, Santa Ana, Cali-fornia, USA) supplemented with 10% fetal bovineserum (FBS; ICN Biomedicals, Ohio, Aurora, USA),30mg/mL

L

-ascorbic acid phosphate megnesiumsalt-

n

-hydrate (Wako Pure Chemical Industries, Osaka,Japan), 60 µg/mL kanamycin (Gibco BRL, GrandIsland, NY, USA) and 5mmol

β

-glycerophosphate(Nacalai Tesque, Kyoto, Japan) at 37

°

C in a humid-ified atmosphere containing 5% carbon dioxide. Themedium was changed every other day.

Cell culture of rabbit and mouse MCC cells

Chondrocytes were isolated from MCC of 2-week-oldrabbits and 17-day-old embryonic mice by a modi-fication of the procedure described by Takigawa

et al

. (1984). After all condyles from both specieswere removed, tissue was minced and incubated incalcium- and magnesium-free phosphate-bufferedsaline (PBS) with 0.1% ethylenediaminetetraaceticacid (EDTA) for 30 min at 37

°

C, followed by 0.15%trypsin in PBS for 30 min and 0.15% collagenase(Boehringer Mannheim, Indianapolis, USA) in PBS for1h. Rabbit condylar chondrocytes were culturedat a density of 1

×

10

6

cells/35 mm dish in a 1:1(v/v) mixture of Ham’s F-12 medium and Dulbecco’smodified Eagle’s medium (DME/F12 medium)containing 10% FBS. After reaching confluence, thecells were trypsinized and inoculated in 48-microwellplates (Corning, New York, USA) at a density of5

×

10

4

/well, and in 10cm tissue-culture dishes(Corning) coated with type I collagen at a densityof 5

×

10

6

cells/dish, respectively. Cells were main-tained in DME/F12 medium containing 10% FBSand 50µg/mL ascorbic acid at 37

°

C in a 5% carbondioxide in air. The culture medium was renewedevery other day.

In contrast, mouse mandibular chondrocytes wereinoculated at a density of 1

×

10

4

cells/0.28cm

2

in aPenicilynder cup (Top Labo-Ware, Osaka, Japan)that was placed in the center of 35-mm dishes(Falcon, Fukushima, Japan) and cultured for up to3 weeks with medium for organ culture as describedbefore. These cultures were then used for immuno-histochemical analyses.

Immunohistochemical procedure

Type I and X collagens and OC were examinedimmunohistochemically to identify the cartilage andbone-type marker proteins. Two-to-three-week cultured-mouse MCC cells were fixed in 4% paraformal-dehyde for 30 min at room temperature and washedthoroughly with PBS (pH7.2). Cultures were reactiv-ated with a STUF MARK 2 unmasking solution kit(Serotec, Raleigh, NC, USA) for 10min and endog-enous peroxidase was blocked by treatment with3% H

2

O

2

in methanol for 10 min at room temperature.After washing with PBS, specimens were incubatedwith rabbit antirat type I collagen antibody (LSL, Tokyo,Japan) diluted 1:200 in PBS containing 0.1% BSA,rabbit antirat type X collagen antibody (MAP, LSL)diluted 1:100 with PBS, or rabbit antimouse OC ata dilution of 1:500 (a gift from Dr S. Ishizuka, TeijinBiomedical Research Institute, Tokyo, Japan) for 1.5 hat 37

°

C. After washing, specimens were incubated withhorseradish-peroxidase-conjugated (HRP-conjugated)goat antirabbit immunoglobulin G second antibodies


Phenotypic switching in chondrocytes

239

(CAPPEL; Organon Teknika, Durham, NC, USA, 1:500dilution) for 1h at 37

°

C. Immunoreactivity was visualizedwith 3,3

′

-diaminobenzidine (DAB) that contained0.05% H

2

O

2

for approximately 10 min, and then coun-terstained lightly with hematoxylin. Control cultureswere incubated directly with second antibodies inthe absence of primary antibodies and processedas above. No significant positive immunoreactivitywas found in controls.

Histological and histochemical observations

For histochemical analysis of mineralization, vonKossa’s and alizarin red stainings were performed.Rabbit MCC cells cultured for 2 weeks were fixedwith 4% paraformaldehyde, washed thoroughly inPBS, and incubated with 5% silver nitrate for 20 min(Ishizeki

et al

., 1996b). For alizarin red staining, thecells in the 48-well plates were fixed with absolutemethanol and stained with 1% alizarin red S (Merck,Darmstadt, Germany) at pH 6.4 (Shukunami

et al

.,1997).

For light microscopic observations, undecalcifiedsemithin sections (1 µm), obtained from epoxy-resin(Epon-812; Taab Laboratories Equipment Ltd, Alder-maston, UK) samples for electron microscopy, werestained with 0.1% toluidine blue (pH 3.0).

For electron microscopy, samples of mouse-organculture were fixed in cold buffered 2.5% glutaralde-hyde (pH 7.2) for 2–4h and postfixed in 1% osmiumtetroxide containing 1.5% potassium ferrocyanide(Ishizeki

et al

., 1996b). After being dehydrated througha graded ethanol series, these specimens wereembedded in Epon 812 according to conventionalprocedures. Ultrathin sections in the absence ofdecalcification were cut with a diamond knife on aLKB-8800 ultrotome (Bromma, Sweden). These sec-tions were then stained with uranyl acetate and leadcitrate prior to examination under a H-7100 (Hitachi,Tokyo, Japan) electron microscope.

Measurement of alkaline phosphatase (ALPase) activity in rabbit MCC cells

The ALPase activity was measured by a modificationof the method by Bessey

et al

. (1946) using

p

-nitrophenyl phosphate as a substrate. Cultures werehomogenized with a glass homogenizer in 0.9%NaCl/0.2% Triton X-100 at 0

°

C and centrifuged for15 min at 12 000

g

. The supernatant was assayed in0.5 mol/L Tris/HCl buffer (pH 9.0) supplemented with0.5 mmol/L

p

-nitrophenyl phosphate and 0.5 mmol/LMgCl

2

. The reaction mixture was incubated at 37

°

Cfor 30 min, and the reaction stopped by the additionof a quarter volume of 1 mol/L NaOH. Hydrolysis of

p

-nitrophenyl phosphate was monitored by a spec-trofluorophotometer as changes in A410.

P

-Nitrophenolwas used as a standard.

Northern blot analysis in rabbit MCC cells

For isolation of total RNA, cells in 10-cm culturedishes were lyzed with solution D (4 mol GuanidineThiocyanate, 25 mmol sodium citrate, 0.5% sarcosyl,0.1 mol 2-mercaptoethanol) and immediately frozen.Total RNA, which was isolated by the phenol/chloro-form extraction method as previously described(Chomczynski & Sacchi, 1987), was separated elec-trophoretically in a 2.2-mol formaldehyde-1% agarosegel and transferred to a nylon membrane using ablotting system (Schleicher and Schuell, Dassel,Germany). The membrane was baked for 2 h at 80

°

C.The following deoxyribonucleic acid (DNA) probeswere used; pKT 1180 containing rat collagen type

α

1(II) cyclic (c) DNA 1.4 kb; type I collagen (I-ACfrom Koken, Toshima, Tokyo), pKT 1809 containingrat collagen type

α

2 (I) cDNA (2 kb) as provided byDr T. Kimura (Osaka University), and rAP 54 contain-ing rat alkaline phosphatase cDNA (2.5 kb) providedby G. A. Rodan (Merk Sharp and Dohme ResearchLaboratory, USA). Inserts for DNA probes werelabeled using a random primer labeling kit (Strata-gene, La Jolla, CA, USA). Membrane was hybridizedwith 32P-labeled DNA overnight, and then washedwith 2

×

sodium chloride sodium citrate (SSC) and0.2% sodium dodecyl sulfate (SDS) for 15 min atroom temperature, and with 0.1

×

SSC and 0.2% SDSfor 15 min at 65

°

C. Autoradiography was carried outat

−

70

°

C using Kodak XAR film (Eastman Kodak,Rochester, New York, USA) and an intensifying filter.In most cases, the nylon membranes were strippedby washing in 5 mmol Tris-HCl, pH 8.0, 0.2 mmolEDTA, 0.05% pyrophosphate, 0.1% Denhardt’s rea-gent for 1–2 h at 65

°

C.

Results

Histological and histochemical features of mouse MCC in organ culture

Phase-contrast microscopic observations demon-strated that calcified matrix appeared initially in thecentral zone after 2 weeks in organ culture (Fig.1a)and at 4 weeks had expanded throughout theperipheral zone (Fig. 1b). Von Kossa’s staining con-firmed that the dark areas as seen by phase-contrast microscopy were in agreement with thecalcification deposited on the extracellular matrixsurrounding chondrocytes (Fig.1c).

Light microscopic analysis showed that after 1week in organ culture, the peripheral zone of MCCis covered with flattened cells and that the centralzone is occupied by abundant toluidine blue-positivecollagenous matrix (Fig. 2a). After 2 weeks in cul-ture, mineralization was initiated at two points in the


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et al.

central and peripheral zones (Fig. 2b). The calcifiedmatrix was more widely expanded toward peripheralzone at 3 weeks (Fig. 2c) and, at 4 weeks, it furtherexpanded into almost all areas of MCC (Fig. 2d).

Electron microscopy of mouse MCC in organ culture

Mandibular chondrocytes prior to organ culture wereshown to have centrally located large round nuclei,rough endoplasmic reticulum, some mitochondriaand glycogen aggregates (Fig. 3a). The nucleus wasclear and euchromatin-rich, and surrounded by arelatively long endoplasmic reticulum. Intercellularspaces were filled with fine collagenous matrix.

After 1 week in organ culture, the chondrocytesat the central area contained large nuclei, several

dense bodies, mitochondria and a well-developedGolgi-endoplasmic reticulum system, and weresurrounded by fine collagenous fibrils (Fig. 3b). After2 weeks in culture, chondrocytes were characterizedby an increase of dense bodies, vacuoles andrelatively long cytoplasmic processes, in additionto mitochondria and rough endoplasmic reticulum(Fig. 3c). These cells appeared to resemble the hype-rtrophic cells in intact cartilage, but it was especiallynoted that pericellular matrix in this stage containednewly formed thick collagen fibrils. At 3 weeks, cellsretained well-developed Golgi apparatus and wereaccompanied with many Golgi elements such asGolgi cisternae and Golgi vesicles (Fig. 3d). TheMCC cells cultured for 4 weeks contained large ovalnuclei, a few mitochondria, dense bodies and Golgi

Figure 1. Phase-contrast micrographs and von Kossa’s staining of mouse mandibular condylar cartilage (MCC) in organ culture.(a) Photomicrograph of explant cultured for 2 weeks. Matrix mineralization is initiated in the hypertrophic cartilage of the central zone.(b) Explant after 4 weeks in organ culture. Note the calcified matrix expanding from the central zone to the peripheral zone. (c) VonKossa’s staining after 2 weeks in culture shows a calcified matrix expanded throughout MCC. Magnification (a,b) ×35, (c) × 150.

Figure 2. Light micrographs of mouse mandibular condylar cartilage (MCC) in organ culture by staining with toluidine blue. (a) After1 week of organ culture, the top of the mandibular condyle is covered with spindle-shaped fibroblastic cells, and the central zone isoccupied by abundant collagenous matrix. (b) Matrix mineralization is initiated at two points in the central and peripheral zones(arrowheads) of condylar cartilage after 2 weeks of culture. (c) After 3 weeks in culture, the matrix mineralization, stained dark bluewith toluidine blue, expanded widely. (d) By 4 weeks in culture, extracellular matrix intensively mineralized and cells changed intospindle-shaped cells. (a–d) Magnification ×80.



241

apparatus, but generally organelles were poorlydeveloped (Fig. 3e). Pericellular spaces filled withuncalcified thick collagen fibrils were narrowed andelongated cytoplasmic processes penetrated intothe calcified extracellular matrix. This morphologicalappearance resembled that of osteoblasts.

Immunohistochemical localization of type I and X collagens and OC

Mouse mandibular chondrocytes cultured for upto 3 weeks were analyzed for immunostaining. Theexpression of type X collagen was identified aroundhypertrophic cells after 2 weeks of culture, but stain-ing was absent in the peripheral cells of cartilagenodules (Fig. 4a). We strongly detected OC in thenodule-forming round cells, and hypertrophicchondrocytes, which were characterized by meta-chromatic and enlarged cell bodies, were slightlyimmunostained after 2 weeks in culture (Fig. 4b). Theexpression of OC protein was initiated substantially

at the same time as type X collagen, but was iden-tified over longer periods than type X collagen.

When the specimens collected at 3 weeks inculture were immunostained for type I collagen,immunoreactivity was detected in association withcells encircled by extracellular matrix located at the topof nodules (Fig. 4c). However, no staining was observedin nodular structures at the early stages of culture.

Histological and histochemical features of rabbit MCC in cell culture

When MCC cells grown for 1 week were observed bystaining with alizarin red, they adopted fibroblasticor polygonal appearances (Fig. 5a). Consequently,these cells formed nodules and cells located on thenodules changed to typical round chondrocytes after2 weeks in culture. On certain nodules, calcifica-tion appeared around some chondrocytes (Fig. 5b).Chondrocytes at 3 weeks in culture hypertrophiedand the intercellular matrix surrounding them became

Figure 3. Electron micrographs of mousemandibular condylar cartilage in organ culture.(a) Intact chondrocyte prior to organ cultureobtained from 1-day-old mouse. This cell,surrounded by fine collagenous fibrils (FC),contains mitochondria (MT), endoplasmicreticulum (ER), a few dense bodies (DB) andglycogen patricles (G) in addition to a largeround nucleus (N). (b) After 1 week in culture, acell at the central zone contains N, MT, DB andwell-developed Golgi apparatus (GA). FC, finecollagenous fibrils. (c) By 2 weeks, the cell thatcontains ER, vacuoles (V) and many DBs formsnewly thick banded collagen fibrils (BC) inthe pericellular space. (d) The MCC cell after3 weeks in culture is surrounded by calcifiedmatrix (CM) and occupied by large oval N andwell-developed organelles such as a fewMT, DB, GA and ER. (e) A cell surrounded byintensively CM at 4 weeks of culture is char-acterized by spindle-shaped morphology andelongated cytoplasmic processes (arrowheads)as osteoblasts. Bars,1 µm.


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et al.

intensively calcified (Fig. 5c). By further increasingthe culture duration, the calcified matrix coveringnodular structures expanded extensively (Fig. 5d).

The changes on standing in rabbit MCC cultureswere characterized by the stainings with alcian bluefor cartilage-type proteoglycans and with alizarin redfor showing calcium deposition (Fig. 6). The alcianblue-positive matrix increased at 13 days in cultureand then decreased gradually, whereas alizarinred-positive areas increased during days 16–27 inagreement with matrix mineralization.

Biochemical features of the rabbit MCC in culture

The ALPase activity revealed a peak at approxim-ately 15 days showing initial mineralization (Fig. 7).After that, the level of ALPase activity decreased toapproximately 50 nmol/min/µg DNA with increasingduration of culture.

Northern blot analysis of the total RNA extractedfrom MCC cells showed that mRNA for type II col-lagen was expressed at high levels at day 7 andthen declined (Fig. 8a), and that ALPase mRNA

Figure 4. Immunohistochemical localization of type X and type I collagens and osteocalcin (OC) in mouse mandibular condylarcartilage cells. (a) High expression of type X collagen is recognized intensively in nodular matrix surrounding hypertrophicchondrocytes after 2 weeks in culture. (b) Staining of OC showing the intracellular immunolocalization in nodule-formingmetachromatic chondrocytes and round cells after 2 weeks in culture. (c) Immunohistochemical distribution of type I collagen by 3weeks of culture. Immunoreactivity is detected around the nodule-forming cells. Bar,30 µm.

Figure 5. Alizarin red staining of rabbitmandibular condylar cartilage (MCC) cells in cellculture. (a) The MCC cells grown for 1 weekadopt fibroblastic and polygonal appearances.(b) After 2 weeks in culture, many cells changeinto typical round chondrocytes and matrixcalcification is initiated in some places(arrowheads). (c) At 3 weeks of culture, cellslocating at the top of nodules induce matrixcalcification around these cells. (d) With anincreasing duration of culture, until 4 weeks, thecalcified matrix expands extensively along thenodular structures. Bar,30 µm.



243

was expressed highly at day 7 and declined gradu-ally to be almost undetectable by day 22 (Fig. 8b). TypeI collagen mRNA that was expressed at a low levelat day 7 increased to a peak level at day 12 anddecreased gradually to day 7 level by day 22 (Fig. 8c).

Discussion and conclusions

We have demonstrated that chondrocytes in mouseand rabbit mandibular condyles evoke osteogenicpotential

in vitro

. Immunohistochemical and histolog-ical analyses revealed that in the cell and organcultures of mouse MCC, type I collagen was pro-duced by cells converting into spindle-shaped cells,accompanied by the expression of type X collagenand OC at a late stage of culture. These findingswere also supported by biochemical analyses in cellcultures of rabbit MCC showing that instead of adecrease in expression of type II collagen mRNA,the expressions of type I collagen and ALPase

Figure 8. Northern blot analysis of total ribonucelic acid (RNA)extracted from rabbit mandibular condylar cartilage cells grownfor 7, 12, 17, and 22 days. Arrows refer to the position ofribosomal RNAs. (a) Note that type II collagen messenger (m)RNA is expressed at a high level at day 7 and declinesthereafter. (b) ALPase mRNA is also expressed highly at day 7and declines gradually to be almost undetectable by day 22.(c) Type I collagen mRNA that was expressed at a low level atday 7 increases to a peak level at day 12 and decreasesgradually to the day 7 level by day 22.

Figure 6.

Histochemical stainings of rabbit mandibular con-dylar cartilage cells in cell culture. Proteoglycan-rich matrix,which was stained positively with alcian blue, appears at 13days in culture, and then gradually decreases. In contrast,alizarin red-positive areas showing matrix mineralizationincrease with time in culture.Magnification

×

0.8.

Figure 7.

Alkaline phosphatase (ALPase) activity in rabbitmandibular condylar cartilage cells. Note that the activity forALPase becomes highest prior to matrix mineralization. TheALPase activity and deoxyribonucleic acid contents weremeasured as described in Materials and Methods. Values aremeans

±

standard deviations for four wells.


244

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et al.

mRNAs increased reversibly. Thus, the present find-ings suggested that cellular modification in MCCunder culture conditions occurred not only in thephenotypic changes but also at a gene level.

The morphology of cells converting to osteoblasticcells under organ culture was distinguished by sev-eral stages. Osteoblastic cells locating in the centralzone of the MCC were surrounded by thick-bandedcollagen fibrils formed in the pericellular matrix. Thick-banded collagen fibrils were easily distinguishablefrom the fine type II collagen fibrils seen in intactcartilage matrix. These fibrils ultrastructurally resem-bled those reported in chondrocyte transformationof Meckel’s cartilage in culture (Ishizeki

et al

., 1996b,1997) and the femur growth plates of chick embryos(Roach

et al

., 1995; Roach, 1997), and were identi-fied as type I collagen in bone-composing collagenmatrix. If type I collagen appeared initially in theprechondral cells covering condyles, it should havebeen identified early in the culture. However, suchcollagen fibrils were not detected either morpholog-ically or biochemically early in the culture, but wereformed in the pericellular spaces during later stageof the culture. Therefore, the switching of fibrillogen-esis to type I collagen is involved in the osteogenicconversion of chondrocytes and is indicative ofosteogenic conversion.

The evidence of the conversion based on mor-phological findings was also supported by immuno-histochemical analyses for type I and type Xcollagens and for OC. Type X collagen, which is aspecific collagen in the hypertrophic chondrocytes,was immunolocalized in the chondrocytes after2 weeks in culture. Therefore, it is clear that thechondrocytes in the present culture converted toosteogenic cells after the terminal differentiation ofchondrocytes. Although OC shows a high affinity forhydroxyapatites crystals and has been used as aprincipal marker protein of bone matrix, it is alsopresent in dentin, cementum and calcified cartilages(Hauschka, 1977; Hauschka & Reid, 1978; Glimcher

et al

., 1979; Linde

et al

., 1982). Thus, the conversionof chondrocytes in the present cultures cannot beproved by just expression of OC, but the sequentialexpression of type I collagen as demonstrated byimmunostaining and Northern blotting strongly sug-gest chondrocyte conversion.

Biochemical findings also support events forcellular transformation in that cells converted fromchondrocytes expressed marker genes for bone(Celeste

et al

., 1986; Weiss

et al

., 1986; Lian

et al

.,1989; Strauss

et al

., 1990; Descalzi-Cancedda

et al

.,1992). In the present study, peaks of expression formRNAs of ALPase and type I collagen followed afterthe expression of type II collagen mRNA (Fig. 8).Moreover, two peaks of expression of type I collagen

were recognized after 7 days and 2 weeks in culture.Mizoguchi

et al

. (1990) reported that secondary-typecartilage, such as MCC, contains type I, II and Xcollagens. Furthermore, Inoue

et al

. (1995) observedbiphasic expression for type I collagen mRNA thatpeaked at the start of culture (days 1–4) and thebeginning of mineralization (days 13–16) in rat MCCcells during long-term culture, and suggested that thesecond peak of type I collagen at days 13–16 mightbe related to the osteogenic differentiation of MCC cells.As we thoroughly removed the perichondrium consistingof the apical progenitor zone in the mandibular con-dyles prior to organ and cell culture, it is unlikely thatosteogenic cells were differentiated directly from per-ichondrium. In addition, the expression of the high levelof type I collagen mRNA was not constantly maintainedduring culture periods, and type I collagen protein wasnot identified immunohistochemically at the early stageof culture. Thus, the first expression of type I collagenmRNA on day 7 is regarded as a temporary reactionfor chondrocytes in response to environmental changesunder culture conditions. In the organ culture, typeI collagen fibrils appeared initially around the spindle-shaped cells: the cells synthesizing thick periodicbanded fibrils were converted from chondrocytes.Therefore, a peak of type I collagen mRNA as detectedbiochemically at 2 weeks in culture might result insuch osteoblastic cells being associated with themorphological conversion of cells into osteoblasticones, as indicated by Inoue

et al

. (1995).In summary, by the combination of morphological

and biochemical analyses, we have clarified thatphenotypic switching occurred in chondrocytes ofMCC

in vitro. Although there are a few reports on theconversion of chondrocytes in growth-plate cartilageof long bones in vivo (Holtrop, 1972; Yoshioka & Yagi,1988; Galotto et al., 1994), they are few in numberin comparison with those of cartilage derived froman ectomesenchymal origin, such as chicken chon-droid bone (Hall, 1972; Beresford, 1981; Lengeléet al., 1996) and mouse Meckel’s cartilage (Ishizekiet al., 1996b, c, 1997), including MCC. Further studyis required to ascertain whether the phenotypicconversion in MCC in the present study is either anintrinsic feature in ectomesenchymal-derived cartilageor is caused by culture conditions.

Acknowledgments

This study was supported in part by a Grant-in-Aidfor Scientific Research (no. 13671909 to Dr T. Nawa)from the Ministry of Education, Science and Cultureof Japan, and a Grant-in-Aid for High-PerformanceBio-Medical Materials Research from the Ministry ofScience, Education, Sports and Culture of Japan (toDr T. Nawa and Dr K. Ishizeki). This study was also


Phenotypic switching in chondrocytes 245

supported in part by a grant (to Dr Y. Hiraki) fromNEDO.

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Phenotypic switching of in vitro mandibular condylar cartilage during matrix mineralization