In VitroChondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

238, 265–272 (1998)

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In Vitro Chondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells Brian Johnstone,1 Thomas M. Hering,* Arnold I. Caplan,† Victor M. Goldberg, and Jung U. Yoo Skeletal Research Center and Department of Orthopaedics, *Department of Medicine, and †Department of Biology, Case Western Reserve University, 2080 Adelbert Road, Cleveland, Ohio 44106

A culture system that facilitates the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal progenitor cells has been developed. Cells obtained in bone marrow aspirates were first isolated by monolayer culture and then transferred into tubes and allowed to form three-dimensional aggregates in a chemically defined medium. The inclusion of 1007 M dexamethasone in the medium induced chondrogenic differentiation of cells within the aggregate as evidenced by the appearance of toluidine blue metachromasia and the immunohistochemical detection of type II collagen as early as 7 days after beginning threedimensional culture. After 21 days, the matrix of the entire aggregate contained type II collagen. By 14 days of culture, there was also evidence for type X collagen present in the matrix and the cells morphologically resembled hypertrophic chondrocytes. However, chondrogenic differentiation was achieved in only approximately 25% of the marrow cell preparations used. In contrast, with the addition of transforming growth factor-b1 (TGF-b1), chondrogenesis was induced in all marrow cell preparations, with or without the presence of 1007 M dexamethasone. The induction of chondrogenesis was accompanied by an increase in the alkaline phosphatase activity of the aggregated cells. The results of RT-PCR experiments indicated that both type IIA and IIB collagen mRNAs were detected by 7 days postaggregation as was mRNA for type X collagen. Conversely, the expression of the type I collagen mRNA was detected in the preaggregate cells but was no longer detectable at 7 days after aggregation. These results provide histological, immunohistochemical, and molecular evidence for the in vitro chondrogenic differentiation of adult mammalian progenitor cells derived from bone marrow. q 1998 Academic Press

cartilage when implanted in vivo [1–4]. A culture system has been developed in which these cells will undergo osteogenic differentiation in vitro [5]. However, attempts to develop in vitro conditions in which mesenchymal progenitor cells isolated from postnatal mammalian bone marrow will progress down the chondrogenic lineage have been less successful. There are studies reporting in vitro chondrogenesis using postnatal mammalian cells [6, 7], but none have demonstrated histologically identifiable cartilage formation, although type II collagen production has been detected, suggesting at least prechondroid tissue production. We have developed a culture system that facilitates the chondrogenic differentiation of postnatal mammalian marrow mesenchymal progenitor cells. This system is an adaptation of the ‘‘pellet’’ culture system that was originally described as a method for preventing the phenotypic modulation of chondrocytes in vitro [8, 9]. More recently, the system has been used in studies of the terminal differentiation of growth-plate chondrocytes [10, 11]. This culture system allows cell–cell interactions analogous to those that occur in precartilage condensation during embryonic development [12]. However, this cell configuration is not sufficient for the induction of chondrogenesis: the chondrogenic differentiation of the marrow-derived progenitor cells required the use of a defined medium to which were added certain bioactive factors, including dexamethasone and TGF-b1. This study describes the development of the system, and the consequent production of hypertrophic chondrocytes by the differentiation of bone marrow-derived mesenchymal progenitor cells. This system provides a means for studying the process of chondrogenesis, including those factors that regulate the progression of cells through the entire chondrogenic lineage.

INTRODUCTION

METHODS

Cells isolated from postnatal mammalian bone marrow have the potential for differentiation into the specialized cells of mesenchymal tissues such as bone and

Cell harvest and colony formation. Rabbit bone marrow was harvested from either the iliac crests or tibias of 30 5-month-old New Zealand White rabbits. The marrow was harvested from the proximal anterior tibial metaphysis or from the posterior superior iliac spine via small skin incisions. An 18-gauge needle was used to penetrate the cortex of the bone and 7–8 ml of marrow was aspirated into a syringe containing 3000 units of heparin. Dulbecco’s modified Eagle’s

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To whom correspondence and reprint requests should be addressed. Fax: (216) 368-1332. E-mail: [email protected].

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0014-4827/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.

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medium (DMEM) with 10% fetal bovine serum (FBS) was added to the aspirate and the number of nucleated cells was determined. The cells were plated out at 20 1 106/100-mm dish and grown for 14 days at 377C, 5% CO2 with medium changes every 4 days. Aggregate culture. On day 14, adherent colonies of cells were trypsinized, counted, and 2 1 105 cell aliquots were spun down at 500g in 15-ml polypropylene conical tubes. The FBS containing medium was then replaced with a defined medium, consisting of DMEM with ITS/ Premix (Collaborative Biomedical Products: insulin (6.25 mg/ ml), transferrin (6.25 mg/ml), selenous acid (6.25 mg/ml), and linoleic acid (5.35 mg/ml), with bovine serum albumin (1.25 mg/ml)). Pyruvate (1 mM) and ascorbate 2-phosphate (37.5 mg/ml) were also added. Aggregates were cultured with or without dexamethasone (1007 M), TGFb1 (0.5 to 10 ng/ml, recombinant human, R&D Systems), or a combination of these agents. For some experiments, the 10% FBS containing medium was not replaced. The pelleted cells were incubated at 377C, 5% CO2 . Within 24 h of incubation, the cells formed an essentially spherical aggregate that did not adhere to the walls of the tube. Medium changes were carried out at 2- to 3-day intervals and aggregates were harvested at time points up to 21 days. Alkaline phosphatase activity. Medium was removed from the aggregate cultures and they were rinsed in Tyrodes solution prior to incubation with 200 ml of 5 mM p-nitrophenylphosphate in 50 mM Tris, 150 mM NaCl, pH 9.0, for 30 min at room temperature. The resulting colorimetric reaction was quantified by determining the absorbance of the substrate solution at 405 nm. Histology and immunohistochemistry. For histological and immunohistochemical analyses, the aggregates were frozen in OCT and 5-mm sections were cut. For histological evaluation, sections were stained with toluidine blue. For immunohistochemistry, the frozen sections were fixed briefly for 10 min in methanol after a brief immersion in distilled water to remove the OCT. Blocking of nonspecific antibody binding sites was done by incubating the slides in 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h. The sections were then incubated with primary antibody for 30 min, diluted in 0.5% BSA in PBS. Two antibodies with epitopes in type II collagen were used: a polyclonal antibody (affinity purified antitype II, Rockland, Inc.) and a monoclonal antibody (C4F6, kindly provided by Clinton Chichester, URI). In addition, an antibody to type X collagen was used (kindly provided by Gary Gibson, Henry Ford Hospital, Detroit, MI). To facilitate antibody access to the collagens, the sections were predigested with chondroitinase ABC (0.1 U/ ml in 0.1 M Tris-acetate, Seikagaku). Reactivity was detected with fluorescence microscopy after incubation for 30 min with an FITClinked secondary antibody (either anti-rabbit Ig or anti-mouse Ig, Cappel) diluted in 0.5% BSA in PBS. RNA isolation. For two separate marrow cell preparations, RNA was prepared from the preaggregate mesenchymal progenitor cell cultures, and subsequent aggregate cultures from the same preparations, with a modification of the method of Chomczynski and Sacci [13]. Cells were lysed in culture dishes (1 ml/10 cm2) with TRIZOL reagent (Life Technologies Inc., Grand Island, NY). For each timepoint chosen, 20 pellets were pooled and homogenized using a Dounce homogenizer in TRIZOL reagent, and RNA was prepared as per kit instructions. RNA was isolated directly from 5-week-old rabbit articular cartilage using the method of Nemeth et al. [14]. RNA was quantified by comparing ethidium bromide fluorescence with a standard series of RNA dilutions [15]. Northern hybridization. RNA samples (15 mg) were electrophoresed through 1% agarose gels containing formaldehyde [15] and were transferred overnight to a nitrocellulose filter [16]. Following transfer, filters were dried and baked over 2 h at 807C. Filters were prehybridized at 427C in a solution containing 50% formamide, 51 SSPE, 21 Denhardt’s reagent, 100 mg/ml salmon sperm DNA, and 0.1% SDS [15] for several hours. Northern blots were sequentially hybridized at 427C in the same solution with 32P-labeled cDNA probes for the a2(I) chain of type I collagen, and the a1(II) chain of type II collagen prepared by random primer labeling, and with a cDNA probe

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for EF-1a [17] to control for differences in RNA loading. Blots were rinsed and then washed three times at 247C with 500 ml of 0.1X SSPE, 0.1% SDS, and twice at 547C (a1(II) probe) or 657C (a2(I) probe) with 500 ml of 0.1X SSPE, 0.1% SDS, and exposed to X-ray film. The a2(I) probe was a cloned 702-bp rabbit-specific RT-PCR product generated from rabbit cartilage RNA in our laboratory. Upper and lower primers, specific for sequence in exons 49 and 52 of the human HUMC1A2 gene [18] were: 5*-GGT GGT TAT GAC TTT GGT TAC-3* and 5*-CAG GCG TGA TGG CTT ATT TGT-3 *, respectively. The a1(II) probe was a 3.8-kb genomic fragment containing exons 45–54 of the human COL2A1 gene [19, 20] previously shown to hybridize to rabbit cartilage type II collagen RNA on a Northern blot (Hering, unpublished result). cDNA synthesis and PCR. RNA from cultured mesenchymal progenitor cells and aggregate cultures (350 ng each) and rabbit articular cartilage (5 mg) was used for oligo d(T)-primed cDNA synthesis using MoMLV-H reverse transcriptase [21]. cDNA synthesized from equivalent amounts (50–100 ng) of preaggregate mesenchymal progenitor cell or aggregate culture RNA, or 125 ng of cartilage RNA, was used as template for PCR amplification per 25-ml reaction volume using primer pairs designed using sequence obtained from the GenBank database: human type II collagen a1(II) chain [22, 23], human type I collagen a2(I) chain [24], and human type X collagen a1(X) [25]. Primer sets were as follows: collagen a1(II): 5*-CTG CTC GTC GCC GCT GTC CTT-3* and 5*-AAG GGT CCC AGG TTC TCC ATC-3 *, collagen a2(I); 5*-GGT GGT TAT GAC TTT GGT TAC-3 * and 5*-CAG GCG TGA TGG CTT ATT TGT-3 *, collagen a1(X); 5*-GCC CAA GAG GTG CCC CTG GAA TAC-3* and 5*-CCT GAG AAA GAG GAG TGG ACA TAC-3*. Calculated optimal annealing temperatures (OLIGO Primer Analysis Software, National Biosciences Inc., Plymouth, MN) were used for each primer pair, and samples were withdrawn for analysis in agarose gels following 30 and 40 cycles of amplification. Expected product sizes were as follows: collagen a1(II), 432 bp (IIA form) and 225 bp (IIB form); collagen a2(I), 702 bp; collagen a1(X), 703 bp. PCR products were analyzed by electrophoresis on a 1% agarose gel containing ethidium bromide [21]. Total PCR reaction products were ligated into a TA cloning vector (pCRII, Invitrogen) and transformed into Escherichia coli. A number of plasmids representing different cloned PCR products were purified and inserts sequenced by the DNA Sequencing Core Facility of the Northeastern Ohio Multipurpose Arthritis Center using a Pharmacia Biotech ALFexpress Automated DNA Sequencer. Rabbit-specific PCR products representing rabbit collagen a1(II) alternatively spliced forms using these primers are 435 bp (IIA form) and 225 bp (IIB form). This partial rabbit IIA sequence has been deposited to the GenBank data base under Accession No. AF027122. Southern blot analysis. Southern blot analysis using labeled oligonucleotide probes internal to amplifying primers was performed as per standard protocols [21] to determine the relative abundance of the collagen IIA and IIB forms in the RT-PCR amplification reaction. Following electrophoresis, samples were transferred from agarose gels to nitrocellulose membranes and were sequentially probed using 5*-end 32P-labeled oligonucleotides. We designed an oligonucleotide probe (probe AB) spanning exons 5 and 6 that would hybridize to both the IIA and IIB splice variants and a probe that would specifically hybridize to the IIA splice variant (probe A), recognizing exon 2. The sequences of these oligonucleotides were as follows: probe AB, 5*TTC ACC TGC AGG TCC CTG AGG-3*; probe A, 5*-ACA CAG ATC CGG CAG GGC TCC-3*. Following hybridization and washing, membranes were exposed to Kodak BioMax film for varying periods of time at 0707C with an intensifying screen.

RESULTS

Aggregate Formation During primary culture, adherent colonies of cells formed. Differences in cell morphology were observed

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between colonies: most cells were fibroblast-like, but some were more flattened. After 14 days in culture the cells were trypsinized to release them from the substratum and used for the aggregate culture studies, after centrifugation into pellets as described above. In the serum-free defined medium, condensation of the cells into a single aggregate was seen within 16 h of incubation after centrifugation. However, cells incubated in DMEM with 10% FBS did not form a clearly identifiable aggregate at any time after centrifugation. Therefore, only the aggregates from the defined medium incubations, with or without additives (dexamethasone, TGF-b1), were available for analysis. Effect of Dexamethasone on the Cell Aggregates Aggregate cultures were set up for marrow cell cultures from 24 rabbits. For each rabbit cell preparation, aggregates were incubated with or without dexamethasone in the defined medium. Duplicate aggregates were harvested at 7, 14, and 21 days after centrifugation and frozen in OCT prior to analysis. In 6 of the 24 marrow cell preparations, aggregates incubated with dexamethasone had metachromatic staining with toluidine blue that was characteristic of cartilage matrix (Fig. 1). Within the metachromatic staining matrix there were cells in lacunae with the appearance of hypertrophic chondrocytes. This was seen as early as day 7, and clearly identifiable by day 14. By day 21, the morphology of some aggregates was entirely cartilaginous. This change in morphology and staining pattern was not observed in any aggregate incubated without dexamethasone. The metachromatic staining pattern of aggregated cells incubated in the presence of dexamethasone suggested that a cartilaginous matrix had been synthesized. To confirm this, immunohistochemistry was carried out with an antibody to type II collagen. Positive immunostaining was observed only in regions of aggregates that had metachromatic staining, which by day 21 comprised the whole aggregate. Aggregates incubated without dexamethasone had no detectable staining for type II collagen.

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the aggregates (data not shown). At 1 ng/ml TGF-b1, chondrogenic differentiation of the central aggregate region had not occurred by 21 days of culture, although differentiation was observed in the outer third of the aggregate; this was even more restricted in the aggregates incubated with 0.5 ng/ml. In order to better define the extent of the chondrogenic differentiation of the marrow-derived cells, we also immunostained representative sections of aggregates with an anti-type X antibody. Type X collagen was detectable throughout the pellet by day 21 (Fig. 2). Alkaline Phosphatase Activity This was measured for the six preparations to which TGF-b1 was added, with or without dexamethasone. The alkaline phosphatase activity in the dexamethasone-treated aggregates was low at day 7 and did not increase over the time in culture (Fig. 3). This correlates with the histological and immunohistochemical findings that dexamethasone alone was not sufficient to induce chondrogenesis in the preparations used for this assay. In contrast, the inclusion of TGF-b1 in the aggregate cultures caused an increase in the measured alkaline phosphatase activity. This increase correlated with the chondrogenic differentiation of the cells. Extracellular Matrix Gene Expression during Aggregate Chondrogenesis

Northern hybridization of the total cellular RNA from preaggregate cells was done with matrix molecule probes for types I and II collagen (Fig. 4). The probe for collagen a1(I) hybridized to bands of the expected size (approximately 5 kb) in the preaggregate cell RNA, as well as in the rabbit fibroblast control RNA. No hybridization to a1(II) mRNA (which would be approximately 5 kb) was detectable upon subsequent rehybridization with the collagen a1(II) probe. RT-PCR analysis of aggregate cultures was performed using primers spanning collagen a1(II) exons 1–7 (Fig. 5). Only the collagen IIB (225 bp) splice variant was detectible following 30 cycles of amplification using RNA purified from rabbit articular cartilage as a template. Amplification using RNA from 7-day aggregate cultures reEffects of TGF-b1 on Aggregate Chondrogenesis vealed PCR products representing both the IIB and the Six rabbit cell preparations were used for testing the IIA (432 bp) splice variants. The identity of these PCR effects of TGF-b1 on chondrogenesis. Aggregates were products was confirmed by sequence analysis. Southincubated with TGF-b1 (10 ng/ml), dexamethasone, or ern blot analysis using labeled oligonucleotide probes both. In this series of aggregates, no chondrogenesis was also performed on the same RT-PCR amplification was observed in those incubated with dexamethasone products. Two bands were visualized when the blot was alone (Fig. 2). However, TGF-b1, either alone or in com- probed with an oligonucleotide designed to hybridize bination with dexamethasone, induced chondrogenesis with exons 5 and 6 (Fig. 5B). For confirmation of speciin the aggregated cells. The aggregates formed in the ficity, a probe designed to hybridize with exon 2, the presence of both dexamethasone and TGF-b1 appeared alternatively spliced exon included in the IIA form, was larger than those incubated with TGF-b1 alone. In sep- used and only the slower migrating band was detected. arate experiments, it was determined that lowering the Further PCR experiments were done to assess the TGF-b1 concentration decreased the chondrogenesis in changes in the expression of types I and X collagen.

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FIG. 1. Rabbit bone marrow-derived cells cultured as aggregates. (A, B, C, D) Immunostaining with type II collagen antibody. (E, F) Toluidine blue staining. (A) 7, (B) 14, and (C, E) 21 days with 1007 M dexamethasone; (D, F) 21 days without dexamethasone.

Samples were withdrawn following 40 cycles of amplification. Collagen type I a2(I) chain mRNA appeared more abundant in preaggregate cell RNA than in day 7 aggregate cells (Fig. 6A). This result correlates with high steady state levels of a1(I) chain mRNA detected by Northern blot analysis of similarly cultured cells (Fig. 4). It was also detected in a rabbit articular cartilage sample included in the analysis. The presence of type I mRNA in the articular cartilage sample could be due

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to cells from the synovium or subchondral bone being included in the harvested tissue. A collagen a1(X) PCR product was also amplified from cartilage RNA, and from day 7 aggregate cells, but no product was detectible in preaggregate cells (Fig. 6B). The detection of type X in the articular cartilage sample was probably due to the inclusion of hypertrophic cells in the harvest since the tissue was taken from 5-week-old rabbits that still possess an articular–epiphyseal cartilage complex [26].

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FIG. 2. Rabbit bone marrow-derived cells cultured as aggregates in defined medium for 21 days: (A, C) with 1007 M dexamethasone; (B, D, E) with 1007 M dexamethasone and 10 ng/ml TGF-b1. (A, B) Toluidine blue staining; (C, D) immunostaining with anti-type II antibody; (E) immunostaining with anti-type X collagen antibody.

DISCUSSION

Mesenchymal progenitor cells with chondrogenic potential are present in many tissues of the body. Those of the bone marrow are of particular interest because of their ease of harvest and the potential for the use of these cells to facilitate cartilage repair [27]. There are numerous studies demonstrating that cells isolated from

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bone marrow can be reimplanted in vivo and undergo osteochondral differentiation [1–4]. Furthermore, in vitro genesis of osseous tissue, in the form of bone nodules, is a well-recognized assay for the osteogenic potential of these cells [28]. However, the formation of cartilage in vitro from postnatal mammalian bone marrow has not been well demonstrated. Successful in vitro chondrogenesis has been demonstrated with avian and

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Dexamethasone is not a specific chondrogenic differentiation factor, as demonstrated by its ability to induce multiple end-phenotypes when added to cultured fetal rat calvarial cells with differentiation potential [32, 34]. In fact, impairment of chondrogenesis in murine neonatal condylar cartilage has been observed in the presence of dexamethasone [35], although it induced chondrogenesis in organoid cultures of murine embryonic cells [33]. The addition of dexamethasone facilitated chondrogenic differentiation in only 25% of the rabbit marrow cell aggregate preparations. The reasons for this are unclear, but it may be related to the number of cells with chondrogenic capacity that are within a given marrow aspirate and subsequently adhere to culture plates. The results of several studies indicate that there appears to be a minimum number FIG. 3. Alkaline phosphatase activity of the pellets as assessed by measuring the absorbance value for the postincubation solution of cells required before chondrogenesis can occur [36– 43]. Such a variation between aggregates is possible at 405 nm. (Dex) dexamethasone. since we are not using clonally isolated populations of cells, and there is cellular heterogeneity in the marrowderived monolayer cultures used for these experiments. embryonic mammalian cells and cell lines [6, 29–33], However, how can we then explain the effect of adding but there has only been one report of induction of chon- TGF-b1, which induced chondrogenesis in all preparadrogenesis from postnatal mammalian cells [7]. In the tions? Perhaps, this cytokine initially increases the propresent study, we describe the chondrogenic differentia- liferation of cells with chondrogenic potential to a point tion of mesenchymal progenitor cells from postnatal where the critical number is reached and the differentimammalian bone marrow. The presence of a metachro- ation proceeds. Alternatively, TGF-b1 may provide the matic-staining matrix, the chondrocytic appearance of cellular stimulus for differentiation, regardless of the the cells, and the detection of type II collagen mRNA and protein signify that the tissue generated by these marrow-derived cells is cartilage. Furthermore, the cells differentiate into their terminal phenotype, the hypertrophic chondrocyte, as indicated by the detection of type X collagen mRNA and protein and the concomitant rise in alkaline phosphatase activity. The induction of the chondrogenesis of these cells required particular culture conditions. The cells were maintained in a format resembling that of a precartilage condensation [12]. In a recent paper, Noble et al. (7) described experiments where the addition of dextran sulfate to porcine bone marrow cells grown to confluence on tissue culture plates caused retraction of the cells into nodular structures in which type II collagen was immunolocalized after 6 days. Thus, as found in our studies, chondrogenesis was induced after the cells formed precartilage condensation-like structures. Of interest is the fact that Noble et al. could achieve initiation of chondrogenesis in serum-containing medium, whereas induction of chondrogenesis does not occur in rabbit bone marrow-derived cell aggregates incubated in medium containing fetal bovine serum. Likewise, if the serum is removed and the aggregates are incubated in a defined medium without dexamethasone or TGFFIG. 4. Northern hybridization of preaggregate rabbit bone-marb1, no chondrogenesis occurs. This implies that the row-derived cell RNA with matrix molecule probes. Total cellular addition of dextran sulfate may have effects on the RNA from the preaggregated cells (lane 1) and from cultured rabbit porcine marrow cells other than simply creating a con- dermal fibroblasts (lane 2) hybridized with probes for (A) collagen densation-like structure. a1(I), (B) collagen a1(II).

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FIG. 5. (A) RT-PCR analysis to determine collagen type IIA and IIB splice variants in rabbit cartilage (lane 1) and day 7 pellet culture (lane 2) RNA. Lane 3 is a 1-kb ladder. (B) Southern blot analysis of the RT-PCR amplification reaction products produced from day 7 aggregate culture extracted RNA, using either an oligonucleotide probe that hybridizes to both collagen type IIA and IIB (lane 1) or a probe specific for IIA (lane 2).

number of cells present with chondrogenic capacity. In the embryo, epithelial–mesenchymal interactions are crucial for differentiation. In the culture system, the addition of the TGF-b1 provides the appropriate external signal, that when coupled with the endogenous factors communicated between cells within the condensation, facilitates chondrogenic differentiation. In those preparations where dexamethasone addition was sufficient to induce differentiation, endogenous TGF-b1 or other inductive cytokines may have been present in high enough concentrations that the dexamethasoneinduced changes were enough to facilitate chondrogenesis. TGF-b1, -b2, and -b3 have been detected in the developing skeleton including the areas where the primitive mesenchymal tissue are undergoing condensation and cartilage formation [44–46]. All rabbit marrow-derived pluripotential cell aggregates treated with TGF-b1 progressed on to form histologically identifiable cartilage, thus supporting the idea that TGF-b1 plays an important role in chondrogenesis. The appearance of the type X collagen in the aggregates as shown by immunohistochemistry as well as PCR demonstrate that these cells will terminally differentiate into hypertrophic chondrocytes. The appearance of type X collagen is a rapid phenomenon occurring soon after the appearance of the type II collagen. This rapid appearance of type X collagen soon after the chondrocytic differentiation of mesenchymal cells has been shown in vitro in avian chondrogenesis studies [47]. When the aggregated cells underwent chondrogenesis, a concomitant elevation in the alkaline phosphatase level was detected. This rise in the alkaline phosphatase activity is consistent with the differentiation of these cells into hypertrophic chondrocytes [48]. However, this result contrasts those of other in vitro studies of the effects of dexamethasone and TGF-b1 on terminal differentiation. Although not necessary for chick embryonic cell chondrogenesis in vitro, dexamethasone supports cell viability but delays the appearance of type X collagen [49]. TGF-b1 has also been

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shown to inhibit growth plate chondrocyte hypertrophy [50]. In aggregate cultures of growth plate chondrocytes, TGF-b1 stimulated cartilage-specific proteoglycan production, but when added at later stages, inhibited the appearance of type X collagen [10, 11]. However, in our system, the cells progress through the chondrogenic lineage to hypertrophy, with type X collagen detected as early as 7 days. This occurred in the presence of dexamethasone and TGF-b1. The contrasting effects of these factors on mesenchymal progenitor cell aggregate cultures compared with chick embryonic and rat growth plate cells may be due to species or cell-type differences or to the dissimilar culture conditions used. When chondrogenesis was achieved, the morphology of the aggregate changed from the appearance of a mesenchymal cell condensation to that of cellular cartilage, such as is seen in embryonic limb formation. Furthermore, at day 7 postaggregation, the presence of type IIA collagen mRNA was detected by RT-PCR. Type IIA collagen is the splice variant of type II collagen that has been found in prechondrocytes and immature chondrocytes [51, 52]. This form has an extra exon (exon 2, coding for 69 amino acids) spliced into it. Type II collagen without this exon (designated IIB) is the form associated with maturing chondrocytes and those found in postnatal cartilage. These observations lead to the suggestion that the aggregate culture system is a model of embryonic chondrogenic differentiation and that the process is a recapitulation of embryonic events. This hypothesis is currently being explored.

FIG. 6. RT-PCR analysis of rabbit articular cartilage (lane 1), preaggregate mesenchymal cells (lane 2) and day 7 aggregate cells (lane 3) to determine the expression of mRNA for (A) collagen type I and (B) collagen type X. The 123-bp ladders are also shown. Arrowheads indicate the positions of the expected products.

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We thank John Kollar and Amad Awadallah for expert technical assistance and R. Tracy Ballock, M.D., for advice on the culture system. This study was supported in part by N.I.H. Grants AR-44390 (B.J.) and AR-37726 (V.G.) and AR-20618 (Northeastern Ohio Multipurpose Arthritis Center).

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Received April 24, 1997 Revised version received October 3, 1997

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