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EX973858
cartilage when implanted in vivo [14]. 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 cellcell 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 78 ml of marrow was aspirated into a
syringe containing 3000 units of heparin. Dulbeccos modified Eagles
To whom correspondence and reprint requests should be addressed. Fax: (216) 368-1332. E-mail: bxj9@po.cwru.edu.
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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 Denhardts 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 4554 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 (50100 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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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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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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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, 2933], 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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