low medium calcium content promotes chondrocyte phenotype in

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low medium calcium content promotes chondrocyte phenotype in
LOW MEDIUM CALCIUM CONTENT PROMOTES CHONDROCYTE PHENOTYPE IN SUSPENSION AND
MONOLAYER CULTURE
+*Gigout, A; *Buschmann, M D;*Jolicoeur, M
+* Ecole Polytechnique, Montreal, QC, Canada
[email protected]
METHODS: Culture media. Cells were cultured in a serum-free
medium, composed of a 1/1 (v/v) mix of Calcium-free HAM’s F12 and
Calcium-free DMEM Low Glucose, supplemented with proline,
glutamine, non-essential amino acids, HEPES, ITS+ (10 g/mL insulin,
5.5 g/mL transferrin, 0.05% BSA, 1.7 mM linoleic acid, 0.5 g/mL
sodium selenite), 10-8 M dexamethasone, 5 10-5M -mercaptoethanol,
fresh ascorbate at 30 g/mL and EGF, PDGF-BB, FGF-2 all 2 ng/mL.
To this basal medium were added different amounts of CaCl2 to obtain
media containing 0 µM, 1 µM, 5µM, 50 µM and 1 mM of Ca2+. Cell
isolation and culture. Cells were isolated from the femoropatellar
groove of a 1-2 months calf knee using sequential digestion with
protease Type XIV and Collagenase CLS2. Released cells were then
filtered and seeded at low density (1.25 104 cells/cm2 hence 2.3 105
cells/dish) in the different culture media, in monolayer or in suspension.
For suspension culture Petri dishes were previously coated with 2%
agarose to prevent cell adhesion. Half of the media volume was changed
every two days. mRNA isolation, reverse transcription and RealTimePCR. Total RNA was isolated in Trizol® and was reverse-transcribed.
Real-Time PCR was done in the RotorGene 3000 (Corbett Research)
where cDNAs coding for collagen type I and type II were amplified in
the presence of SYBRGreen. Fluorescence confocal microscopy. Cells
were fixed/permeabilised in 0.5% glutaraldehyde containing 0.3% Triton
X100 and auto fluorescence reduced with NaBH4 followed by digestion
with chondroitinase ABC and keratanase. Samples were then stained for
actin with Alexa Fluor® 488 phalloidin, and for the nucleus with
Hoechst 33258. Finally, samples were treated against quenching with
catalase and glucose oxydase and mounted in Mowiol 4-88 and observed
with a Zeiss LSM510 confocal microscope.
RESULTS AND DISCUSSION: Our results corroborated the
hypothesis that low calcium helps to maintain chondrocyte phenotype,
even in monolayer culture where decreasing calcium concentration
reduced the ratio of collagen type I to collagen type II mRNA levels
(Fig. 1). At 0 M Ca2+, collagen type I was no longer detectable in our
PCR conditions while collagen II was still detectable early in the PCR
reaction. At 1 mM Ca2+, the collagen type I/II mRNA ratio was lower for
suspension cells than those in monolayer, but collagen type I mRNA
was still detected in suspension. Lowering the calcium content to 5 µM
and below in suspension decreased type I collagen mRNA to
undetectable levels. Importantly, at 5, 1 or 0 M Ca2+, chondrocytes do
not dedifferentiate and were still able to proliferate, albeit at a lower
level than at higher Ca2+concentrations (data not shown).
0.10
Collagen I/collagen II
INTRODUCTION: Calcium plays a crucial role in chondrocyte
differentiation for example in the transformation of hyaline to
hypertrophic cartilage [1], where a high extracellular calcium
concentration increases expression of markers of terminal chondrocyte
differentiation [2]. On the other hand, mRNA levels of aggregan and
type II collagen were found to decrease with increasing calcium (from
0.4 mM to 6 mM) in a chondrocyte cell line RCJ3.1C5.18 [3]. Thus low
calcium appears to influence chondrocyte matrix synthesis and
phenotype. Monolayer culture of chondrocytes is known to induce their
dedifferentiation [4, 5] where cells acquire a fibroblastic morphology
and increase collagen type I expression relative to collage type II.
Therefore, culturing chondrocytes in monolayer in a low calcium
environment could help to maintain the chondrocytic phenotype. To date
however, the lowest Ca2+ concentration studied was 0.1 mM. Here we
propose to examine yet lower concentrations, down to 1 M and to
observe the influence of low extracellular calcium on chondrocyte
morphology and gene expression in monolayer and in suspension
culture, the latter where chondrocytes are known to maintain their
phenotype [6]. We also expected that low calcium levels would lower
cell aggregation in suspension as found with other cell types [7]. We
therefore hypothesized that low calcium levels in medium would: i)
promote collagen type II expression over type I; ii) affect cell
morphology in monolayer; iii) reduce cell aggregation in suspension.
0.08
0.06
0.04
Non-detectable
collagen type I
0.02
0.00
1mM 50 M 1 M 0 M
2+
Ca in suspension
1mM 50 M 1 M 0 M
2+
Ca in monolayer
FIGURE 1. Ratio of collagen type I and collagen type II mRNA
expression. Mean ± SD.
1mM Ca2+
1 M Ca2+
Monolayer
culture
1mM Ca2+
1 M Ca2+
Suspension
culture
FIGURE 2. Chondrocytes stained for the nucleus (in blue) and actin (in
green), after 10 days. Scale bar = 50 m.
Cell morphology was greatly influenced by calcium concentration (Fig
2). In monolayer with 1 mM Ca2+, most cells were elongated with a
fibroblastic morphology and the actin cytoskeleton displayed an
abundance of stress fibers indicating cell attachment points to the Petri
dish surface. In contrast, at 1 M Ca2+in monolayer culture, relatively
few cells were fibroblastic and cells presented far fewer stress fibers. In
suspension, we found that chondrocytes quickly formed aggregates and,
surprisingly, low calcium content did not seem to inhibit this
aggregation. In suspension, cells were found to be tightly packed in
these aggregates, their close proximity suggesting the presence of cellcell contacts. Chondrocytes therefore most probably use calciumindependent adhesion molecules in this aggregation process, in
particular N-CAM (calcium-independent adhesion molecules) [8].
REFERENCES :[1]Reginato et al, 1993, Dev. Dyn., 198, 284-295 [2]
Chang et al., 2002, Endocrinology, 143, 1467-1474 [3] Chang et al.,
1999, Endocrinology, 140, 1911-919 [4]Elima and Vurio, 1989, FEBS
Letters, 258, 195-198 [5] Sailor et al., 1996, JOR, 14, 937-945 [6]
Castagnola et al., 1988, J. Cell Biol., 106, 461-467 [7] Madhusudan et
al., 1993, Biotechnol. Bioeng., 179-187 [8] Tavella et al., 1994, Exp.
Cell Res., 215, 354-362.
51st Annual Meeting of the Orthopaedic Research Society
Poster No: 1441

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