DEVELOPMENTAL BIOLOGY 172, 293 –306 (1995)
Progression and Recapitulation of the Chondrocyte
Differentiation Program: Cartilage Matrix Protein
Is a Marker for Cartilage Maturation
Qian Chen, David M. Johnson, Dominik R. Haudenschild,
and Paul F. Goetinck1
Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical
School, Building 149, 13th Street, Charlestown, Massachusetts 02129
During endochondral bone formation, chondrocytes in the cartilaginous anlage of long bones progress through a spatially
and temporally regulated differentiation program before being replaced by bone. To understand this process, we have
characterized the differentiation program and analyzed the relationship between chondrocytes and their extracellular
environment in the regulation of the program. Our results indicate that, within an epiphyseal growth plate, the zone of
proliferating chondrocytes is not contiguous with the zone of hypertrophic chondrocytes identified by the transcription of
the type X collagen gene. We find that the postproliferative chondrocytes which make up the zone between the zones of
proliferation and hypertrophy specifically transcribe the gene for cartilage matrix protein (CMP). This zone has been termed
the zone of maturation. The identification of this unique population of chondrocytes demonstrates that the chondrocyte
differentiation program consists of at least three stages. CMP translation products are present in the matrix surrounding
the nonproliferative chondrocytes of both the zones of maturation and hypertrophy. Thus, CMP is a marker for postmitotic
chondrocytes. As a result of the changes in gene expression during the differentiation program, chondrocytes in each zone
reside in an extracellular matrix with a unique macromolecular composition. Chondrocytes in primary cell culture can
proceed through the same differentiation program as they do in the cartilaginous rudiments. In culture, a wave of differentiation begins in the center of a colony and spreads to its periphery. The cessation of proliferation coincides with the appearance
of CMP and eventually the cells undergo hypertrophy and synthesize type X collagen. These results reveal distinct switches
at the proliferative– maturation transition and at the maturation– hypertrophy transition during chondrocyte differentiation
and indicate that chondrocytes synthesize new matrix molecules and thus modify their preexisting microenvironment as
differentiation progresses. However, when ‘‘terminally’’ differentiated hypertrophic chondrocytes are released from their
surrounding environment and incubated in pellet culture, they stop type X collagen synthesis, resume proliferation, and
reinitiate aggrecan synthesis. Eventually they cease proliferation and reinitiate CMP synthesis and finally type X collagen.
Thus they are capable of recapitulating all three stages of the differentiation program in vitro. The data suggest a high
degree of plasticity in the chondrocyte differentiation program and demonstrate that the progression and maintenance of
this program is regulated, at least in part, by the extracellular environment which surrounds a differentiating chondrocyte
during endochondral bone formation. q 1995 Academic Press, Inc.
INTRODUCTION
Cartilaginous rudiments of a developing embryo serve as
anlagen from which long bones are formed during endochondral bone formation. During this process the length
and shape of a bone is determined. Limb mesodermal pre1
To whom correspondence should be addressed. Fax: (617) 726
4189.
cursor cells differentiate into chondrocytes of the cartilaginous growth plate and they progress through a chondrocyte
differentiation program, starting as proliferating chondrocytes and ending up as hypertrophic chondrocytes. The hypertrophic chondrocytes are then removed and replaced by
bone. The chondrocyte differentiation program has been
partitioned into different stages based on cell shape and
volume, biosynthetic and metabolic activities, and, most
accurately, on molecular markers. At least two models have
been proposed. According to one model (Castagnola et al.,
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1988) the differentiation program is divided into two stages
and the chondrocytes are considered to be either in the
proliferative or in the hypertrophic state. Thus, this model
proposes that the proliferative stage immediately precedes
the hypertrophic stage during endochondral bone formation. The proliferating chondrocytes synthesize type II collagen and aggrecan (Miller, 1972; Doege et al., 1990). These
markers distinguish the chondrocytes from their mesodermal precursors which synthesize type I collagen. The hypertrophic chondrocytes produce type X collagen, a hypertrophic cartilage-specific molecule (Schmid and Conrad, 1982;
Capasso et al., 1984). The second model proposes that the
chondrocytes of the growth plate are divided into three distinct stages that are identified histologically as the zones of
proliferation (zone 1), maturation (zone 2), and hypertrophy
(zone 3) (Kim and Conrad, 1977; Stocum et al., 1979). According to this model, the transition of the chondrocytes
from the zone of proliferation to the zone of hypertrophy
would be separated by an additional developmental stage,
the stage of maturation. However, no specific molecular
markers have been identified for chondrocytes in this stage.
The molecular markers that characterize the differentiated state of chondrocytes are extracellular matrix (ECM)
macromolecules and the chondrocyte differentiation process is modulated by extracellular cues including the ECM
(Benya and Schaffer, 1982; von der Mark et al., 1992b). In
monolayer culture, chondrocytes which are seeded at low
density will transform their chondrogenic phenotype (round
shape, type II collagen synthesis) to a fibroblastic one (flattened morphology, type I collagen synthesis) under certain
conditions (von der Mark et al., 1977). This transformation
depends on cell shape and attachment, which is mediated
through cell– matrix interaction (Benya and Schaffer, 1982).
The alteration of the chondrocyte differentiation process
also occurs in vivo. For example, articular cartilage normally does not undergo endochondral bone formation.
However, in the osteoarthritic articular cartilage where the
matrix is destroyed, chondrocytes resume proliferation and
type X collagen, a hallmark of cartilage hypertrophy, is synthesized (von der Mark et al., 1992b). Thus, hypertrophy of
chondrocytes is reinitiated (Hoyland et al., 1991). It is unclear what determines chondrocytes to undergo regeneration, dedifferentiation, or hypertrophy (von der Mark et al.,
1992a).
There are three major types of ECM molecules in cartilage: collagens, proteoglycans, and noncollagenous glycoproteins. That these macromolecules play an important
morphogenetic role during endochondral bone formation is
demonstrated most strikingly by the analysis of mutations
which alter the expression of the genes of aggrecan core
(Argraves et al., 1981; Goetinck, 1991) and collagens type
II (Bogaert et al., 1992; Garofalo et al., 1991, 1993; Metsaranta et al., 1992), type IX (Nakata et al., 1993), and type X
(Jacenko et al., 1993). As part of this study we investigated
the gene expression of cartilage matrix protein (CMP) during
endochondral bone formation. CMP is a major noncollagenous protein in the matrix of cartilage (Paulsson and Heine-
gård, 1981). It contains two homologous repeats (CMP-like
domains) that are separated by an EGF-like domain (Argraves et al., 1987; Kiss et al., 1989). CMP-like domains
have also been found in collagen types VI, VII, XII, and XIV;
von Willebrand factor; complement factors B and C2; the a
chains of the integrins Mac-1, p150/95, LFA-1, and VLA-2;
malaria thrombospondin-related anonymous protein; dihydropyridine-sensitive calcium channel; and inter-a-trypsin
inhibitor (Winterbottom et al., 1992; Yamagata et al., 1991;
Gerecke et al., 1993; Parente et al., 1991; Bork and Rohde,
1991). In the matrix of cartilage, CMP interacts with collagen fibrils (Winterbottom et al., 1992) and proteoglycans
(Paulsson and Heinegård, 1979). The collagen binding of
CMP has been localized to the CMP domains. Thus, it has
been suggested that CMP may be involved in collagen fibrillogenesis and in the organization of the matrix of cartilage (Tondravi et al., 1993).
The results of the present study indicate that the CMP
gene is transcribed only in the postproliferative chondrocytes in a zone between the zones of proliferation and hypertrophy of the growth plate. CMP translation products are
present in the matrix surrounding the nonproliferative
chondrocytes of both the zones of maturation and hypertrophy. Thus, CMP is a marker for postmitotic chondrocytes.
As a result of the changes in gene expression during the
differentiation program, chondrocytes in each zone reside
in an extracellular matrix with a unique macromolecular
composition. Chondrocytes in cell culture proceed through
the same temporal progression of proliferation, maturation,
and hypertrophy in that CMP is produced by postproliferative chondrocytes before type X collagen is synthesized.
Finally, hypertrophic chondrocytes that are released from
their surrounding environment are capable of recapitulating
all three stages of the differentiation program in vitro.
MATERIALS AND METHODS
Cell and Organ Culture
Primary cultures of chondrocytes were established as described by Schmid and Conrad (1982). Briefly, tibiotarsi
were dissected from stage 41 (15th day of incubation) chick
embryos (Hamburger and Hamilton, 1951). The bone shell
and the tarsus region were separated from the cartilaginous
growth plate and discarded. To ensure that the selected regions of chondrocytes were derived from the desired zone,
only cells at the center of any one zone were used for cell
or pellet culture. The cells at the boundary with neighboring
zones were discarded. In addition, control experiments were
carried out by immunostaining with mAbs, to ensure that
a cartilage piece was only positive for the molecular marker
that is characteristic for chondrocytes of the desired zone.
For organ culture, cartilage explants are cultured in Ham
F-12 medium containing 10% fetal calf serum (plating medium) (Gibco, Grand Island, NY) at 377C in an atmosphere
of 5% CO2 . For monolayer cell culture and pellet mass
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Chondrocyte Differentiation Program
culture, a piece of cartilage from a defined zone was subjected to enzymatic treatment with 0.1% trypsin (Sigma,
St. Louis, MO), 0.3% collagenase (Worthington, Freehold,
NJ), and 0.1% type I testicular hyaluronidase (Sigma) (dissociation medium). After an incubation of 30 min at 377C this
dissociation medium was removed and replaced with fresh
dissociation medium and incubated at 377C for an additional hour. For monolayer cell culture, chondrocytes were
resuspended in plating medium containing 0.01% testicular
hyaluronidase. After culturing overnight, the medium of
the chondrocytes was replaced with fresh medium without
hyaluronidase. The medium was changed every other day.
Pellet mass cultures were performed as described by Kato
et al. (1988). Briefly, chondrocytes were washed and pelleted
at 1000g in a 15-ml plastic centrifuge tube and incubated
in plating medium at 377C.
Determination of DNA Content
DNA content from organ or pellet mass cultures was determined as described by Labarca and Paigen (Labarca and
Paigen, 1980). Briefly, a piece of cartilage or cell pellet was
homogenized in phosphate-saline buffer (PSEB: 0.05 M
Na2 HPO4 , 2 M NaCl, 0.003 M EDTA, pH 7.4), and sonicated
briefly. Hoechst nuclear dye (Polysciences, Inc., Warrington, PA) was added in 1:100 dilution in PSEB and incubated at room temperature for 1 hr. Fluorescence was measured with a TKO 100 fluorometer from Hoefer (San
Francisco, CA). DNA standards were used and the determinations were made in the linear range of the curve.
In Situ Hybridization
In situ hybridization for mRNAs encoding aggrecan core
protein, link protein, CMP, and type X collagen were performed according to Hayashi et al. (1986) and Linsenmayer
et al. (1991). The slides were viewed by dark-field and
bright-field optics and photographed on a Microphot-FXA
microscope from Nikon (Melville, NY). The probe for CMP
was CMP 1 (Argraves et al., 1987). The probe for aggrecan
core protein was PG 525 (Stirpe et al., 1987). The link protein probe was LPG2 (Deák et al., 1986). The type X collagen
probe was a PstI/PvuII fragment of pYN 3116 (Ninomiya et
al., 1986).
BrdU Incorporation and Detection
Incorporation and detection of BrdU in cartilage tissues
were performed with the 5-bromo-2*-deoxyuridine labeling
and detection kit II from Boehringer-Mannheim (Indianapolis, IN). Labeling cartilage with BrdU was carried out in ovo
or in vitro. Both methods gave identical results. For in ovo
labeling, 40 ml of BrdU (1 ml/g of body weight) were injected
into the aircell of an egg 3 hr before dissection. For in vitro
tissue labeling, a tibiotarsus was dissected out of the embryonic chick leg. Perichondrium and bone shell were then
peeled from the cartilage to ensure penetration of BrdU into
the tissue. Different labeling time periods were tested. One
hour was the minimum time for intensive labeling. For cell
culture labeling, BrdU (1 mM) is added to cell culture 1 hr
before immunostaining.
Immunocytochemistry and Histochemistry
Tissue embedding, processing for cryostat sectioning, and
indirect immunofluorescence for immunocytochemical
analyses were performed as described by Fitch et al. (1982).
For double immunofluorescence staining, cultured cells
were fixed at 0207C with 70% ethanol, 50 mM glycine, pH
2.0, for 20 min. Slides were then washed with phosphatebuffered saline (PBS) and incubated with primary antibodies. After washing with PBS, secondary antibodies were applied together with Hoechst nuclear dye. Slides were
washed and mounted in 95% glycerol in PBS. Double or
triple exposure photography was performed with a microscope from Nikon.
The monoclonal antibody against CMP, 1H1/E2, was
generated as described in detail by Binette et al. (1994).
1H1/E2 (IgG1, k) recognizes purified chick CMP (100 m g/
ml) in ELISA up to a dilution of 1005 of ascites fluid and
does not recognize purified chick collagen type II or purified chick link protein. 1H1/E2 also recognizes CMP as a
single band of 54 kDa (Winterbottom et al., 1992), when
total proteins from cartilage extracts are electrophoresed
on 8% SDS – PAGE gels under reducing conditions. The
other mAbs employed recognize link protein (4B6/A5; Binette et al., 1994), type II collagen (II-II 6B3; Hendrix et
al., 1982), and a helical determinant in type X collagen (XAC9; Schmid and Linsenmayer, 1985). The mAb against
BrdU was purchased from Boehringer-Mannheim. The
polyclonal antibodies (RC1) were generated in rabbit
against purified chicken CMP. They were characterized
by both ELISA and Western blot analysis as recognizing
CMP specifically. Affinity-purified rhodamine or fluorescein-conjugated secondary antibody was purchased
from Jackson ImmunoResearch (West Grove, PA).
Western Blot Analysis
Chick sterna were homogenized in 4 M urea, 50 mM Tris
at pH 7.5. The homogenate was centrifuged and the amount
of protein in the supernatant was measured by the optical
density of the sample at 280 nm. Five micrograms of each
supernatant were analyzed on a discontinuous 8% SDS–
PAGE. The proteins were transferred onto Immobilon–
PVDF membrane (Millipore Corp., Bedford, MA) in 25 mM
Tris, 192 mM glycine, 15% methanol. The membranes were
blocked in 2% bovine serum albumin fraction V (Sigma) in
PBS for 30 min and then probed with the anti-CMP
mAb1H1/E2, or with a polyclonal Ab EM 77 against type
X collagen (Pacifici et al., 1991). Horseradish peroxidaseconjugated goat anti-mouse IgG (H / L) or anti-rabbit IgG
(Bio-Rad Lab, Melville, NY) was used as a secondary antibody. Visualization of the immunoreactive proteins was
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achieved using the ECL Western blotting detection reagents
(Amersham Corp., Heights, IL) and exposing the membrane
to Kodak X-Omat AR film.
RESULTS
The CMP Gene Is Transcribed Only by
Chondrocytes of the Zone of Maturation
To identify different stages in the chondrocyte differentiation program, we used the incorporation of BrdU to visualize proliferating chondrocytes, and probes that allowed the
determination of the transcriptional activity of a number
of genes that encode ECM molecules. A bright-field histological section of a growth plate of a stage 41 embryo is
shown in Fig. 1A. Histologically identifiable zones are indicated. Incorporation of BrdU by the nuclei identifies the
zone of proliferation (P) at the distal end of the tibia (Fig.
1B). Chondrocytes in this region (zone 1) are small and
round, with many mitotic figures in the nuclei (Fig. 2, 1).
In the zone of hypertrophy (zone 3), the size of the cells is
dramatically increased (Fig. 2, 3). This zone (H) is characterized by the presence of the mRNA for type X collagen (Fig.
1F), a hypertrophic cartilage-specific molecule (Schmid and
Linsenmayer, 1983). A comparison of Figs. 1B and 1F indicates that the proliferative zone is not contiguous with the
hypertrophic zone. Therefore, there exists an additional
zone between the zones of proliferation and hypertrophy.
This zone corresponds to the histologically identified zone
of maturation (Kim and Conrad, 1977; Stocum et al., 1979)
and the CMP gene is transcribed only by chondrocytes in
this zone (M) (Fig. 1C). Flattened cells at the beginning of
this zone (2a) give rise to medium-sized cells toward the
end of the zone (2b), fulfilling the transition from the proliferating to the hypertrophic state (see Figs. 1A and 2, 2a and
2b). Transcripts for aggrecan core protein and link protein
are present in chondrocytes of both the proliferative zone
and the zone of maturation, but not in those of the zone of
hypertrophy (Figs. 1D and 1E). Thus, the spatially restricted
transcriptional activity of the CMP gene in the growth plate
indicates that the chondrocytes situated between the chondrocytes that make up the zones of proliferation and hypertrophy are a unique population of cells. These observations
clearly establish the presence of at least three stages in the
differentiation program of chondrocytes.
The Translation Product of the CMP Gene Is a
Marker for Postproliferative Chondrocytes
The distribution of ECM proteins and incorporated BrdU
was examined in sections from the tibiotarsus of a stage 45
chick embryo. BrdU labeling (Fig. 3A) revealed a zone of
proliferating chondrocytes in the epiphyseal growth plate.
At this stage the proliferative zone is shorter than the one
at stage 41 (compare Figs. 3A and 1B). There are many proliferating chondrocytes at the perimeter of the growth plate,
FIG. 1. Micrographs of sections of a tibiotarsus from a stage 41
chick embryo. Bright-field histochemical staining (H&E) (A), immunofluorescence micrograph with a mAb against incorporated
BrdU (B), and dark-field micrographs of in situ hybridizations with
cDNAs for CMP (C), the core protein of aggrecan (D), link protein
(E), and type X collagen (F). P and 1, zone of proliferation (zone 1);
M and 2, zone of maturation (zone 2); 2a, first half of the mature
zone; 2b, second half of the mature zone; H and 3, zone of hypertrophy (zone 3). Bar, 400 mm.
reflecting the rapid appositional growth of the rudiment
(Fig. 3A). Proliferating cells of the blood or bone marrow
could also be identified (Fig. 3A, white arrows). CMP could
be shown by immunofluorescence to be present in the two
postproliferative zones of maturation and hypertrophy as
well as in the tarsus (Fig. 3C). CMP is absent not only from
the proliferative zone, but also from the proliferating chondrocytes at the perimeter of the tibia (compare Figs. 3C and
3A). Type X collagen was present only in the hypertrophic
zone (Fig. 3D). In contrast to the specific localization of
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Chondrocyte Differentiation Program
FIG. 2. Morphology of chondrocytes during in vivo differentiation in the growth plate of a stage 41 embryonic chick tibiotarsus (bottom)
compared with the changes of the proliferative zone explants incubated in culture (top). The sections are from the proliferative zone (1),
first half of the mature zone (2a), second half of the mature zone (2b), and the hypertrophic zone (3) in vivo or the explant from the
proliferative zone incubated in culture for 1 hr (1h), 20 hr (1d), and 5 days (5d). All the sections are stained with hematoxylin and eosin.
Arrows in (1) indicate dividing chondrocytes. Bar, 10 mm.
CMP and type X collagen, link protein was detected
throughout the growth plate (Fig. 3B). Both CMP and link
protein were present in the matrix surrounding chondrocytes of the hypertrophic region even though the genes for
these proteins are not transcribed by the chondrocytes of
that zone (Figs. 3C and 3B). Thus, the translation product
of the CMP gene is a marker for nonproliferating chondrocytes of either the mature or the hypertrophic zone. These
results indicate that the synthetic repertoire of chondrocytes changes as differentiation proceeds and, as a result, the
composition of matrix is modified during the progression of
the differentiation program.
Spatial and Temporal Expression of the
Differentiation Program in Primary Chondrocyte
Cultures
To test whether the spatial organization of chondrocytes
into distinct zones in vivo reflects the manifestation of their
differentiation process temporally, we examined the differentiation time sequence of chondrocytes in monolayer cell
culture. Chondrocytes were isolated from the core of the
proliferative zone of tibiae from stage 41 chick embryos and
cultured. After 3 days of culture, CMP can be detected in
the cultures, but only in a subset of the chondrocytes. The
FIG. 3. Fluorescence micrographs of sections from a tibiotarsus of a stage 45 chicken embryo. Reactions with monoclonal antibodies
against incorporated BrdU (A), link protein (B), CMP (C), and type X collagen (D) are shown. P, zone of proliferation; M, zone of maturation;
H, zone of hypertrophy. White arrows point to the proliferation of cells from blood or bone marrow. Bar, 300 mm.
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Chen et al.
FIG. 4. Fluorescence staining of primary cultures of chondrocytes isolated from the proliferative zone of the epiphyseal growth plate.
The presence of the cells are indicated by the blue nuclei staining with the Hoechst dye. (A, B, and C) From 3-day cultures. (D) is from
a 15-day culture. (A) Cells stained with mAb 1H1/E2 against CMP (red). Notice the red staining only in the center of the colonies. (B)
Cells doubly stained with mAb II-II 6B3 against type II collagen (red) and RC-1 against CMP (green). The solid arrow points to the type
II collagen staining in the cells. The open arrow points to the CMP staining. The overlapping region of type II (red) and CMP (green) is
yellow, which is only at the center of the colony. (C) Cells doubly stained with anti-BrdU (red) and RC1 against CMP (green). The solid
arrow points to the BrdU staining in the nuclei of the cells at the periphery of the colony, suggesting that they are proliferating cells. The
open arrow points to the blue nuclei (BrdU negative) in the center of the colony. These cells are CMP positive (green). (D) Chondrocytes
after 15 days of culture. Only part of a colony is shown. Cells doubly stained with RC1 against CMP (green) and mAb X-AC9 against
type X collagen (red). The solid arrow points to the filamentous staining of CMP connecting the cells in the colony. The open arrow
points to the punctate staining of type X collagen in the center of the colony. Bar, 225 nm.
CMP-positive cells are located at the center of the colonies
(Fig. 4A). The cells at the periphery of the colonies do not
stain for CMP. The CMP distribution pattern is in contrast
to that of type II collagen, which can be detected in the
cells throughout the colonies (Fig. 4B). To test whether the
CMP-negative cells at the periphery are in fact the proliferating chondrocytes, the cultures were incubated in the presence of BrdU for 1 hr. The peripheral cells which lack CMP
actively incorporate BrdU (Fig. 4C), indicating that they are
proliferating chondrocytes. BrdU incorporation cannot be
detected in the chondrocytes at the center of the colonies
which produce CMP (Fig. 4C), indicating that these are postproliferative cells. After 15 days of incubation, chondrocytes have stopped dividing, as indicated by the lack of BrdU
incorporation (data not shown). CMP is laid down in the
extracellular space as filamentous material throughout the colonies (Fig. 4D). At this time, type X collagen can be detected
as punctate fluorescence signals within the colonies. Thus, in
the culture system, the chondrocytes go through the same differentiation program as they do in vivo. A wave of differentiation spreads from the center of the colonies to the periphery
as proliferative cells differentiate into mature and subsequently
into hypertrophic chondrocytes.
Removal of the Extracellular Environment from
Proliferative Chondrocytes Prolongs Their
Proliferation and Delays Their Maturation
The data presented above suggest that there is a correlation between the state of differentiation of chondrocytes
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FIG. 5. Diagram describing the method used to generate differentiation stage-specific organ cultures and pellet mass cultures. P, proliferative zone; M, mature zone; H, hypertrophic zone. These three zones were separated by microdissection. The cartilage explants were either
directly incubated as organ cultures or digested with collagenase, testicular hyaluronidase, and trypsin to eliminate the extracellular
environment. These chondrocytes were washed and pelleted and incubated as pellet cell cultures. The proliferative zone and the hypertrophic zone were tested in this experiment.
and their ECM expression. To test if this state is directly
influenced by the extracellular environment surrounding
differentiating chondrocytes, an in vitro culture system was
devised in which pieces of cartilage are taken from the core
of the zones of proliferation and hypertrophy (Fig. 5). In one
set of experiments the chondrocytes are maintained as an
isolated piece of cartilage surrounded by their original extracellular matrix (organ culture). These are compared with the
chondrocytes that are enzymatically removed from their
surrounding matrix and cultured as a pellet mass culture
(pellet culture). The organ and pellet cultures were carried
out with chondrocytes that originated from the same region
of the growth plate. For each comparison, therefore, the
chondrocytes are from the same developmental stage.
When a piece of cartilage from the proliferative zone is
maintained as an organ culture in the presence of serum
the DNA content remains constant during 12 days of incubation (Fig. 6, solid squares). This observation indicates that
the chondrocytes have stopped proliferating in culture. The
morphology of the chondrocytes of the cartilaginous pieces
cultured for 1 hr, 1 day, or 5 days is very similar to that
seen in the mature (zones 2a and 2b) and the hypertrophic
(zone 3) zones, respectively (compare Fig. 2, 1h to 2a, 1d to
2b, and 5d to 3). These changes in morphology suggest that
the chondrocytes have undergone differentiation in culture.
This rapid process of differentiation in vitro from the proliferative through the mature to the hypertrophic stage was
demonstrated at the molecular level by the incorporation of
BrdU (marker for proliferation) and the expression of CMP
(marker for maturation) and type X collagen (marker for
hypertrophy) (Fig. 7). The incorporation of BrdU by the cells
during the first hour of incubation confirms their identity
as proliferating chondrocytes (Fig. 7A). There is little staining for the postmitotic chondrocyte marker CMP at the end
of the first hour (Fig. 7B), and there is no staining for type
X collagen (data not shown). However, after 1 day of incubation (20 hr), there is strong staining for CMP (Fig. 7D), no
detectable BrdU incorporation (Fig. 7C), and still no staining
for type X collagen (data not shown). These changes suggest
that the chondrocytes have differentiated to the stage of
maturation. Type X collagen can be first detected at the 3rd
day of incubation (data not shown). After 5 days of incubation, type X collagen can be detected throughout the cultured piece (Fig. 7F) and there is no BrdU incorporation
detected (Fig. 7E), indicating that the chondrocytes have
reached the hypertrophic stage. The results also suggest that
the constant level of DNA in the cultured pieces of cartilage
is the result of the maintenance of living cells and not the
result of a balance between proliferation and cell death. In
contrast, in organ cultures without serum, DNA content in
the cultured pieces decreases by 50% during incubation (Fig.
6, solid diamonds). However, in these cultures the chondrocytes go through the same differentiation sequence as the
cells cultured in the presence of serum (data not shown).
Therefore, in organ culture, serum is necessary for preventing cell death or tissue degradation, but not required
for chondrocyte differentiation.
To compare quantitatively the appearance of CMP and
type X collagen during incubation of the cartilage explants
from the proliferative zone, an equal amount of extracts (5
mg) from cartilage explants cultured for 0, 1, and 5 days are
analyzed by Western blot. As shown in Fig. 7G, a band of
CMP trimers (200 kDa) is barely detectable at the beginning
of incubation (0 day). It is clearly detected after 1 day of
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FIG. 6. Effects of serum and extracellular environment on the proliferation of primary chondrocytes derived from the proliferative zone.
A cartilage explant from zone 1 of a stage 41 chick growth plate was incubated in the presence (solid square) or absence (solid diamond)
of serum. Alternatively, the chondrocytes were released from their extracellular environment and incubated as a pellet culture in the
presence of serum (open square). The 100% value is the DNA content of a zone 1 cartilage explant at the beginning of incubation. The
curves are representative of three different experiments.
incubation and is present at the 5th day of incubation. In
comparison, type X collagen (59 kDa) is not detected at
either 0 or 1 day of incubation, but is detected at the 5th
day of incubation. Thus, when chondrocytes from the proliferative zone are cultured as an organ, proliferation ceases
and differentiation starts during the 1st day of incubation.
In contrast, in pellet mass cultures of proliferative chondrocytes that are removed from their extracellular environment, proliferation continues for 10 days (Fig. 6, open
square). These chondrocytes synthesize a new surrounding
matrix (data not shown) before undergoing subsequent maturation and hypertrophy. Therefore, the transient removal
of the extracellular environment from proliferating cells in
situ prolongs the proliferative period and delays the progression of the differentiation program.
Hypertrophic Chondrocytes Can Recapitulate the
Chondrocyte Differentiation Program
The behavior of postmitotic, ‘‘terminally’’ differentiated
hypertrophic chondrocytes was examined in organ and in
pellet mass cultures. Pieces of hypertrophic cartilage were
taken from the core of late hypertrophic zones. Chondrocytes in these pieces are large in size (see Fig. 2) and they
actively transcribe the type X collagen gene (Fig. 9C). To
eliminate the complication of cell death, hypertrophic
chondrocytes were incubated only in serum containing medium. In organ culture, the DNA content of the incubated
pieces of hypertrophic cartilage remains constant (Fig. 8,
solid squares). However, in pellet mass cultures, the postmitotic hypertrophic chondrocytes that were deprived of their
extracellular surrounding in situ resume proliferation and
double their DNA content by 6 days (Fig. 8, open squares).
To determine if the resumption of proliferation represents
the beginning of the recapitulation of the differentiation
program, we examined, by in situ hybridization, the expression of the genes encoding aggrecan core protein and CMP
during the incubation of the chondrocytes in pellet cultures.
These two genes are not active in hypertrophic chondrocytes (Figs. 9A and 9B). At Day 4 of incubation, the transcription of the genes for aggrecan core protein and CMP is
reinitiated throughout the pellet mass culture derived from
the hypertrophic zone (Figs. 9D and 9E). The presence of
transcripts for aggrecan core protein is still present at Day
7 (Fig. 9G) and the transcription of CMP seems to reach a
peak at this time. Both the density and intensity of the
grains for CMP (hybridization signals) increase (Fig. 9H).
This suggests that there is an elevation in the transcriptional activity of the CMP gene by an increased number of
cells. There is no longer any increase in DNA content (Fig.
8, open squares) at this time. In contrast, the transcription of
the type X collagen gene, a marker for hypertrophic cartilage
(Fig. 9C), is greatly down-regulated at 4 days of incubation
(Fig. 9F). At Day 12 of incubation, the expression for type
X collagen is reinitiated (Fig. 9I). The histochemical staining
of a Day 12 pellet mass culture shows the de novo synthesized matrix material that is deposited between the cells
by the chondrocytes during the incubation period (Fig. 9J,
arrow).
The sections of the pellet cultures and organ cultures of
hypertrophic chondrocytes were examined by immunostaining with mAbs against incorporated BrdU and type X
collagen. At Day 4 of incubation, there are many BrdUpositive nuclei in the pellet culture (Fig. 10A) and none in
the organ culture (Fig. 10C). However, at Day 7 of incubation, there are only a few BrdU-positive nuclei in the pellet
culture (Fig. 10E) and still none in the organ culture (Fig.
10G). These results confirm the measurement of DNA content in both pellet and organ cultures (Fig. 8). Furthermore,
type X collagen is distributed throughout both pellet and
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Chondrocyte Differentiation Program
content and incorporation of BrdU), maturation (CMP expression), and hypertrophy (type X expression).
DISCUSSION
The Cellular Organization of the Growth Plate
FIG. 7. Fluorescence micrographs of sections from the zone 1 cartilage explant cultured for 1 hr (A and B); 20 hr (C and D); and 5
days (E and F). Scale, 1:25. Sections were reacted with mAbs against
incorporated BrdU (A, C, and E), against CMP (B and D), and against
type X collagen (F). (G) Western blot analysis of expression of CMP
and type X collagen by cartilage explants of the proliferative zone
cultured for 0, 1, and 5 days. Each lane contains 5 mg of protein
from extracts of cultured cartilage explants.
organ cultures (Figs. 10B, 10D, 10F, and 10H). Since transcripts for type X collagen are present in the hypertrophic
chondrocytes at the beginning of incubation (Fig. 9C), but
are not detected in the pellet cultures at Day 4 of incubation
(Fig. 9F), type X collagen in the matrix of the pellet cultures
at Day 4 must have been synthesized by the cells which
are type X collagen mRNA-positive at the beginning of the
incubation. This confirms the cells in the pellet culture
which are capable of proliferation as hypertrophic chondrocytes. These data indicate that the nonproliferative terminally differentiated hypertrophic chondrocytes have the capacity to undergo ‘‘retrodifferentiation’’ in response to
changes in the extracellular environment. These cells are
then capable of recapitulating the differentiation program
by reinitiating the stages of proliferation (increase of DNA
The chondrocytes of the growth plate progress through
a differentiation program which can be partitioned into a
number of stages. These stages are clearly reflected in the
spatial organization of the chondrocytes in the different
zones of the growth plate. The generally accepted model,
as presented in many textbooks (Ross and Romrell, 1989;
Gilbert, 1994), describes the proliferative zone in a growth
plate as immediately preceding the hypertrophic zone. Our
study clearly shows that, at least in chick embryos, this is
not the case. The distribution of proliferating chondrocytes
in a growth plate was determined by monitoring the localization of BrdU incorporated by dividing chondrocytes at
the G1-S phase transition. The zone of hypertrophic chondrocytes was identified by the expression of type X collagen,
a commonly accepted marker of hypertrophic chondrocytes
(Schmid and Linsenmayer, 1983). Our results clearly indicate that the proliferating chondrocytes make up a zone
which is not contiguous with the hypertrophic zone. Therefore, there exists a population of nonproliferating chondrocytes that are not yet hypertrophic. Here we identify the
transcription of the CMP gene as a molecular marker for
chondrocytes of that zone. Our observations are consistent
with a second model which proposes that the chondrocytes
of a growth plate are divided into at least three distinct
stages that have been identified histologically as the zones
of proliferation, maturation, and hypertrophy (Stocum et
al., 1979; Oohira et al., 1974). The zones that we identify
FIG. 8. Effects of the extracellular environment on the proliferation of primary chondrocytes derived from the hypertrophic zone.
A cartilage explant of zone 3 (solid square) or the cell pellet mass
derived from zone 3 (open square) was incubated in serum containing medium. The 100% value is the DNA content of a zone 3
cartilage explant at the beginning of incubation. The curves are
representative of three different experiments.
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Chen et al.
FIG. 9. Micrographs of sections of chondrocyte pellet mass cultures. Bright-field micrographs of in situ hybridizations with cDNA
probes for the core protein of aggrecan (A, D, and G), CMP (B, E, and
H), and type X collagen (C, F, and I). (A, B, and C) From hypertrophic
cartilage before culture; (D, E, and F) from 4-day cultures; (G and
H) from 7-day cultures; (I and J) from 12-day cultures. Bright-field
histochemical staining (H&E) of Day 12 culture (J). The arrow
points to the extracellular matrix areas between the chondrocytes.
Bar, 20 mm.
as the zones of proliferation and maturation are referred to,
in the generally accepted model, as the resting zone and the
zone of proliferation, respectively. This discrepancy may
have resulted from a lack of appropriate markers for identifying chondrocytes of the zones of proliferation, maturation, and hypertrophy when the first model was proposed.
Since the definition of the developmental stages of the same
regions of a growth plate differs between the two models,
many studies on endochondral bone formation that use the
first model should be reinterpreted.
CMP Is a Molecular Marker of Cartilage
Maturation
In this study, we have characterized the molecular events
during the three stages of endochondral bone formation fur-
ther by determining the pattern of transcription of genes
that encode chondrocyte matrix molecules. Our report is
the first to identify CMP as a product of and a marker for
mature chondrocytes. A restricted pattern of expression of
the CMP gene in the growth plate has also been reported
in human and mouse (Aszódi et al., 1994; Mundlos and
Zabel, 1994). However, in both reports it is assumed that
the proliferative zone is contiguous with the hypertrophic
zone, and the zone of proliferation was not identified experimentally in either study. In human embryonic long bones
the mRNA for CMP was reported to be present in the upper
‘‘hypertrophic’’ and lower ‘‘proliferative’’ zone (Mundlos
and Zabel, 1994). This region is likely to correspond with
the zone of maturation as defined in the present study. In
another report (Aszódi et al., 1994) the expression of a
chicken genomic clone was monitored by immunohistochemical means in transgenic mice. High levels of chicken
CMP were reported to be present in the zone of proliferating
chondrocytes and not in the zone of hypertrophic chondrocytes. It is difficult to compare the interpretation of these
results with those of the present study since the proliferating chondrocytes were not identified in the transgenic animals. The reported absence of the translation product of the
chicken CMP gene in the hypertrophic zone of the
transgenic mice differs from our observation (Chen et al.,
unpublished) that mouse CMP is present in the hypertrophic region of normal mice.
It has been reported that CMP is not detected in the
superficial layer of chondrocytes covering the joint articular surface (Franzen et al., 1987; Mundlos and Zabel, 1994),
but is detected in the deep layer of articular cartilage
(Mundlos and Zabel, 1994). This observation is reminiscent of the distribution pattern of CMP in the embryonic
growth plate described here. Therefore, we suggest that
the difference in CMP distribution is due to the different
stages of cartilage development, rather than the different
types of cartilage. Consistent with this idea are our previous results (Stirpe and Goetinck, 1989), which indicated
that the transcripts for CMP are first detected at stage 26
during chick limb development, later than those for type
II collagen (stage 23 and those for link protein and aggrecan
core protein (stage 25).
The Differentiation Program of Chondrocytes
The results of the analysis of the spatial arrangement of
chondrocytes in the growth plate is corroborated by the
study of chondrocytes in monolayer cell culture in which
differentiation events happen sequentially. The analysis in
vitro offered an advantage since immunostaining of chondrocytes in culture allows the characterization of differentiation events in individual cells. Chondrocytes isolated from
the proliferative zone of the epiphyseal growth plate go
through the stages of proliferation, maturation, and hypertrophy in vitro as they do in vivo. It is demonstrated by our
cell culture experiment that CMP is only made by those
cells that are no longer proliferating (lack of BrdU incorpora-
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Chondrocyte Differentiation Program
FIG. 10. Immunofluorescence micrographs of sections from organ cultures or pellet mass cultures of hypertrophic chondrocytes. (A and B)
From 4-day pellet cultures; (C and D) from 4-day organ cultures; (E and F) from 7-day pellet cultures; (G and H) from 7-day organ cultures.
(A, C, E, and G) Reacted with a mAb against BrdU; (B, D, F, and H) reacted with X-AC9, a mAb against type X collagen. Bar, 50 mm.
tion). Therefore, CMP contributes to the formation of the
cartilage matrix established by postproliferative chondrocytes, before the matrix is further modified by type X collagen synthesized by hypertrophic chondrocytes. These results indicate that the properties of the cartilage matrix
change during the course of differentiation. Furthermore,
this modification of the matrix is controlled, at least in
part, at the mRNA level. This is illustrated by the in situ
hybridization data of the exclusive synthesis of mRNA for
CMP by mature chondrocytes and of mRNA for type X by
hypertrophic chondrocytes.
The program of chondrocyte differentiation is summarized
in Fig. 11. When chondrogenesis is initiated, the genes for
collagens type II (Miller, 1972) and IX (Linsenmayer et al.,
1991), aggrecan core protein, and link protein (Figs. 1D and
1E) are activated and the gene for type I collagen is repressed
(Linsenmayer et al., 1973). The switch from mesodermal precursor cells to proliferating chondrocytes is the first step in
the cartilage differentiation program. The gene for CMP is
activated when chondrocytes stop proliferating and undergo
maturation. This step represents the second switch in the
program. When chondrocytes undergo hypertrophy, the genes
that encode CMP, link protein, and aggrecan core protein are
repressed and the gene for type X collagen is activated. This
represents the third switch in the program. These three
switches represent key transition points in chondrogenesis
and endochondral bone formation. The first switch separates
chondrocytes from their mesodermal precursors and the second switch separates proliferating chondrocytes from postmitotic mature chondrocytes. In the ‘‘permanent’’ cartilage such
as articular cartilage and the cartilage of the caudal portion of
the sternum, chondrocyte differentiation stops at the maturation stage. If the third switch is activated, chondrocytes proceed to the hypertrophic stage. Therefore, this switch separates the permanent cartilage from the transitional cartilage
which serves as a model for endochondral bone formation.
The Chondrocyte Differentiation Program
Is Reversible
In the present study we also show that the chondrocyte
differentiation program is dynamic. It can be delayed, re-
FIG. 11. The chondrocyte differentiation program indicating possible points for dedifferentiation and retrodifferentiation. The relationship
of the program with the expression of genes encoding extracellular matrix molecules is indicated. AGG, aggrecan; LP, link protein; CMP,
cartilage matrix protein.
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Chen et al.
versed, and recapitulated. Our data suggest that the surrounding extracellular environment is essential for maintaining the state of differentiation of chondrocytes. The extracellular cues from this microenvironment may include
both ECM molecules and growth factors. It has been shown
that growth factors such as TGF-b and b-FGF are important
in modulating and preventing chondrocyte hypertrophy
during endochondral bone formation (Ballock et al., 1993;
Kato et al., 1988). Furthermore, growth factors have been
shown to bind to the extracellular matrix network. Thus,
the transient removal of the surrounding matrix may also
affect the localized activities of growth factors. Alternatively, the reinitiation of chondrocyte proliferation and the
temporary loss of the differentiated phenotype could result
from an alteration of cell – matrix interactions originally
present in cartilage and maintained by the matrix architecture. This hypothesis is also consistent with our data which
indicate that, when matrix is resynthesized and deposited
following initial cell isolation and incubation in pellet culture, chondrocyte proliferation ceases and differentiation
proceeds.
Finally, we have demonstrated that the activation and
repression of genes during endochondral bone formation are
reversible events and that terminally differentiated chondrocytes can reverse their differentiated state and recapitulate the entire differentiation program. The mechanism for
the retrodifferentiation from postmitotic, differentiated hypertrophic chondrocytes to proliferating chondrocytes is different from the dedifferentiation of proliferating chondrocytes to fibroblast-like cells (Fig. 11). Retrodifferentiation
involves the switch between proliferation and differentiation within the chondrocyte differentiation program,
whereas dedifferentiation involves changes to a nonchondrogenic phenotype. Our observations suggest that the terminally differentiated chondrocytes are developmentally
flexible in that they still have the potential to regenerate
ECM and form a ‘‘new’’ cartilage. Although our data are the
first to report the reversal of differentiation from terminally
differentiated chondrocytes, similar examples have been reported in muscle cells during newt limb regeneration (Lo
et al., 1993). The retrodifferentiation from postmitotic myotubes to myoblasts is shown to be mediated by retinoblastoma protein (Rb) (Gu et al., 1993) or p107 in the Rb-deficient cell lines (Schneider et al., 1994). The regulatory pathway of chondrocyte differentiation remains to be fully
explored and understood. However, this report, together
with other previous studies (Cancedda et al., 1992; Bruckner
et al., 1989; Schmid et al., 1990), provides a basis for elucidating the complicated and yet well-regulated mechanisms
of chondrocyte differentiation during bone development
and repair.
ACKNOWLEDGMENTS
We thank Yoshi Ninomiya for providing the cDNA probe for
collagen type X, Tom Linsenmayer for mAbs against collagen types
II and X, Maurizio Pacifici for the pAb against type X collagen, and
Janet Cravens for the production of mAb against CMP. Supported
by NIH Grant HD 22016 to P.F.G. Q.C. is a recipient of a postdoctoral fellowship from the Arthritis Foundation.
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Received for publication January 27, 1995
Accepted August 16, 1995
Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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