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MICROSCOPY RESEARCH AND TECHNIQUE 43:156–173 (1998)
Chondrogenic Potential of Skeletal Cell Populations:
Selective Growth of Chondrocytes and Their Morphogenesis
and Development In Vitro
LOUIS C. GERSTENFELD,1* CYRIL D. TOMA,2 JONATHAN L. SCHAFFER,3 AND WILLIAM J. LANDIS4
1Musculoskeletal
Research Laboratory, Boston University Medical Center, Boston, Massachusetts 02118
of Orthopedic Surgery, University of Vienna Medical School, Vienna, Austria
3Department of Orthopedic Surgery, Brigham and Woman’s Hospital and Harvard Medical School, Boston, Massachusetts 02115
4Department of Orthopedic Surgery, Children’s Hospital and Harvard Medical School, Boston, Massachusetts 02115
2Department
KEY WORDS
type X collagen; hypertrophic cartilage; bone formation
ABSTRACT
Most vertebrate embryonic and post-embryonic skeletal tissue formation occurs
through the endochondral process in which cartilage serves a transitory role as the anlage for the
bone structure. The differentiation of chondrocytes during this process in vivo is characterized by
progressive morphological changes associated with the hypertrophy of these cells and is defined by
biochemical changes that result in the mineralization of the extracellular matrix. The mechanisms,
which, like those in vivo, promote both chondrogenesis in presumptive skeletal cell populations and
endochondral progression of chondrogenic cells, may be examined in vitro. The work presented here
describes mechanisms by which cells within presumptive skeletal cell populations become restricted
to a chondrogenic lineage as studied within cell populations derived from 12-day-old chicken embryo
calvarial tissue. It is found that a major factor associated with selection of chondrogenic cells is the
elimination of growth within serum-containing medium. Chondrogenesis within these cell populations appears to be the result of permissive conditions which select for chondrogenic proliferation
over osteogenic cell proliferation. Data suggest that chondrocyte cultures produce autocrine factors
that promote their own survival or proliferation. The conditions for promoting cell growth,
hypertrophy, and extracellular matrix mineralization of embryonic chicken chondrocytes in vitro
include ascorbic acid supplementation and the presence of an organic phosphate source. The
differentiation of hypertrophic chondrocytes in vitro is associated with a 10–15-fold increase in
alkaline phosphatase enzyme activity and deposition of mineral within the extracellular matrix.
Temporal studies of the biochemical changes coincident with development of hypertrophy in vitro
demonstrate that proteoglycan synthesis decreases 4-fold whereas type X collagen synthesis
increases 10-fold within the same period. Ultrastructural examination reveals cellular and
extracellular morphology similar to that of hypertrophic cells in vivo with chondrocytes embedded in
a well formed extracellular matrix of randomly distributed collagen fibrils and proteoglycan.
Mineral deposition is seen in the interterritorial regions of the matrix between the cells and is
apatitic in nature. These characteristics of chondrogenic growth and development are very similar
in vivo and in vitro and they suggest that studies of chondrogenesis in vitro may provide a valuable
model for the process in vivo. Microsc. Res. Tech. 43:156–173, 1998. r 1998 Wiley-Liss, Inc.
INTRODUCTION
Much of skeletal formation during vertebrate embryogenesis and postnatal growth occurs by a process
referred to as the endochondral sequence. This is a
developmentally regulated series of stages that proceeds in a highly coordinated temporal and spatial
manner, in which there is sequential recruitment and
differentiation of cells that form cartilage, vascular, and
bone tissues. During the initial phases of this process,
mesenchymal stem cells are recruited, and they condense and differentiate into chondrocytes to produce an
initially unmineralized and avascular cartilage model
of a developing bone. Subsequently, the chondrocytes
within this model further differentiate and their elaborated matrix mineralizes to form calcified cartilage in
specific areas. The mineralization proceeds and continr 1998 WILEY-LISS, INC.
ues in the presence of vascularization of the tissue. This
is initiated by means of blood vessels accompanied by
undifferentiated mesenchymal cells entering cartilage
regions containing hypertrophic chondrocytes. Chondrocytes within lacunae localized in these mineralized
areas die and perivascular cells adjacent to cores of
calcified cartilage secrete new matrix that results in
bone deposition. The process is completed when metaphyseal remodeling occurs as the mineralized extracellular matrix of both bone and cartilage is ultimately
Contract grant sponsor: NIH; Contract grant numbers: HD 22400, AR41452,
N01-HD-2–3144; Contract grant sponsor: Max Kade Foundation.
*Correspondence to: Dr. Louis C. Gerstenfeld, Musculoskeletal Research
Laboratory, Boston University Medical Center, 715 Albany St., Housman 2,
Boston, MA 02118.
Received 16 July 1998; accepted in revised form 19 July 1998
CHONDROGENIC POTENTIAL OF SKELETAL CELLS
resorbed and replaced with bone alone (Farnum and
Wilsman, 1987; Ham and Cormack, 1979; Holtrop,
1972; Hunziker et al., 1984; Shapiro et al., 1977).
In normal vertebrate development in vivo, an endochondral phase seldom occurs during the formation of
the calvaria and the facial bones of the cranial skeleton.
These tissues are generated by direct induction through
a process known as membranous bone formation. On
the other hand, cartilage tissue is present in some
instances, including that of articular cartilage surfaces
that form on the maxillary processes of the jaw by direct
differentiation of ‘‘secondary’’ cartilage within the periosteum (Thorogood, 1979). In addition, specific experimental manipulation in vivo such as that causing
calcium deficiency by growing chick embryos in a
shell-less environment has been shown to give rise to
cartilage formation within the calvaria (Jacenko and
Tuan, 1986). Such changes to either cartilage or bone
during vertebrate development would not be unanticipated considering that cell populations derived from
embryonic calvaria from several species exhibit the
potential to differentiate into one of several multiple
cell lineages including those of osteoblasts (Aronow et
al., 1990; Bellows and Aubin, 1989; Gerstenfeld et al.,
1987), adipocytes (Bellows et al., 1994; Beresford et al.,
1992; Kodama et al., 1982; Yamaguchi and Kahn, 1991),
myoblasts (Grigoriadis et al., 1988; Yamaguchi and
Kahn, 1991), and chondrocytes (Asahina et al., 1993;
Bellows et al., 1989; Rifas et al., 1982; Toma et al.,
1997). These results indicate that, although not normally observed, there is a subset of cells within the cell
populations of the cranial bones that retains chondrogenic potential and can express a cartilage phenotype
in vitro. The selection of cells having a cartilage phenotype in cultured calvarial cell populations has been
shown to occur as a result of a variety of experimental
conditions including treatment of the cultures with
osteogenic protein (OP-1) (Asahina et al., 1993) or
corticosteroids (dexamethasone) (Grigoriadis et al.,
1988); growth of the cells on gelatin bone matrix
(Terashima and Urist, 1975); or growth of the cells in
the absence of serum (Cole et al., 1992; Rifas et al.,
1982; Toma et al., 1997). These observations suggest
that the epigenetic factors that regulate normal lineage
progression of skeletal cells and presumably direct the
changes so noted might be both identified and studied
in vitro.
Studies from several groups also indicate that both
genetic factors (Gerstenfeld et al., 1989) and environmental factors (Eavey et al., 1988) alter chondrocyte
phenotype and promote cell hypertrophy. With respect
to the latter, chondrocyte hypertrophy can be stimulated in vitro by culturing cells in a three-dimensional
(collagen or agarose) gel, selecting an appropriate cell
plating density (Solursh and Meier, 1974), or supplementing the cells with appropriate nutrient and vitamin D3 metabolites (Gerstenfeld et al., 1990) or ascorbic
acid (Gerstenfeld and Landis, 1991; Leboy et al., 1989).
The identification of the required growth conditions has
led to research studies that have examined the phenotypic developmental progression of chondrocyte hypertrophy and morphogenesis in vitro.
The present report examines the mechanism(s) and
contributory role of developmental versus environmental factors in the selection of cells to differentiate into
157
the chondroblastic lineage and to undergo further
hypertrophic differentiation. For this purpose, 12-dayold embryonic chicken calvaria and vertebrae have
been used as tissue sources for presumptive skeletal
cells and committed growth chondrocytes, respectively.
The results detail underlying mechanisms of cell type
selection and limitation for the development of chondrogenic and osteogenic lineages, define phenotypic changes
that accompany hypertrophic differentiation, and describe chondrocyte and matrix morphogenesis during
the hypertrophic progression of events.
MATERIALS AND METHODS
Cell Culture
Skeletal cell populations were isolated by three sequential trypsin-collagenase treatments of 12-day-old
chicken calvaria as previously described (Gerstenfeld et
al., 1996). Only cells released from the third consecutive digest were used for experimental determinations.
Cells were suspended in minimal essential medium
(MEM) supplemented with 10% fetal bovine serum
(FBS) (Sigma Chemical Co., St. Louis, MO) and plated
at 1.25 x 105 cells/60 mm diameter and 5 x 105 cells/100
mm diameter culture dishes. After 24 hours, cells
were washed once in phosphate buffered saline
(PBS) with a subsequent change of medium to either
MEM supplemented with 10% NU-Serum IV Culture
Supplement (Collaborative Biomedical Products, Bedford, MA) (Wong and Tuan, 1993) or 10% FBS. Cultures
were maintained in MEM/10% FBS or MEM/10% NUSerum for 2–3 weeks with medium changes every 2
days until cells reached confluence. At that time, the
medium was changed to BGJb (Fitton-Jackson
modification) supplemented with 10% FBS or 10%
NU-Serum. After another 2 days, the medium was
supplemented with 10 mM ␤-glycerophosphate (␤GPO4)
and 2 days later this medium was changed into ‘‘mineralizing medium,’’ containing 10% FBS or 10% NUSerum, 10 mM ␤GPO4, and 12.5 µg/ml ascorbic acid.
The time at which the cultures were introduced to the
mineralizing medium was denoted as day 0. Thereafter,
the medium was changed every 3 days until the experiments were terminated. All time points are referenced
to day 0.
In studies focusing on growth chondrocyte maturation and hypertrophic development, committed chondrocytes were prepared from vertebral bodies of 12-day-old
embryonic chickens. In these experiments, the chondrocyte cultures were grown under conditions that were
permissive for growth chondrocyte differentiation as
previously described (Gerstenfeld and Landis, 1991). In
other experiments, committed chondrocytes were prepared from the cephalic halves of 17-day embryonic
chicken sterna and these cultures were grown under
conditions identical to those used for the vertebral
chondrocytes. In other investigations, conditioned medium supplementation experiments were carried out
using medium from the sternal chondrocyte cultures in
the following manner: Cephalic chondrocytes were selected and grown for at least 2 weeks in culture and,
beginning at the end of the second week, medium was
collected (this was termed ‘‘conditioned medium’’). Collected fresh from the sternal chondrocyte cultures
through the duration of the experiments, this condi-
158
L.C. GERSTENFELD ET AL.
tioned medium was mixed at a ratio of either one or
three parts to either three or one part, respectively, of
Dulbecco’s modified eagle’s medium (DME) (Sigma
Chemical Co.) containing 10% FBS, 10 mM ␤GPO4, and
12.5 µg/ml ascorbic acid. Calvarial cells from 12- or
17-day-old embryonic chickens were then grown for 2
weeks in 1:3 or 3:1 DME:conditioned supplement. This
medium was replenished with conditioned medium
every 2 days over the time period.
Bromodeoxyuridine Labeling
BrdU labeling was as previously described (Gerstenfeld et al., 1996). Six days after their initiation, cell
cultures prepared from 12-day-old embryonic chicken
calvaria were pulsed for 3 hours with BrdU (Sigma
Chemical Co.) at a final concentration of 20 mM. At the
end of the pulse period, cell layers were fixed for 30
minutes at 4°C with 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2. Cell layers were then reacted for
60 minutes with anti-BrdU antibody supplemented
with nuclease (Amersham Inc., Arlington Heights, IL),
diluted 1:10 in PBS buffer containing 0.05% bovine
serum albumin (BSA) (w/v) and 0.5% Tween (v/v).
Secondary antibody reactions followed with anti-mouse
FTIC-conjugated IgG.
Isolation and Analysis of mRNA
Total RNA and DNA were isolated using Tri-ReagentTM, (Molecular Research Center, Cincinnati, OH)
according to the manufacturer’s instructions. RNA was
resolved on 1% agarose gels containing 2.2 M formaldehyde (Thomas, 1980) and 5 µg of total RNA based on
OD260 were loaded per gel lane. Equal loading of RNA
was verified by ethidium bromide staining of the gel
before blotting onto Biotrans nylon membranes (ICN
Biomedical Inc., Aurora, OH). Chicken cDNAs used for
these studies were pro ␣1(I) collagen (Lehrach et al.,
1979), pro ␣1(II) collagen (Young et al., 1984), osteocalcin (Neugebauer et al., 1995), osteopontin (Moore et al.,
1991), and aggrecan (Sai et al., 1986). Type X collagen
was detected using a 790 bp sequence beginning at the
splice junction of exon 3 of a chicken genomic type X
collagen clone (gcCOLX; L.C. Gerstenfeld, unpublished
data). This sequence corresponds to the sequence published by LuValle et al. (1988).
32P-radiolabeled cDNA probes were synthesized by
random priming and hybridization was carried out at
65°C in 50 mM PIPES, 100 mM NaCl, 50 mM Na
phosphate, pH 7.0, 1 mM EDTA buffer containing 5%
SDS (w/v) and 60 mg/ml single stranded salmon sperm
DNA. Hybridization was carried out for 18 hours in a
rotating hybridization oven (Robins Scientific, Sunnyvale, CA). Autoradiograms were quantitated using
an LKB Ultrascan II laser scanning densitometer (LKB,
Broma, Sweden) and values were normalized to 18S
ribosomal RNA obtained by hybridization of each blot to
a conserved nucleotide sequence probe of the 18S
ribosomal subunit (Ambion Corp., Austin, TX).
Analysis of Collagen and Proteoglycan Synthesis
Collagen protein synthesis was analyzed by pulse
labeling with 3H-proline. Cultures were labeled for 24
hours using 5 ml of 50 µCi/ml 3H-proline (110 mCi/
mmol) (Amersham Inc.) after 6 days of growth in
mineralizing medium. The total cell layer-associated
proteins and secreted medium proteins were processed
in the presence of proteolytic inhibitors as described
previously (Gerstenfeld et al., 1984). Fifty thousand
cpm of medium-labeled proteins and 100,000 cpm of cell
layer-associated proteins were applied per lane for each
gel. Collagens were resolved by electrophoresis on
5–10% continuous gradient SDS-polyacrylamide gels
(Laemmli, 1970) and fluorography was carried out
(Bonner and Laskey, 1974). Quantitation of fluorographs was performed on an LKB Ultrascan II laser
scanning densitometer (LKB, Broma, Sweden).
For proteoglycan analysis, cell cultures were grown
in 33 mm tissue culture wells. At day 6 after the change
to complete medium, replicates (n ⫽ 6) were labeled
with 50 µCi/ml Na2[35S]O4 (21 mCi/mmol) in 1.5 ml for
24 hours. Proteoglycans were precipitated overnight
with 1.3% potassium acetate (v/v) in 95% ethanol.
Samples were collected by centrifugation at 16,000g in
a microcentrifuge for 30 minutes, resuspended, and
precipitated a second time. Incorporation was determined by filtration of the precipitate onto glass filters
followed by scintillation counting (Gerstenfeld et al.,
1990).
Statistical Analysis
Biochemical assays were performed with a minimum
of 6 replicates and were repeated with at least 2–3
separate preparations of cells. All values were normalized to DNA content. Data were tabulated as the
mean ⫾ standard deviation (or standard error of the
mean where appropriate) with a minimum sample size
of 6. Statistical analysis consisted of ANOVA, Student’s
t-test of significance, and the Tukey HSD test using
Version 6.11 of SAS for Windows software package (SAS
Institute Inc., Cary, NC). Values of p ⬍ 0.05 were
considered significant. Mean ratios for cell count data
were compared using an independent samples Student’s t-test (Bland and Altman, 1995). For all calculations, two-tailed values of p ⬍ 0.05 were considered
statistically significant.
Histological and Ultrastructural Analysis
of Chondrocyte Cultures
Alkaline phosphatase expression was examined by
histochemical staining. Duplicate cultures were fixed
with 2% paraformaldehyde in 0.1 M cacodylate buffer
at pH 7.4 for 10 minutes, rinsed with fresh buffer, and
stained for alkaline phosphatase activity using naphtol
AS-MX phosphate and Fast Red TR salts (Sigma Chemical Co.). Matrix calcification was determined by hematoxylin/eosin and von Kossa (phosphate) staining. For
ultrastructural analysis, samples (cells and matrix)
were fixed and embedded directly in culture plates
using either aqueous (glutaraldehyde-OsO4) or anhydrous (ethylene glycol) techniques (Landis et al., 1977).
Specimens were sectioned (⬃80 nm) with diamond
knives on a Reichert Ultracut S ultramicrotome (Leica,
Deerfield, IL) and floated briefly on an aqueous or
anhydrous (ethylene glycol) trough fluid (Landis et al.,
1977). Some sections were stained with 8% uranyl
acetate in absolute ethanol and with Sato’s lead.
CHONDROGENIC POTENTIAL OF SKELETAL CELLS
159
Fig. 1. Selection of chondrogenesis by growth of presumptive skeletal
cell populations in serum-substituted medium. Left (top to bottom):
Histochemical analysis of 12-day-old embryonic chicken calvarial cell
cultures grown 6 days in medium containing either 10% fetal bovine
serum (FBS) or 10% serum substitute (serum subt., SS). Cell layers were
stained for either alkaline phosphatase (Apase) activity or proteoglycans
based on alcian blue reactivity. Representative staining characteristics of culture dish regions are denoted. Right (top): Bar graph of
alcian blue staining nodules in cultures grown in SS (solid bars) or
10% FBS (open bars). The number of alcian blue staining nodules was
counted in 6 dishes and compared in 2 separate experiments. Error
bars denote one standard deviation of these data. Right (middle):
Analysis of proteoglycan synthesis in cultures grown 12 days in SS
(solid bars) or 10% FBS (open bars). Cells were labeled on day 6 with
50 µCi/ml Na2[35S]O4 (21 mCi/mmol) in 1.5 ml for 24 hours and counted as
noted in Materials and Methods. Error bars denote one standard deviation
(n ⫽ 10) for two separate experiments. Right (bottom): Analysis of
alkaline phosphatase enzyme activity (by p-nitrophenol, p-NP conversion) of cultures grown in SS (solid bars) or 10% FBS (open bars). Enzyme
activity was measured on day 6. The results in all panels were determined
from 12 wells in 2 separate experiments (exp 1 and exp 2). Error bars
denote one standard deviation. Left panel magnification, 100x.
Ultrastructural features of such sections were examined by conventional transmission microscopy at 60 or
80 kV in either a Philips EM 300 (Philips Electronic
Instruments, Inc., Mahwah, NJ) or a JEOL 100C
(JEOL USA, Peabody, MA) electron microscope. Mineral in unstained sections was studied by selected area
160
L.C. GERSTENFELD ET AL.
Fig. 2. Comparison of chondrogenic and osteogenic expression in
12-day-old embryonic calvarial cultures grown under conditions permissive for chondrogenesis. A: RNAs were isolated from cells after 12
days of culture growth in mineralizing medium supplemented with
either SS (serum substitute) or 10% FBS. Northern blot analysis was
then carried out for bone and cartilage mRNAs, including aggrecan
(AG), osteopontin (OPN), osteocalcin (OC), and ␣1(I), ␣1(II), and ␣1(X)
collagen chains. The efficiency and uniformity of blot transfers are
demonstrated by hybridization profiles to an 18S ribosomal probe and
staining with ethidium bromide (EtBr). B: Collagen protein synthesis
in cultures grown for 12 days in complete medium supplemented with
either FBS or SS. The electrophoretic positions of 3H-proline-labeled
␣1(I ⫹ II), ␣2(I), pro ␣1(X), and ␣1(X) collagen chains isolated from
either cell layers or medium are indicated. The horizontal line
marking to the left of some blots indicates the position of 27S mRNA
used as a molecular weight indicator.
electron diffraction at 80 or 100 kV in the Philips
instrument and by electron probe microanalysis at 25
kV in a STEM-modified JSM-50A scanning electron microscope (JEOL USA) (Landis and Glimcher,
1978, 1982). All microscopes were equipped with a
liquid nitrogen anticontaminator to limit specimen
artifacts.
35SO
RESULTS
Chondrogenic Selection Within Presumptive
Skeletal Cell Populations Isolated From
12-Day-Old Embryonic Chicken Calvaria
The growth of 12-day-old embryonic chicken calvarial
cell populations in medium in which FBS was replaced
with a serum substitute has been shown to promote
chondrogenesis (Toma et al., 1997). As Figure 1 shows,
histological staining of whole cultures with alcian blue
identified the presence of chondrogenic cell nodules
throughout the cell layers in such serum-substituted
samples. The number of alcian blue staining nodules
could be counted and compared between cultures grown
in the absence or presence of medium containing FBS
(Fig. 1, top right). These results demonstrate that the
total number of chondrogenic nodules increased approximately 5–10-fold in the cultures grown in serum substitute. Chondrogenesis was biochemically determined by
4 labeling of sulfated proteoglycans (Fig. 1, middle
right), and these results independently confirm an
⬃8–20-fold increase in the chondrogenic phenotype of
the serum-substituted cultures.
Differences in alkaline phosphatase enzyme activity
were assessed in the two sets of cultures. At early time
points, histochemical staining for alkaline phosphatase
was seen only in the cells localized between the chondrocyte nodules in the cultures containing serum substitute (Fig. 1, middle left). In contrast, alkaline phosphatase staining was intense throughout the entire cell
layer of the cultures containing FBS (Fig. 1, bottom
left). Such histochemical results suggest that the enzyme activity would be much higher in the cultures
grown in FBS. However, biochemical measurement of
total enzyme activity was considerably higher in cultures grown in serum-substituted medium after 12
days (Fig. 1, bottom right). Histochemical staining after
12 days was also seen throughout the cell layers of
cultures grown in either FBS or serum substitute (data
not shown). Thus, since both hypertrophic chondrocytes
and osteoblasts express alkaline phosphatase, this assay by itself cannot discriminate between osteogenic
cells and hypertrophic chondrocytes. Moreover, as the
chondrogenic cells progress toward hypertrophy, they
CHONDROGENIC POTENTIAL OF SKELETAL CELLS
will express very high levels of alkaline phosphatase
(see below).
The expression and hypertrophic progression of the
chondrogenic phenotype were also examined by growing the 12-day-old embryonic chicken calvarial cultures
under conditions that both permitted chondrogenesis
and promoted mineralization. In these studies, a number of genes that are specific for either the chondrogenic
or osteogenic cells were examined (Fig. 2A). A definitive
increase in aggrecan and type II and X collagen gene
expression (chondrogenesis markers) was seen in cultures grown in serum-substituted medium. However,
as was previously demonstrated (Toma et al., 1997), the
expression of type X collagen was observed only after
longer periods of growth in serum-substituted medium
that had been specifically supplemented with ascorbic
acid and ␤GPO4 (see below). While chondrogenic cells
were selectively increased when grown in serum substitute, there was only a slight diminishment in the
osteogenic phenotype of the same cultures as determined from osteopontin and osteocalcin gene expression (osteogenesis markers), demonstrating that the
cultures maintained their overall osteogenic differentiation.
The relationship between levels of mRNA expression
and new collagen synthesis was assessed in the cultures by pulse labeling with 3H-proline after 12 days of
growth in mineralizing medium (Fig. 2B). Type I collagen synthesis was detected in cultures grown in both
serum-substituted and FBS-containing medium as evidenced by the detection of the ␣2(I) collagen chain. In
both cultures ␣2(I) was detected as a major secretory
product based on its prevalence in the medium. An
increased type II collagen expression was indicated by a
greatly increased ␣1 to ␣2 ratio seen in serumsubstituted cultures. In comparing the cultures grown
under the different conditions, proline labeled bands at
the position of pro ␣1(X) and its processed form were
detected only in the cultures grown in serum-substituted medium. Consistent with previous analysis (Gerstenfeld and Landis, 1991), type X collagen was shown
to increase preferentially in its accumulation in the cell
layers of the mineralizing culture.
The mechanisms by which various growth conditions
promote the selection of specific cell lineages within a
population of presumptive skeletal cells may be examined by determining the total number of cells within the
population that express osteogenic or chondrogenic
lineage markers (Gerstenfeld et al., 1996; Toma et al.,
1997). In the current study, this was carried out by
immunolabeling the cell populations grown under the
different culture conditions with markers selective for
chondrogenic (type II collagen) and osteogenic (osteocyte SB-5) cells (Fig. 3). Analysis shows dense nodules
of birefringent rounded cells cultured with serum substitute selectively labeled for type II collagen but few
such chondrogenic cells in cultures grown under normal conditions in the absence of serum substitute (Fig.
3A, first and second rows of panels). In contrast, within
cell populations grown under either culture condition,
the terminal osteogenic marker SB-5 was present on
cells throughout both cultures (Fig. 3A, third row of
panels).
In order to determine if there was selective proliferation of chondrogenic cells compared to osteogenic cells,
proliferative cells were labeled by BrdU incorporation
161
(Fig. 3A, bottom panels). There was selective incorporation of BrdU within the chondrogenic nodules, while in
comparison few cells were labeled in the internodular
regions composed primarily of osteogenic cells. These
results show that the chondrogenic cells were more
proliferative than the osteogenic cells.
The percentage of cells that expressed either osteogenic or chondrogenic markers was determined from
cell counts taken from random microscopic fields at
identical magnifications and these results are summarized in Figure 3B. Under control growth conditions in
medium supplemented with FBS, ⬃15% of the cells in
the embryonic cultures expressed a chondrogenic phenotype while about ⬃40% of the cells in these cultures
were positive for the expression of the osteogenic SB-5
marker. When these same cultures were grown in
medium containing serum substitute, there was a
⬃3.5-fold increase in the cells that expressed the chondrogenic phenotype such that ⬃50% of the cells were
now positive. This increase in the number of chondrogenic cells was associated with a slight decrease in the
number of cells that expressed the osteocyte specific
marker to ⬃35% of the cell population. This decrease
was not statistically significant compared to the cell
numbers determined from FBS cultures. Thus, growth
of these cells in serum substitute did not change the
overall percentage of cells that expressed an osteogenic
phenotype based on their immunoreactivity for SB-5.
The results presented in Figure 3 suggest that chondrocytes may produce autocrine factors that are selective for their own survival or further proliferation.
Preliminary studies carried out to test this hypothesis
are depicted in Figure 4.
In these experiments, committed growth chondrocytes derived from the cephalic portions of 17-day-old
embryonic chicken sterna were grown in culture and
their medium was collected. This conditioned medium
was added as a supplement to the medium of 12-day-old
chicken calvarial cells grown in FBS. The 12-day-old
embryonic calvarial cell populations were allowed to
grow in the presence of different ratios of conditioned:
FBS medium and the chondrogenic phenotype of the
cultures was subsequently examined for expression of
type X collagen within the cell layers. Figure 4 shows a
comparison of the type X collagen labeling seen in these
cultures supplemented in ratios of 1:3 or 3:1 and
without conditioned medium. Increased labeling intensity and numbers of observable type X collagen-reactive
chondrocyte nodules are found, dependent on the ratio
of conditioned medium that was added. Immunostained
cephalic chondrocyte cultures grown under conditions
promoting chondrocyte hypertrophy were used as a
positive control. Calvarial cell cultures from 17-day-old
embryonic chickens were used as negative controls
since previous studies demonstrated such cell populations lacked the ability to undergo chondrogenesis in
response to growth in serum-substituted medium (Toma
et al., 1997). This latter control was also used to address
the question of whether there was adsorption of the
type X collagen to the osteoblast cell layers because of
type X presence in the conditioned medium isolated
from the cephalic chondrocyte cultures. No immunofluorescent labeling with type X collagen antibody was
apparent for the 17-day-old embryonic chicken calvarial cell populations supplemented with a 3:1 ratio of
chondrocyte-conditioned medium. Type X collagen was
Fig. 3 A.
CHONDROGENIC POTENTIAL OF SKELETAL CELLS
163
Fig. 3. Chondrogenesis in presumptive skeletal cell populations.
A: (page 162) Comparisons of chondrogenic and osteogenic phenotypic
markers in 12-day-old chicken calvarial cell cultures grown in either
SS (serum substitute) or 10% FBS. The osteocyte cell surface marker
SB-5 and type II collagen were used as independent determinants for
osteogenic or chondrogenic differentiation, respectively, for the cultured cells. Proliferative cells were labeled with BrdU. The photomicrographs depict cellular expression of the various markers carried out
after 6 days of growth in complete medium. Top: Phase contrast
micrographs of cells cultured with FBS or SS followed by matched
immunochemical analysis with type II collagen (all panels, magnification, 100x). Intense staining of dense chondrogenic nodules may be
noted. Immunochemical analysis for SB-5 expression shows numerous
labeled cells in cultures grown with FBS or SS (magnification, 200x).
Osteocyte processes can be clearly observed (magnification, 400x,
inset, left panel). Similar numbers of SB-5 positive cells may be seen
throughout the cultures. Bottom: Nuclear uptake of BrdU. Labeling
is more pronounced within a chondrogenic nodule in a culture grown
in SS than in an inter-nodular area of a culture supplemented with
FBS (magnification, 400x). B: (above) A graphic depiction of the
percentage of cells expressing chondrogenic and osteogenic phenotypes in calvarial cultures from 12-day-old chicken embryos. Quantitative data are from Toma et al. (1997). The percent of immunologically
labeled cells for type II collagen (COL II) or SB-5 was calculated from
10 random micrographic fields such as represented in A (middle) for
FBS or serum substitute (SS) cultures. Error bars denote one standard
deviation of the measurements. Differences in type II collagen are
significant at p ⫽ .005.
found in cultures grown for 12 days; its presence can be
attributed to induction of new chondrogenic differentiation.
Finally, it is interesting to note the distinctive morphological location of the type X collagen that was seen
within these cultures. Type X collagen labeled a dense
fibrillar organization restricted to the chondrocyte nodules with a discernible pericellular distribution around
individual cells within the 12-day calvarial cell cultures. A similar more heavily labeled distribution of the
type X collagen was found in the cephalic chondrocyte
cultures isolated from 17-day-old embryonic chickens.
Labeling of these cultures was much more extensive
compared to that of calvarial cells and was observed
over the entire cell layer. This result was most likely
caused by the higher levels of synthesis of the cephalic
cultures.
data. The progression of these cultures grown under
different conditions was assessed qualitatively by histochemical analysis of individual culture wells (Fig. 5)
and measured quantitatively (Table 1). Cultures grown
in both DME and BGJb media containing ascorbic acid
promote the endochondral sequence based on the observed increased alkaline phosphatase activity and
mineral deposition that was seen in these cultures (Fig.
5A and Table 1). While the inclusion of organic phosphate (␤GPO4) increases mineral deposition in cultures
grown in both DME and BGJb, it only enhanced alkaline phosphatase activity when included in BGJb. Overall proteoglycan deposition in the cultures did not
appear to vary based on alcian blue staining (Fig. 5A).
There was, however, a marked increase in both DNA
content and total protein synthesis in cultures grown in
BGJb medium. Results of an examination of the temporal sequence of endochondral progression are shown in
Figure 5B. Ascorbic acid and ␤GPO4 introduced into the
culture system at day 6 causes the cells to undergo
rapid mineral deposition as determined from alkaline
phosphatase and von Kossa staining.
In previous studies of osteoblast (Gerstenfeld et al.,
1987) and chondrocyte cultures (Gerstenfeld and Landis, 1991), ascorbic acid supplementation was shown to
promote both collagen deposition and cellular differentiation. Figure 6 demonstrates that culture supplemen-
Hypertrophic Progression of Growth
Chondrocytes In Vitro
Conditions can be described that promote the progression in vitro of the endochondral sequence of the
chondrogenic program. Committed chondrocytes isolated from the vertebral columns (a tissue undergoing
endochondral replacement in vivo (Shapiro, 1992)) of
12-day-old embryonic chickens were used in these
studies. Figures 5 and 6 and Table 1 present relevant
164
L.C. GERSTENFELD ET AL.
Fig. 4. Autocrine selection of chondrogenic growth of 12-day-old
embryonic calvarial cell populations. Type X collagen matrix deposition in 12-day-old embryonic chicken calvarial cultures grown in the
presence of conditioned medium from hypertrophic chondrocytes
derived from 17-day-old embryonic chicken cephalic sterna. The
identity and distribution of type X collagen extracellular matrix
accumulation within chondrogenic nodules were determined by immunolocalization. Top: Calvarial cell cultures grown for 12 days in
complete medium containing 10% FBS without conditioned medium
(control) or with a 1:3 or 3:1 ratio of conditioned medium from cephalic
sternal chondrocyte cultures. Bottom: Negative and positive controls,
respectively, of 17-day-old chicken embryo calvarial cell populations
treated with a 3:1 ratio of chondrocyte-conditioned medium (left) and
cephalic sternal chondrocyte cultures after 12 days of growth in
mineralizing medium. All photomicrographs: magnification, 400x.
tation with ascorbic acid promotes chondrocyte hypertrophic differentiation as demonstrated by the induction
of high levels of type X collagen mRNA expression and
protein synthesis. Figure 7 summarizes the overall
temporal relationships between proteoglycan and collagen synthesis to mineral deposition and relates these to
aggrecan and types II and X collagen mRNA expression. Here, it is apparent that proteoglycan synthesis
and message levels fall rapidly after a few days of
culture time while collagen content increases and then
drops and mineral continuously increases. The rise and
fall in collagen synthesis is mirrored by a preceding
type II collagen message expression. Type X expression
gradually rises throughout the culture period.
The morphogenesis of the chondrocytes and their
matrices under culture growth conditions permissive
for endochondral progression is shown in Figures
8–11. Corresponding to the changes in gene expression
and biochemistry described above, there are changes in
the structure of the chondrocytes and their extracellular regions that likewise reflect a progression through
the endochondral sequence. The temporal development
of the cultures morphologically shows increasing numbers of cells having rounded shapes and residence
within lacunae (Fig. 8). Changes in cell size, shape,
distribution, and cytoplasmic content in the cultures
are apparent (Figs. 8–11) with larger chondrocytes
being associated with not only more mature cultures
(grown for longer periods) but also the relatively deeper
regions of a culture dish where mineralization is becoming prominent (Figs. 8, 10). Representative calvarial
chondrocytes from 12-day-old embryonic chickens and
grown for 17–18 days in culture are generally marked
by typical intracellular organelles, including a single
nucleus, extensive endoplasmic reticulum, vesicles, and
vacuoles (Fig. 9). Many chondrocytes, particularly the
larger ones, are observed within a highly staining
pericellular region characterized by the thin fibrils of
CHONDROGENIC POTENTIAL OF SKELETAL CELLS
165
Fig. 5. Histochemical analysis of proteoglycan, alkaline phosphatase, and mineral deposition in cultures of vertebral chondrocytes
grown under different conditions. A: All cultures were grown for 15
days in BGJb or DME with or without ␤-glycerophosphate (␤-GPO4).
On day 15, separate 33 mm wells were analyzed using histochemical
staining to assess proteoglycan deposition (alcian blue), mineral
deposition (von Kossa silver nitrate), or alkaline phosphatase (Apase,
fast red). B: Time course over 20 days of mineral accumulation and
alkaline phosphatase activity in 33 mm culture wells containing
vertebral chondrocytes grown in BGJb medium supplemented with
␤-GPO4. Images are of the full 33 mm wells.
type II collagen and small condensations suggestive of
proteoglycans (aggrecan) (Fig. 11). Mineral in the form
of dense, irregularly shaped deposits is exclusively
extracellular in location and is found overlying the
collagen (Fig. 10), frequently following individual fibrils
and in clear association with them (Fig. 11). There are
many cytoplasmic processes that invade the extracellular regions of the cultures (Fig. 11) and these appear to
terminate in small vesicles (Fig. 11). Consistent association of such vesicles with mineral is equivocal. With
time, the cultures mineralize more completely, and the
numerous mineral deposits can be identified structurally as apatitic on the basis of their electron diffraction
character (data not presented; see Gerstenfeld and
Landis, 1991).
by which mesenchymal stem cells are restricted to the
osteogenic vs. the chondrogenic lineage was examined
in skeletal cell populations isolated from embryonic
chicken calvaria. The use of cell populations from
calvaria is particularly informative since this tissue
undergoes direct bone induction in the absence of an
endochondral stage. Thus, the appearance of chondrocytes in these cell populations arises through the
induction and differentiation of the mesenchymal stem
cells to chondrocytes. The data presented and reviewed
here indicate that the developmental restriction of
skeletal cells of the calvaria was not a result of positive
selection for osteogenic differentiation but a negative
selection against the progressive growth of chondrogenic cells in the absence of either a permissive or
inductive environment. This conclusion is based on the
findings that the 12-day-old embryonic calvarial cell
populations selectively proliferated to chondrogenic
cells but were little changed in their percentage of
osteogenic cells when they were induced to undergo
chondrogenesis.
DISCUSSION
The data presented here focus on the growth conditions in vitro that either induce chondrogenesis from
presumptive or uncommitted cells or promote chondrocyte maturation and terminal differentiation toward a
hypertrophic phenotype. The underlying mechanism(s)
166
L.C. GERSTENFELD ET AL.
Fig. 6. Effect of ascorbic acid on expression of types II and X
collagen. A: Northern blot analysis of type II and type X collagen
mRNA expression in 12-day-old embryonic chicken cultures of vertebral chondrocytes grown 15 days in culture, in the absence (⫺) or
presence (⫹) of ascorbic acid. Five micrograms of total RNA were
applied to each lane. Horizontal lines to the left of the blots indicate
the position of 27S mRNA as a molecular weight marker. B: Collagen
synthesis in ascorbic acid treated (⫹/⫺) cultures. Intact procollagen
and collagen ␣ chains for type II and type X of either cell layer or
medium were separately extracted as described in Materials and
Methods. The electrophoretic positions of various 3H-proline-labeled
collagen chains of interest are indicated.
TABLE 1. Culture conditions and their effects on culture parameters*
Mineralization1
DNA content (µg/33-mm well)
Apase activity (nM p-nitrophenol/µg DNA/30 min)
Total protein synthesis (3H-leucine/µg DNA)2
Percent collagen synthesis3
DME
DME ⫹ Asc
DME ⫹ Asc ⫹ ␤GPO4
BGJb ⫹ Asc
BGJb ⫹ Asc ⫹ ␤GPO4
⫺
5.8 ⫾ 0.9
15.0 ⫾ 4.0
1.07 ⫻ 105
34 ⫾ 8
⫹
6.2 ⫾ 0.5
140.0 ⫾ 30.0
ND
48 ⫾ 12
⫹⫹
7.4 ⫾ 0.6
162.0 ⫾ 26.5
ND
53 ⫾ 14
⫹⫹⫹
10.6 ⫾ 0.6
148.0 ⫾ 25.0
6.8 ⫻ 104
58 ⫾ 14
⫹⫹⫹⫹
13.7 ⫾ 0.7
403.0 ⫾ 46.0
4.1 ⫻ 104
62 ⫾ 15
*Reprinted with permission from Gerstenfeld, L.C., and Landis, W.J. (1991) Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell
culture system. J. Cell Biol., 112:501–513.
1Gross extracellular matrix calcification was assessed qualitatively by von Kossa staining.
2Total protein synthesis was assessed after 24 hours of labeling of day 15 cultures and represents the addition of both medium and cell layer nondialyzable TCA
precipitable counts per minute.
3Percent collagen synthesis was determined by collagenase treatment of day 15 cultures after 24 hours 3H-proline labeling and samples represent the addition of both
medium and cell layer-associated nondialyzable counts per minute.
ND ⫽ Not determined; Asc ⫽ ascorbic acid; ␤GPO4 ⫽ ␤-glycerophosphate.
It is interesting to speculate that the balance between chondrogenesis and osteogenesis may be controlled through epigenetic signals that promote presumptive skeletal cell populations to differentiate
independently toward either the osteogenic or chondrogenic lineage. Thus, while intrinsic genetic regulation
controls the gross anatomic patterning of the skeletal
organs, micropatterning and growth control probably
reside in a wide variety of extrinsic mediators that may
be related to structural functions of the organs or their
physiological role in mineral homeostasis. Studies of
secondary cartilage formation of the articulating surfaces of the vertebrate maxillary process have shown
that during embryogenesis the absence of movement of
this joint inhibited secondary growth cartilage formation (Thorogood, 1979). Correlative to this finding is the
report that bone repair in rigidly fixed fractures of
minimized micro-motion results in less cartilage formation and direct induction of new bone formation (Probst
and Spiegel, 1997). In addition, systemic factors such as
calcium stasis (Jacenko and Tuan, 1986), levels of
1,25(OH)2D3 (Gawande and Tuan, 1990), or the presence of corticosteroids such as dexamethasone (Bellows
et al., 1989) have all been shown to affect the balance of
osteogenic versus chondrogenic differentiation. Finally,
the presence of local morphogens such as OP-1 (Asahina et al., 1993), transforming growth factor-␤ (TGF␤)
(Derynck, 1994), and basic fibroblast growth factor
CHONDROGENIC POTENTIAL OF SKELETAL CELLS
167
Fig. 7. Comparison of chondrocyte mineral deposition, collagen synthesis, proteoglycan synthesis, and gene expression over a
20-day growth period in vitro. Top: Total
collagen synthesis (l), proteoglycan synthesis (●), and mineral deposition (o) were determined from triplicate 33 mm culture dishes of
duplicate experiments with 12-day-old embryonic chicken vertebral chondrocytes. Bottom: Comparison of type II collagen (l), type
X collagen (o), and aggrecan (●) mRNA gene
expression over 21 days. The left axis of the
plot pertains to quantitative levels of aggrecan (Ag) and the right axis to quantitative
levels of either type II or type X collagen.
Error bars denote total range of experimental
variation.
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L.C. GERSTENFELD ET AL.
Fig. 8. A light micrograph of a 12-day-old embryonic chicken
calvarial chondrocyte culture grown for 18 days in mineralizing
medium. The culture has been sectioned to show its full thickness
profile. The tissue was fixed in glutaraldehyde and osmium tetroxide
and stained with toluidine blue. The interface between the culture
dish and the cultured cells and matrix is marked by the relatively
straight line along the bottom of the micrograph. The culture consists
of numerous cells, each having a generally round shape and residing in
single lacunae. The size, shape, and disposition of the cells are similar
to those features of growth plate chondrocytes observed in situ. The
extracellular matrices vary in their intensity of staining as do some of
the cells. Pericellular staining marks the lacunae of certain cells
(arrowheads) and, in the deeper portions of the culture, nearer the
culture dish bottom, there is deposition of considerable mineral
(arrows). Magnification, 1,200x; bar ⫽ 10 µm.
(FGF) (Rousseau et al., 1994) are also suggested to
influence the balance between chondrogenesis and osteogenesis for presumptive skeletal cell populations.
The apparent local control in the balance of chondrogenic and osteogenic differentiation has led to the
concept that these processes are coupled in some manner (Gerstenfeld and Shapiro, 1996). For example,
promotion of chondrogenesis by an absence of serum in
growth medium is consistent with studies showing that
either embryonic calvarial cell cultures or undifferentiated limb bud cells in serum-free conditions become
chondrogenic rather than osteogenic (Cole et al., 1992;
Rifas et al., 1982; Toma et al., 1997; Wong and Tuan,
1995). Unlike many other cell types, chondrocytes
appear to be specifically adapted to grow in serum-free
conditions in vitro (Ballock and Reddi, 1994; Bruckner
et al., 1989; Quarto et al., 1992). These data might also
suggest that serum contains factors that either inhibit
chondrogenesis or promote selective growth of osteogenic cells.
Chondrocytes themselves may produce factors that
promote their own differentiation or growth. This conclusion is supported by studies that have shown that
chondrogenesis can occur when cells are grown at high
plating densities or in micromass cultures, even in the
presence of serum supplementation (Osdoby and
Caplan, 1979). Other studies demonstrate that chondrocytes cultured at high densities produce autocrine
factors that enable them to survive at low cell densities
in the absence of serum (Ishizaki et al., 1994), and it is
interesting to speculate that growth in serum substitute reported here and elsewhere (Toma et al., 1997;
Wong and Tuan, 1995) may have either promoted
chondrogenesis or maintained critical numbers of chondrogenic cells that allow their survival and expansion
in culture. Further work reports that calvarial cell
populations isolated from 14-day avian embryos and
grown in vitro produce factors that inhibit limb bud
mesenchymal cells from undergoing chondrogenesis
(Wong and Tuan, 1995). Moreover, the limb bud mesenchymal cells produce factors that stimulate selected
calvarial cell populations to undergo chondrogenesis
(Wong and Tuan, 1995). The results in the present
study provide yet additional evidence that chondrocytes produce factors that promote chondrogenic differentiation. All the above results lead to the conclusion
that, under normal growth conditions in vivo, osteogenic differentiation predominates as a result of regulated selection against progressive chondrogenic differentiation, which itself is probably under the control
primarily of autocrine and paracrine factors produced
by the local precursor cell population.
Considerable data has accumulated pertaining to the
culture conditions that promote growth chondrocyte
differentiation. From the research summarized here
and that of several other groups (Gerstenfeld and
Landis 1991; Leboy et al., 1989; Sandell and Daniel,
1988), one of the most critical factors leading to maintenance of the chondrocyte phenotype, progressive culture growth, and hypertrophic differentiation of these
cells is the inclusion of ascorbic acid in the growth
media. The role of ascorbic acid as an essential cofactor
in collagen synthesis is well documented (Lyons and
Schwartz, 1984), and the biochemical data presented
here show that ascorbic acid increased both total collagen synthesis and matrix accumulation. Ultrastructural analysis further demonstrates that over the period of chondrocyte growth in vitro, these cells
elaborated an extensive extracellular matrix that retained morphological features comparable to those seen
in vivo, including increasing collagen content with
time. Recent studies of both chondrocytes and osteoblast growth in vitro describe the crucial role the
extracellular matrix (ECM) plays in stimulating cellular differentiation (Franceschi et al., 1994; Gerstenfeld
et al., 1988; Gerstenfeld and Landis, 1991). These
reports and the present data suggest that the differentiation of specialized connective tissue cells not only is
controlled by extrinsic soluble regulators but also is
dependent on some form of feedback regulatory process
related to the assembly and deposition of their matrices. While the exact mechanism(s) by which the ECM
may exert its effects on cellular differentiation in this
manner are incompletely understood, considerable information has accumulated to support the concept that
both the maintenance of normal cell morphology and
specific receptor interactions with the ECM are quite
CHONDROGENIC POTENTIAL OF SKELETAL CELLS
169
Fig. 9. An electron micrograph of a calvarial chondrocyte culture
grown for 17 days in mineralizing medium. The region contains a
number of cells typically having a single nucleus and nucleolus as well
as cytoplasmic organelles marked by numerous vesicles and vacuoles
and extensive endoplasmic reticulum. The cells reside within lacuna
(L) defined by relatively electron transparent pericellular spaces (S).
The extracellular matrix beyond the pericellular regions of the tissue
is characterized principally by collagen fibrils (C). Mineral (M) appears as electron dense, small punctate and larger irregularly shaped
extracellular deposits. This specimen is torn (T) in places as a result of
sectioning through the mineral. Magnification, 2,850x; bar ⫽ 5.0 µm.
important in promoting normal skeletal cell differentiation (Franceschi, 1992).
While the data presented here define growth conditions that promote growth chondrocyte differentiation,
they also summarize several fundamental features that
characterize synthetic changes associated with hypertrophic cartilage maturation and matrix formation.
One of the most striking is an inverse relationship
between the levels of collagen and proteoglycan synthesis in comparison to mineral deposition. Thus, increasing collagen synthesis may be correlated with mineral
accumulation while proteoglycan synthesis levels decrease. Such a relationship is consistent with earlier
work (Gerstenfeld and Landis, 1991) and with the
suggestions that proteoglycans inhibit mineral nucleation (Buckwalter et al., 1987) while the development of
a collagenous matrix is a prerequisite to mineral deposition (Glimcher, 1992). It is also apparent that the
synthetic levels in collagen and proteoglycan observed
here are primarily controlled at a pre-translational
level as reflected by comparisons in collagen and aggrecan mRNA. Further, while overall collagen synthesis is
increasing, it is largely a result of increasing levels of
type X collagen expression. The reciprocal relationship
found here in the expression of types II and X collagen
is comparable to the reported temporal patterns of
expression for these genes observed during the endochondral differentiation of appendicular growth cartilage in vivo (Schmid and Linsenmayer, 1985).
Both the cells and extracellular matrices within the
cultures described here develop and retain a number of
ultrastructural features that have their close correlates
with the biochemical results presented above as well as
the structural characteristics of chondrocytes and matrix in vivo. These include, for example, the presence of
high levels of alkaline phosphatase as mineralization
occurs and progresses in culture. Microscopically, alkaline phosphatase and other enzymes may be identified
by specific immunoreactivity of culture sections or
inferred principally through the observation of electron
dense condensates appearing within extracellular
vesicles. In this paper, the many such dense vesicles are
present in the extracellular regions of calvarial chondrocytes as cultures of these cells grow and mineralize.
Their detection follows the progressive changes in
alkaline phosphatase measured with nitrophenol. A
second correlate is reflected in the overall chondrocyte
culture thickness that is elaborated and increases with
time. The changes observed can be attributed to a
significant degree of hydration of the culture matrix
through water retention mediated by proteoglycans.
Evidence for these macromolecules is clear in micro-
170
L.C. GERSTENFELD ET AL.
Fig. 10. Microscopic view of the hypertrophic state in vitro. The
lower portions of a culture nearer to the culture dish bottom may be
marked by calvarial cells (C) undergoing hypertrophy and having
sparse cytoplasmic elements. The extracellular matrix at these loca-
tions contains many mineral deposits (M) overlying or otherwise in
apparent association with collagen fibrils of narrow diameter. This
micrograph is taken from a culture grown for 17 days. Magnification,
4,400x; bar ⫽ 5.0 µm.
graphs taken from regions spanning wide portions of
the culture volume, a result that is consistent with the
known presence, location, and distribution of proteoglycans in vivo. In addition, the ultrastructural studies
here reveal numerous type II collagen fibrils comprising the culture matrices and whose appearance corresponds to their biochemical detection in the same
culture samples as well as their documented presence
in vivo. Perhaps the most important observation made
in this work is that these culture systems will mineralize and they do so in a reproducible manner. As such,
these systems are distinct from others that mineralize
unpredictably or not at all or elaborate mineral that is
abnormal. In the present cultures, the mineral is
apatitic, having a chemical nature identical by electron
diffraction means to that found in vivo. The mineral is
also discretely deposited and is associated principally
with the many type II collagen fibrils comprising the
matrices. In these aspects, the chondrocyte cultures
again follow the manner of mineralization that has
been reported extensively, based on many studies of
growth cartilage in vivo.
In summary, the data reviewed here indicate that
studies in vitro have led to considerable understanding
of mechanisms that regulate chondrocyte differentiation, morphogenesis, extracellular matrix formation,
and mineralization. These data suggest that chondrogenesis occurs in three stages: the differentiation of
chondrogenic cells within a population of presumptive
skeletal cells; the maintenance and expansion of these
chondrocytes; and chondrocyte terminal maturation.
The progressive differentiation of chondrocytes in vitro
demonstrates that the cells develop in a manner comparable to that found in vivo. Chondrocyte maturation is
characterized by an increasing expression of type X
collagen and alkaline phosphatase and a decreasing
expression of type II collagen and aggrecan. These
changes are accompanied by correlative morphogenetic
modulations including chondrocyte progression to a
hypertrophic phenotype and the development of a ma-
CHONDROGENIC POTENTIAL OF SKELETAL CELLS
171
Fig. 11. Electron micrograph of the chondrocyte-matrix interface.
An enlargement of a region from a 17-day-old cell culture shows
cytoplasmic organelles, including mitochondria (arrows), the Golgi
apparatus (G), dense vacuoles (DV), endoplasmic reticulum (ER), and
other elements. The cell appears to be secreting numbers of thin
collagen fibrils adjacent to the cell envelope and its infoldings. The
narrow diameter and faint periodicity (arrowheads) of the fibrils are
consistent with structural features of type II collagen. Cell processes
(P) sectioned into a variety of profiles, possible vesicles (V) containing
alkaline phosphatase, highly dense condensations of putative proteoglycans (PG), and mineral deposits (M) may be observed in the
extracellular matrix. The mineral is located over the collagen network
in the matrix and some deposits follow individual collagen fibrils
(double arrowheads). The cellular and matrix characteristics illustrated here are similar to those described for cartilage in vivo.
Magnification, 25,600x; bar ⫽ 1.0 µm.
trix characterized ultimately by extensive apatitic mineral deposition corresponding to that observed in vivo.
ated figures. Other antibodies were obtained from the
Developmental Studies Hybridoma Bank, which is
maintained by the Department of Pharmacology and
Molecular Sciences at Johns Hopkins University School
of Medicine, Baltimore, MD, and the Department of
Biology at the University of Iowa, Des Moines, IA,
under contract NO1-HD-2–3144 from the National
Institutes of Health.
ACKNOWLEDGMENTS
This investigation was supported in part by NIH
grant HD22400 (L.C.G.), NIH AR41452 (W.J.L.), and
the Max Kade Foundation (C.D.T.). The authors express thanks to Ms. Karen J. Hodgens (Children’s
Hospital, Boston, MA) for superb technical assistance
and manuscript preparation and to Drs. Tom Linsenmayer (Tufts University School of Medicine, Boston,
MA), Scott Bruder (Osiris Corp., Baltimore, MD), and
Arnold Caplan (Case Western Reserve University, Cleveland, OH) for generously providing type X collagen and
osteoblast lineage-specific antibodies, respectively,
which were used in these studies. The authors are also
indebted to Mr. Christopher George (Boston University
Medical Center) for assistance with the computer gener-
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