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EXPERIMENTAL CELL RESEARCH 238, 265–272 (1998) ARTICLE NO. EX973858

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 mesen- chymal progenitor cells has been developed. Cells ob- tained 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 10 07 M dexamethasone in the medium induced chondrogenic

differentiation of cells within the aggregate as

denced by the appearance of toluidine blue metachro- masia and the immunohistochemical detection of type II collagen as early as 7 days after beginning three- dimensional 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, chon-

drogenic differentiation was achieved in only

cartilage when implanted in vivo [1–4]. A culture sys- tem has been developed in which these cells will un- dergo osteogenic differentiation in vitro [5]. However, attempts to develop in vitro conditions in which mesen- chymal progenitor cells isolated from postnatal mam- malian bone marrow will progress down the chondro- genic lineage have been less successful. There are stud- ies reporting in vitro chondrogenesis using postnatal mammalian cells [6, 7], but none have demonstrated

  • evi- histologically identifiable cartilage formation, although type II collagen production has been detected, sug- gesting at least prechondroid tissue production. We have developed a culture system that facilitates the chondrogenic differentiation of postnatal mamma- lian marrow mesenchymal progenitor cells. This system is an adaptation of the ‘‘pellet’’ culture system that was originally described as a method for preventing the phe-

  • approxi- notypic modulation of chondrocytes in vitro [8, 9]. More recently, the system has been used in studies of the ter- minal differentiation of growth-plate chondrocytes [10, 11]. This culture system allows cell–cell interactions analogous to those that occur in precartilage condensa- tion 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

mately 25% of the marrow cell preparations used. In contrast, with the addition of transforming growth fac- tor-b1 (TGF-b1), chondrogenesis was induced in all marrow cell preparations, with or without the pres- ence of 10 07 M dexamethasone. The induction of chon- drogenesis was accompanied by an increase in the al-

kaline 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 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 study-

collagen. Conversely, the expression of the type I colla- gen mRNA was detected in the preaggregate cells but was no longer detectable at 7 days after aggregation. These results provide histological, immunohistochem-

ical, and molecular evidence for the in vitro

  • chondro- ing the process of chondrogenesis, including those factors that regulate the progression of cells through the entire

genic differentiation of adult mammalian progenitor

cells derived from bone marrow. 1998 Academic Press chondrogenic lineage.

INTRODUCTION

METHODS

Cells isolated from postnatal mammalian bone mar- Cell harvest and colony formation. Rabbit bone marrow was har-

row have the potential for differentiation into the spe- cialized cells of mesenchymal tissues such as bone and

1 To whom correspondence and reprint requests should be ad-

vested 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

dressed. Fax: (216) 368-1332. E-mail: bxj9@po.cwru.edu. syringe containing 3000 units of heparin. Dulbecco’s modified Eagle’s

  • 265 0014-4827/98 $25.00 Copyright 1998 by Academic Press All rights of reproduction in any form reserved.

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JOHNSTONE ET AL.

medium (DMEM) with 10% fetal bovine serum (FBS) was added to for EF-1a [17] to control for differences in RNA loading. Blots were

the aspirate and the number of nucleated cells was determined. The rinsed and then washed three times at 24 C with 500 ml of 0.1X cells were plated out at 20 1 10 6 /100-mm dish and grown for 14 days SSPE, 0.1% SDS, and twice at 54 C (a1(II) probe) or 65 C (a2(I)

at 37 C, 5% CO

2 with medium changes every 4 days.

Aggregate culture. On day 14, adherent colonies of cells were tryp-

sinized, counted, and 2 1 10 5 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. Ag- gregates were cultured with or without dexamethasone (10 07 M), TGF-

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. Up- per 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 , respec- tively. 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).

b1 (0.5 to 10 ng/ml, recombinant human, R&D Systems), or a combina- cDNA synthesis and PCR. RNA from cultured mesenchymal pro- tion of these agents. For some experiments, the 10% FBS containing genitor cells and aggregate cultures (350 ng each) and rabbit articular

medium was not replaced. The pelleted cells were incubated at 37 C, cartilage (5 mg) was used for oligo d(T)-primed cDNA synthesis using

5% CO 2 . Within 24 h of incubation, the cells formed an essentially

MoMLV-H reverse transcriptase [21]. cDNA synthesized from equiv-

spherical aggregate that did not adhere to the walls of the tube. Me- alent amounts (50–100 ng) of preaggregate mesenchymal progenitor dium changes were carried out at 2- to 3-day intervals and aggregates cell or aggregate culture RNA, or 125 ng of cartilage RNA, was used

were harvested at time points up to 21 days. as template for PCR amplification per 25-ml reaction volume using

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 im-

munohistochemical 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 immer-

sion in distilled water to remove the OCT. Blocking of

nonspecic

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 anti- type 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 colla-

gens, 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

linked 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 prepara- tions, with a modification of the method of Chomczynski and Sacci [13]. Cells were lysed in culture dishes (1 ml/10 cm 2 ) with TRIZOL

reagent (Life Technologies Inc., Grand Island, NY). For each time-

point 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 artic-

ular cartilage using the method of Nemeth et al. [14]. RNA was

quantified by comparing

ethidium bromide uorescence with a stan-

dard series of RNA dilutions [15]. Northern hybridization. RNA samples (15 mg) were electropho-

resed 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 80 C. Filters were prehybridized at 42 C in a solution containing 50% formamide, 51 SSPE, 21 Denhardt’s reagent, 100 mg/ml salmon sperm DNA, and

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 repre- senting different cloned PCR products were purified and inserts se- quenced by the DNA Sequencing Core Facility of the Northeastern Ohio Multipurpose Arthritis Center using a Pharmacia Biotech ALFexpress Automated DNA Sequencer. Rabbit-specific PCR prod- ucts representing rabbit collagen a1(II) alternatively spliced forms

  • FITC- 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 oli- gonucleotide 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 32 P-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, mem- branes were exposed to Kodak BioMax film for varying periods of time at 070 C with an intensifying screen.

RESULTS

0.1% SDS [15] for several hours. Northern blots were sequentially Aggregate Formation

hybridized at 42 C in the same solution with 32 P-labeled cDNA

probes

for the a2(I) chain of type I collagen, and the a1(II) chain of type II

During primary culture, adherent colonies of cells

collagen prepared by random primer labeling, and with a cDNA probe formed. Differences in cell morphology were observed

IN VITRO CHONDROGENESIS

267

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 substra- tum 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 incuba- tion after centrifugation. However, cells incubated in DMEM with 10% FBS did not form a clearly identifi- able aggregate at any time after centrifugation. There- fore, only the aggregates from the defined medium in- cubations, with or without additives (dexamethasone,

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 aggre- gates 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 repre- sentative sections of aggregates with an anti-type X antibody. Type X collagen was detectable throughout the pellet by day 21 (Fig. 2).

TGF-b1), were available for analysis. Alkaline Phosphatase Activity

Effect of Dexamethasone on the Cell Aggregates

Aggregate cultures were set up for marrow cell cul- tures from 24 rabbits. For each rabbit cell preparation, aggregates were incubated with or without dexametha- sone 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 tolu- idine blue that was characteristic of cartilage matrix (Fig. 1). Within the metachromatic staining matrix there were cells in lacunae with the appearance of hy- pertrophic 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 cartilagi- nous. 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 sug-

gested that a cartilaginous matrix had been

synthe-

sized. To confirm this, immunohistochemistry was car-

ried out with an antibody to type II collagen.

Positive

immunostaining was observed only in regions of aggre-

gates that had metachromatic staining, which by

day

21 comprised the whole aggregate. Aggregates incu-

bated without

dexamethasone had no detectable stain-

ing for type II collagen.

This was measured for the six preparations to which TGF-b1 was added, with or without dexamethasone. The alkaline phosphatase activity in the dexametha- sone-treated aggregates was low at day 7 and did not increase over the time in culture (Fig. 3). This corre- lates 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 hy- bridization to a1(II) mRNA (which would be approxi- mately 5 kb) was detectable upon subsequent rehybrid- ization 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 fol- lowing 30 cycles of amplification using RNA purified from rabbit articular cartilage as a template. Amplifi-

cation using RNA from 7-day aggregate cultures re-

Effects of TGF-b1 on Aggregate Chondrogenesis vealed PCR products representing both the IIB and the

Six rabbit cell preparations were used for testing the effects of TGF-b1 on chondrogenesis. Aggregates were incubated with TGF-b1 (10 ng/ml), dexamethasone, or both. In this series of aggregates, no chondrogenesis was observed in those incubated with dexamethasone alone (Fig. 2). However, TGF-b1, either alone or in com- bination with dexamethasone, induced chondrogenesis in the aggregated cells. The aggregates formed in the presence of both dexamethasone and TGF-b1 appeared larger than those incubated with TGF-b1 alone. In sep-

IIA (432 bp) splice variants. The identity of these PCR products was confirmed by sequence analysis. South- ern blot analysis using labeled oligonucleotide probes was also performed on the same RT-PCR amplification products. Two bands were visualized when the blot was probed with an oligonucleotide designed to hybridize with exons 5 and 6 (Fig. 5B). For confirmation of speci- ficity, a probe designed to hybridize with exon 2, the alternatively spliced exon included in the IIA form, was 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.

268

JOHNSTONE ET AL.

268 JOHNSTONE ET AL. FIG. 1. Rabbit bone marrow-derived cells cultured as aggregates. (A, B, C,

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 10 07 M dexamethasone; (D, F) 21 days without dexamethasone.

Samples were withdrawn following 40 cycles of amplifi-

to cells from the synovium or subchondral bone being

cation. Collagen type I a2(I) chain mRNA appeared included in the harvested tissue. A collagen a1(X) PCR

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

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].

IN VITRO CHONDROGENESIS

269

IN VITRO CHONDROGENESIS 269 FIG. 2. Rabbit bone marrow-derived cells cultured as aggregates in defined medium

FIG. 2. Rabbit bone marrow-derived cells cultured as aggregates in defined medium for 21 days: (A, C) with 10 07 M dexamethasone; (B, D, E) with 10 07 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

bone marrow can be reimplanted in vivo and undergo

osteochondral differentiation [1–4]. Furthermore, in Mesenchymal progenitor cells with chondrogenic po- vitro genesis of osseous tissue, in the form of bone nod- tential are present in many tissues of the body. Those ules, is a well-recognized assay for the osteogenic poten- of the bone marrow are of particular interest because of tial of these cells [28]. However, the formation of carti-

their ease of harvest and the potential for the use of lage in vitro from postnatal mammalian bone marrow these cells to facilitate cartilage repair [27]. There are has not been well demonstrated. Successful in vitro numerous studies demonstrating that cells isolated from chondrogenesis has been demonstrated with avian and

270

JOHNSTONE ET AL.

270 JOHNSTONE ET AL. FIG. 3. Alkaline phosphatase activity of the pellets as assessed by measuring

FIG. 3.

Alkaline phosphatase activity of the pellets as assessed

by measuring the absorbance value for the postincubation solution

at 405 nm.

Dexamethasone is not a specific chondrogenic differ- entiation factor, as demonstrated by its ability to in- duce multiple end-phenotypes when added to cultured fetal rat calvarial cells with differentiation potential [32, 34]. In fact, impairment of chondrogenesis in mu- rine neonatal condylar cartilage has been observed in the presence of dexamethasone [35], although it in- duced 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 of cells required before chondrogenesis can occur [36–

(Dex) dexamethasone. 43]. Such a variation between aggregates is possible since we are not using clonally isolated populations of cells, and there is cellular heterogeneity in the marrow- derived monolayer cultures used for these experiments.

embryonic mammalian cells and cell lines [6, 29–33], but there has only been one report of induction of chon- drogenesis from postnatal mammalian cells [7]. In the

However, how can we then explain the effect of adding TGF-b1, which induced chondrogenesis in all prepara- tions? Perhaps, this cytokine initially increases the pro-

present 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 differenti- mammalian 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 hyper- trophic 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 precarti- lage condensation [12]. In a recent paper, Noble et al. (7) described experiments where the addition of dex- tran sulfate to porcine bone marrow cells grown to con- fluence 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 initia- tion 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 TGF-

270 JOHNSTONE ET AL. FIG. 3. Alkaline phosphatase activity of the pellets as assessed by measuring

b1, no chondrogenesis occurs. This implies that the addition of dextran sulfate may have effects on the

FIG. 4. Northern hybridization of preaggregate rabbit bone-mar- row-derived cell RNA with matrix molecule probes. Total cellular 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).

IN VITRO CHONDROGENESIS

271

IN VITRO CHONDROGENESIS 271 FIG. 5. (A) RT-PCR analysis to determine collagen type IIA and IIB

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

shown to inhibit growth plate chondrocyte hypertrophy [50]. In aggregate cultures of growth plate chondro- cytes, TGF-b1 stimulated cartilage-specific proteogly- can production, but when added at later stages, inhib- ited the appearance of type X collagen [10, 11]. How- ever, in our system, the cells progress through the chondrogenic lineage to hypertrophy, with type X colla- gen detected as early as 7 days. This occurred in the presence of dexamethasone and TGF-b1. The con- trasting effects of these factors on mesenchymal pro- genitor 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 cul- ture conditions used.

a probe specic for IIA (lane 2). When chondrogenesis was achieved, the morphology

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 exter- nal signal, that when coupled with the endogenous fac- tors communicated between cells within the condensa- tion, facilitates chondrogenic differentiation. In those preparations where dexamethasone addition was suf- ficient to induce differentiation, endogenous TGF-b1 or other inductive cytokines may have been present in high enough concentrations that the dexamethasone- induced changes were enough to facilitate chondrogen- esis. TGF-b1, -b2, and -b3 have been detected in the developing skeleton including the areas where the primitive mesenchymal tissue are undergoing conden- sation and cartilage formation [44–46]. All rabbit mar- row-derived pluripotential cell aggregates treated with TGF-b1 progressed on to form histologically identifi- able cartilage, thus supporting the idea that TGF-b1 plays an important role in chondrogenesis. The appearance of the type X collagen in the aggre- gates as shown by immunohistochemistry as well as PCR demonstrate that these cells will terminally differ- entiate into hypertrophic chondrocytes. The appear- ance of type X collagen is a rapid phenomenon oc- curring soon after the appearance of the type II colla- gen. This rapid appearance of type X collagen soon after the chondrocytic differentiation of mesenchymal cells has been shown in vitro in avian chondrogenesis stud- ies [47]. When the aggregated cells underwent chondro- genesis, a concomitant elevation in the alkaline phos- phatase level was detected. This rise in the alkaline phosphatase activity is consistent with the differentia- tion 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, dexa- methasone supports cell viability but delays the ap- pearance of type X collagen [49]. TGF-b1 has also been

of the aggregate changed from the appearance of a mes- enchymal cell condensation to that of cellular cartilage, such as is seen in embryonic limb formation. Further- more, 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 chon- drocytes [51, 52]. This form has an extra exon (exon 2, coding for 69 amino acids) spliced into it. Type II colla- gen without this exon (designated IIB) is the form asso- ciated with maturing chondrocytes and those found in postnatal cartilage. These observations lead to the sug- gestion 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.

IN VITRO CHONDROGENESIS 271 FIG. 5. (A) RT-PCR analysis to determine collagen type IIA and IIB

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. Arrow- heads indicate the positions of the expected products.

272

JOHNSTONE ET AL.

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 Multi- purpose Arthritis Center).

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