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Prolonged exposure of human chondrocytes to ascorbic acid modifies cellular behavior in an agarose gel.

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THE ANATOMICAL RECORD 238:31-37 (1994)
Prolonged Exposure of Human Chondrocytes to Ascorbic Acid
Modifies Cellular Behavior in an Agarose Gel
Department of Anatomical Sciences, University of Oklahoma Health Sciences Center,
College of Medicine BMSB 553, Oklahoma City, Oklahoma 73190
Using an agarose gel culture system, the response of adult
human chondrocytes to prolonged exposure of ascorbic acid was evaluated
using histochemical, immunocytochemical and morphological techniques.
The response of these cells to ascorbic acid was different from those previously reported in the literature. Many chondrocytes branched within the
agarose gel with continued exposure to ascorbic acid while other chondrocytes maintained a round configuration typical of chondrocytes in vivo.
Fibronectin and type I collagen were closely associated with the cell processes of the branching cells. Type I1 collagen and an alcian blue-staining
matrix were associated with the rounded cells but not with the branched
cells. These data suggest that the chondrocytes are able to express both
dedifferentiated and redifferentiated phenotypes with ascorbic acid under
these culture conditions. In addition, human chondrocytes were cultured in
a collagen gel and began branching within 1 hour of culture. It is possible
that an accumulation of type I collagen in the pericellular matrix of ascorbic acid treated cultures may enhance and explain the branching seen in
these cultures. Studies by others have indicated that ascorbic acid may
enhance, reduce, and/or modify the cartilage matrices produced by chondrocytes. These controversial reports in the literature are presumably due
to variations between species and the culture methods employed.
0 1994 Wiley-Liss, Inc.
Key words: Chondrocytes, Tissue culture, In Vitro
Numerous in vitro studies have addressed the role of in monolayer-cultured bovine and chicken chondroascorbic acid on typical hyaline cartilage matrix syn- cytes treated with ascorbic acid (Daniel et al., 1984;
thesis. Although various animal chondrocytes have Levenson, 1969). Proteoglycan synthesis was enhanced
been cultured in the presence of ascorbic acid, it is not in bovine articular chondrocytes cultured in agarose
surprising that the response of cells varies with the (Aydelotte e t al., 1986) and reduced when chick sternal
species and culture methods. In some instances carti- chondrocytes were cultured in agarose and treated
lage matrix production is enhanced or unchanged with ascorbic acid (Bounelis and Daniel, 1983). In adwhile in others it is modified andlor reduced. Ascorbic dition, preliminary observations from this laboratory
acid caused a switch from type I1 collagen to type I (Aulthouse, 1991) have suggested that alcian blue
collagen in bovine and rabbit chondrocytes cultured in stained proteoglycans were reduced when human cosmonolayer (Daniel et al., 1984; Sandell and Daniel, tochondral chondrocytes, cultured in agarose, were ex1988; Layman et al., 1972). Sandell and Daniel (1988) posed to ascorbic acid. In light of these contradictory
also reported that collagen synthesis of chick sternal findings, it was important to extend the preliminary
chondrocytes, cultured in monolayer, did not switch studies to determine what effect ascorbic acid had on
phenotypes. Ascorbic acid has been shown to be essen- cartilage matrix synthesis of human chondrocytes cultial during mouse chondrogenesis in vitro and was re- tured in a three dimensional agarose gel.
ported, by Dozin et al. (1992), to cause mouse embryMATERIALS AND METHODS
onic chondrocytes to express type I1 collagen in liquid
suspension culture only in the presence of ascorbic
1. Cell Culture
Human costochondral cartilage was obtained at auWith respect to proteoglycan synthesis, several stud- topsy and individuals with recognized skeletal abnories utilizing rabbit chondrocytes cultured in monolayer malities were excluded. Twelve cases ranging in age
have indicated that ascorbic acid enhanced the production and accumulation of cartilage specific proteoglycans pericellularly (Wright et al., 1988; Jouis e t al.,
1985; McDevitt et al., 1988). This pericellular accumuReceived May 11, 1993; accepted August 5, 1993
lation of proteoglycans and collagen was also observed
from one day to six years were evaluated. Primary cultures were established as previously described (Aulthouse et al., 1989). In brief, released chondrocytes were
plated in Dublecco’s Modified Eagle’s Media, (DMEM)
containing 4.5 g/L glucose, (Gibco, Grand Island, NY)
with 10% fetal calf serum, FCS (Irvine Scientific, Santa
Ana, CA), 1% L-glutamine (Irvine Scientific, Santa
Ana, CA), and with 0.1% penicillin-streptomycin
(Gibco, Grand Island, NY), in 24 multiwell tissue culture dishes (Corning, Corning, NY). At confluence, primary cultures were passaged to tissue culture flasks.
Cells were rinsed twice with Hanks’ balanced salt solution (HBSS) (Irvine Scientific, Santa Ana, CA) and
dissociated in 0.2% trypsin (Gibco, Grand Island, NY)
with 5 mM ethylenediamine-tetraacetic acid (EDTA)
(Sigma Chemical Co., St. Louis, MO). The cells were
then resuspended in 4 ml of media and passaged to 25
cm2 tissue culture flasks. Cells were passaged one to
five times.
cian 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% ETOH
with 3% acetic acid and finally 70% ETOH (Aydelotte
et al., 1986).
111. Quantificationof Cell Clusters as an
Expression of Chondrogenesis
Alcian blue stained whole cultures were photographed with a n IMT-2 Olympus inverted microscope
(EA4 objective). All single cells and cell clusters associated with alcian blue staining were counted. Using
the Crunch Statistical program (Crunch Software Corporation, Oakland, CA) 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 treatment groups. Four different
cases were analyzed. Three companion cultures were
established for each treatment group. The treatment
groups included cultures that were fed twice weekly
and served as untreated normal controls, cultures
treated daily with 50 pg/ml ascorbic acid, and cultures
with no treatment but had daily complete media
Cells in agarose
Cells were dissociated from monolayer, centrifuged,
resuspended, and maintained in 5 ml of complete
DMEM. The cell suspension was allowed to rest for 1h r
at 4°C and then filtered through 2 ply, No. 20, nitex
(Tetko Inc., Elmsford, NY). The cell suspension was
IV. Transmission Electron Microscopy
centrifuged and resuspended at 5 x lo5 cells in 1 ml of
were rinsed 3 times in PBS and fixed for 1
0.5% low temperature agarose (Biorad Cat#162-0017,
temperature in a mixture of 1.5% glutarRichmond, CA) in DMEM. A 10 pl drop of cell suspension was plated onto 35 mm tissue culture dishes aldehyde and 1.5%paraformaldehyde in 0.1 M sodium
(Corning) that were coated with 1% high temperature cacodylate buffer, pH 7.2, with 0.15% ruthenium red.
agarose (Biorad cat# 162-0100, Richmond, CA). The Cultures were rinsed with 0.1 M sodium cacodylate
cultures were subsequently refrigerated at 4°C for 15 buffer with ruthenium red for two 15-min intervals and
min to allow the low temperature agarose to gel. Cul- without ruthenium red for 15 min and then overnight.
tures were treated daily with 50 pg/ml L-ascorbic acid After postfixation for 1 hour in 2% OsO, in 0.1 M so(Sigma Chemical Co., St. Louis, MO) with complete dium cacodylate buffer, the cultures were rinsed in the
media change. To evaluate the effects of daily media buffer and dehydrated in a graded series of ethanol.
change on chondrogenesis, parallel cultures were fed Specimens were then processed through propylene oxdaily by complete media change without ascorbic acid. ide-Epon 812 resin, embedded, and sectioned with a
In addition, untreated normal control cultures (fed Reichert-Jung Ultracut E microtome. Thin sections
twice weekly) were established. Cultures were evalu- were stained with 25% uranyl acetate in methanol and
2% Reynold’s lead citrate, and viewed with a Siemens
ated after 3 weeks.
102 transmission electron microscope.
Cells in collagen
V. lmmunocytochemistry Used to Identify
Cells were dissociated from monolayer culture and
Matrix Macromolecules
suspended in a type I collagen gel (Collaborative ReCultures were rinsed 3 times in PBS and fixed in
search) (1 mg/ml) at a final concentration of 5 x lo5
cells/ml. One hundred microliters of cell suspension two, 5 min changes of methano1:acetone (1:l) and airwere plated in 100 mm Corning tissue culture dishes dried overnight. After rehydration in PBS, the cultures
and incubated for 1 hour at 37°C and fed 2 ml of were incubated overnight a t 4°C in monoclonal antiDMEM. Cultures were observed and photographed a t 1 body to one of the following monoclonal antibodies
to 2 hours and at 5 days. Some cultures were digested [purchased from the Developmental Studies Hybridwith 0.1% collagenase (Worthington Biochemical oma Bank, University of Iowa, Iowa City, IA]: human
Cooporation) for 15 min at 37°C. The released chondro- type I1 collagen (CIICI, undiluted, Holmdahl et al.,
cytes were subsequently resuspended in 0.5% low tem- 1986), human type I procollagen (M38, undiluted, Mcperature agarose as described previously. The agarose Donald et al., 1986) or human fibronectin (HFN 7.1,
cultures (5 x lo5 cells/ml) were maintained for 2 undiluted, Schoen et al., 1982). Cultures incubated in
weeks and treated with ascorbic acid a s described conditioned medium from the NS1 myeloma cell line
before fusion served a s negative controls. The cultures
were rinscd and incubated for 1 hour a t room temper11. Light Microscopic Detection of Cartilage Proteoglycans
ature in fluorescein-conjugated rabbit anti-mouse IgG
With Alcian Blue
(1:100, Cappel, Organon Teknika Corporation, West
Cultures were rinsed in phosphate buffered saline Chester, PA). After final rinsing, the cultures were
(PBS) and then simultaneously fixed and stained for 24 mounted in 80% glycerine in PBS and photographed
hours in a mixture of 2.5% glutaraldehyde, 0.05% al- through a n Olympus Vanox microscope (DPlanApo 20
UV PL Olympus objective) equipped for fluorescence
Human costochondral chondrocytes were cultured
for 1to 3 weeks in a n agarose gel and treated daily by
complete media change with 50 pg/ml ascorbic acid or
0 p,g/ml ascorbic acid (complete media change without
treatment). Some cultures not treated with ascorbic
acid were fed twice weekly and served a s untreated
normal controls. There were no differences between
cultures fed by complete media change daily or fed by
complete media change twice weekly.
Cultures from both feeding schedules (twice weekly
or daily) contained numerous clusters of isogenous cells
that were surrounded with alcian blue-staining matrices. An alcian blue-staining matrix was first associated
with several single cells at 1 week and, by 3 weeks of
culture, numerous chondrogenic clusters were surrounded by a n alcian blue-staining matrix (Figs.
lA,B). There was no significant difference in the number of clusters between these 2 treatment groups. In
contrast, cultures that had been treated daily with 50
pg/ml ascorbic acid had a significant reduction of chondrogenic clusters when compared to cultures not
treated with ascorbic acid (Fig. 2). Moreover, single
cells with or without a n alcian blue-staining matrix
were predominant in these cultures. Chondrocytes
grown for 3 weeks in agarose with daily (Fig. 1B) or
bi-weekly (Figs. lA, 3A) media changes showed no
morphological differences. In both instances, the cells
retained their typically round chondrocyte-like appearance. However, cultures treated daily with ascorbic
acid contained cells with extended cell processes (Figs.
lC, 3B). This change in cell morphology was not reversible. Once branching was established, continued
culture in the absence of ascorbic acid did not cause the
cells to return to the rounded state. With stereomicroscopy and Hoffman modulation contrast optics, i t was
possible to determine that the cells were branching
within the agarose and not spreading onto the plastic
surface of the tissue culture dish.
Typically, fibronectin and type I collagen are not associated with adult human chondrocytes cultured for 3
weeks in agarose, however, immunocytochemistry revealed that both fibronectin (Fig. 4A,B) and type I
procollagen (Fig. 4C,D) were associated with these
branches. Branching cells occasionally were seen wrapping around alcian blue-staining clusters (Fig. 3B).
Type I procollagen and fibronection were found in the
branching cells but not in the central groups staining
with alcian blue (Fig. 4A,C). Round cells not labeled
with fibronectin or type I procollagen stained positive
for type I1 collagen (data not shown). These data demonstrate that both the dedifferentiated and differentiated chondrocyte phenotype were present in cultures
treated with ascorbic acid. Numerous forms of cell
branching were apparent, ranging from single to
multibranched. Cell processes were closely associated
with bundles of thick-banded collagen which accumulated over time (Fig. 5). Cells not treated with ascorbic
acid lacked thick-banded collagen pericellularly, although delicate collagen fibrils were apparent along
with proteoglycans. These observations are consistent
Fig. 1. Human chondrocytes cultured for 3 weeks in an agarose gel
stained with alcian blue. A: Untreated normal control culture. B
Culture treated daily with 0 pgiml ascorbic acid. C: Culture treated
daily with 50 pgiml ascorbic acid (original magnification, x 13.2).
with those previously reported by this laboratory
(Aulthouse et al., 1989).
When adult human chondrocytes were cultured in a
type I collagen gel, the cells did not maintain the round
configuration a s seen in agarose culture but were
branched. In many cells, branching was detected as
early as 1 hour after plating. Cell branching became
more extensive over time and, by 5 days, nearly all
9 8 0
-0 60
Fig. 2.The quantification of chondrogenic clusters as a n expression
of chondrogenic activity after 3 weeks exposure to ascorbic acid. Representative alcian blue stained cultures from each treatment group
were analyzed and quantitated. NC = untreated normal control cultures with media change twice weekly. 0 pgiml AA = control cultures
with daily media change receiving 0 pgiml ascorbic acid (AA). 50
pg/ml AA = cultures treated daily with 50 p g h l ascorbic acid. Values are mean 2 SEM. NC > 0 p d m l AA (p > 0.05, not significant);
NC > 50 pg/ml AA (P5 0.01); 0 pgiml AA > 50 pg/ml AA (P 5 0.01).
cells had a t least one cell process (Fig. 6). The typical
morphology of branching cells in the collagen lattice
was bipolar or multipolar. After culture for 5 days in a
type I collagen gel no alcian blue-staining material was
detected. Following release of the branched chondrocytes from the collagen gel with 0.1% collagenase, the
cells rounded up by retracting their processes. Subsequent culture of these cells in a n agarose gel (without
ascorbic acid treatment) allowed the cells to maintain
their round configuration. After 2 to 3 weeks these cells
had reexpressed the typical cartilage matrix (type I1
collagen and proteoglycans) as detected by immunocytochemistry and staining with alcian blue (data not
shown). However, when chondrocytes were released
from collagen gels and cultured in a n agarose gel, then
treated daily with 50 pg/ml ascorbic acid, the branching pattern was re-established.
Fig. 3. Micrograph of representative cells from 3 week alcian blue
stained agarose cultures, A: Untreated normal control cultures. Note
that the cells are round and that a n alcian blue-staining matrix is
seen surrounding the cell clusters. B Culture treated daily with 50
Previous observations published from this laboratory
(Aulthouse, 1991) indicate that daily treatment of human chondrocytes with 50 pg/ml ascorbic acid reduces
the production of alcian blue-staining proteoglycans.
The present study further characterizes the response of
human chondrocytes to ascorbic acid when cultured in
agarose for 3 weeks. We found that cultures treated
daily with 50 pg/ml ascorbic acid had a significant reduction in chondrogenic isogenous cell nests. Moreover,
single cells predominated in the treated cultures, suggesting a decrease in mitotic activity. In addition, the
morphology of the cells changed with ascorbic acid
Typically, chondrocytes grown in agarose are round.
However, daily exposure to ascorbic acid caused many
cells to branch and extend cell processes. It is apparent
that treatmentwith ascorbic acid and not daily media
changes caused these modifications in
ImmunocYtochemistrY revealed t h a t both fibronectin
and type I collagen were associated with the cell processes. These matrices are typical of human chondrocytes grown in monolayer implying a dedifferentiated
or fibroblast-like phenotype. Statistical analysis
showed a significant decrease in chondrogenic activity,
reduction of clusters, in cultures treated with ascorbic
acid when compared to untreated controls. Moreover,
human fibroblasts cultured under the same conditions,
in agarose with daily ascorbic acid treatment, do not
branch (data not shown). Therefore, it is unlikely that
there is a contaminating population of fibroblasts
within the agarose culture which were stimulated to
branch with ascorbic acid treatment.
Ascorbic acid has been associated with phenotypic
switching of cultured cells. Layman e t al. (1972) reported that when rabbit chondrocytes are cultured in
the presence of ascorbic acid they switched phenotype
by producing collagen characteristic of skin and bone.
Ascorbic acid has also been found to facilitate phenotype switching or transdifferentiation in other cells beside chondrocytes. For example, Itoh (1976) showed
that cultured neural retinal cells of chick embryos dif-
pgiml of ascorbic acid. Note that cell branching is obvious and in some
instances the cell processes are wrapping around the alcian blue
stained matrix (original magnification, X 66).
Fig. 4. Immunofluorescent detection of fibronectin and type I procollagen in human chondrocytes cultured in agarose and treated daily
with 50 pg/ml ascorbic acid for 3 weeks. A: Fibronectin is associated
with the cell processes of branching cells and is lacking in the rounded
cells. B Phase contrast of A. C: Type I procollagen is associated with
the cell processes of branching cells and is lacking in the rounded
cells. D Phase contrast of C (original magnification, x 50).
ferentiated into lentoid bodies and pigmented cells in
the presence ascorbic acid. Similary, Itoh and Eguchi
(1986) reported that in certain culture conditions including ascorbic acid, chick pigmented epithelial cells
transdifferentiated into lens cells.
It is unlikely that ascorbic acid is causing the cells to
completely dedifferentiate or switch phenotype because
they continue to produce cartilage specific matrices,
type I1 collagen and alcian blue-staining proteoglycans. This simultaneous production of both types I and
I1 collagen is similar to that seen with cultured bovine
chondrocytes treated with ascorbic acid (Daniel et al.,
1984; Sandell and Daniel, 1988). However, it was reported that while type I1 collagen was deposited in the
pericellular matrix, both types I and I11 collagen were
detected only in the culture media. This differs from
the results of the current study in that type I collagen
was closely associated with the branching cells. This
suggests that the agarose may be facilitating the interaction and entrapment of the extracellular matrices.
Indeed, it has been reported by others that ascorbic
acid treatment enhanced a pericellular accumulation
of matrices (Jouis et al., 1985; McDevitt et al., 1988;
Wright et al., 1988). It is interesting that an alcian
blue-staining matrix, i.e., proteoglycan presence, is not
a requirement for cell branching since single cells
without alcian blue halos are also branched.
It is possible that the production and accumulation of
type I collagen in the pericellular matrix may facilitate
cell branching. It is interesting to note that adult human chondrocytes cultured in a collagen gel begin to
branch as early as 1hour in culture, and that culture in
a collagen gel did not appear to influence the re-expression of the chondrogenic phenotype when the cells
were further cultured in an agarose gel. The behavior
of cells in the collagen gel was reminiscent of that seen
in monolayer. Nonetheless, the culture of chicken limb
bud mesenchymal cells in a collagen gel supports chondrogenesis. Solursh and coworkers (1982) have shown
that single mesenchymal cells maintain a rounded configuration and produce type I1 collagen and pericellular
alcian blue-staining.
While numerous studies have been conducted to
evaluate the influence of ascorbic acid on the chondrogenic phenotype in culture, there have been no reports
of a modification of the cellular morphology in response
Fig. 5. Transmission electron micrograph of human chondrocytes
cultured in agarose and treated daily with 50 pg/ml ascorbic acid. A
Cell processes from chondrocytes cultured for 5 weeks. B: Cell pro-
cesses from chondrocytes cultured for 10 weeks. Note that the collagen is closely associated with the cell processes and is thick and
banded (original magnification, x 8,000).
to ascorbic acid treatment. It seems plausible that variations between species combined with numerous culture techniques may explain the disparity in the literature regarding the effects of ascorbic acid on
chondrocytes. It is reasonable to suggest that, as observed with the bovine chondrocytes, ascorbic acid is
stimulating both types I and I1 collagen synthesis in
the human chondrocyte cultured in agarose. This
would account for the simultaneous expression of both
the differentiated and dedifferentiated phenotypes.
Moreover, the production and accumulation of type I
collagen would facilitate and explain the branching of
cells as seen in both the cells grown in agarose with
ascorbic acid treatment and the cells grown in a collagen gel.
Special thanks to Dr. Sarah Johnson and colleagues
for their help in acquiring tissue; Mr. Tod Dow, Mr.
Melville Vaughan, Ms. Nancy Halliday, and Dr. James
Tomasek for technical assistance; Mr. Ben Han for photographic assistance; Mr. John Nordquist for assistance with transmission electron microscopy; and Dr.
Jane M. Jacob for editorial comments. This research is
supported in part by research grants; OCAST grant
Fig. 6. Human chondrocytes cultured in a collagen gel were photographed at 2 hours (A) and a t 5 days
(B). At 2 hours numerous cells have extended cell processes and by 5 days most chondrocytes have a
bipolar or muitibranched morphology (original magnification. X 66).
HN9-019, Presbyterian Harris Research Foundation,
and Presbyterian Health Foundation #85.
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