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Influence of retinol on human chondrocytes in agarose culture.

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THE ANATOMICAL RECORD 23252-59 (1992)
Influence of Retinol on Human Chondrocytes in Agarose Culture
AMY LYNN AULTHOUSE, CECILIA M. CARUBELLI, TOD M. DOW,
CHRISTINE ZIEGELMAYER, AND MICHAEL BECK
University of Oklahoma Health Sciences Center, College of Medicine, Anatomical Sciences,
Oklahoma City, Oklahoma (A.LA.,C.M.C., T.M.D.); University of Mainz, Department of
Pediatrics, Mainz, Germany (C.Z., M B J
ABSTRACT
Vitamin A and its congeners, collectively called retinoids, are
known to have teratogenic potential and have induced craniofacial and limb malformations in numerous animal species. More importantly, retinoids are recognized as teratogenic to fetuses of pregnant women who have taken such preparations for dermatologic disorders. Information gathered from the study of animal
models suggests that retinoids interfere with cartilage differentiation. If chondrogenesis in limb development is disturbed it may contribute to limb reductions and
malformations. In vitro studies using various animal systems have shown that
cartilage matrix macromolecules are altered to resemble those secreted by mesenchymal cells. The response of human chondrocytes to retinoids in vitro is not
known. Culture of human chondrocytes in agarose maintains the cartilage phenotype and therefore serves as a model system to evaluate the influence of retinoids
directly on human chondrogenesis. The studies presented in this paper were done
t o determine if the expression of specific matrix macromolecules of human chondrocytes in agarose culture is altered by retinol treatment. Immunocytochemistry
demonstrated enhanced labeling of type I collagen while type I1 collagen labeling
was reduced in cultures treated with retinol. In addition, morphometric analyses
indicated a decrease in the size and number of chondrogenic clusters and that
individual cells synthesized less alcian blue matrix when compared to parallel
control cultures. The size of the proteoglycan monomers, glycosaminoglycan side
chains as well as the disaccharide composition were not affected. However, there
was a reduction in the quantity of proteoglycan monomers produced.
Vitamin A and its analogues can exert major effects
on the control of pattern formation and differentiation.
It has been shown that retinoic acid when applied directly to developing chick limb buds causes duplication
of digits (Tickle et al., 1985) and proximo-distal serial
repetition of stump tissue in regenerating limbs (Maden, 1982, 1983). Retinoic acid has been shown to
mimic the activity of the zone of polarizing activity
(ZPA), a posterior region of the limb bud known to
cause duplications when grafted anteriorly (Tickle et
al., 1982). Retinoic acid has been described to act as a
morphogen. Morphogens are hypothetical signal substances emitting from an organizing center. The cells
then respond to different concentrations by adopting
different pathways of differentiation (Slack, 1987).
Hence, certain retinoids may be responsible for proper
development of chick limb buds when in specific concentrations and locations. However, recent studies by
Wanek et al. (1991) have suggested that retinoic acid
acts by converting anterior limb mesenchymal cells to
ZPA cells which in turn would bring about digit duplication. Moreover, Noji et al. (1991) have reported that
it is unlikely that exogenous retinoic acid is identical
with a morphogen secreted from a graphed ZPA but
that there could be an “endogenous retinoic a c i d that
may serve as a morphogen.
Although retinoids may be instrumental in deter0 1992 WILEY-LISS. INC
mining proper skeletal development, they have also
been shown to be teratogenic when given during specific times and concentrations in development. The effects of excess retinol given to pregnant mice and rats
during certain periods of gestational growth resulted in
pups with skeletal defects. Rat pups displayed various
craniofacial anomalies while mouse pups showed numerous craniofacial and limb defects (Pillans et al.,
1988; Takakubo and Eto, 1986).
Retinoids are commonly used in humans in the treatment of various dermatologic disorders. Specifically,
isotretinoin (Accutane) and etretinate (Tigason) have
been administered orally to treat severe acne and psoriasis. Since the release of these drugs, numerous cases
of spontaneous abortions and fetal abnormalities have
been reported. Fetal craniofacial and limb malformations have been associated with maternal ingestion of
isotretinoin during pregnancy (Barnhart, 1989; Lammer et al., 1985; McBride, 1985; Willhite et al., 1986).
Multiple synostoses, facial dysmorphia, syndactylies,
Received April 9, 1991; accepted June 24, 1991.
Address reprint requests to Amy Lynn Aulthouse, Ph.D., University of Oklahoma Health Sciences Center, College of Medicine, Department of Anatomical Sciences, P.O. Box 26901, Oklahoma City,
OK 73190.
INFLUENCE OF RETINOL ON HUMAN CHONDROCYTES
absence of terminal phalanges, malformations of hip,
ankle, and forearm, and other skeletal defects have
been reported when etretinate was taken during pregnancy (Barnhart, 1989; Grote et al., 1985; Happle et
al., 1984; Willhite e t al., 1986).
Animal models have shown that retinoids interfere
with limb development resulting in limb reduction
(Kochhar, 1973; Takakubo and Eto, 1986; Willhite et
al., 1986). Both in vivo and in vitro animal studies have
demonstrated that retinoids interfere with chondrogenesis and subsequently with limb development.
These reports have demonstrated that chondrogenesis
is interrupted concurrent with a switch in collagen
phenotype, a decrease in proteoglycan synthesis, and
a n increase in fibronectin synthesis.
Numerous cases of limb reduction in humans have
been reported (Lammer et al., 1985; Willhite et al.,
1986). Currently, human studies are restricted to interpretation of clinical manifestations because no system has yet been developed to evaluate the effect(s) of
retinoids directly on human chondrocytes. We utilized
an agarose culture system to directly study the response of human chondrocytes to retinol treatment.
METHODS AND MATERIALS
Culturing Techniques
Primary cell culture
Human costochondral cartilage was obtained a t autopsy. Individuals with recognized skeletal abnormalities were excluded. Six cases with ages ranging from 4
days to 6 years were evaluated. Primary cultures were
established as previously described (Aulthouse et al.,
1989). In brief, released chondrocytes were plated in
Dulbecco’s Modified Eagles Media (DMEM; Gibco,
Grand Island, NY) with 10% fetal calf serum (FCS;
Irvine Scientific, Santa Ana, CA), 1%L-glutamine (Irvine Scientific), and with 0.1% penicillin-streptomycin
(Gibco), in 24 multiwell tissue culture dishes (Corning,
Corning, NY).
53
(Corning) that were coated with 1% high temperature
agarose (Biorad Cat# 162-0100). The cultures were
subsequently refrigerated at 4°C for 15 minutes to allow the 0.5% low temperature agarose to gel. The cultures were fed twice weekly for 1-4 weeks. Cultures
were treated daily with 10 ng all-trans retinol (Sigma
Chemical Co.) dissolved in absolute ethanol. A stock
solution, 1mgll ml, of retinol was kept dark and stored
at -20°C. To evaluate the effects of ethanol on chondrogenesis, parallel cultures were treated with equivalent volumes of absolute ethanol. In addition, untreated normal control cultures were established.
There were no detectable differences between ethanoltreated and untreated normal controls. To evaluate the
potential of cells to recover after exposure to retinol,
some cultures were treated for 2 weeks with retinol and
then allowed to rest for 2 weeks without further treatment.
Light Microscopy Detection of Cartilage Proteoglycans
With Alcian Blue
Treated and control cultures were rinsed in phosphate-buffered saline (PBS) and then simultaneously
fixed and stained for 24 hours in a mixture of 2.5%
glutaraldehyde, 0.05% alcian blue with 25 mM sodium
acetate (pH 5.6), and 0.4 M magnesium chloride. Fixation was followed by brief rinses with 3% acetic acid,
then 25% and 50% ethanol equivalent volumes (ETOH)
with 3% acetic acid and finally 70% ETOH (Aydelotte
e t al., 1986).
Quantification of Alcian Blue Staining Material of
Single Cells
In order to quantitate the amount of alcian blue
staining material produced by individual cells, single
cells were randomly selected from cultures that had
been treated daily with 10 nglml retinol, or equivalent
volumes of ethanol, or that were untreated. The
amount of alcian blue surrounding a single cell was
determined by subtracting the perimeter of the cell
Monolayer culture
from the total perimeter of the alcian blue halo surThe biochemical assays required quantities of cells rounding the cell. A Zeiss Interactive Digital Analysis
in excess of those obtained in primary cultures. There- System (ZIDAS, Zeiss, Oberkochen, West Germany)
fore, cells in primary cultures were grown to conflu- was used for these measurements. Using the Crunch
ence and passaged to tissue culture flasks. Cells were Statistical program (Crunch Software Corporation,
rinsed twice with Hank’s balanced salt solution (HBSS) Oakland, CA), the means were calculated and a one(Irvine Scientific) and dissociated in 0.2% trypsin way analysis of variance was performed. The Newman(Gibco) with 5 mM ethylenediamine-tetraacetic acid Keuls post-hoc test was performed to compare the dif(EDTA) (Sigma Chemical Co., St. Louis, MO). The cells ferent treatment groups (4 weeks retinol, 4 weeks
were then resuspended in 4 ml of media and passaged ethanol, and 4 weeks untreated).
to 25 cm2 tissue culture flasks. Cells were passaged one
to five times.
Agarose culture
Cells were dissociated from monolayer, centrifuged,
resuspended, and maintained in 5 ml of complete
DMEM. The cell suspension was allowed to rest for 1
hour at 4°C and then filtered through 2 ply, No. 20,
nitex (Tetko Inc., Elmsford, NY). The cell suspension
was centrifuged and resuspended at 5 x lo5 cells in 1
ml of 0.5% low temperature agarose (Biorad Cat#1620017, Richmond, CA) in DMEM. For alcian blue quantitation and immunocytochemistry, 10 ~l of cell suspension was plated in 35 mm tissue culture dishes
Quantification of Cell Clusters as an Expression
of Chondrogenesis
Alcian blue stained whole cultures were photographed with a n IMT-2 Olympus inverted microscope
(E A 4 objective). All single cells and cell clusters associated with alcian blue staining were counted. Using
the Crunch Statistical program the means were calculated and a one-way analysis of variance was performed. The Newman-Keuls post-hoc test was performed to compare the different groups of treatment (4
weeks retinol, 4 weeks ethanol, 4 weeks untreated, and
2 weeks retinol followed by 2 weeks untreated).
A.L. AULTHOUSE ET AL.
54
Fig. f , Whole cultures stained with alcian blue after culture in agarose for 3 weeks. A: Normal untreated
control culture. B: Culture treated daily with absolute ethanol (equivalent volume given in (C). C:
Culture treated daily with 10 ng/ml all-trans retinol. A-C, X 15.
lmmunocyiochemistiy Used to identify
Matrix Macromolecules
Retinol-treated, ethanol-treated, and untreated control agarose cultures were rinsed three times in PBS
and fixed in two, 5 minute changes of methano1:acetone
(1:l) and air dried overnight. After rehydration in PBS,
the cultures were incubated overnight at 4°C in monoclonal antibody to one of the following monoclonal antibodies purchased from the Developmental Studies
Hydridoma Bank, University of Iowa, Iowa City, IA:
human type I1 collagen (CIICI, undiluted, Holmdahl et
al., 1986) or human type I procollagen (M38, undiluted,
McDonald et al., 1986). Cultures incubated in conditioned medium from NS1 myeloma cell line before fusion served as negative controls. The cultures were
rinsed and incubated for 1hour at room temperature in
fluorescein-conjugated rabbit anti-mouse IgG (1:100,
Cappel, Organon Teknika Corporation, West Chester,
PA). After final rinsing, the cultures were mounted in
80% glycerine in PBS and photographed through a n
Olympus Vanox microscope (DPlanApo 20 UV PL
Olympus objective) equipped for fluorescence microsCOPY.
Proteoglycan Biochemistry
The following biochemical assays were conducted on
untreated (normal controls), ethanol-treated (volume
equivalents), and all-trans retinol-treated chondrocytes grown in agarose culture.
CL column (1.0 x 100 cm, Pharmacia, Uppsala, Sweden). The agarose was rinsed with PBS and the proteoglycans were extracted with 4 M guanidinium chloride
(GnC1) in 0.1 M sodium acetate buffer, pH 5.8, containing protease inhibitors (0.1 M 6-aminohexanoic acid,
10 mM EDTA, 5 mM benzamidine, and 10 mM N-ethylmaleimide; all from Sigma Chemical Co.) a t 4°C for
24 hours. Subsequently, the agarose was separated
from the extract by centrifugation for 1hour at 30,000
rpm (Sorvall Ultracentrifuge OTD65B, TV 865 Sorvall
Vertical Rotor, Dupont Sorvall Instrument, Wilmington, DE). The supernatant was then dialyzed against
water and precipitated with ammonium sulfate (48
hours at 4°C). The precipitate was then centrifuged for
1 hour at 30,000 rpm. The sample was then resuspended in 4 M GnCl buffer. Fifty microliters of the
crude GnCl extract was next subjected to gel chromatography on a Sepharose 2B-CL column (1.0 x 100 cm,
Pharmacia).
The equilibration and elution buffer was 0.1 M sodium acetate, pH 5.8, with 4 M GnC1, and protease
inhibitors. The excluded and total volumes were determined with high-molecular-weight DNA from bacteriophage lambda D (Sigma Chemical Co.) and Na235S0,,
respectively. One milliliter fractions were collected and
assayed for radioactivity.
Determination of the hydrodynamic size of
glycosaminoglycan chain lengths
Proteoglycan containing fractions collected from the
Sepharose 2B-CL column were further analyzed. Glycosaminoglycan chains were detached from proteoglyDetermination of the hydrodynamic size of the
can
monomers by beta elimination (Truppe and Kresse,
proteoglycan monomers
1978). The samples were dialyzed against distilled waCells were suspended in agarose as described above ter, evaporated and resuspended in 500 p1 0.15 M
for 3 weeks (5 x lo5 cells in 0.5 ml agarose in medium). NaOH, and incubated for 4 hours at 37°C. An equal
After incubating for 48 hours in sulfate-free medium volume of 8 M GnCl was added to each sample. The
(SERVA West Germany), the cells were exposed to 100 hydrodynamic size was then determined by gel chropCi Na>S04 (Amersham-Buchler, Frankfurt/M, West matography on a 1.0 x 100 cm Sephacryl S-300 column
Germany) in 1.5 ml sulfate-free medium for 48 hours. (Pharmacia) as described by Beck et al. (1988). The
Proteoglycans that were secreted into the medium and equilibration and elution buffer was 0.1 M sodium acretained in the agarose were analyzed. The medium etate, pH 5.8, with 4 M GnCl and protease inhibitors.
was removed, precipitated with ammonium sulfate as The excluded and total volumes were determined with
described below, and then applied to a Sepharose 2B- Dextran blue and Na235S04,respectively.
INFLUENCE OF RETINOL ON HUMAN CHONDROCYTES
'60
O!
N^
50
50
E
E
v
40
40
30
30
20
20
10
10
6
0
0
NC
ROL
55
Glycosaminoglycan composition
Proteoglycan monomers (50 pl about 1,000-2,000
cpm) collected from Sepharose 2B-CL fractions were
digested with 20 mU chondroitin AC lyase (Sigma) in
0.1 M TrisiHCL, pH 7.3, with 0.1 M sodium acetate at
37°C for 12 hours as described by Beck et al., 1988. The
digest was then subjected to descending paper chromatography in butan-1-ol/l M NH,/acetic acid (2:1:3 by
volume). One centimeter strips, cut into small pieces
were eluted in 1 ml distilled water and measured with
a Kontron MR 300 Liquid Scintillation Counter.
RESULTS
Light Microscopy
A
-
N
Whole cultures (untreated, ethanol-treated, and
retinol-treated) stained with alcian blue are seen in
Figure 1. There are fewer alcian blue staining chondrogenic clusters in retinal-treated cultures when compared to ethanol and untreated cultures. Untreated
normal control and ethanol-treated cultures have numerous, large clusters of isogenic groups of chondrocytes. The retinol-treated cultures tend to have fewer
clusters that often contain only one or two cells.
15
Quantification of Aician Blue Staining Matrices
NC
non
ROL
6
200 T
$
+
5
-
y.-
T 200
180
180
160
160
140
140
120
120
;100
m
p
100
80
80
c
60
60
6
40
40
20
20
D
0
0
NC
ETOH
ROL
ROL2
C
Fig. 2. A Morphometric values recorded after 4 weeks treatment in
culture of a typical case as alcian blue staining material per single
cell (ABS mm'). NC, untreated; ETOH, ethanol equivalent volumes;
ROL, 10 ng retinol. Values are the mean ? SEM. NC > ROL ( P 5
0.01); NC > ETOH (P 4 0.01); ETOH > ROL (not significant). B:
Morphometric values of cell size (mm') from cells recorded in (A). NC,
untreated; ETOH, ethanol equivalent volumes; ROL, 10 ng retinol.
Values are the mean & SEM. There were no significant differences
between cell sizes. C: Representative case demonstrates the potential
for recovery after retinol treatment. The chondrogenic capacity is expressed in the number of chondrogenic clusters. NC, untreated;
ETOH, ethanol equivalent volumes (4weeks treatment); ROL, 10 ng
retinol (4 weeks treatment); ROL2, 10 ng retinol (2 weeks treatment
followed by 2 weeks no treatment). Values are the mean f SEM. NC
> ROL (P 5 0.01); ETOH > ROL (P4 0.01); NC, > ROL2 (P 5 0.01),
ROL2 > ROL (not significant).
The amount of alcian blue staining material surrounding individual cells was quantitated to determine
if differences between untreated and treated cultures
existed. Morphometric analysis indicated that individual cells in cultures treated with 10 ng retinol produce
significantly less alcian blue matrix (mm2)when compared to normal (untreated) controls and ethanoltreated cultures (Fig. 2A). The cell size was not affected
by retinol treatment (Fig. 2B).
The potential for cultures to recover from exposure to
retinol was evaluated by quantitating alcian blue associated cell clusters as an expression of chondrogenesis. It was found that untreated control cultures and
ethanol-treated cultures (4weeks treatment) had significantly more chondrogenic clusters than the retinoltreated cultures (4 weeks treatment). Moreover, cultures that were treated with retinol for 2 weeks
followed by 2 weeks of no treatment had significantly
less chondrogenic clusters than sister cultures that
were untreated or were treated with ethanol for 4
weeks. However, these cultures had more chondrogenic
clusters than cultures that had been treated continuously for 4 weeks, suggesting a potential for recovery in
vitro (Fig. 2C).
lmmunocytochemistry
Four week cultures were analyzed for the presence of
type I and I1 collagens. Retinol-treated cultures had a
strong label for type I procollagen (Fig. 3E). In contrast, both untreated normal control cultures (Fig. 3A)
and ethanol-treated cultures (Figure 3C) had no detectable immunofluorescence for type I procollagen. While
type I1 collagen is readily detectable by 4 weeks in
normal untreated cultures (Fig. 4A) it is also present in
ethanol-treated cultures (Fig. 4C). Cultures treated
with retinol, however, showed a marked decrease in
fluorescent labeling (Fig. 4E).
56
A.L. AULTHOUSE ET AL.
Fig. 3. Immunofluorescent detection of type I procollagen in human
chondrocytes cultured for 4 weeks in agarose. A. Untreated control
culture. Cell cluster (location indicated by arrowhead) is negative for
type I procollagen. B Phase contrast of A. C: Culture treated with
absolute ethanol (equivalent volume given in (El. Cell cluster (location indicated by arrowhead) is negative for type I procollagen. D:
Phase contrast of C. E: Culture treated with 10 ng/ml all-trans
retinol. Cell cluster (arrowhead) is positive for type I procollagen. F:
Phase contrast of E. Cell clusters are typically surrounded by alcian
blue staining matrix. The refractile ring seen in the phase contrast
photographs corresponds to the outer boundaries of alcian blue matrix. A-F, x 146.
Proteoglycan Analysis
gel chromatography, i t was found that the size of glycosaminoglycan chains was not affected by retinol (Fig.
5B). There were no detectable differences between ethanol-treated and untreated normal controls in the size
of proteoglycan monomers or glycosminoglycan side
chains. Analysis of disaccharide composition by paper
chromatography showed the ratio of chondroitin-
It was found using Sepharose 2B-CL gel chromatography, that the size of the proteoglycan monomer was
not affected by retinol treatment. However, the quantity of labeled monomers was reduced (Fig. 5A). The
size of the glycosaminoglycan side chains was also determined. With beta elimination and Sephacryl S-300
INFLUENCE O F RETINOL ON HUMAN CHONDROCYTES
57
Fig. 4. Immunofluorescent detection of type I1 collagen in human
chondrocytes cultured for 4 weeks in agarose. A Untreated control
culture. Cell cluster (arrowhead) is positive for type I1 collagen. B:
Phase contrast of A. C: Culture treated with absolute ethanol (equivalent volume given in El. Cell cluster (arrowhead) is positive for type
I1 collagen. D: Phase contrast of C. E: Culture treated with 10 ngiml
all-trans retinol. Cell cluster (location indicated by arrowhead) has a
reduced staining intensity when compared to A and C . F: Phase contrast of E. A-F, x 146.
6-sulfate to chondroitin-4-sulfate to be approximately
1:3 in both untreated and retinol-treated cultures. The
proteoglycan composition was identical in both the media and agarose.
aggregate into the precartilage blastema and in the
second phase, a cartilage matrix is elaborated which is
replaced by a calcified bone matrix during endochrondral bone formation. Possible mechanisms for the teratogenesis of retinoids may relate to defects in the
formation of the precartilage condensation and/or subsequent defects in chondrocyte growth and elaboration
of matrix and/or enhanced matrix degradation.
DISCUSSION
Appendicular skeletal development may be divided
into two phases. In the first phase, mesenchymal cells
58
A.L. AULTH()USE ET AL.
Sapharose 2B-CL
i
VO
2oT
i . O091
16
KO,=
0-0
0
10
0.4
10 ng Rol
20
30
40
50
70
60
80
90
100
Ffaction weight (9)
A
Sephocryl S-300
7
4
0-0
E
KO,=
0 ng Rol
@-0 10 ng
0.3
Rol
*
2t
@C)
&
1
@
&
0
-
0
10
20
30
40
50
60
70
80
I
90
100
Fraction weight (4)
6
Fig. 5. Size exclusion chromatography of Na2?3O, labeled proteoglycans extracted from human chondrocyte cultures. Untreated cultures, 0 ng ROL (open circles), and cultures treated with retinol for 3
weeks, 10 ng ROL (solid circles). There were no differences between
untreated and ethanol-treated cultures. In addition there were no
differences between proteoglycans obtained from the media and the
agarose. The graphs show the proteoglycans extracted from agarose.
A: The hydrodynamic size of proteoglycan monomers is the same in
both untreated and retinol-treated cultures. There are less labeled
proteoglycans with retinol treatment. B: The size of the glycosaminoglycan side chains of the retinol-treated cultures is the same as in the
untreated cultures.
Animal studies using mice and hamsters have indicated that retinoic acid induced teratogenesis occurs
during organgogenesis suggesting that blastema formation is unaffected (Pillans et al., 1988; Willhite e t
al., 1986). Similarly, evaluation of clinical cases demonstrates that in humans teratogenesis occurs in organogenesis and not in initial stages of blastema formation (Willhite et al., 1986). The possible degradative
effects of retinoids on cartilage matrix have also been
evaluated. Initial investigations suggested that retinoids caused degradation of cartilage matrix (McElligott, 1962) probably through lysosomal proteolytic enzymes (Fell and Dingle, 1963; Lucy et al., 1961).
However, i t has since been shown using membrane stabilizers and proteolytic inhibitors that retinoids acted
by interfering with matrix synthesis (Shapiro and
Poon, 1976; Solursh and Meier, 1973).
Recently, in vitro studies have shown that retinoids
may cause a decrease in andlor a modification of the
synthesis of cartilage specific matrix macromolecules.
In other animal studies, a decrease in proteoglycan
synthesis was reported in chick sternal chondrocytes
(Horton e t al., 1987; Solursh and Meier, 1973) and also
in rabbit articular chondrocytes (Benya and Padilla,
1986). Similarly, in cultured rabbit chondrocytes type
I1 collagen synthesis decreased while type 111collagen
and fibronectin synthesis increased (Benya and Padilla, 1986). Type I1 collagen synthesis decreased while
fibronectin and type I collagen increased in chick cultured chondrocytes (Horton et al., 1987). In addition,
Horton et al. (1987) reported that alterations in the
matrix components seen in cultured chick sternal
chondrocytes were correlated with changes in specific
mRNA transcripts. Nuclear runoff experiments indicated changes in the transcriptional activities of the
collagen I1 and collagen I11 genes following retinoic
acid treatment. From these data it was surmized that
retinoic acid produces a change in chondrocyte phenotype by altering the pattern of gene expression.
Our data support the findings of previous in vitro
animal studies which concluded that chondrocytes exposed to retinol express a hybrid phenotype. The cartilage specific matrix macromolecules are expressed in a
reduced fashion while type I collagen synthesis was
enhanced. The expression of type I collagen suggests
that the cells may be maintained in a modulated phenotype. However, the continued synthesis of cartilage
specific proteoglycans and type I1 collagen by these
chondrocytes suggests that the expression of a cartilage specific matrix is possible. It has been shown by
von der Mark e t al. (1977) that both types I and I1
collagen may be expressed simultaneously. It is noteworthy in our system that a n alcian blue staining matrix was associated with cells t h a t stained positive for
type I procollagen or type I1 collagen.
Some in vitro studies have indicated that retinoids
maintain chondrocytes in a mesenchymal-like state.
Alcian blue staining nodules are not produced in micromass cultures from chick (Hassell et al., 1987; Pacifici et al., 1980), mouse (Hassell et al., 1978; Lewis et
al., 1987; Pennypacker et al., 19781, or rat (Gallandre
and Kistler, 1980) limb buds and the cells are maintained in a mesenchymal morphology, i.e., flat and
stellate. It has been further demonstrated that retinoic
acid maintains the synthesis of the mesenchymal glycoprotein, fibronectin, in both chick (Hassell et al.,
1987) and mouse (Lewis et al., 1987) chondrocyte cultures. This does not seem to be the case with human
chondrocytes cultured under these conditions because
of the continued production of cartilage specific matrices.
It is possible that this alteration of matrix (reduced
cartilage specific matrix macromolecules and increased
type I collagen) coupled with a decrease in the size and
number of isogenous groups may modify the establishment of a normal cartilaginous template. This culture
system may serve as a model system to further evaluate the importance of cartilage in appendicular skele-
INFLUENCE OF RETINOL ON HUMAN CHONDROCYTES
togenesis. Indeed retinol and other retinoids may elicit
limb malformations by interfering with this early
stage in the development of the appendicular skeleton.
ACKNOWLEDGMENTS
We thank Dr. Sarah Johnson and colleagues for their
help in acquiring tissue, Mr. Ben Han for photographic
assistance, and Dr. Daniel L. Feeback for editorial comments. This research is supported in part by research
grants; OCAST grant HN9-019, OU College of Medicine Alumni Association, Presbyterian Harris Research Foundation, and Presbyterian Health Foundation #85.
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1986 Characterization of the antibody response in mice with type
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Horton, W.E., Y. Yamada, and J.R. Hassell 1987 Retinoic acid rapidly
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